BACKGROUND
over the past few decades, many neuropeptides have been identified in the neural and neuroendocrine structures of both vertebrate and invertebrate species  <cit> .
in vertebrates, more than 50 % of the identified neuropeptides and peptide hormones are amidated, whereas more than 90 % of bioactive peptides in the brains of invertebrates  are amidated  <cit> .
most neuropeptides in tissues require post-translational modifications, specifically the α-amidation of the carboxyl group of the c-terminal amino acid.
such amidation represents an essential step for peptide hormones to acquire complete biological activity, receptor recognition, and signal transduction at target cells  <cit> .
c-terminal amidation of peptides, a necessary step for the conversion of prohormones into functional products, is mediated by the activity of the peptidylglycine α-amidating monooxygenase , a type i membrane protein that is localized to the trans-golgi network and to secretory granules in neural and endocrine tissues  <cit> .
pam is a bifunctional enzyme containing two specific domains:  peptidylglycine α-hydroxylating monooxygenase   <cit>  and  peptidyl-α-hydroxyglycine α-amidating lyase   <cit> .
the cellular bioactivity of pam depends on ascorbic acid, copper, and molecular oxygen  <cit> .
mice lacking pam do not survive beyond mid-gestation  <cit> .
both of its enzymes act sequentially to generate an active α-amidated peptide product and glyoxylate   <cit> .fig.
<dig> schematic representation of the α-amidation process in cells.
the α-amidation process is a highly specific enzyme-dependent post-translational modification need for converting prohormones into functional peptide products in neurosecretory cells.
panel a depicts the specific processing of large propeptide precursor proteins containing active peptide sequences  via pc1/pc <dig> proconvertase activity alone  or together with the functional activity peptidylglycine α-amidating monooxygenase  .
panel b illustrates the sequential steps of pam-related bifunctional activity  which includes the peptidylglycine α-hydroxylating monooxygenase  activity depicted in step  <dig> and the peptidyl-α-hydroxyglycine-α-amidating lyase  activity depicted in step  <dig>  as shown, two molecules of ascorbate are oxidized to form two molecules of semidehydroascorbate during the reduction of two enzyme-bound cu atoms  into two enzyme-bound cu atoms  as previously described  <cit>
although a substantial amount of data is available on the reaction mechanism of phm  and its relevance to physiology   <cit>  and medicine   <cit> , few details concerning the activity and relevance of the pal domain of pam have been reported  <cit> .
phm is a di-copper, ascorbate-dependent monooxygenase that catalyzes the stereospecific α-hydroxylation of a glycine -extended precursor peptide by molecular oxygen   <cit> ; whereas pal catalyzes the n-dealkylation of the peptidyl-α-hydroxyglycine intermediate generated by phm  <cit> .
in vertebrate species , phm and pal are encoded by the same rna transcript  <cit> , whereas in non-vertebrate species , each domain appears to be encoded by a separate gene  <cit> .
although the α-amidation of almost all amino acids found in nature has been observed , α-amidation of active neuropeptides occurs most frequently on non-polar residues such as phenylalanine    <cit> , leucine    <cit> , methionine    <cit> , and glycine    <cit> .
in addition, amidation of some residues such as isoleucine  3/stresscopin) occurs rarely  <cit> .
peptides containing the α-amidated isoform of a valine residue represent the smallest group of functional amide neuropeptides displaying pleiotropic bioactivity  that have been identified in neural structures in both vertebrate and non-vertebrate species .
this group of neuropeptides includes distinct bioactive peptide hormones such as, α-msh  <cit> , metorphamide/adenorphin  <cit> , secretin  <cit> , urocortins   <cit>  and enterins  <cit> .
early research showed that small and large peptide molecules can be separated via reverse-phase liquid chromatography  based on the hydrophobicity of the peptide structure and its amino acid composition  <cit> .
this separation can be followed by sequence analysis of dansylated amino acids via the edman technique using dansyl chloride as the reagent to label amino acid residues  <cit> .
these studies led to the discovery of two amidated opioid peptides, adrenorphin and amidorphin  <cit> .
with the advent of recombinant dna technology and cloning procedures  <cit>  together with proteomic analysis using mass spectrometry techniques   <cit> , the search for mrna transcripts and mrna-translated protein products has led to the identification of many functional peptides  <cit> .
in combination with mass spectrometry, monoclonal antibodies  became powerful biological tools used in clinical settings  <cit>  and animal studies  <cit>  to detect post-translational modifications of small molecules in tissues  <cit> .
in the present study, we used splenocyte/myeloma cell fusion technology to generate stable hybridomas secreting mabs against the structural α-amidated isoform of a valine residue with the goal of identifying and characterizing putative novel c-terminal valine amide neuropeptides in the mammalian cns and neurosecretory tissues, as well.
immunohistochemical  analyses showed that valine amidated neuropeptides, such as, α-msh  <cit> , metorphamide/adrenorphin  <cit> , secretin  <cit> , and ucn  <cit>  are widely distributed throughout the mammalian cns and neuroendocrine system  <cit> , including the immune system  <cit> .
in general, antibodies generated to characterize the anatomical distribution of valine-amide peptide immunoreactivity in the brain or neuroendocrine tissues required the synthesis of a fragment or peptide motif encoded along the complete amino acid sequence of the peptide of interest, and used then, as the antigenic epitope to which the antibody is targeted  <cit> .
to date, no ihc studies using specific antibodies raised against small isovariants of a single haptenic molecule, for instance, the free α-carboxylic acid or the structural α-amidated isoform of an amino acid residue  have been studied or reported.
thus, the present paper shows the identification of a single mab  that displays a high selectivity, sensitivity and specificity for the structural α-amidated isoform of the valine residue when tested against distinct amidated and non-amidated peptide antigens in different immunoassays.
moreover, this mab exhibited a great capability in detecting endogenous valine amide peptide immunoreactivity along the neuroaxis of the rat brain.
our data demonstrates the high versatility of this mab, which makes it an amazing biological “tool” for biomedical research and clinical settings.
materials and methods
peptide synthesis
synthetic peptides with the primary sequences -gly-gly-gly-val-conh <dig>  and -gly-gly-gly-val-coo−  were synthesized by peninsula labs .
both isovariants of the valine residue, the α-amidated  and the free α-carboxylic acid  isoforms, were covalently linked to the c-terminal domain of the peptide backbone and were supplied as lyophilized material at 98 % purity.
both isovariants of the valine residue were used as antigenic epitopes in synthetic peptide constructs and in naturally occurring bioactive peptides to determine the cross-reactivity of the antibodies to each epitope in specific immunoassays.
one crucial advantage of using these peptides in immunoassays is that the  residue at the n-terminal domain of these synthetic peptides may be used as a radiolabeled tracer in specific radioimmunoassays  when the peptides are labeled with  .
moreover, these short oligopeptide constructs, which were synthesized by condensing a triplet of glycine residues  into a peptide backbone, were designed to maintain optimal backbone flexibility, linear conformation, and solubility in aqueous solution, as has been shown for other polymeric structures containing a short linker backbone of glycine residues  <cit> .
immunoconjugates
the structural α-amidated isovariant of the valine residue  was covalently linked to keyhole limpet hemocyanin  according to standard coupling procedures as previously reported  <cit> , and this complex was used as an immunoconjugate for immunization  <cit> .
the free α-carboxylic acid isovariant of valine  , the synthetic peptides -gly2–4-val-conh <dig> and -gly2–4-val-coo−, the fibronectin/fibrin-related peptide rgdv-coo−, metorphamide/adrenorphin , and the wasp venom-derived ionophoric peptide mastoparan x   were similarly conjugated to bovine serum albumin  using  <dig>  % glutaraldehyde  as a cross-linker  <cit> .
the cross-linking reactions were terminated via the addition of  <dig>  m glycine  to the reaction mixture.
the immunoconjugates were dialyzed using a 12 kda cut-off nitrocellulose membrane  for 72 h against 10 l of phosphate-buffered saline   to remove non-reactive aldehydes.
finally, the each immunoconjugate was diluted to 1 mg/ml.
the purified immunoconjugates of bsa-linked peptide were frozen and stored at –20 °c for use as adsorbent antigens in elisas and dot-blot assays  <cit> .
animals
all animal studies and procedures were performed in accordance with the national institute of health guide for the care and use of laboratory animals  and were approved by the animal care and bioethics committee of the national institute of psychiatry, mexico city .
immunization
female balb/c mice  were purchased from charles river laboratories  and used for immunization and collection of specific antisera according to standard procedures as described previously  <cit> .
the animals, which were designated as r1–r <dig>  received an initial subcutaneous injection of  <dig>  ml of a 1: <dig> emulsion of  <dig> μg klh-val-conh <dig> immunoconjugate in  <dig>  ml of pbs : <dig>  ml of complete freund´s adjuvant .
three subcutaneous booster injections consisting of  <dig>  ml of a 1: <dig> emulsion of  <dig> μg klh-val-conh <dig> immunoconjugate in  <dig>  ml of pbs : <dig>  ml of incomplete freund’s adjuvant  were administered, followed by a final intraperitoneal booster injection of klh-val-conh <dig> in  <dig>  ml of pbs : <dig>  ml of incomplete freund’s adjuvant 30 days after the previous immunization.
blood  was collected from the tail vein of the mice every 2 weeks after each immunization and was centrifuged in an eppendorf 5804r centrifuge at 1200 rpm for 10 min at 4 °c.
the collected sera were stored at −20 °c until further use.
elisa for polyclonal antibodies
the titers of polyclonal valine amide antisera  from vaccinated mice were determined via standard antibody-capture elisa  <cit>  in microtiter 96-well elisa plates .
each well was coated with  <dig>  μg of bsa--gly2–4-val-conh <dig> or bsa-rgdv-coo− immunoconjugate in the presence of  <dig>  m sodium bicarbonate.
after overnight incubation, the coated wells were blocked with elisa buffer  for 2 h at room temperature , washed with elisa buffer, and incubated overnight at 4 °c in 50 μl of vaa diluted 1:10–1: <dig>  the wells were subsequently incubated for 2 h at rt in 50 μl of pbs  containing a 1: <dig> dilution of horseradish peroxidase -conjugated goat anti-mouse immunoglobulin g   and  <dig>  % tween- <dig>  color development was performed using the chromogenic reagent o-phenylenediamine dihydrochloride .
the plates were measured at 490 nm in an automated elisa plate reader .
preimmune sera from the mice were used as controls.
each assay was performed in triplicate.
vaccinated mice displaying antisera titers greater than 1: <dig> dilution whose sera yielded an a <dig> reading greater than two standard deviations  beyond the mean of the non-specific value obtained using preimmune serum were selected as candidates for cell fusion and for the generation of stable productive hybridomas.
cell fusion and generation of productive hybridomas
hyperimmune mice displaying high antisera titers based on elisa received an intraperitoneal pre-fusion booster of 50 μg of the klh-val-conh <dig> immunoconjugate in 200 μl of pbs  in the absence of adjuvant 7 days before lymphoid cells were harvested  <cit> .
mice were killed by cervical dislocation; splenocytes were collected under aseptic conditions  and were promptly fused with the murine myeloma sp2/ <dig> cells  using 50 % polyethylene glycol  <dig> /pbs  according to standard cell fusion protocols as described previously  <cit> .
the murine myeloma cell line sp2/ <dig> is a variant line derived from the fusion of mopc- <dig> and balb/c mouse splenocytes  <cit> ; it is frequently used for the generation of stable hybridomas due to its high growth rate, rapid replication rate, high capacity for cell fusion, and cloning efficiency  <cit> .
the fused cells were resuspended at a concentration of 2 ×  <dig> cells/ml in a selective enriched hybridoma medium .
the fused cells were seeded on a thymocyte feeder layer  in 20 × 96-well plates .
myeloma cells and hybridoma colonies displaying logarithmic growth to a density of 5–6 × 106 cells/ml were diluted to 1 × 105 cells/ml using cryopreservation medium  and stored at −196 °c under liquid nitrogen in cryogenic vials .
elisa for mabs
elisas were used to identify mabs against val-conh <dig> secreted by positive colonies using the same protocols as those described for the screening of polyclonal antisera from mice except that bsa-val-conh <dig>  bsa-metorphamide/adrenorphin, bsa-mastoparan-x, bsa-val-coo−, and bsa-rgdv-coo− were used as adsorbent antigens in the wells, that 50 μl of various dilutions  of the supernatants collected from hybridoma colonies were added to the coated wells, and that the plates were incubated overnight at 4 °c.
val-conh2-ir signal was detected at 490 nm in an automated elisa plate reader  using a 1: <dig> dilution of hrp-conjugated goat anti-mouse igg  and the chromogenic reagent opd .
val-conh <dig> mab specificity was determined based on the absence of cross-reactivity against the free α-carboxylic acid form of the valine residue  and/or against the c-terminal amide form of the leucine residue  in mastoparan x. preimmune sera from the mice were used as controls in all assays.
expansion of hybridomas
productive hybridomas secreting antibodies were recovered and subjected to expansion and subcloning procedures at limiting cell dilutions as previously described by hockfield et al.
<cit> .
isotyping
isotyping of the mabs was performed using an isoquick kit for mouse monoclonal isotyping .
dot–blot assays
dot–blot assays were used to assess the mab specificities against both isovariants of the valine residue in the tested antigens according to standard procedures described by hockfield et al.
<cit>  and adapted by loi et al.
<cit> .
briefly, both isovariants of the valine residue and synthetic bsa-conjugated peptides were tested as potential cross-reactive antigens.
immunoconjugates of the bsa-conjugated antigenic peptides tyr-gly2-4-val-conh <dig>  tyr-gly2-4-val-coo−, rgdv-coo−, metorphamide/adrenorphin, and mastoparan x  were diluted in 50 % methanol to obtain stock solutions at concentrations of 10−2–10−9 m. two microliters of each antigenic bsa-peptide immunoconjugate at 10−12–10−16 m were spotted onto nitrocellulose membranes  .
after spotting the peptides, the dried membranes were soaked in a preblocking solution containing 1 % teleostean gelatin , 10 mm pbs, and  <dig>  % tween- <dig>   for 1 h at rt.
the membranes were washed  <dig> times for 5 min with the preblocking solution and incubated overnight at 4 °c in the presence of a 1: <dig> dilution of hybridoma supernatants containing a mab in 10 mm pbs .
the membranes were then washed and incubated for 2 h at rt in a solution containing  <dig>  % tween- <dig> and 10 mm pbs  in the presence of hrp-conjugated goat anti-mouse igg  diluted 1: <dig> .
the membranes were washed  <dig> times for 5 min with  <dig>  % tween- <dig> in 10 mm pbs  to remove excess secondary antibody.
signal detection was performed using a chemiluminescence kit .
solid-phase ria
a solid-phase ria for the α-amidated isovariant of the valine residue was adapted from standard procedures described by hockfield et al.
<cit> .
briefly, the n-terminal  residue of the synthetic peptide tyr-gly2-4-val-conh <dig> was iodinated with  using the iodo-gen protein labeling procedure  according to the manufacturer’s protocol, and the peptide was purified via high-performance liquid chromatography using hypersil wide-pore 5-μm, c- <dig>  2 × 150 mm columns.
the solid-phase ria was prepared by loading 100 μl of blocking solution containing 500 ng protein a  and  <dig>  m nahco <dig>  in immunolon ii removable wells , followed by incubation overnight at 4 °c.
the coated wells were washed  <dig> times for 10 min with ria buffer .
fifty microliters of ria buffer containing a hybridoma supernatant at 1: <dig> dilution were added to the wells in quadruplicate, and the plates were incubated overnight at 4 °c .
after removing the antibody solution and washing with ria buffer, 50 μl of ria buffer containing competitive peptides  and the radiolabeled peptide tracer were applied to the adsorbed anti-val-conh <dig> mabs for 2 h at rt.
typical standard displacement curves were generated using approximately  <dig> c.p.m.
of the radiolabeled tracer -tyr-gly2-4-val-conh <dig> and the non-labeled peptides used in the competitive ria .
the treated wells were washed and counted for 4 min in a 10-channel gamma counter .
peptide fractions from neural and endocrine tissues
tissue extraction and preparation of whole peptide fractions from various neural and endocrine tissues were performed according to standard procedures and the protocols previously described by asai et al.
<cit> .
briefly, male wistar rats  pre-anesthetized with lethal doses of sodium pentobarbital  were decapitated, and the abdominal cavity was opened for surgical extraction of the pancreas and the adrenal glands.
rat brain regions such as the cortex, the hippocampus, the striatum, the hypothalamus, the thalamus, the cerebellum , the pituitary and the spinal cord and endocrine glands including the adrenal gland and the pancreas were rapidly dissected on ice.
the brain regions and the glands were promptly homogenized using a tissue homogenizer  at 800 rpm in an ice–cold acid:acetone solution  and centrifuged at  <dig> ×g for 1 h at 4 °c in a sorvall rc28s centrifuge .
the hcl:acetone solution was removed from the crude peptide fraction supernatants via vacuum evaporation .
the dried pellets were resuspended in 5 % tfa solution and loaded on sep-pak-rp c- <dig> columns   pre-activated with 100 % tfa for solid-phase peptide extraction.
the peptide fractions from tissue homogenates of the brain and the endocrine glands were eluted from the columns using 20 % tfa:h2o, dried via vacuum evaporation and stored at −20 °c.
the thawed pellets were resuspended in a solution containing 50 mm tris–hcl, ph  <dig> , and  <dig>  mm cacl <dig>  and 50-µl aliquots of ria buffer containing a 1:5–1: <dig> dilution of a purified peptide fraction were individually assessed for the p18c <dig> mab via solid-phase ria using the radioactively labeled peptide -tyr-gly2-4-val-conh <dig>  the labeled tracer was used to quantify the abundance of val-conh2-ir in the tissues.
peptide immunoreactivity in the samples is expressed as nmol/μg protein .
protein concentration determination
the protein concentrations in the peptide fractions extracted from the rat brain and neuroendocrine tissues were determined according to standard procedures using the micro-bca protein assay kit  based on the instructions provided by the manufacturer.
the absorbance at 570 nm was measured using a microwell plate reader .
the protein concentration of the samples was expressed as µg/ml .
tissue preparation
tissue preparation for ihc detection of val-conh2-ir was performed according to standard procedures previously described by anton et al.
<cit> .
briefly, six male wistar rats  were anesthetized with sodium pentobarbital   and transcardially perfused with 250 ml of 10 mm pbs /heparin  solution, followed by perfusion with 800 ml of 4 % paraformaldehyde/10 mm pbs  solution at 4 °c.
the flow rate was held constant  using a hamilton double-pumping system .
fixed tissues  were cryoprotected in 30 % sucrose/10 mm pbs  solution for 3 days at 4 °c.
the fixed brain tissues were sliced into 40-μm-thick sagittal sections using a cryostat ; alternatively, the fixed neuroendocrine tissues were sliced into 40-μm-thick coronal sections.
the sliced sections were collected in 12-well plates , allowed to float freely in 10 mm pbs / <dig>  % sodium azide solution, and stored at 4 °c until use.
storage for up to 1 month in the aforementioned solution does not result in loss of immunoreactivity  <cit> .
immunohistochemistry
ihc analysis of val-conh2-ir in brain and neuroendocrine tissue slices was performed according to standard procedures described previously  <cit>  with minor modifications.
briefly, both brain and neuroendocrine tissue sections were processed while free-floating and were initially washed in 10 mm pbs  followed by 10 % nabh <dig> in 10 mm pbs for 15 min.
after three additional 10-min washes in 10 mm pbs, the tissue sections were permeabilized with  <dig>  % tween- <dig> in 10 mm pbs for 20 min and washed with 10 mm pbs .
then, endogenous peroxidase activity was blocked for 40 min in a  <dig>  % hydrogen peroxidase/ <dig>  % tween-20/10 mm pbs  solution.
following three additional 10-min washes in 10 mm pbs, the sections were incubated for 4 h in a preblocking solution containing dialyzed 10 % horse serum, 1 % bsa,  <dig>  % tween- <dig>  and 10 mm pbs .
the sections were then incubated on an orbital shaker for 16 h at 4 °c in the primary valine amide mab p18c <dig> diluted 1: <dig> in the preblocking solution .
after  <dig> 10-min washes with 10 mm pbs, the sections were incubated for 2 h in a donkey anti-mouse biotinylated  secondary antibody  diluted 1: <dig> in pre-blocking solution .
after  <dig> and  <dig> 10-min washes with the preblocking solution and with  <dig>  % tween-20/10 mm pbs  solution, respectively, the sections were incubated in the avidin–biotin complex , followed by 310-min washes with  <dig>  % tween- <dig> in 10 mm pbs and by  <dig> 10-min washes with 10 mm pbs .
color development was enhanced by a 5–10 min incubation in a  <dig>  %  <dig> ′-diaminobenzidine tetrahydrochloride / <dig>  % h2o2/ <dig>  % nickel sulfate/10 mm pbs  solution.
the sections were then washed in 10 mm pbs, mounted on slides, and air-dried overnight.
the prepared slices were cleaned with xylene and cover-slipped with entellan .
microscopic analysis of the brain sections was performed under bright-field illumination using a das leica dmr qwin microscope.
positive immunoreactivity detected by the pc18c <dig> mab in tissue slices is referred as valine amide-like immunoreactivity .
the neuroanatomical areas displaying positive val-conh2-ir signal were identified according to the atlas of the rat brain by paxinos and watson   <cit> .
controls
as a control of labeling specificity, adjacent slices from rat neuroendocrine tissues and brain sections were preabsorbed with 10 μm of the valine amide residue  .
the intensity and the density of positive pc18c <dig> mab-immunoreactive signal in the brain were compared to those of val-conh2-ir detected in the pituitary and adrenal glands as shown in fig.
2a–c.
both tissues were as used internal standards, and the immunoreactive signals were arbitrarily graded using a similar grading scale to the scale used to map the distribution of orl- <dig> receptor immunoreactivity in the rat brain  <cit>  as follows: very intense ; intense ; moderate ; low ; and not detected .fig.
<dig> pre-absorption control assays of valine amide immunoreactivity in tissues.
the panels show bright-field photomicrographs  of val-conh2–ir in neuroendocrine glands and sagittal sections of the rat brain using the specific pc18c <dig> mab.
panel a illustrates the expression of val-conh2–ir in the pituitary gland before  and after pre-absorption with 10 μm of the isovariant of the valine residue  .
note the absence of pc18c <dig> mab-ir in the tissue after treatment with the val-conh <dig> residue.
nl neural lobe, il intermediate lobe, al anterior lobe .
panel b illustrates the expression of val-conh2-ir in the adrenal gland before  and after pre-absorption with 10 μm of the isovariant val-conh <dig> .
pc18c <dig> mab-ir was localized to the core region of the adrenal gland  .
panel c shows the expression of val-conh2-ir in a representative sagittal section of the rat brain before  and after pre-absorption with 10 μm of the isovariant val-conh <dig> .
the intensity of immunoreactive signals was arbitrarily graded using a scale previously described in  <cit>  .
scale bar = ×40
subtraction analysis
immunoreactive signals produced from peptide antibodies used to map the anatomical distribution of c-terminal valine amide neuropeptides  in the rat cns and neuroendocrine tissues were used as a reference to subtract from pc18c <dig> mab-ir signals detected in the rat cns.
positive pc18c <dig> mab-ir signals in brain areas that did not match with previously identified c-terminal amide–peptide immunoreactivity, were considered as potential brain areas expressing putative novel valine amide neuropeptides.
positive val-conh2-ir signals detected in subtracted brain regions were arbitrarily graded as follows: very intense ; intense ; moderate ; low ; or not detected  .fig.
<dig> subtracted brain regions expressing putative valine amide peptide
immunoreactivity.
p18c5-mab-ir detected along the neuroaxis of the rat brain  was subtracted from brain areas expressing peptide immunoreactivity for α-msh, metorphamide, secretin, and urocortins 1- <dig>  as shown, moderate-to-high intensity mab-ir signals detected in subtracted brain areas were localized to the cortex  and the main and accessory olfactory nuclei .
subcortical areas displaying mab-ir signal included the subiculum , the core  and shell  of the nucleus accumbens , the substantia innominata of the septum , the amygdalo-hippocampal  and amygdalo-anterior nuclei  of the limbic system, the lateral posterior nucleus of the thalamus  and the lateral hypothalamic  nuclei.
brainstem areas included the substantia nigra , the superior colliculus , the retrorubral nucleus , the solitary tract  and the flocculus of the cerebellum .
with the exception of the areas described above, only the lh and the fl showed intense immunolabeling similar to that detected in cortical layers of the cb, the il of the pituitary and the adrenal medulla .
as shown, these brain areas were arbitrarily graded as displaying low val-conh2-peptide immunoreactive signals because they corresponded to areas containing the amidated peptides used as references in this study .
the neuroanatomical areas showing putative valine amide peptide immunoreactivity were identified based on the rat brain atlas of paxinos and watson  <cit>
statistical analysis
student’s t test was used to establish significant differences between samples assessed via ria or elisa.
the significance level was set at p ≤  <dig> .
RESULTS
identification of hyperimmune vaccinated mice
immunization of female balb/c mice  with the klh-val-conh <dig> immunoconjugate after five consecutive booster injections produced animals exhibiting antibody responses against the α-amidated isoform of the valine residue  .
vaas collected from five  of the seven vaccinated mice displayed significantly high cross-reactive signal against the bsa-tyr-gly2-4-val-conh <dig> adsorbed antigen based on elisa; these signals were twofold higher than the non-specific a <dig> values detected for the pre-immune control serum at the tested dilutions .
however, the vaas from the vaccinated animals did not display significantly higher cross-reactive signal than the controls for the free α-carboxylic acid isoform of the valine residue in the bsa-rgdv-coo− antigen .
the criteria for the selection of the samples for the cell fusion procedures were based on the detection of high immunoreactive signals  against the α-amidated isoform of the valine residue in the bsa-tyr-gly2-4-val-conh <dig> immunoconjugate at the highest antisera dilution tested  in vaccinated animals and on the lack of immunoreactivity for the carboxylic acid form of the same residue in the bsa-rgdv-coo− antigen .
among the five hyperimmune animals  selected for the generation of hybridoma-secreting mabs, mouse r <dig> exhibited the highest cross-reactivity against bsa-tyr-gly2-4-val-conh <dig>  displaying high titers of antibodies at vaa dilutions of 1: <dig>  and 1: <dig>  .fig.
<dig> identification of valine amide antibodies in mice.
animals defined as r1–r <dig>  were vaccinated with an initial subcutaneous injection with a 1: <dig> emulsion of 50 µg of keyhole limpet hemocyanin -val-conh <dig> immunoconjugate and complete freund’s adjuvant, followed by booster injections with incomplete freund’s adjuvant.
sera were collected and used to identify high-titer valine amide antisera  in the vaccinated animals using a standard elisa.
the wells were coated with synthetic immunoconjugates of bovine serum albumin --gly2–4-val-conh <dig> or bsa-rgdv-coo.
the bsa-rgdv-coo−-coated wells displayed no significant signal .
assays were performed in triplicate using two serial dilutions of mouse antisera .
the ordinate describes the immunoreactive signals detected as absorbance at 490 nm  in the assay.
the values are expressed as the mean ± sem
productive hybridomas
cell fusion of activated splenocytes from mouse r <dig> with the murine myeloma sp2/ <dig> cells yielded  <dig> stable hybridomas  from a total of  <dig> seeded cells .
elisa revealed three stable hybridoma-forming colonies , designated as p15a <dig>  p17c <dig>  and p18c5; these three colonies secreted mabs that recognized the cognate epitope of val-conh <dig> in both the bsa-val-conh <dig> and bsa-tyr-gly2-4-val-conh <dig> immunoconjugates .
the hybridomas secreting mabs displaying cross-reactivity against the α-amidated isoform of the valine residue represented  <dig>  % of the total productive hybridomas formed after cell fusion.
isotyping of the immunoglobulin class of the mabs secreted by these three hybridomas showed that they belonged to the igg <dig> class.
elisa
elisas were evaluated as an initial step in characterizing the specificity of the three distinct antibodies  against the structural α-amidated form of the valine residue.
immunoconjugates of amidated and non-amidated val- and leu-containing peptides linked to bsa were used as adsorbed antigens in the assays .
the p15a <dig> and p17c <dig> mabs displayed no differences in cross-reactivity against the adsorbed antigens of bsa-val-conh <dig>  bsa-tyr-gly2-4-val-conh <dig>  metorphamide , bsa-val-coo−, or mastoparan x  at the tested antibody dilutions  .
however, the p18c <dig> mab displayed a distinct pattern of reactivity against the α-amidated and free carboxylic acid isovariants of the valine residue in the tested antigens, as depicted in fig.
<dig>  as shown, the p18c <dig> mab exhibited high evident cross-reactivity against the α-amidated form of the valine residue in bsa-val-conh <dig>  at both low and high antibody dilutions .
notably, a less apparent immunoreactive response was detected against the metorphamide/adrenorphin peptide antigen.
cross-reactivity with the latter antigen decayed rapidly at the initial working dilutions of the antibody , in contrast to the slow decay of immunoreactive signal for the bsa-val-conh <dig> antigen at higher dilutions .
however, the p18c <dig> mab cross-reacted with the antigenic isovariants of the valine residue in the bsa-val-coo−, bsa-tyr-gly2-4-val-conh <dig>  bsa-tyr-gly2-4-val-coo−, and bsa-rgdv-coo− immunoconjugates, including the α-amidated isoform of leu in the mastoparan x peptide antigen.
the immunoreactive signals against these antigens were  <dig> - and  <dig> -fold lower than those against the bsa-val-conh <dig> antigen at the lowest  and highest  antibody dilutions, respectively.fig.
<dig> characterization of p18c <dig> mab specificity.
the vaccinated animals displaying the highest titers based on elisa were used for the generation of stable productive hybridomas.
supernatants from the resulting hybridoma-producing colonies were screened via standard elisa to assess the cross-reactivity of hybridoma-secreting mabs against the isovariants of the val  or leu  residues contained in the c-terminal domain of bsa-conjugated peptides and were used as adsorbed antigens together with bsa-val-conh <dig> and bsa-val-coo− immunoconjugates in the assay.
the p18c <dig> mab was tested at increasing dilutions  in elisas  or at a specific dilution  in dot-blot assays .
bsa-val-conh <dig>  bsa-val-coo− and bsa- peptide immunoconjugates were used as adsorbed antigens in the elisa or as spotted antigens  in the dot–blot assay.
elisa  was performed in triplicate using serial twofold dilutions of the p18c <dig> mab .
the absorbance of the immunoreactive signals in positive wells  was measured at λ = 490 nm
dot–blot assays
dot–blot assays were used to confirm and validate the specificity of the selected antibodies  against the conjugated bsa-peptide antigens  tested in the assay .
in general, the results of the dot–blot assays showed that the intensity of ihc staining from each hybridoma-secreted mab was proportional to the concentration of the peptide antigen spotted on the membrane filter .
the intensity of the detected immunoreactive signals in the dot–blots was compared to that of the control filters using only a secondary mouse-igg antibody, as depicted in fig.
5b.
as shown, p18c <dig> mab  displayed a high specificity and selectivity for the α-amidated isoform of the valine residue among the peptide antigens tested in the assay, as shown in fig.
5b.
this mab exhibited very intense immunoreactive signals against the metorphamide and yggg-val-conh <dig> peptide antigens at the highest peptide concentration tested , showing moderate immunoreactive signals against the peptide at lower peptide concentrations .
however, a weak immunoreactive signal was observed against mastoparan x at the highest concentration tested , and no signals were observed at lower concentrations .
interestingly, no immunoreactive signals against both yggg-val-coo− and rgdv-coo− antigens were detected at any of the peptide concentration tested.
thus, the p18c <dig> mab showed superior antibody selectivity against the α-amidated form of the valine residue.
based on the high specificity and selectivity of the p18c <dig> mab for the α-amidated form of the valine residue in peptide antigens based on elisas and dot–blot assays, a sensitive solid-phase ria for p18c <dig> mab was developed and used to further assess the specificity and selectivity of this hybridoma-secreted mab.
validation of the sensitivity of this ria enabled further identification and characterization of putative novel valine–amide neuropeptides from brain and neurosecretory tissues of rodents in subsequent studies.
solid-phase ria
a fmol-sensitive solid-phase ria for the p18c <dig> mab was developed using both the structural α-amidated isoform and the free carboxylic acid form of the valine residue conjugated to bsa, including both the c-terminal amide peptides and synthetic non-amidated peptides, using the synthetic -tyr-gly2-4-val-conh <dig> as the labeled tracer .
a representative ria showing typical displacement curves by synthetic -tyr-gly2-4-val-conh <dig> using the p18c <dig> mab is depicted in fig.
<dig>  as shown, the p18c <dig> mab recognized non-labeled tyr-gly2-4-val-conh <dig> over the tested concentration range, with an ic <dig> of  <dig>  ±  <dig>  fmol/well; the smallest measured displacement value, detected at ic <dig>  was  <dig>  ±  <dig>  fmol/well, and the highest was  <dig>  ±  <dig>  fmol/well at ic <dig> based on this assay.
in the same context, metorphamide was bound at an ic <dig> of  <dig>  ±  <dig>  fmol/well, and its lowest and highest displacement values were detected at the ic <dig>  and the ic <dig> , respectively.
similar displacement values were observed for bsa-val-conh <dig>  the p18c <dig> mab detected bsa-val-conh <dig> at an ic <dig> of  <dig>  ±  <dig>  fmol/well, an ic <dig> of  <dig>  ±  <dig>  fmol/well, and an ic <dig> of  <dig>  ±  <dig>  fmol/well.
moreover, no significant cross-reactivity against mastoparan x was observed for the concentration range of competitive peptides  tested in this assay.
furthermore, the p18c <dig> mab failed to recognize the structural free carboxylic acid isoform of the valine residue in both tyr-gly2-4-val-coo− and rgdv-coo− over the tested concentration range of the competitive peptide antigens , except for the lowest measurable displacement values detected at the ic <dig> for val-coo− peptides  and for mastoparan x .fig.
<dig> specific solid-phase radioimmunoassay for the p18c <dig> mab.
the p18c <dig> mab was used to generate a reliable and sensitive solid-phase ria for detecting the structural α-amidated isoform of the valine residue in synthetic peptide and non-peptide antigens.
as shown, the p18c <dig> mab was highly sensitive in detecting the val-conh <dig> residue in metorphamide/adrenorphin, bsa-val-conh <dig> and tyr-gly2-4-val-conh <dig> antigens within the tested concentration range, displaying ic <dig> values of  <dig>  ±  <dig>  fmol/well,  <dig>  ±  <dig>  fmol/well, and  <dig>  ±  <dig>  fmol/well, respectively.
however, no cross-reactivity  was detected against antigens expressing the val-coo− residue in tyr-gly2-4-val-coo− or rgdv-coo−, including mastoparan x, within the range of competitive antigen concentrations  tested in the assay
p18c <dig> mab immunoreactivity in control tissues
isotyping analysis of the p18c <dig> mab showed that it is classified as an igg <dig> antibody.
as shown in fig.
2a–c, p18c <dig> mab immunostaining was abolished in control tissues of the hypophysis, the adrenal gland, and representative sagittal sections of the rat brain when the antibody was pre-absorbed with  <dig> μm of the synthetic val-conh <dig> compound.
the intermediate lobe of the hypophysis  displayed intense p18c <dig> mab immunolabeling .
pre-absorption with the synthetic amidated valine compound completely abolished p18c <dig> mab staining in this region .
furthermore, the chromaffin cell-rich area of the adrenal gland  displayed intense p18c <dig> mab immunolabeling .
pre-absorption with the synthetic amidated valine compound completely blocked immunostaining in the core area of the adrenal gland  in adjacent tissue sections using the p18c <dig> mab .
in control sections of the rat brain , the p18c <dig> mab produced intense immunolabeling in several areas .
pre-absorption with the synthetic valine amide resulted in complete abolishment of p18c <dig> mab immunostaining in adjacent brain sections .
p18c <dig> mab immunoreactivity in the rat cns
val-conh2-ir was distributed widely throughout the neuroaxis of the rat brain after immunostaining using the p18c <dig> mab  .
moderate to intense immunolabeling was predominantly localized to the neuropil, displaying scattered stained neuron-like cell bodies appearing in several regions within the rat brain.
a brief summary of the distribution of p18c <dig> mab immunoreactivity in the rostrocaudal areas of the rat cns is described below.fig.
<dig> distribution of pc18c <dig> mab-ir in the rat brain.
the figure illustrates the rostrocaudal distribution of pc18c <dig> mab-ir in 40-μm-thick sagittal slices in fixed rat brain tissue .
bright-field photomicrographs  of sagittal sections of the rat brain display lateral-to-medial aspects of the rat brain regions displaying val-conh2-ir.
the neuroanatomical areas showing pc18c <dig> mab-ir were identified according to the rat brain atlas of paxinos and watson  <cit> .
cortex: fra frontal association cortex, m <dig> primary motor cortex, m <dig> secondary motor cortex, s1hl primary somatosensory cortex, pta parietal association cortex, v1m primary visual cortex , v2mm secondary visual cortex mediomedial, v2ml secondary visual cortex mediolateral, rsa retrosplenial agranular cortex, rsga retrosplenial granular a cortex, cg <dig> cingular cortex area 1– <dig>  olfactory system: ob olfactory bulb, aob accessory olfactory bulb, aod anterior accessory olfactory nucleus, dorsal, aov anterior accessory olfactory nucleus, ventral, aol anterior accessory olfactory nucleus, lateral, aom anterior accessory olfactory nucleus, medial, aoe anterior accessory olfactory nucleus, external, tu olfactory tubercle, baot bed nucleus of the accessory olfactory tract, lot nucleus of the lateral olfactory tract.
hippocampus: hi hippocampal c1–c <dig> fields and dentate gyrus, fi fimbria, s subiculum.
basal ganglia: cpu caudoputamen, acbc accumbens nucleus core, acbsh accumbens nucleus shell, vp ventral pallidum.
septum and septal areas: bst bed nucleus of the stria terminalis, bstl bed nucleus of the stria terminalis, lateral, bstm bed nucleus of the stria terminalis, medial, bac bed nucleus of the anterior commissure, ls lateral septal nucleus, lsd lateral septal nucleus, dorsal part, si substantia innominata, hdb nucleus of the horizontal limb of the diagonal band.
amygdala: me medial amygdaloid nucleus, plco posterolateral cortical amygdaloid nucleus, pmco posteromedial cortical amygdaloid nucleus, aco anterior cortical amygdaloid nucleus, ahi amygdalo-hippocampal area.
diencephalon: th thalamus, avvl anteroventral thalamic nucleus, ventrolateral, sth subthalamic nucleus , lpmr lateral posterior thalamic nucleus, mediorostral, md mediodorsal thalamic nucleus.
hypothalamus: ah anterior hypothalamic area, lh lateral hypothalamic area, so supraoptic nucleus, sor supraoptic nucleus, retrochiasmatic, arc arcuate hypothalamic nucleus.
mammillary bodies: mm medial mammillary nucleus.
mesencephalon: sn substantia nigra, avt ventrotegmental area, vltg ventrolateral tegmental area, r red nucleus, sc superior colliculus, ic inferior colliculus, lpag lateral periaqueductal gray.
pons and medulla: lc locus coeruleus, lpb lateral parabrachial nucleus, mpb medial parabrachial nucleus, rr retrorubral nucleus, pn pontine nucleus , cgpn central gray of the pons, ldtg laterodorsal tegmental nucleus, lso superior olive, lateral nucleus, iod inferior olive, dorsal nucleus, inta interposed cerebellar nucleus, anterior, intp interposed cerebellar nucleus, posterior, cu cuneate nucleus, lve lateral vestibular nucleus, spve spinal vestibular nucleus, mvepc medial vestibular nucleus, parvocellular, mvemc medial vestibular nucleus, magnocellular, vll ventral nucleus of the lateral lemniscus, sol solitary tract.
cb cerebellum.
see text for additional details.
scale bar = ×40
telencephalon
several telencephalic regions exhibited moderate to high immunolabeling, which was predominantly distributed in thin and dense fiber processes, oval-to-round somata, and diffuse fine puncta .
pc18c <dig> mab immunoreactivity  was detected in the primary and secondary motor cortices , parietal sensorimotor cortical fields, and the primary and secondary visual cortices  , as well as the retrosplenial agranular  and granular a  cortices .
within the olfactory system, p18c <dig> mab-ir was observed in both the main olfactory bulb  and the accessory olfactory bulbs .
in the hippocampus , p18c <dig> mab-ir was found in the striatum pyramidale and radiatum, along the ca1–ca <dig> fields of ammon´s horn, and, particularly, in the granular cell layer of the dentate gyrus -, as well as in the fimbria  and the subiculum  .
p18c <dig> mab-ir was also detected in the basal ganglia , septal areas , the lateral  and medial bst , the bed nucleus of the anterior commissure , the lateral septum , the dorsal part of the ls , the substantia innominata , and the nucleus of the horizontal limb of the diagonal band ] and the amygdala , which displayed moderate to high immunostaining that was predominantly distributed in sparse neuropil, stained round-to-oval cell bodies, and fine dendritic puncta .
both the medial amygdaloid nucleus  and the amygdalo-hippocampal area  exhibited low levels of mab-ir .
diencephalon
p18c <dig> mab-ir was observed in several diencephalic structures, including the hypothalamus , which displayed moderate to intense mab immunostaining, .
both the thalamus  i.e., the anteroventral , lateral posterior  and mediodorsal nuclei ] and the subthalamic nuclei  , including several mammillary nuclei i.e., the medial mammillary , lateral mammillary , and submammilothalamic nuclei ], displayed low to moderate mab immunolabeling .
the stained regions included the retrochiasmatic supraoptic  nucleus of the hypothalamus .
mesencephalon
along the mesencephalon, p18c <dig> mab-ir was detected in the substantia nigra  , the superior colliculus  , the inferior colliculus   , the ventral tegmental area , the red nucleus  and the lateral periaqueductal gray area  .
these mesencephalic areas generally displayed low to moderate mab immunolabeling that was predominantly localized within a network of fiber processes dispersed among stained somata .
cerebelum
pc18c <dig> mab-ir was predominantly distributed along the purkinje cell layer of the cerebellar cortical layers; cells in this area displayed highly stained somata-like cell bodies, fine dendritic puncta, and a very dense network of protruding labeled purkinje cell axon fibers .
low to moderate mab-ir was detected in both the flocculus  and the white matter of the cb, including the medial cerebellar peduncle  .
pons and medulla
the pc18c <dig> mab showed low to moderate immunostaining in nuclei and tracts along the rostrocaudal aspects of the pontine and medullary areas of the rat cns .
within the most lateral aspects of the pontine/medullary areas of the brain, low to moderate mab-ir was detected at the retrorubral nucleus , the ventral nucleus of the lateral lemniscus , the lateral nucleus of the superior olive , the cuneate nucleus , and the lateral vestibular nucleus  .
in addition, the pontine nucleus , both the medial  and lateral parabrachial nuclei , both the anterior  and posterior parts of the interposed cerebellar nuclei , and the spinal vestibular nucleus  showed positive immunostaining .
within the medial aspects of these caudal regions of the rat brain, positive mab-ir was detected at the locus coeruleus , the parvocellular  and magnocellular parts of the medial vestibular nuclei , and both the laterodorsal tegmental nucleus  and the dorsal part of the inferior olive , including the central gray matter of the pons  and the solitary tract  .
valine amide peptide immunoreactivity in the rat brain
the reports describing the immunoreactive distribution of α-msh, metorphamide, secretin and ucn1- <dig> in the rat brain  were used to compare each peptide immunoreactivity with the rostrocaudal distribution of p18c <dig> mab-ir in the rat cns, as depicted in fig.
7a–e, respectively.
subtraction analysis
p18c5-mab-ir detected along the rostrocaudal areas of the rat brain  was compared with the distribution of valine amide peptide immunoreactivities detected in the rat brain.
the immunoreactive signals obtained for each peptide were subtracted from pc18c <dig> mab-ir signals detected along the neuroaxis of the rat brain.
the subtracted brain areas expressing val-conh2-ir material are depicted in fig.
<dig>  most of these areas showed moderate-to-high pc18c <dig> mab-ir signals, with the exception of the cerebellar lobes  and the lateral hypothalamus  which exhibited an intense p18c5-mab-ir signals, similar to immunoreactive signal displayed by the il of the hypophysis, used as the internal control tissue.
as shown in fig.
<dig>  valine amide peptide immunoreactivity  was detected in cortical areas of the telencephalon such as m <dig>  the hindlimb  and trunk regions of the primary somatosensory cortex , the parietal association cortex , v2mm, and v2ml.
in addition, cvap-ir was observed in the main accessory ob  and corresponding nuclei, described as the anterior accessory olfactory nucleus dorsal , the nucleus ventral , the nucleus lateral  and the nucleus medialis .
furthermore, vap-ir was detected along the olfactory tubercle , within the bed nucleus of the accessory olfactory tract  and within the nucleus of the lateral olfactory tract .
in addition, cvap-ir was detected in telencephalic structures such as s of the hi, nacc, and the substantia innominata of the ls, including both the ahi and the amygdalo-anterior area  of the limbic system.
in the diencephalon, vap-ir was found in lpmr of the thalamus and in the magnocellular portion of the lh.
within the mesencephalon, vap-ir was primarily localized to the lateral portion of the substantia nigra  and zo of the sc, whereas in the pontine and medullary areas, cvap-ir was primarily localized to rr, sol and the fl of the cb.
peptide fractions displaying vap-ir
several regions of the rat brain and endocrine glands were used to prepare purified peptide fractions from the subtracted brain areas to quantify the content of vap-ir  using the specific solid-phase ria generated for the p18c <dig> mab .
in addition, neuroendocrine glands  were used as control tissues in the assay.
as shown in fig.
<dig>  the rat pituitary  displayed the highest abundance of vap-ir , which was  <dig> -fold greater than the amount observed in the brainstem .
however, low amounts of val-conh2-ir were detected in the cortex  and in the adrenal glands  ; these levels were  <dig> - and  <dig> -fold lower, respectively, than the levels measured in the pituitary.
in the same context, both the cb  and the pancreas  contained low levels of val-conh2-ir, specifically 90- and 56-fold lower, respectively, than those in the pituitary.fig.
<dig> quantification of endogenous valine amide peptide-immunoreactive material in tissues.
the abundance of valine amide peptide-immunoreactive material was estimated in purified peptide fractions prepared from subtracted brain regions  and neuroendocrine glands , which displayed moderate-to-high pc18c <dig> mab immunolabeling  based on the solid-phase ria developed for the pc18c <dig> mab .
the plot shows that the highest abundance of val-conh2-ir material was found in the pituitary  , followed by the brainstem  , the adrenal gland  , and the cortex  .
inset depicts a representative solid-phase ria for the pc18c <dig> mab used for the quantification of valine amide-immunoreactive material in the assays.
tyr-gly-val-conh <dig> was used as a standard to construct a typical standard curve and was detected at a concentration of  <dig>  fmol/well at the ic <dig> value, whereas metorphamide/adrenorphin  was detected at a concentration as low as  <dig>  fmol/well at the ic <dig> value
discussion
the α-amidation process is an essential step for the conversion of prohormones into mature peptides that possess complete biological activity, which includes receptor recognition and transduction of peptide hormone signals in target cells  <cit> .
the α-amidation process has been shown to occur most frequently on neutral amino acids  in neuropeptides, which constitute the largest group  of functional peptide messengers identified in vertebrate and/or invertebrate species.
such peptides display pleiotropic activities across species  <cit> .
however, valine amide peptide hormones represent the smallest group of functional amidated peptides identified to date, and which include the enterochromaffin cell-related peptide hormone, secretin, which belongs to the pacap/vip/glucagon peptide hormone family  <cit> ; the pomc-derived peptide- α-msh -  <cit> ; the proenkephalin a-derived metorphamide/adrenorphin  <cit> ; the stress and appetite regulatory urocortins   <cit>  and the enterins, a family of brain peptides found in the enteric nervous system of aplysia, which modulate both feeding non-feeding behaviors in this marine specie  <cit> .
in order to search for neural structures expressing putative novel valine amide peptide immunoreactive material in the brain and neurosecretory tissues of mammals, we employed splenocyte/myeloma cell fusion procedures, with the aim to generate stable hybridoma-producing mabs able of recognizing the structural α-amidated isoform of the valine residue  in constructed immunoconjugates .
cell fusion procedures between activated splenocytes from the hyperimmune female balb/c mouse -r4— and the murine myeloma cell line sp2/ <dig>  led to the identification of three hybridoma-producing mabs  designated as p15a <dig>  p17c <dig>  and p18c <dig> and which displayed different levels of cross-reactivities against the structural α-amidated and/or the free carboxylic acid isovariants of the valine residue expressed in distinct antigens tested in different immunoassays  used to validate the specificity and selectivity of these mabs .
the efficiency of the cell fusion procedure from the total cells seeded for fusion was  <dig>  %, a value considered within the efficient rates reported in the generation of productive hybridomas  <cit> .
as shown, only p18c <dig> mab showed a predominant cross-reactivity against the α-amidated isoform of the valine residue in the tested antigens in elisa and dot-blot assays  however, p18c <dig> mab showed to be exquisitely selective for the structural α-amidated isoform of the valine residue in the constructed synthetic antigens  tested in the fmol-sensitive solid phase ria  but unable to recognize the free carboxylic acid of same residue in both rdgv, tyr-gly2-4-val-coo− peptide antigens ]  <cit> .
the high specificity and selectivity exhibited by p18c <dig> mab in recognizing the condensed amide  substituent at the carbonyl group  of the asymmetric carbon  in the amino acid, may be due to the sort of complementarity determining regions  encoded along both the heavy and light chains of the antibody, enhancing its preferential affinity and recognition for the structural amide  epitope over the functional carbonyl group  of the amino acid  <cit> .
in same line, specific mabs were shown to display predominant specificities and selectivity in the recognition of small antigens  such as, acetyllysine and –methyllysine antigenic epitopes in proteins  <cit> , or mabs shown to discriminate among isovariants  of the immune mediator, interleukin, il- <dig>  <cit> .
based on our immunoassay results, we explored the capability of the p18c <dig> mab to detect valine amide peptide immunoreactivity along the neuroaxis of the rat brain and rat neuroendocrine tissues  .
our results showed that p18c <dig> mab to was able to recognize the structural α-amidated isoform of the valine residue in endogenous peptide material, as demonstrated for abolishment of mab immunostaining in tested tissues  after preadsorbing the antibody with the synthetic valine amidated residue  .
as shown in fig.
<dig>  the intermediate lobe  of the pituitary  showed an intense mab immunostaining when compared to absence of immunolabeling in both al and the pl in same neuroendocrine tissue .
this neurosecretory area contains the “pituitary melanotrophs” shown to synthesize and release high concentrations of pomc-derived peptides  into the bloodstream  <cit> .
thus, the detection of p18c <dig> mab-ir in the il of the hypophysis reflects the post-translational processing of pomc-derived peptides and conversion of mature c-terminal amidated peptide products, via the activity peptidylglycine α-amidating monooxygenase  .
in same line, the detection of p18c <dig> mab-ir at the core  of the adrenal gland supports the cellular expression of bioactive metorphamide/adrenorphin peptide released from the proenkephalin a protein precursor from the chromaffin cells  <cit> .
p18c <dig> mab-immunoreactivity showed a wide distribution along the neuroaxis of the rat brain .
most of the rostrocaudal areas expressing a low-to moderate or conspicuous immunostaining were matched with brain areas previously shown to contain the most prevalent bioactive valine amide neuropeptides α-msh   <cit> ; secretin   <cit> ; adrenorphin   <cit>  and ucns   <cit> , suggesting that these peptides exert pleiotropic effects in time and tissue-specific manner, in both central and peripheral tissues after targeting their specific cell surface receptors  <cit> .
the properties and functions of valine amide peptide in the brain and peripheral tissues may be search in the web and in some reviews described herein  and is out of the scope of the present paper to detail the relationship between brain structure and valine amide peptide bioactivities.
data obtained from the subtraction analysis revealed the expression of mab-immunolabeling in specific brain areas that did not match with the neuronal distribution of valine amide neuropeptides .
these results suggest that such areas represent neural structures able to synthesize putative novel valine amidated peptide products encoded in large propeptide precursor protein with functions yet to be elucidated.
furthermore, these areas suggest that neurons operating within each immunolabeled brain region or nuclei must express same molecular machinery and posttranslational enzyme-dependent mechanisms, in order to allow the biosynthesis and conversion of prohormones into functional c-terminal amide neuropeptides  at the expense of the functional bioactivities of pc1/pc <dig> proconvertases  <cit>  and the peptidylglycine α-amidating monooxygenase   <cit> , respectively.
this hypothesis is supported by the specific tissue-dependent processing of pomc-derived peptide hormones   <cit>  in the brain and peripheral tissues  <cit> , including the processing of proenkephalin a in the brain and neurosecretory tissues such as, the chromaffin cells of the adrenal medulla   <cit> .
moreover, based on the specificity and selectivity of the p18c <dig> mab for detecting immunoreactive peptide material in the rat brain and neuroendocrine tissues; we explored the feasibility of this mab to determine the abundance of val-conh2-ir in peptide fractions isolated from immunostained subtracted regions using our fmol-sensitive solid-phase ria raised for p18c <dig> mab .
as shown, both cortex , brainstem  and cb were used as representative subtracted brain areas encoding putative valine amide peptide material, in addition of the pancreas, the pituitary and adrenal glands, used as neuroendocrine control tissues .
furthermore, the highest amount of val-conh2-ir peptide material was detected in the pituitary  followed by the brainstem , the adrenal gland  and the cortex  .
the relative abundance of immunoreactive peptide material determined in both bs, ad and the cx were  <dig> ,  <dig> and  <dig>  %, respectively, from that detected in the pituitary.
in same line, both cerebellum  and pancreas  showed lowest content of estimated val-conh2-ir .
differences in the relative amount of peptide immunoreactivity estimated for each neural tissue could rely on the cell concentration of valine amide neuropeptides stored  before their release into the extacellular milieu  <cit>  at the time of tissue extraction and preparation of peptide fractions from tissue homogenates.
although at this stage during mab screening, the exact amount of putative novel valine amide neuropeptides per tissue  may be impossible to obtain.
however, both recombinant dna technology and cloning procedures  <cit>  may lead to the identification and characterization of putative novel cdnas encoding valine amide peptide sequences, as demonstrated for the “enterins”, a family of structurally related nonapeptides and decapeptides that are present in the gut and cns of aplysia, and most of which share the hsfvamide sequence at the c terminus  <cit> .
CONCLUSIONS
the present work describes an experimental strategy used to generate murine monoclonal antibodies able to differentiate between two isovariants of the valine residue.
one single mab designated as, p18c <dig> mab, showed a unique specificity and selectivity for the structural α-amidated isoform of this neutral amino acid in natural  and synthetic  peptide antigens, as revealed from our immunoassays, besides of demonstrating a great capability to detect putative—yet unidentified—valine amide peptide immunoreactive material in specific areas of the rat brain.
the ability of this mab to distinguish between two small structural epitopes in same residue, posit for its wide applications in both research and clinical settings.
for instance, p18c <dig> mab may be used to generate specific biological “kits” used to purify and detect valine amidated peptide products in tissue and/or biological fluids.
furthermore, this mab may be used in immunotherapy settings to reduce tumor growth and tumorigenesis; or used as a detoxifying–neutralizing reagent from blood poisoning compounds such as, toxic amidated peptide molecules found in food, plants and/or animal species.
overall, this work portraits the potential applications of a single mab, who displays exquisite properties when reacting against valine amide peptide antigens, including its capability to identify brain regions expressing putative novel valine amide neuropeptides, with functions yet to be discovered.
authors’ contributions
ba conceived the study.
ba and pl participated in the design and coordination of the study, data analysis, and drafting of the manuscript.
jah participated in the design and synthesis of the immunoconjugates.
mm and rh, together with ba, conducted all immunoassays related to the generation of hyperimmune mice, cell fusion and the generation and expansion of productive hybridomas and collected data for the manuscript.
jc and jah participated in the elisas for monoclonal and polyclonal antibodies, as well as protein determination and the collection of data for the present manuscript.
jc and mep, together with pl performed dot-blot assays, rias, tissue extraction, and preparation of peptide fractions from brain regions and neuroendocrine tissues.
both investigators were involved in the collection of data for the present manuscript.
ra and af participated in the immunohistochemistry studies.
both investigators ba and pl were involved in the collection of data.
as performed the subtraction analysis of positive immunoreactive areas for the p18c <dig> mab and performed the statistical analysis for this study.
all authors helped to draft the manuscript, read and approved the final manuscript, and agreed on its submission.
all authors read and approved the final manuscript.

 over the past few decades, many neuropeptides have been identified in the neural and neuroendocrine structures of both vertebrate and invertebrate species. in vertebrates, more than 50 % of the identified neuropeptides and peptide hormones are amidated, whereas more than 90 % of bioactive peptides in the brains of invertebrates are amidated. most neuropeptides in tissues require post-translational modifications, specifically the α-amidation of the carboxyl group of the c-terminal amino acid. such amidation represents an essential step for peptide hormones to acquire complete biological activity, receptor recognition, and signal transduction at target cells. c-terminal amidation of peptides, a necessary step for the conversion of prohormones into functional products, is mediated by the activity of the peptidylglycine α-amidating monooxygenase, a type i membrane protein that is localized to the trans-golgi network and to secretory granules in neural and endocrine tissues. pam is a bifunctional enzyme containing two specific domains: peptidylglycine α-hydroxylating monooxygenase and peptidyl-α-hydroxyglycine α-amidating lyase. the cellular bioactivity of pam depends on ascorbic acid, copper, and molecular oxygen. mice lacking pam do not survive beyond mid-gestation. both of its enzymes act sequentially to generate an active α-amidated peptide product and glyoxylate.fig.  schematic representation of the α-amidation process in cells. the α-amidation process is a highly specific enzyme-dependent post-translational modification need for converting prohormones into functional peptide products in neurosecretory cells. panel a depicts the specific processing of large propeptide precursor proteins containing active peptide sequences via pc1/pc proconvertase activity alone or together with the functional activity peptidylglycine α-amidating monooxygenase. panel b illustrates the sequential steps of pam-related bifunctional activity which includes the peptidylglycine α-hydroxylating monooxygenase activity depicted in step and the peptidyl-α-hydroxyglycine-α-amidating lyase activity depicted in step as shown, two molecules of ascorbate are oxidized to form two molecules of semidehydroascorbate during the reduction of two enzyme-bound cu atoms into two enzyme-bound cu atoms as previously described although a substantial amount of data is available on the reaction mechanism of phm and its relevance to physiology and medicine, few details concerning the activity and relevance of the pal domain of pam have been reported. phm is a di-copper, ascorbate-dependent monooxygenase that catalyzes the stereospecific α-hydroxylation of a glycine -extended precursor peptide by molecular oxygen; whereas pal catalyzes the n-dealkylation of the peptidyl-α-hydroxyglycine intermediate generated by phm. in vertebrate species, phm and pal are encoded by the same rna transcript, whereas in non-vertebrate species, each domain appears to be encoded by a separate gene. although the α-amidation of almost all amino acids found in nature has been observed, α-amidation of active neuropeptides occurs most frequently on non-polar residues such as phenylalanine, leucine, methionine, and glycine. in addition, amidation of some residues such as isoleucine 3/stresscopin) occurs rarely. peptides containing the α-amidated isoform of a valine residue represent the smallest group of functional amide neuropeptides displaying pleiotropic bioactivity that have been identified in neural structures in both vertebrate and non-vertebrate species. this group of neuropeptides includes distinct bioactive peptide hormones such as, α-msh, metorphamide/adenorphin, secretin, urocortins and enterins. early research showed that small and large peptide molecules can be separated via reverse-phase liquid chromatography based on the hydrophobicity of the peptide structure and its amino acid composition. this separation can be followed by sequence analysis of dansylated amino acids via the edman technique using dansyl chloride as the reagent to label amino acid residues. these studies led to the discovery of two amidated opioid peptides, adrenorphin and amidorphin. with the advent of recombinant dna technology and cloning procedures together with proteomic analysis using mass spectrometry techniques, the search for mrna transcripts and mrna-translated protein products has led to the identification of many functional peptides. in combination with mass spectrometry, monoclonal antibodies became powerful biological tools used in clinical settings and animal studies to detect post-translational modifications of small molecules in tissues. in the present study, we used splenocyte/myeloma cell fusion technology to generate stable hybridomas secreting mabs against the structural α-amidated isoform of a valine residue with the goal of identifying and characterizing putative novel c-terminal valine amide neuropeptides in the mammalian cns and neurosecretory tissues, as well. immunohistochemical analyses showed that valine amidated neuropeptides, such as, α-msh, metorphamide/adrenorphin, secretin, and ucn are widely distributed throughout the mammalian cns and neuroendocrine system, including the immune system. in general, antibodies generated to characterize the anatomical distribution of valine-amide peptide immunoreactivity in the brain or neuroendocrine tissues required the synthesis of a fragment or peptide motif encoded along the complete amino acid sequence of the peptide of interest, and used then, as the antigenic epitope to which the antibody is targeted. to date, no ihc studies using specific antibodies raised against small isovariants of a single haptenic molecule, for instance, the free α-carboxylic acid or the structural α-amidated isoform of an amino acid residue have been studied or reported. thus, the present paper shows the identification of a single mab that displays a high selectivity, sensitivity and specificity for the structural α-amidated isoform of the valine residue when tested against distinct amidated and non-amidated peptide antigens in different immunoassays. moreover, this mab exhibited a great capability in detecting endogenous valine amide peptide immunoreactivity along the neuroaxis of the rat brain. our data demonstrates the high versatility of this mab, which makes it an amazing biological “tool” for biomedical research and clinical settings. materials and methods peptide synthesis synthetic peptides with the primary sequences -gly-gly-gly-val-conh and -gly-gly-gly-val-coo− were synthesized by peninsula labs. both isovariants of the valine residue, the α-amidated and the free α-carboxylic acid isoforms, were covalently linked to the c-terminal domain of the peptide backbone and were supplied as lyophilized material at 98 % purity. both isovariants of the valine residue were used as antigenic epitopes in synthetic peptide constructs and in naturally occurring bioactive peptides to determine the cross-reactivity of the antibodies to each epitope in specific immunoassays. one crucial advantage of using these peptides in immunoassays is that the residue at the n-terminal domain of these synthetic peptides may be used as a radiolabeled tracer in specific radioimmunoassays when the peptides are labeled with. moreover, these short oligopeptide constructs, which were synthesized by condensing a triplet of glycine residues into a peptide backbone, were designed to maintain optimal backbone flexibility, linear conformation, and solubility in aqueous solution, as has been shown for other polymeric structures containing a short linker backbone of glycine residues. immunoconjugates the structural α-amidated isovariant of the valine residue was covalently linked to keyhole limpet hemocyanin according to standard coupling procedures as previously reported, and this complex was used as an immunoconjugate for immunization. the free α-carboxylic acid isovariant of valine, the synthetic peptides -gly2–4-val-conh and -gly2–4-val-coo−, the fibronectin/fibrin-related peptide rgdv-coo−, metorphamide/adrenorphin, and the wasp venom-derived ionophoric peptide mastoparan x were similarly conjugated to bovine serum albumin using  % glutaraldehyde as a cross-linker. the cross-linking reactions were terminated via the addition of  m glycine to the reaction mixture. the immunoconjugates were dialyzed using a 12 kda cut-off nitrocellulose membrane for 72 h against 10 l of phosphate-buffered saline to remove non-reactive aldehydes. finally, the each immunoconjugate was diluted to 1 mg/ml. the purified immunoconjugates of bsa-linked peptide were frozen and stored at –20 °c for use as adsorbent antigens in elisas and dot-blot assays. animals all animal studies and procedures were performed in accordance with the national institute of health guide for the care and use of laboratory animals and were approved by the animal care and bioethics committee of the national institute of psychiatry, mexico city. immunization female balb/c mice were purchased from charles river laboratories and used for immunization and collection of specific antisera according to standard procedures as described previously. the animals, which were designated as r1–r received an initial subcutaneous injection of  ml of a 1: emulsion of μg klh-val-conh immunoconjugate in  ml of pbs:  ml of complete freund´s adjuvant. three subcutaneous booster injections consisting of  ml of a 1: emulsion of μg klh-val-conh immunoconjugate in  ml of pbs:  ml of incomplete freund’s adjuvant were administered, followed by a final intraperitoneal booster injection of klh-val-conh in  ml of pbs:  ml of incomplete freund’s adjuvant 30 days after the previous immunization. blood was collected from the tail vein of the mice every 2 weeks after each immunization and was centrifuged in an eppendorf 5804r centrifuge at 1200 rpm for 10 min at 4 °c. the collected sera were stored at −20 °c until further use. elisa for polyclonal antibodies the titers of polyclonal valine amide antisera from vaccinated mice were determined via standard antibody-capture elisa in microtiter 96-well elisa plates. each well was coated with  μg of bsa--gly2–4-val-conh or bsa-rgdv-coo− immunoconjugate in the presence of  m sodium bicarbonate. after overnight incubation, the coated wells were blocked with elisa buffer for 2 h at room temperature, washed with elisa buffer, and incubated overnight at 4 °c in 50 μl of vaa diluted 1:10–1: the wells were subsequently incubated for 2 h at rt in 50 μl of pbs containing a 1: dilution of horseradish peroxidase -conjugated goat anti-mouse immunoglobulin g and  % tween- color development was performed using the chromogenic reagent o-phenylenediamine dihydrochloride. the plates were measured at 490 nm in an automated elisa plate reader. preimmune sera from the mice were used as controls. each assay was performed in triplicate. vaccinated mice displaying antisera titers greater than 1: dilution whose sera yielded an a reading greater than two standard deviations beyond the mean of the non-specific value obtained using preimmune serum were selected as candidates for cell fusion and for the generation of stable productive hybridomas. cell fusion and generation of productive hybridomas hyperimmune mice displaying high antisera titers based on elisa received an intraperitoneal pre-fusion booster of 50 μg of the klh-val-conh immunoconjugate in 200 μl of pbs in the absence of adjuvant 7 days before lymphoid cells were harvested. mice were killed by cervical dislocation; splenocytes were collected under aseptic conditions and were promptly fused with the murine myeloma sp2/ cells using 50 % polyethylene glycol /pbs according to standard cell fusion protocols as described previously. the murine myeloma cell line sp2/ is a variant line derived from the fusion of mopc- and balb/c mouse splenocytes; it is frequently used for the generation of stable hybridomas due to its high growth rate, rapid replication rate, high capacity for cell fusion, and cloning efficiency. the fused cells were resuspended at a concentration of 2 ×  cells/ml in a selective enriched hybridoma medium. the fused cells were seeded on a thymocyte feeder layer in 20 × 96-well plates. myeloma cells and hybridoma colonies displaying logarithmic growth to a density of 5–6 × 106 cells/ml were diluted to 1 × 105 cells/ml using cryopreservation medium and stored at −196 °c under liquid nitrogen in cryogenic vials. elisa for mabs elisas were used to identify mabs against val-conh secreted by positive colonies using the same protocols as those described for the screening of polyclonal antisera from mice except that bsa-val-conh bsa-metorphamide/adrenorphin, bsa-mastoparan-x, bsa-val-coo−, and bsa-rgdv-coo− were used as adsorbent antigens in the wells, that 50 μl of various dilutions of the supernatants collected from hybridoma colonies were added to the coated wells, and that the plates were incubated overnight at 4 °c. val-conh2-ir signal was detected at 490 nm in an automated elisa plate reader using a 1: dilution of hrp-conjugated goat anti-mouse igg and the chromogenic reagent opd. val-conh mab specificity was determined based on the absence of cross-reactivity against the free α-carboxylic acid form of the valine residue and/or against the c-terminal amide form of the leucine residue in mastoparan x. preimmune sera from the mice were used as controls in all assays. expansion of hybridomas productive hybridomas secreting antibodies were recovered and subjected to expansion and subcloning procedures at limiting cell dilutions as previously described by hockfield et al.. isotyping isotyping of the mabs was performed using an isoquick kit for mouse monoclonal isotyping. dot–blot assays dot–blot assays were used to assess the mab specificities against both isovariants of the valine residue in the tested antigens according to standard procedures described by hockfield et al. and adapted by loi et al.. briefly, both isovariants of the valine residue and synthetic bsa-conjugated peptides were tested as potential cross-reactive antigens. immunoconjugates of the bsa-conjugated antigenic peptides tyr-gly2-4-val-conh tyr-gly2-4-val-coo−, rgdv-coo−, metorphamide/adrenorphin, and mastoparan x were diluted in 50 % methanol to obtain stock solutions at concentrations of 10−2–10−9 m. two microliters of each antigenic bsa-peptide immunoconjugate at 10−12–10−16 m were spotted onto nitrocellulose membranes. after spotting the peptides, the dried membranes were soaked in a preblocking solution containing 1 % teleostean gelatin, 10 mm pbs, and  % tween- for 1 h at rt. the membranes were washed times for 5 min with the preblocking solution and incubated overnight at 4 °c in the presence of a 1: dilution of hybridoma supernatants containing a mab in 10 mm pbs. the membranes were then washed and incubated for 2 h at rt in a solution containing  % tween- and 10 mm pbs in the presence of hrp-conjugated goat anti-mouse igg diluted 1:. the membranes were washed times for 5 min with  % tween- in 10 mm pbs to remove excess secondary antibody. signal detection was performed using a chemiluminescence kit. solid-phase ria a solid-phase ria for the α-amidated isovariant of the valine residue was adapted from standard procedures described by hockfield et al.. briefly, the n-terminal residue of the synthetic peptide tyr-gly2-4-val-conh was iodinated with using the iodo-gen protein labeling procedure according to the manufacturer’s protocol, and the peptide was purified via high-performance liquid chromatography using hypersil wide-pore 5-μm, c- 2 × 150 mm columns. the solid-phase ria was prepared by loading 100 μl of blocking solution containing 500 ng protein a and  m nahco in immunolon ii removable wells, followed by incubation overnight at 4 °c. the coated wells were washed times for 10 min with ria buffer. fifty microliters of ria buffer containing a hybridoma supernatant at 1: dilution were added to the wells in quadruplicate, and the plates were incubated overnight at 4 °c. after removing the antibody solution and washing with ria buffer, 50 μl of ria buffer containing competitive peptides and the radiolabeled peptide tracer were applied to the adsorbed anti-val-conh mabs for 2 h at rt. typical standard displacement curves were generated using approximately c.p.m. of the radiolabeled tracer -tyr-gly2-4-val-conh and the non-labeled peptides used in the competitive ria. the treated wells were washed and counted for 4 min in a 10-channel gamma counter. peptide fractions from neural and endocrine tissues tissue extraction and preparation of whole peptide fractions from various neural and endocrine tissues were performed according to standard procedures and the protocols previously described by asai et al.. briefly, male wistar rats pre-anesthetized with lethal doses of sodium pentobarbital were decapitated, and the abdominal cavity was opened for surgical extraction of the pancreas and the adrenal glands. rat brain regions such as the cortex, the hippocampus, the striatum, the hypothalamus, the thalamus, the cerebellum, the pituitary and the spinal cord and endocrine glands including the adrenal gland and the pancreas were rapidly dissected on ice. the brain regions and the glands were promptly homogenized using a tissue homogenizer at 800 rpm in an ice–cold acid:acetone solution and centrifuged at ×g for 1 h at 4 °c in a sorvall rc28s centrifuge. the hcl:acetone solution was removed from the crude peptide fraction supernatants via vacuum evaporation. the dried pellets were resuspended in 5 % tfa solution and loaded on sep-pak-rp c- columns pre-activated with 100 % tfa for solid-phase peptide extraction. the peptide fractions from tissue homogenates of the brain and the endocrine glands were eluted from the columns using 20 % tfa:h2o, dried via vacuum evaporation and stored at −20 °c. the thawed pellets were resuspended in a solution containing 50 mm tris–hcl, ph, and  mm cacl and 50-µl aliquots of ria buffer containing a 1:5–1: dilution of a purified peptide fraction were individually assessed for the p18c mab via solid-phase ria using the radioactively labeled peptide -tyr-gly2-4-val-conh the labeled tracer was used to quantify the abundance of val-conh2-ir in the tissues. peptide immunoreactivity in the samples is expressed as nmol/μg protein. protein concentration determination the protein concentrations in the peptide fractions extracted from the rat brain and neuroendocrine tissues were determined according to standard procedures using the micro-bca protein assay kit based on the instructions provided by the manufacturer. the absorbance at 570 nm was measured using a microwell plate reader. the protein concentration of the samples was expressed as µg/ml. tissue preparation tissue preparation for ihc detection of val-conh2-ir was performed according to standard procedures previously described by anton et al.. briefly, six male wistar rats were anesthetized with sodium pentobarbital and transcardially perfused with 250 ml of 10 mm pbs /heparin solution, followed by perfusion with 800 ml of 4 % paraformaldehyde/10 mm pbs solution at 4 °c. the flow rate was held constant using a hamilton double-pumping system. fixed tissues were cryoprotected in 30 % sucrose/10 mm pbs solution for 3 days at 4 °c. the fixed brain tissues were sliced into 40-μm-thick sagittal sections using a cryostat; alternatively, the fixed neuroendocrine tissues were sliced into 40-μm-thick coronal sections. the sliced sections were collected in 12-well plates, allowed to float freely in 10 mm pbs /  % sodium azide solution, and stored at 4 °c until use. storage for up to 1 month in the aforementioned solution does not result in loss of immunoreactivity. immunohistochemistry ihc analysis of val-conh2-ir in brain and neuroendocrine tissue slices was performed according to standard procedures described previously with minor modifications. briefly, both brain and neuroendocrine tissue sections were processed while free-floating and were initially washed in 10 mm pbs followed by 10 % nabh in 10 mm pbs for 15 min. after three additional 10-min washes in 10 mm pbs, the tissue sections were permeabilized with  % tween- in 10 mm pbs for 20 min and washed with 10 mm pbs. then, endogenous peroxidase activity was blocked for 40 min in a  % hydrogen peroxidase/  % tween-20/10 mm pbs solution. following three additional 10-min washes in 10 mm pbs, the sections were incubated for 4 h in a preblocking solution containing dialyzed 10 % horse serum, 1 % bsa,  % tween- and 10 mm pbs. the sections were then incubated on an orbital shaker for 16 h at 4 °c in the primary valine amide mab p18c diluted 1: in the preblocking solution. after 10-min washes with 10 mm pbs, the sections were incubated for 2 h in a donkey anti-mouse biotinylated secondary antibody diluted 1: in pre-blocking solution. after and 10-min washes with the preblocking solution and with  % tween-20/10 mm pbs solution, respectively, the sections were incubated in the avidin–biotin complex, followed by 310-min washes with  % tween- in 10 mm pbs and by 10-min washes with 10 mm pbs. color development was enhanced by a 5–10 min incubation in a  % ′-diaminobenzidine tetrahydrochloride /  % h2o2/  % nickel sulfate/10 mm pbs solution. the sections were then washed in 10 mm pbs, mounted on slides, and air-dried overnight. the prepared slices were cleaned with xylene and cover-slipped with entellan. microscopic analysis of the brain sections was performed under bright-field illumination using a das leica dmr qwin microscope. positive immunoreactivity detected by the pc18c mab in tissue slices is referred as valine amide-like immunoreactivity. the neuroanatomical areas displaying positive val-conh2-ir signal were identified according to the atlas of the rat brain by paxinos and watson. controls as a control of labeling specificity, adjacent slices from rat neuroendocrine tissues and brain sections were preabsorbed with 10 μm of the valine amide residue. the intensity and the density of positive pc18c mab-immunoreactive signal in the brain were compared to those of val-conh2-ir detected in the pituitary and adrenal glands as shown in fig. 2a–c. both tissues were as used internal standards, and the immunoreactive signals were arbitrarily graded using a similar grading scale to the scale used to map the distribution of orl- receptor immunoreactivity in the rat brain as follows: very intense; intense; moderate; low; and not detected.fig.  pre-absorption control assays of valine amide immunoreactivity in tissues. the panels show bright-field photomicrographs of val-conh2–ir in neuroendocrine glands and sagittal sections of the rat brain using the specific pc18c mab. panel a illustrates the expression of val-conh2–ir in the pituitary gland before and after pre-absorption with 10 μm of the isovariant of the valine residue. note the absence of pc18c mab-ir in the tissue after treatment with the val-conh residue. nl neural lobe, il intermediate lobe, al anterior lobe. panel b illustrates the expression of val-conh2-ir in the adrenal gland before and after pre-absorption with 10 μm of the isovariant val-conh. pc18c mab-ir was localized to the core region of the adrenal gland. panel c shows the expression of val-conh2-ir in a representative sagittal section of the rat brain before and after pre-absorption with 10 μm of the isovariant val-conh. the intensity of immunoreactive signals was arbitrarily graded using a scale previously described in. scale bar = ×40 subtraction analysis immunoreactive signals produced from peptide antibodies used to map the anatomical distribution of c-terminal valine amide neuropeptides in the rat cns and neuroendocrine tissues were used as a reference to subtract from pc18c mab-ir signals detected in the rat cns. positive pc18c mab-ir signals in brain areas that did not match with previously identified c-terminal amide–peptide immunoreactivity, were considered as potential brain areas expressing putative novel valine amide neuropeptides. positive val-conh2-ir signals detected in subtracted brain regions were arbitrarily graded as follows: very intense; intense; moderate; low; or not detected.fig.  subtracted brain regions expressing putative valine amide peptide immunoreactivity. p18c5-mab-ir detected along the neuroaxis of the rat brain was subtracted from brain areas expressing peptide immunoreactivity for α-msh, metorphamide, secretin, and urocortins 1- as shown, moderate-to-high intensity mab-ir signals detected in subtracted brain areas were localized to the cortex and the main and accessory olfactory nuclei. subcortical areas displaying mab-ir signal included the subiculum, the core and shell of the nucleus accumbens, the substantia innominata of the septum, the amygdalo-hippocampal and amygdalo-anterior nuclei of the limbic system, the lateral posterior nucleus of the thalamus and the lateral hypothalamic nuclei. brainstem areas included the substantia nigra, the superior colliculus, the retrorubral nucleus, the solitary tract and the flocculus of the cerebellum. with the exception of the areas described above, only the lh and the fl showed intense immunolabeling similar to that detected in cortical layers of the cb, the il of the pituitary and the adrenal medulla. as shown, these brain areas were arbitrarily graded as displaying low val-conh2-peptide immunoreactive signals because they corresponded to areas containing the amidated peptides used as references in this study. the neuroanatomical areas showing putative valine amide peptide immunoreactivity were identified based on the rat brain atlas of paxinos and watson statistical analysis student’s t test was used to establish significant differences between samples assessed via ria or elisa. the significance level was set at p ≤ .  identification of hyperimmune vaccinated mice immunization of female balb/c mice with the klh-val-conh immunoconjugate after five consecutive booster injections produced animals exhibiting antibody responses against the α-amidated isoform of the valine residue. vaas collected from five of the seven vaccinated mice displayed significantly high cross-reactive signal against the bsa-tyr-gly2-4-val-conh adsorbed antigen based on elisa; these signals were twofold higher than the non-specific a values detected for the pre-immune control serum at the tested dilutions. however, the vaas from the vaccinated animals did not display significantly higher cross-reactive signal than the controls for the free α-carboxylic acid isoform of the valine residue in the bsa-rgdv-coo− antigen. the criteria for the selection of the samples for the cell fusion procedures were based on the detection of high immunoreactive signals against the α-amidated isoform of the valine residue in the bsa-tyr-gly2-4-val-conh immunoconjugate at the highest antisera dilution tested in vaccinated animals and on the lack of immunoreactivity for the carboxylic acid form of the same residue in the bsa-rgdv-coo− antigen. among the five hyperimmune animals selected for the generation of hybridoma-secreting mabs, mouse r exhibited the highest cross-reactivity against bsa-tyr-gly2-4-val-conh displaying high titers of antibodies at vaa dilutions of 1: and 1:.fig.  identification of valine amide antibodies in mice. animals defined as r1–r were vaccinated with an initial subcutaneous injection with a 1: emulsion of 50 µg of keyhole limpet hemocyanin -val-conh immunoconjugate and complete freund’s adjuvant, followed by booster injections with incomplete freund’s adjuvant. sera were collected and used to identify high-titer valine amide antisera in the vaccinated animals using a standard elisa. the wells were coated with synthetic immunoconjugates of bovine serum albumin --gly2–4-val-conh or bsa-rgdv-coo. the bsa-rgdv-coo−-coated wells displayed no significant signal. assays were performed in triplicate using two serial dilutions of mouse antisera. the ordinate describes the immunoreactive signals detected as absorbance at 490 nm in the assay. the values are expressed as the mean ± sem productive hybridomas cell fusion of activated splenocytes from mouse r with the murine myeloma sp2/ cells yielded stable hybridomas from a total of seeded cells. elisa revealed three stable hybridoma-forming colonies, designated as p15a p17c and p18c5; these three colonies secreted mabs that recognized the cognate epitope of val-conh in both the bsa-val-conh and bsa-tyr-gly2-4-val-conh immunoconjugates. the hybridomas secreting mabs displaying cross-reactivity against the α-amidated isoform of the valine residue represented  % of the total productive hybridomas formed after cell fusion. isotyping of the immunoglobulin class of the mabs secreted by these three hybridomas showed that they belonged to the igg class. elisa elisas were evaluated as an initial step in characterizing the specificity of the three distinct antibodies against the structural α-amidated form of the valine residue. immunoconjugates of amidated and non-amidated val- and leu-containing peptides linked to bsa were used as adsorbed antigens in the assays. the p15a and p17c mabs displayed no differences in cross-reactivity against the adsorbed antigens of bsa-val-conh bsa-tyr-gly2-4-val-conh metorphamide, bsa-val-coo−, or mastoparan x at the tested antibody dilutions. however, the p18c mab displayed a distinct pattern of reactivity against the α-amidated and free carboxylic acid isovariants of the valine residue in the tested antigens, as depicted in fig.  as shown, the p18c mab exhibited high evident cross-reactivity against the α-amidated form of the valine residue in bsa-val-conh at both low and high antibody dilutions. notably, a less apparent immunoreactive response was detected against the metorphamide/adrenorphin peptide antigen. cross-reactivity with the latter antigen decayed rapidly at the initial working dilutions of the antibody, in contrast to the slow decay of immunoreactive signal for the bsa-val-conh antigen at higher dilutions. however, the p18c mab cross-reacted with the antigenic isovariants of the valine residue in the bsa-val-coo−, bsa-tyr-gly2-4-val-conh bsa-tyr-gly2-4-val-coo−, and bsa-rgdv-coo− immunoconjugates, including the α-amidated isoform of leu in the mastoparan x peptide antigen. the immunoreactive signals against these antigens were - and -fold lower than those against the bsa-val-conh antigen at the lowest and highest antibody dilutions, respectively.fig.  characterization of p18c mab specificity. the vaccinated animals displaying the highest titers based on elisa were used for the generation of stable productive hybridomas. supernatants from the resulting hybridoma-producing colonies were screened via standard elisa to assess the cross-reactivity of hybridoma-secreting mabs against the isovariants of the val or leu residues contained in the c-terminal domain of bsa-conjugated peptides and were used as adsorbed antigens together with bsa-val-conh and bsa-val-coo− immunoconjugates in the assay. the p18c mab was tested at increasing dilutions in elisas or at a specific dilution in dot-blot assays. bsa-val-conh bsa-val-coo− and bsa- peptide immunoconjugates were used as adsorbed antigens in the elisa or as spotted antigens in the dot–blot assay. elisa was performed in triplicate using serial twofold dilutions of the p18c mab. the absorbance of the immunoreactive signals in positive wells was measured at λ = 490 nm dot–blot assays dot–blot assays were used to confirm and validate the specificity of the selected antibodies against the conjugated bsa-peptide antigens tested in the assay. in general, the results of the dot–blot assays showed that the intensity of ihc staining from each hybridoma-secreted mab was proportional to the concentration of the peptide antigen spotted on the membrane filter. the intensity of the detected immunoreactive signals in the dot–blots was compared to that of the control filters using only a secondary mouse-igg antibody, as depicted in fig. 5b. as shown, p18c mab displayed a high specificity and selectivity for the α-amidated isoform of the valine residue among the peptide antigens tested in the assay, as shown in fig. 5b. this mab exhibited very intense immunoreactive signals against the metorphamide and yggg-val-conh peptide antigens at the highest peptide concentration tested, showing moderate immunoreactive signals against the peptide at lower peptide concentrations. however, a weak immunoreactive signal was observed against mastoparan x at the highest concentration tested, and no signals were observed at lower concentrations. interestingly, no immunoreactive signals against both yggg-val-coo− and rgdv-coo− antigens were detected at any of the peptide concentration tested. thus, the p18c mab showed superior antibody selectivity against the α-amidated form of the valine residue. based on the high specificity and selectivity of the p18c mab for the α-amidated form of the valine residue in peptide antigens based on elisas and dot–blot assays, a sensitive solid-phase ria for p18c mab was developed and used to further assess the specificity and selectivity of this hybridoma-secreted mab. validation of the sensitivity of this ria enabled further identification and characterization of putative novel valine–amide neuropeptides from brain and neurosecretory tissues of rodents in subsequent studies. solid-phase ria a fmol-sensitive solid-phase ria for the p18c mab was developed using both the structural α-amidated isoform and the free carboxylic acid form of the valine residue conjugated to bsa, including both the c-terminal amide peptides and synthetic non-amidated peptides, using the synthetic -tyr-gly2-4-val-conh as the labeled tracer. a representative ria showing typical displacement curves by synthetic -tyr-gly2-4-val-conh using the p18c mab is depicted in fig.  as shown, the p18c mab recognized non-labeled tyr-gly2-4-val-conh over the tested concentration range, with an ic of  ±  fmol/well; the smallest measured displacement value, detected at ic was  ±  fmol/well, and the highest was  ±  fmol/well at ic based on this assay. in the same context, metorphamide was bound at an ic of  ±  fmol/well, and its lowest and highest displacement values were detected at the ic and the ic, respectively. similar displacement values were observed for bsa-val-conh the p18c mab detected bsa-val-conh at an ic of  ±  fmol/well, an ic of  ±  fmol/well, and an ic of  ±  fmol/well. moreover, no significant cross-reactivity against mastoparan x was observed for the concentration range of competitive peptides tested in this assay. furthermore, the p18c mab failed to recognize the structural free carboxylic acid isoform of the valine residue in both tyr-gly2-4-val-coo− and rgdv-coo− over the tested concentration range of the competitive peptide antigens, except for the lowest measurable displacement values detected at the ic for val-coo− peptides and for mastoparan x.fig.  specific solid-phase radioimmunoassay for the p18c mab. the p18c mab was used to generate a reliable and sensitive solid-phase ria for detecting the structural α-amidated isoform of the valine residue in synthetic peptide and non-peptide antigens. as shown, the p18c mab was highly sensitive in detecting the val-conh residue in metorphamide/adrenorphin, bsa-val-conh and tyr-gly2-4-val-conh antigens within the tested concentration range, displaying ic values of  ±  fmol/well,  ±  fmol/well, and  ±  fmol/well, respectively. however, no cross-reactivity was detected against antigens expressing the val-coo− residue in tyr-gly2-4-val-coo− or rgdv-coo−, including mastoparan x, within the range of competitive antigen concentrations tested in the assay p18c mab immunoreactivity in control tissues isotyping analysis of the p18c mab showed that it is classified as an igg antibody. as shown in fig. 2a–c, p18c mab immunostaining was abolished in control tissues of the hypophysis, the adrenal gland, and representative sagittal sections of the rat brain when the antibody was pre-absorbed with μm of the synthetic val-conh compound. the intermediate lobe of the hypophysis displayed intense p18c mab immunolabeling. pre-absorption with the synthetic amidated valine compound completely abolished p18c mab staining in this region. furthermore, the chromaffin cell-rich area of the adrenal gland displayed intense p18c mab immunolabeling. pre-absorption with the synthetic amidated valine compound completely blocked immunostaining in the core area of the adrenal gland in adjacent tissue sections using the p18c mab. in control sections of the rat brain, the p18c mab produced intense immunolabeling in several areas. pre-absorption with the synthetic valine amide resulted in complete abolishment of p18c mab immunostaining in adjacent brain sections. p18c mab immunoreactivity in the rat cns val-conh2-ir was distributed widely throughout the neuroaxis of the rat brain after immunostaining using the p18c mab. moderate to intense immunolabeling was predominantly localized to the neuropil, displaying scattered stained neuron-like cell bodies appearing in several regions within the rat brain. a brief summary of the distribution of p18c mab immunoreactivity in the rostrocaudal areas of the rat cns is described below.fig.  distribution of pc18c mab-ir in the rat brain. the figure illustrates the rostrocaudal distribution of pc18c mab-ir in 40-μm-thick sagittal slices in fixed rat brain tissue. bright-field photomicrographs of sagittal sections of the rat brain display lateral-to-medial aspects of the rat brain regions displaying val-conh2-ir. the neuroanatomical areas showing pc18c mab-ir were identified according to the rat brain atlas of paxinos and watson. cortex: fra frontal association cortex, m primary motor cortex, m secondary motor cortex, s1hl primary somatosensory cortex, pta parietal association cortex, v1m primary visual cortex, v2mm secondary visual cortex mediomedial, v2ml secondary visual cortex mediolateral, rsa retrosplenial agranular cortex, rsga retrosplenial granular a cortex, cg cingular cortex area 1– olfactory system: ob olfactory bulb, aob accessory olfactory bulb, aod anterior accessory olfactory nucleus, dorsal, aov anterior accessory olfactory nucleus, ventral, aol anterior accessory olfactory nucleus, lateral, aom anterior accessory olfactory nucleus, medial, aoe anterior accessory olfactory nucleus, external, tu olfactory tubercle, baot bed nucleus of the accessory olfactory tract, lot nucleus of the lateral olfactory tract. hippocampus: hi hippocampal c1–c fields and dentate gyrus, fi fimbria, s subiculum. basal ganglia: cpu caudoputamen, acbc accumbens nucleus core, acbsh accumbens nucleus shell, vp ventral pallidum. septum and septal areas: bst bed nucleus of the stria terminalis, bstl bed nucleus of the stria terminalis, lateral, bstm bed nucleus of the stria terminalis, medial, bac bed nucleus of the anterior commissure, ls lateral septal nucleus, lsd lateral septal nucleus, dorsal part, si substantia innominata, hdb nucleus of the horizontal limb of the diagonal band. amygdala: me medial amygdaloid nucleus, plco posterolateral cortical amygdaloid nucleus, pmco posteromedial cortical amygdaloid nucleus, aco anterior cortical amygdaloid nucleus, ahi amygdalo-hippocampal area. diencephalon: th thalamus, avvl anteroventral thalamic nucleus, ventrolateral, sth subthalamic nucleus, lpmr lateral posterior thalamic nucleus, mediorostral, md mediodorsal thalamic nucleus. hypothalamus: ah anterior hypothalamic area, lh lateral hypothalamic area, so supraoptic nucleus, sor supraoptic nucleus, retrochiasmatic, arc arcuate hypothalamic nucleus. mammillary bodies: mm medial mammillary nucleus. mesencephalon: sn substantia nigra, avt ventrotegmental area, vltg ventrolateral tegmental area, r red nucleus, sc superior colliculus, ic inferior colliculus, lpag lateral periaqueductal gray. pons and medulla: lc locus coeruleus, lpb lateral parabrachial nucleus, mpb medial parabrachial nucleus, rr retrorubral nucleus, pn pontine nucleus, cgpn central gray of the pons, ldtg laterodorsal tegmental nucleus, lso superior olive, lateral nucleus, iod inferior olive, dorsal nucleus, inta interposed cerebellar nucleus, anterior, intp interposed cerebellar nucleus, posterior, cu cuneate nucleus, lve lateral vestibular nucleus, spve spinal vestibular nucleus, mvepc medial vestibular nucleus, parvocellular, mvemc medial vestibular nucleus, magnocellular, vll ventral nucleus of the lateral lemniscus, sol solitary tract. cb cerebellum. see text for additional details. scale bar = ×40 telencephalon several telencephalic regions exhibited moderate to high immunolabeling, which was predominantly distributed in thin and dense fiber processes, oval-to-round somata, and diffuse fine puncta. pc18c mab immunoreactivity was detected in the primary and secondary motor cortices, parietal sensorimotor cortical fields, and the primary and secondary visual cortices, as well as the retrosplenial agranular and granular a cortices. within the olfactory system, p18c mab-ir was observed in both the main olfactory bulb and the accessory olfactory bulbs. in the hippocampus, p18c mab-ir was found in the striatum pyramidale and radiatum, along the ca1–ca fields of ammon´s horn, and, particularly, in the granular cell layer of the dentate gyrus -, as well as in the fimbria and the subiculum. p18c mab-ir was also detected in the basal ganglia, septal areas, the lateral and medial bst, the bed nucleus of the anterior commissure, the lateral septum, the dorsal part of the ls, the substantia innominata, and the nucleus of the horizontal limb of the diagonal band ] and the amygdala, which displayed moderate to high immunostaining that was predominantly distributed in sparse neuropil, stained round-to-oval cell bodies, and fine dendritic puncta. both the medial amygdaloid nucleus and the amygdalo-hippocampal area exhibited low levels of mab-ir. diencephalon p18c mab-ir was observed in several diencephalic structures, including the hypothalamus, which displayed moderate to intense mab immunostaining,. both the thalamus i.e., the anteroventral, lateral posterior and mediodorsal nuclei ] and the subthalamic nuclei, including several mammillary nuclei i.e., the medial mammillary, lateral mammillary, and submammilothalamic nuclei ], displayed low to moderate mab immunolabeling. the stained regions included the retrochiasmatic supraoptic nucleus of the hypothalamus. mesencephalon along the mesencephalon, p18c mab-ir was detected in the substantia nigra, the superior colliculus, the inferior colliculus, the ventral tegmental area, the red nucleus and the lateral periaqueductal gray area. these mesencephalic areas generally displayed low to moderate mab immunolabeling that was predominantly localized within a network of fiber processes dispersed among stained somata. cerebelum pc18c mab-ir was predominantly distributed along the purkinje cell layer of the cerebellar cortical layers; cells in this area displayed highly stained somata-like cell bodies, fine dendritic puncta, and a very dense network of protruding labeled purkinje cell axon fibers. low to moderate mab-ir was detected in both the flocculus and the white matter of the cb, including the medial cerebellar peduncle. pons and medulla the pc18c mab showed low to moderate immunostaining in nuclei and tracts along the rostrocaudal aspects of the pontine and medullary areas of the rat cns. within the most lateral aspects of the pontine/medullary areas of the brain, low to moderate mab-ir was detected at the retrorubral nucleus, the ventral nucleus of the lateral lemniscus, the lateral nucleus of the superior olive, the cuneate nucleus, and the lateral vestibular nucleus. in addition, the pontine nucleus, both the medial and lateral parabrachial nuclei, both the anterior and posterior parts of the interposed cerebellar nuclei, and the spinal vestibular nucleus showed positive immunostaining. within the medial aspects of these caudal regions of the rat brain, positive mab-ir was detected at the locus coeruleus, the parvocellular and magnocellular parts of the medial vestibular nuclei, and both the laterodorsal tegmental nucleus and the dorsal part of the inferior olive, including the central gray matter of the pons and the solitary tract. valine amide peptide immunoreactivity in the rat brain the reports describing the immunoreactive distribution of α-msh, metorphamide, secretin and ucn1- in the rat brain were used to compare each peptide immunoreactivity with the rostrocaudal distribution of p18c mab-ir in the rat cns, as depicted in fig. 7a–e, respectively. subtraction analysis p18c5-mab-ir detected along the rostrocaudal areas of the rat brain was compared with the distribution of valine amide peptide immunoreactivities detected in the rat brain. the immunoreactive signals obtained for each peptide were subtracted from pc18c mab-ir signals detected along the neuroaxis of the rat brain. the subtracted brain areas expressing val-conh2-ir material are depicted in fig.  most of these areas showed moderate-to-high pc18c mab-ir signals, with the exception of the cerebellar lobes and the lateral hypothalamus which exhibited an intense p18c5-mab-ir signals, similar to immunoreactive signal displayed by the il of the hypophysis, used as the internal control tissue. as shown in fig.  valine amide peptide immunoreactivity was detected in cortical areas of the telencephalon such as m the hindlimb and trunk regions of the primary somatosensory cortex, the parietal association cortex, v2mm, and v2ml. in addition, cvap-ir was observed in the main accessory ob and corresponding nuclei, described as the anterior accessory olfactory nucleus dorsal, the nucleus ventral, the nucleus lateral and the nucleus medialis. furthermore, vap-ir was detected along the olfactory tubercle, within the bed nucleus of the accessory olfactory tract and within the nucleus of the lateral olfactory tract. in addition, cvap-ir was detected in telencephalic structures such as s of the hi, nacc, and the substantia innominata of the ls, including both the ahi and the amygdalo-anterior area of the limbic system. in the diencephalon, vap-ir was found in lpmr of the thalamus and in the magnocellular portion of the lh. within the mesencephalon, vap-ir was primarily localized to the lateral portion of the substantia nigra and zo of the sc, whereas in the pontine and medullary areas, cvap-ir was primarily localized to rr, sol and the fl of the cb. peptide fractions displaying vap-ir several regions of the rat brain and endocrine glands were used to prepare purified peptide fractions from the subtracted brain areas to quantify the content of vap-ir using the specific solid-phase ria generated for the p18c mab. in addition, neuroendocrine glands were used as control tissues in the assay. as shown in fig.  the rat pituitary displayed the highest abundance of vap-ir, which was -fold greater than the amount observed in the brainstem. however, low amounts of val-conh2-ir were detected in the cortex and in the adrenal glands; these levels were - and -fold lower, respectively, than the levels measured in the pituitary. in the same context, both the cb and the pancreas contained low levels of val-conh2-ir, specifically 90- and 56-fold lower, respectively, than those in the pituitary.fig.  quantification of endogenous valine amide peptide-immunoreactive material in tissues. the abundance of valine amide peptide-immunoreactive material was estimated in purified peptide fractions prepared from subtracted brain regions and neuroendocrine glands, which displayed moderate-to-high pc18c mab immunolabeling based on the solid-phase ria developed for the pc18c mab. the plot shows that the highest abundance of val-conh2-ir material was found in the pituitary, followed by the brainstem, the adrenal gland, and the cortex. inset depicts a representative solid-phase ria for the pc18c mab used for the quantification of valine amide-immunoreactive material in the assays. tyr-gly-val-conh was used as a standard to construct a typical standard curve and was detected at a concentration of fmol/well at the ic value, whereas metorphamide/adrenorphin was detected at a concentration as low as fmol/well at the ic value discussion the α-amidation process is an essential step for the conversion of prohormones into mature peptides that possess complete biological activity, which includes receptor recognition and transduction of peptide hormone signals in target cells. the α-amidation process has been shown to occur most frequently on neutral amino acids in neuropeptides, which constitute the largest group of functional peptide messengers identified in vertebrate and/or invertebrate species. such peptides display pleiotropic activities across species. however, valine amide peptide hormones represent the smallest group of functional amidated peptides identified to date, and which include the enterochromaffin cell-related peptide hormone, secretin, which belongs to the pacap/vip/glucagon peptide hormone family; the pomc-derived peptide- α-msh -; the proenkephalin a-derived metorphamide/adrenorphin; the stress and appetite regulatory urocortins and the enterins, a family of brain peptides found in the enteric nervous system of aplysia, which modulate both feeding non-feeding behaviors in this marine specie. in order to search for neural structures expressing putative novel valine amide peptide immunoreactive material in the brain and neurosecretory tissues of mammals, we employed splenocyte/myeloma cell fusion procedures, with the aim to generate stable hybridoma-producing mabs able of recognizing the structural α-amidated isoform of the valine residue in constructed immunoconjugates. cell fusion procedures between activated splenocytes from the hyperimmune female balb/c mouse -r4— and the murine myeloma cell line sp2/ led to the identification of three hybridoma-producing mabs designated as p15a p17c and p18c and which displayed different levels of cross-reactivities against the structural α-amidated and/or the free carboxylic acid isovariants of the valine residue expressed in distinct antigens tested in different immunoassays used to validate the specificity and selectivity of these mabs. the efficiency of the cell fusion procedure from the total cells seeded for fusion was  %, a value considered within the efficient rates reported in the generation of productive hybridomas. as shown, only p18c mab showed a predominant cross-reactivity against the α-amidated isoform of the valine residue in the tested antigens in elisa and dot-blot assays however, p18c mab showed to be exquisitely selective for the structural α-amidated isoform of the valine residue in the constructed synthetic antigens tested in the fmol-sensitive solid phase ria but unable to recognize the free carboxylic acid of same residue in both rdgv, tyr-gly2-4-val-coo− peptide antigens ]. the high specificity and selectivity exhibited by p18c mab in recognizing the condensed amide substituent at the carbonyl group of the asymmetric carbon in the amino acid, may be due to the sort of complementarity determining regions encoded along both the heavy and light chains of the antibody, enhancing its preferential affinity and recognition for the structural amide epitope over the functional carbonyl group of the amino acid. in same line, specific mabs were shown to display predominant specificities and selectivity in the recognition of small antigens such as, acetyllysine and –methyllysine antigenic epitopes in proteins, or mabs shown to discriminate among isovariants of the immune mediator, interleukin, il-. based on our immunoassay results, we explored the capability of the p18c mab to detect valine amide peptide immunoreactivity along the neuroaxis of the rat brain and rat neuroendocrine tissues. our results showed that p18c mab to was able to recognize the structural α-amidated isoform of the valine residue in endogenous peptide material, as demonstrated for abolishment of mab immunostaining in tested tissues after preadsorbing the antibody with the synthetic valine amidated residue. as shown in fig.  the intermediate lobe of the pituitary showed an intense mab immunostaining when compared to absence of immunolabeling in both al and the pl in same neuroendocrine tissue. this neurosecretory area contains the “pituitary melanotrophs” shown to synthesize and release high concentrations of pomc-derived peptides into the bloodstream. thus, the detection of p18c mab-ir in the il of the hypophysis reflects the post-translational processing of pomc-derived peptides and conversion of mature c-terminal amidated peptide products, via the activity peptidylglycine α-amidating monooxygenase. in same line, the detection of p18c mab-ir at the core of the adrenal gland supports the cellular expression of bioactive metorphamide/adrenorphin peptide released from the proenkephalin a protein precursor from the chromaffin cells. p18c mab-immunoreactivity showed a wide distribution along the neuroaxis of the rat brain. most of the rostrocaudal areas expressing a low-to moderate or conspicuous immunostaining were matched with brain areas previously shown to contain the most prevalent bioactive valine amide neuropeptides α-msh; secretin; adrenorphin and ucns, suggesting that these peptides exert pleiotropic effects in time and tissue-specific manner, in both central and peripheral tissues after targeting their specific cell surface receptors. the properties and functions of valine amide peptide in the brain and peripheral tissues may be search in the web and in some reviews described herein and is out of the scope of the present paper to detail the relationship between brain structure and valine amide peptide bioactivities. data obtained from the subtraction analysis revealed the expression of mab-immunolabeling in specific brain areas that did not match with the neuronal distribution of valine amide neuropeptides. these results suggest that such areas represent neural structures able to synthesize putative novel valine amidated peptide products encoded in large propeptide precursor protein with functions yet to be elucidated. furthermore, these areas suggest that neurons operating within each immunolabeled brain region or nuclei must express same molecular machinery and posttranslational enzyme-dependent mechanisms, in order to allow the biosynthesis and conversion of prohormones into functional c-terminal amide neuropeptides at the expense of the functional bioactivities of pc1/pc proconvertases and the peptidylglycine α-amidating monooxygenase, respectively. this hypothesis is supported by the specific tissue-dependent processing of pomc-derived peptide hormones in the brain and peripheral tissues, including the processing of proenkephalin a in the brain and neurosecretory tissues such as, the chromaffin cells of the adrenal medulla. moreover, based on the specificity and selectivity of the p18c mab for detecting immunoreactive peptide material in the rat brain and neuroendocrine tissues; we explored the feasibility of this mab to determine the abundance of val-conh2-ir in peptide fractions isolated from immunostained subtracted regions using our fmol-sensitive solid-phase ria raised for p18c mab. as shown, both cortex, brainstem and cb were used as representative subtracted brain areas encoding putative valine amide peptide material, in addition of the pancreas, the pituitary and adrenal glands, used as neuroendocrine control tissues. furthermore, the highest amount of val-conh2-ir peptide material was detected in the pituitary followed by the brainstem, the adrenal gland and the cortex. the relative abundance of immunoreactive peptide material determined in both bs, ad and the cx were, and  %, respectively, from that detected in the pituitary. in same line, both cerebellum and pancreas showed lowest content of estimated val-conh2-ir. differences in the relative amount of peptide immunoreactivity estimated for each neural tissue could rely on the cell concentration of valine amide neuropeptides stored before their release into the extacellular milieu at the time of tissue extraction and preparation of peptide fractions from tissue homogenates. although at this stage during mab screening, the exact amount of putative novel valine amide neuropeptides per tissue may be impossible to obtain. however, both recombinant dna technology and cloning procedures may lead to the identification and characterization of putative novel cdnas encoding valine amide peptide sequences, as demonstrated for the “enterins”, a family of structurally related nonapeptides and decapeptides that are present in the gut and cns of aplysia, and most of which share the hsfvamide sequence at the c terminus.  the present work describes an experimental strategy used to generate murine monoclonal antibodies able to differentiate between two isovariants of the valine residue. one single mab designated as, p18c mab, showed a unique specificity and selectivity for the structural α-amidated isoform of this neutral amino acid in natural and synthetic peptide antigens, as revealed from our immunoassays, besides of demonstrating a great capability to detect putative—yet unidentified—valine amide peptide immunoreactive material in specific areas of the rat brain. the ability of this mab to distinguish between two small structural epitopes in same residue, posit for its wide applications in both research and clinical settings. for instance, p18c mab may be used to generate specific biological “kits” used to purify and detect valine amidated peptide products in tissue and/or biological fluids. furthermore, this mab may be used in immunotherapy settings to reduce tumor growth and tumorigenesis; or used as a detoxifying–neutralizing reagent from blood poisoning compounds such as, toxic amidated peptide molecules found in food, plants and/or animal species. overall, this work portraits the potential applications of a single mab, who displays exquisite properties when reacting against valine amide peptide antigens, including its capability to identify brain regions expressing putative novel valine amide neuropeptides, with functions yet to be discovered. authors’ contributions ba conceived the study. ba and pl participated in the design and coordination of the study, data analysis, and drafting of the manuscript. jah participated in the design and synthesis of the immunoconjugates. mm and rh, together with ba, conducted all immunoassays related to the generation of hyperimmune mice, cell fusion and the generation and expansion of productive hybridomas and collected data for the manuscript. jc and jah participated in the elisas for monoclonal and polyclonal antibodies, as well as protein determination and the collection of data for the present manuscript. jc and mep, together with pl performed dot-blot assays, rias, tissue extraction, and preparation of peptide fractions from brain regions and neuroendocrine tissues. both investigators were involved in the collection of data for the present manuscript. ra and af participated in the immunohistochemistry studies. both investigators ba and pl were involved in the collection of data. as performed the subtraction analysis of positive immunoreactive areas for the p18c mab and performed the statistical analysis for this study. all authors helped to draft the manuscript, read and approved the final manuscript, and agreed on its submission. all authors read and approved the final manuscript. 
alpha -amidation occurs in almost all species and amino acids identified in nature. our results demonstrate the versatility of a single mab able to differentiate between two structural subdomains of a single amino acid. these brain regions varied in the relative amount of putative novel valine amide peptide immunoreactive material estimated through a fmol-sensitive solid-phase radioimmunoassay raised for p18c mab. c-terminal valine amide neuropeptides constitute the smallest group of functional peptide compounds identified in neurosecretory structures in vertebrate and invertebrate species. p18c mab-immunoreactivity exhibited a wide distribution along the neuroaxis of the rat brain, particularly in brain areas that did not cross-match with the neuronal distribution of known valine amide neuropeptides. p18c mab displayed the highest specificity and selectivity against c-terminal valine amidated peptide antigens in different immunoassays. the α-amidated isoform of valine residue was conjugated to klh-protein carrier and used to immunize mice. three productive hybridoma were tested against peptides antigens containing both the c-terminal α-amidated and free α-carboxylic acid isovariant of the valine residue. this mab offers a wide spectrum of potential applications in research and medicine, whose uses may extend from a biological reagent to a detoxifying reagent or as a specific immunoreagent in immunotherapy settings among many others. hyperimmune animals displaying high titers of valine amide antisera were used to generate stable hybridoma-secreting mabs. alpha -amidation of peptides is a mechanism required for the conversion of prohormones into functional peptide sequences that display biological activities, receptor recognition and signal transduction on target cells.
 alpha -amidation of peptides is a mechanism required for the conversion of prohormones into functional peptide sequences that display biological activities, receptor recognition and signal transduction on target cells. alpha -amidation occurs in almost all species and amino acids identified in nature. c-terminal valine amide neuropeptides constitute the smallest group of functional peptide compounds identified in neurosecretory structures in vertebrate and invertebrate species. methods the α-amidated isoform of valine residue was conjugated to klh-protein carrier and used to immunize mice. hyperimmune animals displaying high titers of valine amide antisera were used to generate stable hybridoma-secreting mabs. three productive hybridoma were tested against peptides antigens containing both the c-terminal α-amidated and free α-carboxylic acid isovariant of the valine residue.  p18c mab displayed the highest specificity and selectivity against c-terminal valine amidated peptide antigens in different immunoassays. p18c mab-immunoreactivity exhibited a wide distribution along the neuroaxis of the rat brain, particularly in brain areas that did not cross-match with the neuronal distribution of known valine amide neuropeptides. these brain regions varied in the relative amount of putative novel valine amide peptide immunoreactive material estimated through a fmol-sensitive solid-phase radioimmunoassay raised for p18c mab.  our results demonstrate the versatility of a single mab able to differentiate between two structural subdomains of a single amino acid. this mab offers a wide spectrum of potential applications in research and medicine, whose uses may extend from a biological reagent to a detoxifying reagent or as a specific immunoreagent in immunotherapy settings among many others. keywords valineleucineantibodyantiserahybridomaα-amidationcross-reactivityimmunoassayimmunoconjugateneuropeptideissue-copyright-statement© the author 2015 