Growth hormone releasing hormone complementary peptides

The present invention provides synthetic peptides and fragments and analogs thereof complementary to growth hormone releasing hormone, and antibodies raised against such peptides. The present invention also includes methods of using such peptides and antibodies.

BACKGROUND OF THE INVENTION 
This invention relates to biologically important synthetic Growth Hormone 
Releasing Hormone complementary peptides and antibodies thereto. 
Growth Hormone Releasing Hormone (GHRH), also known as Growth Hormone 
Releasing Factor, is a hypothalamic peptide which positively regulates the 
synthesis and secretion of growth hormone in the anterior pituitary. GHRH 
was originally isolated and structurally characterized from human 
pancreatic tumors that caused acromegaly. Since then, GHRH has been 
isolated from several different species, including rat, pig, cow, man, 
sheep, and goat (Bohlen et al., Biochem. Biophys. Res. Comm. 125: 
1005-1012, 1984). 
GHRH has been widely studied. The amino acid sequence of rat hypothalamic 
GHRH has been determined (Bohlen et al., 1984, supra). Also, the cDNA to 
rat GHRH has been reported (Mayo et al., Nature 314: 464-467, 1985). Human 
GHRH is reported to have high homology with rat GHRH and a GH-stimulating 
effect on the rat pituitary gland (Baird et al., Neuroendocrinology 42: 
273-276, 1986). Ling et al., Biochem. Biophys. Res. Comm. 123: 854-861, 
(1984) tested the capacity of a series of C-terminal deleted analogs of 
synthetic human GHRH to release growth hormone and report that the minimal 
biologically important core of GHRH with full intrinsic activity comprises 
the fragment (3-21). 
Biro suggested (Biro, Medical Hypotheses, 7:969-1007, 1981) that 
protein-protein interactions are based on binding of restricted portions 
of the proteins that are primarily formed by "informational complementary 
(ic)" amino acids. He also suggested that these specific complementary 
amino acids are encoded by complementary DNA sequences; further, 
interaction between complementary amino acid sequences would occur both in 
parallel and in antiparallel alignment of the peptides. Although Biro 
(1981) investigated only the 5'-3' direction, his data also support the 
significance of the 3'-5' direction by emphasizing the importance of 
palindrome nucleic acid sequences in encoding the specifically interacting 
peptide sequences. 
Model peptides designed to have minimal homology to the naturally occurring 
peptide but having the same hydropathic pattern have been demonstrated to 
exhibit biological activity (See, e.g., Kaiser and Kezdy, Science, 223: 
249-255, 1984). Blalock and Smith reported that codons for hydrophilic and 
hydrophobic amino acids on one strand of DNA are complemented by codons 
for hydrophobic and hydrophilic amino acids on the other DNA strand, 
respectively, and that codons for slightly hydrophilic ("uncharged") amino 
acids are complemented by codons for amino acids of the same character 
(Biochem. Biophys. Res. Comm. 121: 203-207, 1984). These workers theorize 
that the two complementary strands of the DNA encode two peptides having 
hydropathic anti-complementarity. It has been reported that the 
hydropathic anti-complementarity of a number of amino acids (and hence 
that of the peptides) based on the genetic code occurs when complementary 
codons are read in the 5'-3' as well as in the 3'-5' direction (Bost et 
al., Proc. Natl. Acad. Sci. 82: 1372-1375, 1985a; Bost et al., Biochem. 
Biophys. Res. Comm. 128: 1372-1380, 1985b; Blalock and Bost, Biochem. J. 
234: 679-683, 1986). 
Bost et al., 1985a, supra, have reported that a peptide ("HTCA") 
corresponding to the complementary (5'-3') RNA sequence of ACTH (1-24) 
mRNA is capable of binding synthetic ACTH as determined by ELISA. Blalock 
and Bost, supra, have reported that both 3'-5' and 5'-3' complementary 
peptides bind .sup.125 I-ACTH in a solid phase binding assay. Similar 
binding was reported for 5'-3' complementary peptides of .gamma.-endorphin 
(Bost et al., 1985a, supra). Antibodies raised against the complementary 
peptide, HTCA, have been reported to stimulate corticosterone secretion of 
adrenocortical cells in vitro (Bost et al., 1985a, supra). It is also 
reported that, using the same antibodies in immune affinity 
chromatography, the ACTH-receptor was purified and its molecular structure 
and .sup.125 I-ACTH binding characteristics were determined (Bost and 
Blalock, Molec. Cell. Endocrinol. 44: 1-9, 1986). According to Bost et 
al., 1985b, supra, messenger RNA sequences complementary to the mRNA 
sequences for the receptors of epidermal growth factor (EGF), 
interleukin-2 (IL-2) and transferrin (TF) encode peptides having high 
homologies with the amino acid sequence of their respective ligands, if 
the transcription is carried out in 3'-5' direction. Gorcs et al., 
Peptides, 7: 1137-1145 (1986) report possible recognition of the GnRH 
receptor by an antiserum against a peptide encoded by nucleotide sequence 
complementary to mRNA of a GnRH precursor peptide. 
GHRH peptides have applications to the fields of animal husbandry, clinical 
medicine and basic research. For example, it was determined that 
administration of human GHRH to lactating holstein cows increases the 
secretion of growth hormone consistently and causes an apparent increase 
in feed to milk conversion (Enright et al., J. Dairy Sci. 69: 344-351, 
1986). GHRH peptides are useful in vitro, e.g., as unique research tools 
for understanding how growth hormone secretion is regulated at the 
pituitary level and are also be useful in vivo, e.g., to treat symptoms 
related to growth hormone deficiencies, to increase the rate and extent of 
growth in commercial animals, to increase milk yield in commercial 
animals. 
SUMMARY OF THE INVENTION 
The present invention provides biologically important synthetic peptides 
and fragments thereof, complementary to GHRH. The present invention also 
includes antibodies raised against such peptides and methods of using such 
peptides and antibodies. 
One peptide, according to this invention, is complementary in the 3'-5' 
direction to residues 14-25 of GHRH and has the sequence: 
H-Asp-Pro-Val-Asn-Ile-Arg-Ala-Phe-Asp-Asp-Val-Leu-Y, wherein Y is OH or 
NH.sub.2 (hereinafter referred to as 3'-5' CP). Another peptide of this 
invention is complementary in the 5'-3' direction to residues 18-43 of 
GHRH and has the following sequence: 
H-Val-Glu-Pro-Gly-Ser-Leu-Phe-Leu-Val-Pro-Leu-Pro-Leu-Leu-Pro-Val-His-Asp- 
Phe-Val-Gln-Gln-Phe-Ala-Gly-Ile-Y, wherein Y is OH or NH.sub.2 (hereinafter 
referred to as 5'-3' CP). 
The GHRH complementary peptides of the present invention or fragments 
thereof, or antibodies raised against them may be used (1) in vitro, e.g., 
for various assays, and (2) may be administered in vivo to mammals, 
including humans, to increase Growth Hormone ("GH") release for 
therapeutic purposes and for the purpose of increasing growth of poultry 
and livestock, increasing milk production in cows and possibly increasing 
wool production in sheep.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides biologically important complementary 
peptides ("CP") overlapping the sequence of human GHRH (18-25). As 
mentioned above, Ling et al. found a gradual decrease in relative 
GH-releasing potency of C-terminal deleted GHRH fragments until reaching 
hGHRH (1-27) (12% relative potency). A sharp decrease in biological 
activity was found with shorter fragments, e.g., hGHRH (1-24): 0.02% 
relative potency; hGHRH (1-19): No activity. Hence, the sequence of hGHRH 
(20-27) is essential for the receptor ligand interaction. The amino acid 
sequence of both the 3'-5' CP and 5'-3' CP was derived from the mRNA (Mayo 
et al., supra) for rat GHRH. 
Both the 3'-5' and 5'-3' CP contain a 6 amino acid-long sequence that is 
identical with a sequence in rat GHRH and human GHRH in hydropathic 
nature. If one aligns the amino acid sequences of the 3'-5' and 5'-3' CP 
in antiparallel direction, one can find a high homology in their amino 
acid distribution in the region that corresponds to GHRH (22-27) (FIG. 
1b). The hydrophobic amino acids are the same in both the 3'-5' and 5'-3' 
CP, while the hydrophilic ones are closely related but have a different 
charge (Asp vs. Gln). On the contrary, other peptides in the glucagon 
family have "uncharged" amino acids in position 25 (Trp in gastric 
inhibitory peptide and glucagon; Gly in secretin, Ser in vasoactive 
intestinal peptide [VIP]and peptide histidine-isoleucine-27 [PHI-27]) and 
in position 22 (Tyr in VIP and PHI-27). 
The peptides of the present invention can be synthesized by any suitable 
method, such as by exclusively solid-phase techniques, by partial 
solid-phase techniques, by fragment condensation, by classical solution 
couplings, or by the employment of recently developed recombinant DNA 
techniques. 
The 12 amino acid long 3'-5' CP of GHRH and the 26 amino acid long 5'-3' CP 
of GHRH (See FIG. 1a) were synthesized at the Molecular Resource Center, 
Macromolecular Synthesis Laboratory, University of Tennessee, Memphis, Dr. 
T. C. Cooper, Director. Applied Biosystems Model 430A, a fully automatic 
instrument-reagent system for solid phase peptide synthesis, was used. The 
Model 430A utilized an optimized system based on R. B. Merrifield's 
concept of solid phase peptide synthesis (Virender et al., Anal. Biochem., 
117: 147-157, 1981). Typically, solid phase synthesis occurs from the `C-` 
to the `N-terminal` of the peptide sequence. The alpha-carboxyl group of 
the C-terminal amino acid residue is covalently attached to an insoluble 
polystyrene resin bead through an organic linker. The alpha-amino group of 
this amino acid, and all the other amino acids used in synthesis, are 
protected by an organic moiety. 
A general synthesis cycle consists of: deprotection of the resin-bound 
alpha-amino group, then washing, neutralization and washing of the resin. 
Next in the cycle is the formation of a peptide bond between the 
deprotected alpha-amino group and the activated carboxyl of the next 
alpha-amino protected amino acid of the sequence. This cycle is repeated 
until the desired sequence is complete. When synthesis is complete, the 
peptide is deprotected and cleaved from its polymer support; it is then 
separated from the resin and purified. 
The GHRH binding capacity of both complementary peptides was tested in an 
ELISA system as described in the examples below. The 3'-5' CP has a 
GH-stimulating effect as shown in the examples below. 
Polyclonal antibodies ("anti 5'-3' CP IgG") were raised in rabbits against 
the 5'-3' CP and purified using techniques known to those skilled in the 
art. The 5'-3' CP was coupled to a carrier protein, keyhole limpet 
hemocyanin (KLH), and used to immunize rabbits. Sera obtained from the 
rabbits was purified using Protein A affinity-chromatography. Polyclonal 
antibodies were also raised against the 3'-5' CP and purified using 
similar techniques. 
As shown in the examples below, purified polyclonal anti 5'-3' CP IgG has a 
GH-stimulating effect. Anti 5'-3' CP IgG stimulates GH secretion from 
adult male rat pituitaries in cell culture. Thus, interaction of the anti 
5'-3' GP IgG preparation and the pituitary cells has a similar effect on 
GH secretion as the interaction of the GHRH receptor and GHRH. 
The 3'-5' and 5'-3' CP, fragments thereof, or analogs thereof having well 
known substitutions and/or additions, as well as non-toxic salts of any of 
the foregoing, and antibodies to such peptides, may be used for research 
purposes or diagnostically, or may be administered therapeutically to 
mammals, including humans. Antibodies provided by the present invention 
may be used for preparative procedures, e.g., to purify the GHRH receptor 
by affinity chromatography. 
The 3'-5' CP or 5'-3' CP, fragments thereof, or antibodies against them, 
("the active ingredient") can be used for the in vivo treatment of 
mammalian species by physicians and/or veterinarians. The amount of said 
active ingredient will, of course, depend upon the severity of the 
condition being treated, the route of administration chosen and the 
specific activity of the active ingredient, and ultimately will be decided 
by the attending physician or veterinarian. Such amount of active 
ingredient or IgG as determined by the attending physician or veterinarian 
is also referred to herein as a "treatment effective" amount. 
The active ingredient may be administered by any route appropriate to the 
condition being tested. Preferably, the peptide is injected into the 
bloodstream of the mammal being treated. It will be readily appreciated by 
those skilled in the art that the preferred route will vary with the 
condition being treated. 
While it is possible for the active ingredient to be administered as the 
pure or substantially pure compound, it is preferable to present it as a 
pharmaceutical formulation or preparation. 
The formulations of the present invention, both for veterinary and for 
human use, comprise the 3'-5' CP or 5'-3' CP, a fragment thereof, analog 
thereof or antibody thereto, as described above, together with one or more 
pharmaceutically acceptable carriers therefor and optionally, other 
therapeutic ingredients. The carriers must be "acceptable" in the sense of 
being compatible with the other ingredients of the formulation and not 
deleterious to the recipient thereof. Such carriers are well known to 
those skilled in the art of pharmacology. Desirably, the formulation 
should not include oxidation agents and other substances with which 
peptides are known to be incompatible. The formulations may conveniently 
be presented in unit dosage form and may be prepared by any of the methods 
well known in the art of pharmacy. All methods include the step of 
bringing into association the active ingredient with a carrier which may 
constitute one or more accessory ingredients. In general, the formulations 
are prepared by uniformly and intimately bringing into association the 
active ingredient with liquid carriers or finally divided solid carriers 
or both, and then, if necessary, shaping the product into the desired 
formulation. 
Formulations suitable for parenteral administration conveniently comprise 
sterile aqueous solutions of the active ingredient, which solutions are 
preferably isotonic with the blood of the recipient. Such formulations may 
be conveniently prepared by dissolving solid active ingredient in water to 
produce an aqueous solution, and rendering said solution sterile. The 
formulations may be presented in unit or multi-dose containers, for 
example, sealed ampules or vials. 
The invention will be further understood with reference to the following 
examples which are purely exemplary in nature and are not meant to be 
utilized to limit the scope of the invention. 
EXAMPLE 1 
Synthesis of 3'-5' and 5'-3' CP of GHRH Peptides 
The syntheses of 3'-5' CP amide, with the sequence: 
H-Asp-Pro-Val-Asn-Ile-Arg-Ala-Phe-Asp-Asp-Val-Leu-NH.sub.2 and 5'-3' CP 
amide, with the sequence: 
EQU H-Ile-Gly-Ala-Phe-Gln-Gln-Val-Phe-Asp-His-Val-Pro-Leu-Leu-Pro-Leu-Pro-Val-L 
eu-Phe-Leu-Ser-Gly-Pro-Glu-Val-NH.sub.2 
were performed on an Applied Biosystems Model 430A Peptide Synthesizer 
which is totally microprocessor controlled, utilizing software version 
1.2. This machine is a solenoid controlled, gas-driven (prepurified 
nitrogen or argon) synthesizer. The aminomethyl resin was purchased from 
Applied Biosystems, Inc. and had an amino acid substitution of about 
0.6-0.7 millimoles/gm. of resin. This resin consists of 1% cross-linked 
polystyrene to which had been attached a phenylacetamidomethyl (PAM) 
group. The carboxyl terminal amino acid, leucine or isoleucine, in these 
cases, was attached to the PAM resin and contained an amino terminal 
blocked with the TBOC (t-butoxylcarbonyl)-protecting group. 
After deprotection of this group on the machine utilizing trifluroacetic 
acid, the peptide was built in a stepwise manner. TBOC-protected amino 
acids were purchased in pre-weighed amounts (approximately 2 mmoles) in 
sealed cartridges from Applied Biosystems. The chemical forms of the amino 
acids used in these peptides are listed below: 
______________________________________ 
t-BOC--L--Aspartic acid 
t-BOC--L--Glutamine 
(O--Benzyl) t-BOC--L--Glutamate (O--BZ) 
t-BOC--L--Asparagine 
t-BOC--L--Glycine 
t-BOC--L--Alanine 
t-BOC--L--Serine (Benzyl) 
t-BOC--L--Arginine (Tosyl) 
t-BOC--L--Histidine (Tos) 
t-BOC--L--Leucine.H.sub.2 O 
t-BOC--L--Valine 
t-BOC--L--Isoleucine 
t-BOC--L--Phenylalanine 
t-BOC--L--Proline 
______________________________________ 
O-Benzyl, Benzyl and Tosyl (p-toluenesulfonyl) refer to the type of amino 
and hydroxyl protecting groups present on the amino acid derivatives. 
Briefly, the resin-bound amino acid is deprotected by the addition of 
trifluroacetic acid, neutralized, and washed extensively. T-BOC protected 
amino acids are dissolved in suitable solvents and transferred to the 
activator vessel of the instrument. DCC (Dicyclohexylcarbodiimide) is then 
added to the dissolved amino acid, and a symmetric anhydride is formed, 
called a PSA (protected symmetric anhydride). A by-product of the 
reaction, dicyclohexylurea, forms a precipitate. The equation for this 
reaction is given below: 
##STR1## 
There are three exceptions to the use of amino acid PSA's in the Model 430A 
cycles; asparagine, glutamine, and arginine are coupled as 
1-hydroxybenzotriazole (HOBT) esters. These esters are utilized because 
symmetric anhydrides of these amino acids are unstable and undergo 
unacceptable side reactions. In addition to being HOBT-esters, these amino 
acids are double coupled, that is, two cartridges of these amino acids are 
required for each cycle. After the initial coupling, the resin is washed, 
then the coupling is repeated, to increase the yields. 
After the PSA is prepared, the amino acid is transferred to the 
concentrator vessel, where it is purged with nitrogen to remove volatile 
dichloromethane (DCM). N,N-Dimethylformamide (DMF) is then added to the 
amino acid. Individual PSA's have varying stabilities in DCM/DMF mixtures, 
so the temperatures are carefully regulated by the program. HOBT-esters 
are not purged since they are unstable. 
Certain individual amino acids require special treatment. Histidine is one 
such amino acid. lt is purchased as the DCHA (dicyclohexylamine) salt and 
passed through a suitably prepared AG-50-X8(H.sup.+) ion exchange column 
in DCM just before use. T-BOC-his(Tos) is unstable in DCM at room 
temperature, and thus must be placed on the machine within 4 hours of use. 
Two other alterations in the cycle are required with histidine. Since the 
amino acid is supplied as a solution, no DCM is delivered to the 
cartridge, and the purge cycle is shortened to 6.5 minutes because of its 
instability. 
As the amino acids are activated and the solvents are evaporated and 
changed, the resin is treated with trifluoroacetic acid (TFA) in DCM 
twice, then washed with DCM to remove some of the TFA. The resin is then 
neutralized with diisopropylethylamine (DIEA) in DMF and washed with DMF. 
DMF is the solvent of choice for coupling of activated amino acids to the 
growing peptide chain. 
After the peptide is complete, the T-BOC group is then removed with the 
standard deprotection step and washed with DCM. 
Synopsis of one single couple cycle: 
Addition of the first amino acid residue of the first peptide, valine, was 
carried out in the following manner: 
1. The amino acid cartridge was punctured with a needle assembly and 
approximately 3 ml of dichloromethane (DCM) was delivered to the powdered 
amino acid. The solution was mixed with nitrogen bubbles for approximately 
2 minutes, and the solution transferred to the activator vessel. 
2. One millimole of 0.5M dicyclohexylcarbodiimide (DCC) in dichloromethane 
was delivered to the activator vessel followed by gas purging to mix. The 
by-product of this reaction, dicyclohexylurea, begins to precipitate 
almost immediately. After 8 minutes, the solution is filtered through a 
glass frit, and delivered to the concentrator vessel. 
3. During the activation and concentration cycles, the resin, in the 
reaction vessel, was treated in the following way: 
a. 33% trifluoroacetic acid (TFA) in DCM for 2.5 minutes 
b. 50% TFA in DCM for 18 minutes 
c. Three DCM washes 
d. 10% DIEA in DMF for 3 minutes 
e. Five DMF washes 
The deprotection steps are identical to the above for all amino acids. 
4. In the concentrator vessel, the DCM solution was purged with nitrogen 
gas for a total of about 16 minutes, and approximately 4 ml of DMF was 
added. The temperature was automatically controlled at 15.degree. C. or 
below. After the last DMF wash of the resin, the activated amino acid in 
DMF was delivered to the reaction vessel and coupled for about 25 minutes 
with vigorous vortexing. Other amino acids utilizing this coupling time 
are histidine, leucine, isoleucine, phenylalanine and proline. Others such 
as aspartic acid, glycine, serine and alanine use about 18 minutes. 
Arginine and asparagine are coupled twice for 42 minutes each. 
5. As the peptide chain lengthens, longer coupling times are required and 
are automatically incorporated into the compiled program as it progresses. 
6. After coupling was completed, the resin was drained and washed five 
times with DCM. Activation of the next amino acid and deprotection of the 
resin for the next cycle was begun. 
Synopsis of a double couple cycle: 
1. Two amino acid cartridges were required for the double couple cycles of 
arginine and asparagine. These were placed one after another in the 
guideway of the Model 430A synthesizer. 
2. The first amino acid cartridge was punctured with a needle assembly and 
approximately 4 ml of HOBT (1-Hydroxybenzotriazole) in 
N,N-Dimethylformamide (DMF) (2 mmole) was delivered to the cartridge to 
dissolve the amino acid. Asparagine requires the addition of 0.3 ml of 
DCM, and arginine requires 1.5 ml of DCM for complete dissolution. After 
mixing (6.5 min for asparagine, 8 min for arginine), the solution was 
transferred to the activator vessel. 
3. The HOBT-ester double couple cycles all employ the same transfer 
process. The HOBT/amino acid mixture is added to 4 ml (2 mmoles) of DCC 
(dicyclohexylcarbodiimide) in the activator vessel. After precipitation of 
DCU, the HOBT-ester is transferred to the concentrator vessel after being 
filtered through a glass frit. 
4. The solution is then directly transferred to the reaction vessel, 
without gas purging. 
5. During the previously described activation phase, the resin was 
deprotected using the following schedule: 
a. 33% TFA in DCM for 2.5 minutes 
b. 50% TFA in DCM for 18 minutes 
c. Three DCM washes 
d. 10% DIEA in DMF for 3 minutes 
e. Five DMF washes 
f. Begin first coupling period 
6. Coupling then takes place for about 42 minutes with vigorous vortexing. 
The resin is then washed according to the following schedule: 
g. Three DMF washes 
h. 10% DIEA in DMF for 45 seconds 
i. One DMF wash 
j. Three DCM washes 
7. The second amino acid cartridge is prepared in the same manner as the 
first, and coupled for another 42 minutes. The resin is then drained, 
washed with DMF, then 5 times with DCM. 
EXAMPLE 2 
ELISA Using 3'-5' and 5'-3' 
The principle of the assay is shown in FIG. 2. 
Reagents: 
Buffers and dilutions 
Coating buffer: 41.25 mM NaHCO.sub.3 /8.75 mM Na.sub.2 CO.sub.3, pH 9.6, 
containing 0.1 g/l thimerosal. 
Wash buffer No. 1: 12.38 mM Na.sub.2 HPO.sub.4 /2.62 mM KH.sub.2 PO.sub.4, 
pH 7.4 containing 135 mM NaCl, 1.0 g/l sodium azide, 5000 USP units/l 
heparin and 0.5 ml/l Tween. 
Diluting buffer No. 1: Wash buffer No. 1 containing 1% (w/v) BSA and 1% 
(v/v) normal goat serum (NGS). 
Wash buffer No. 2: 10.0 mM TRIS/HCl buffer (pH 7.4) containing 135 mM NaCl, 
1.0 g/l sodium azide, 5000 USP units heparin and 0.5 ml/l Tween 20. 
Diluting buffer No. 2: Wash buffer No. 2 containing 1% (w/v) BSA. 
Substrate buffer: 50 mM diethanolamine, 0.5 mM MgCl.sub.2, 0.1 mM 
ZnCl.sub.2, 1.0 g/l sodium azide, 10N HCl to adjust the pH to 9.5. 
Amplifier buffer: 16.5 mM Na.sub.2 HPO.sub.4, 3.5 mM KH.sub.2 PO.sub.4, 1.0 
g/l sodium azide, pH 7.2. 
Substrate: 0.1 mM .beta.-NADP dissolved in substrate buffer. 
Stock solutions of enzymes: Alcohol dehydrogenase (ADH, Sigma, A-3263) and 
diaphorase (Sigma, D-2381) were dissolved in H.sub.2 O in a concentration 
of 20 and 15 mg/ml, respectively. Aliquots were stored frozen at 
-70.degree. C. 
Amplifier: 96 ml amplifier buffer, 4 ml absolute ethanol, 0.5 g BSA, 28 mg 
para-iodo-nitrotetrazolium violet (INT, Sigma 1-8377). After the INT had 
been dissolved, 200 .mu.l of ADH and 200 .mu.l of diaphorase solution was 
added to 20 ml solution immediately before use. 
All materials used for coating of microELISA plates (Immunolon-2, 
Dynatech), were dissolved in coating buffer. 
All other reagents were diluted with diluting buffer No. 1 with the 
exception of the streptavidin-alkaline phosphatase conjugate (SA-AP, 
Bethesda Research Laboratories) which was diluted in diluting buffer No. 
2. 
Antisera. Rat GHRH antiserum (rabbit) was purchased from Peninsula 
Laboratories, Inc., Belmont, Calif. (Lot. No. 008769). This antibody has 
no cross-reactivity with human pancreatic GHRH (hpGHRH) (1-44) NH.sub.2, 
hpGHRH (1-40), porcine GHRH, PHI-27 and VIP. The lyophilized powder was 
rehydrated in distilled water; further dilutions were made with buffer. 
Antirabbit IgG (goat) was purchased from Sigma (Cat. No. R-4626). 
Biotin-labeling of anti-rabbit IgG. Five mg of the anti-rabbit IgG (goat) 
(GARGG) was dissolved in 5 ml freshly prepared, preservative-free 0.1M 
sodium bicarbonate solution (pH 8.4); 5 mg N-hydroxy-succinimidobiotin 
(NHSB, Sigma H-1759) was dissolved in 1.0 ml dimethyl sulfoxide (DMSO). 
One ml of NHSB/DMSO solution was added to 5 ml GARGG. The compounds were 
incubated for 4 hrs at room temperature (RT), then the solution was 
dialyzed against 2.times.1..0 1 PBS (pH 7.4, containing 0.1% sodium azide) 
for 24 hrs each, at 4.degree. C. Finally the biotin-labelled GARGG 
(bio-GARGG) was aliquoted and stored frozen at -70.degree. C. until used. 
Detection system. The detection system (FIG. 2a) is based on Self's enzyme 
amplification method (Self, 1985; Johansson, Stanley and Self, 1985; 
Johansson, Ellis, Bates, Plumb and Stanley, 1986). This method allows 
measurement of 43.10.sup.-20 ml thyroid stimulating hormone (TSH)/well; 
thus, it exceeds the theoretical sensitivity (10.sup.-14 M) of the 
iodine-125 detection system (Johansson, Ellis, Bates, Plumb and Stanley, 
1986; Jackson and Ekins, 1986). Avidin has an extremely high affinity to 
biotin (dissociation constant 10.sup.-15 M) and is a useful tool in ELISA 
systems (Guesdon, Ternynck and Avrameas, 1979; Vilja, Krohn and Tuohimaa, 
1985). 
FIG. 2b depicts a solid-phase immunoassay in which a CP is bound to the 
solid phase and the antibody binds to GHRH, when GHRH forms a GHRH-CP 
complex, i.e., where the CP binds at a different site from the antibody. 
FIG. 2c depicts a solid-phase immunoassay where GHRH is bound to the solid 
phase and the antibody and the CP bind GHRH at the same site; thus the 
binding can be shown as a competition between the antibody and the CP for 
the binding site of GHRH. To exclude (or at least reduce) the non-specific 
binding between the peptides and/or the proteins based on electrostatic 
charge differences, 5 USP units/ml sodium heparin was added to the buffers 
(Pesce, Apple, Sawtell and Michael, 1986). 
The ELISA was carried out as follows: 
Enzyme-linked Immunoassay: 
1. Coating. Materials (e.g., CP's) were dissolved at the concentrations 
indicated in the figures in coating buffer. 120 .mu.l/well, 4.degree. C., 
16 hrs. 
2. Wash: 3.times.200 .mu.l wash buffer No. 1. 
3. Block for non-specific binding: diluting buffer No. 1, 150 .mu.l/well, 
37.degree. C., 1 hr. 
4. Wash: 1.times.200 .mu.l wash buffer No. 1. 
5. GHRH in diluting buffer No. 1, 110 .mu.l/well, 37.degree. C., 1 hr. 
(FIG. 2b). 
6. Wash: 4.times.200 .mu.l wash buffer No. 1. 
7. Anti-GHRH serum diluted in diluting buffer No. 1, 110 .mu.l/well, 
37.degree. C., 1 hr. 
8. Wash: 4.times.200 .mu.l wash buffer No. 1. 
9. Bio-GARGG, 110 .mu.l/well, 37.degree. C., 1 hr. 
10. Wash: 4.times.200 .mu.l wash No. 2. 
11. SA-AP in diluting buffer No. 2, 100 .mu./well, 37.degree. C., 1 hr. 
12. Wash: 6.times.200 .mu.l wash buffer No. 2. 
13. Substrate: 120 .mu.l/well, RT, 30 min. 
14. Amplifier: 190 .mu.l/well, RT, 30 min. 
15. Stop the reaction by 25 .mu.l/well 1.0N HCl. 
16. Double wavelength spectrophotometry. Specific absorption at 492 nm. 
When the wells were coated by GHRH, steps 5-6 were omitted, and the 
anti-GHRH serum was coincubated with CPs. 
2.1: Determination of optimum dilutions of GHRH and anti-GHRH. The plate 
was coated with a dilution series of GHRH (range: 15.6-1000 ng/ml). The 
dilution of the antiserum recommended for RIA by Peninsula was considered 
1:1. Each concentration of GHRH was detected by a dilution series of 
antiGHRH (range: 1:2-1:32). The results are plotted in FIG. 3. Dilutions 
of antiGHRH 1:2 and 1:4 gave a high optical density that exceeded the 
range of the ELISA-reader. Similarly, at the dilution of 1:8, 0.5 and 1.0 
.mu.g/ml GHRH resulted in extremely high optical density. 
2.2: Binding studies according to FIG. 2b. The plate was coated with 
increasing amount of 5'-3' CP or oxytocin (OT) as a control peptide, as 
indicated in FIG. 4. There was a clearcut decreasing tendency in optical 
density as the concentration of the peptides was increased. If there is a 
binding between the 5'-3' CP and GHRH, and the bound GHRH is capable of 
binding the antibody, there should be an increase in the optical density 
as the concentration of 5'-3' CP was increased (GHRH concentration and the 
antiGHRH dilution were constant: 31.25 ng/ml and 1:8, respectively). 
However, we found the contrary. On the other hand, oxytocin (OT) at 
extremely high concentrations could bind GHRH. We consider this 
interaction non-specific; thus, OT had higher non-specific binding to GHRH 
than 5'-3' CP. The lack of increase in optical density in the case of 
5'-3' could be for two different reasons: (1) there is no GHRH-binding 
capacity at all; (2) 5'-3' CP and the anti-GHRH antibody bind GHRH at the 
same site. Experiment 2.3 was designed to answer this question. 
The same experiment was carried out for the 3'-5' CP using the same (w/v) 
concentrations. Again, there was no increase in the optical density (data 
not shown). 
2.3: Binding studies according to FIG. 2c. The plate was coated with 62.5 
ng/ml GHRH. 5'-3' CP was coincubated with an equal volume of 1:4 antiGHRH 
serum; thus, the final dilution of the antibody was 1:8. Final 
concentrations of the peptide are indicated in FIG. 5. OT (a peptide 
having non-specific binding activity for GHRH) was used as control. Both 
OT and 5'-3' CP could displace the antibody from GHRH binding, however, CP 
had a significantly higher potency than OT. The difference in the slopes 
of the displacement curves probably represents specific binding between 
5'-3' CP and GHRH, or the antibody might have cross-reactivity with the 
complementary peptide. However, the latter is excluded by the results of 
Experiment 2.2. 
EXAMPLE 3 
Immunization of Rabbits with the 5'-3' CP 
3.1 Coupling of the peptide to carrier protein. 1.2 mg keyhole limpet 
hemocyanin (KLH, Sigma H-2133) was dissolved in 15 mM KH.sub.2 PO.sub.4 
/Na.sub.2 HPO.sub.4 PBS, pH 7.2. The solution was centrifuged, and the 
supernatant was used to dissolve 3 mg 5'-3' CP. While constantly stirring, 
1.5 ml 20 mM glutaraldehyde (E.M. grade, Polysciences cat #1909) was added 
dropwise to the peptide solution and incubated at room temperature for 60 
min. The reaction product was dialyzed against 2.times.2 1 of PBS at 
4.degree. C. The final volume was divided into 5 equal aliquots and kept 
frozen at -20.degree. C. until further use. 
3.2 Immunization protocol. The rabbits were bled before the beginning of 
the immunization to obtain pre-immune sera for control experiments. For 
the initial injection, two aliquots were emulsified in three volumes of 
complete Freund's adjuvant. The emulsion was injected subcutaneously into 
three albino Myrtle's rabbits in equal doses, 0.1 ml/injection site. Three 
boosters were given subcutaneously at 10-day intervals using incomplete 
Freund's adjuvant, 0.1 ml/injection site. 
Twenty-four days after the initial exposure, the rabbits were bled from the 
auricular vein twice a week. Thirty-fifty ml of blood was collected each 
time. The samples were allowed to clot overnight at 4.degree. C., then 
centrifuged at 2000 rpm for 30 min. The sera were harvested and kept 
frozen in aliquots at -70.degree. C. until further use. 
3.3 Evaluation of the immune response. The antibody production was 
monitored using enzyme-linked immunosorbent assay (ELISA). Dynatech 
Immulon-1 micro-ELISA plates were coated with various concentrations of 
the 5'-3' CP or with coating buffer as control. The solid phase peptide 
was exposed to various dilutions of the antiserum obtained from a rabbit 
45 days after the beginning of the immunization. The IgG bound to the 
solid phase was detected with biolinylated goat anti-rabbit gamma globulin 
(bioGARGG) and streptevidin-alkaline phosphatase conjugate. The 
.beta.-NADP.sup.+ substrate and the alcohol dehydrogenase/diaphorase 
enzyme amplification technique was used, which converts the 
p-iodonitrotetrazolium violet to formazan. The color end product was read 
at 490-405 nM (dual wavelength) using the Bio-Tek EL 310 automatic 
EIA-reader. 
3.4 Immunization of the rabbits with 5'-3' CP. FIG. 6 demonstrates that the 
rabbit serum contains high titer antibodies against the 5'-3' CP. Based on 
this experiment, we chose 1.0 .mu.g/ml 5'-3' CP for coating to monitor the 
timecourse of the immune response. This experiment was then repeated using 
various dilutions of the sera obtained throughout the immunization process 
in order to establish the time course of the immune response. Dilutions of 
the pre-immune serum and the "infinite dilution" (diluting buffer without 
serum) were used as control. 
During the immunization, it came to our attention that the rabbits which 
produced high titer antibodies to the 5'-3' CP grew larger than the 
others. 
______________________________________ 
Rabbit No. 
1 2 3 4 5 6 
______________________________________ 
Peptide 
3'-5'CP 3'-5'CP 3'-5'CP 
5'-3'CP 
5'-3'CP 
5'-3'CP 
BW[g] 5262 5700 5070 6260 (died) 5445 
______________________________________ 
As shown in FIG. 7, the pre-immune serum did not bind to the solid phase 
5'-3' CP even in the highest concentration used. Anti5'-3' CP antibodies 
gradually appeared in the serum during the immunization protocol, and the 
binding was related to the dilution. 
The antiserum showed very low cross-reactivity with GHRH, LHRH, glucagon or 
3'-5' CP (data not shown); thus, it is specific for the antigen (5'-3' 
CP). 
EXAMPLE 4 
Purification of the IgG Fraction from Rabbit Sera 
4.1 Purification of IgG fraction. The IgG fraction was purified from the 
pre-immune serum and the immune serum of the rabbit that grew the largest 
(Rabbit 4, Example 3 above). 
4.2 Coupling of Protein A to Affi-Gel 10 beads. Ten ml of Affi-Gel 10 
(BIO-RAD) was washed in 3.times.30 ml anhydrous isopropanol, then in 
3.times.30 ml ice-cold distilled water. 
5 ml Protein A (Sigma P-6650) was dissolved in 1.0 ml coupling buffer (0.1M 
NaHCO.sub.3, pH 8.2, 4.degree. C.) and added to 2 ml Affi-gel in the same 
buffer. The mixture was shaken overnight at 4.degree. C., then the 
remaining binding sites were blocked with 200 .mu.l 1.0M ethanolamine 
hydrochloride (pH 8.0), 2 hrs at 4.degree. C. with constant shaking. The 
column was packed in a BIO-RAD Econo-Column (ID-1.0 cm, length=5 cm), then 
washed extensively with 0.1M NaHCO.sub.3 (pH 8.0). Finally the column was 
washed with PBS containing 0.1% NaN.sub.3 and kept at 4.degree. C. until 
further use. 
4.3 Coupling of KLH to Affi-Gel 10 beads. The procedure was basically the 
same as above. Fifteen mg KLH was dissolved in 10 ml coupling buffer and 
added to 4 ml Affi-Gel 10. The remaining binding sites were blocked with 
0.4 ml 1.0M ethanolamine HCl. 
4.4 Affinity-chromatography. One ml of the pre-immune serum was diluted 
with 1.0 ml PBS. The diluted serum was passed through the Protein A column 
ten times, then the column was washed extensively with PBS. Twenty drop 
fractions were collected, and the protein concentration was monitored with 
UV-spectrophotometry using a Beckman DU-40 instrument. Rabbit gamma 
globulin (Sigma G-0261) was used for calibration. The absorption spectrum 
was scanned, and the peak appeared at .lambda.=290 nm. When the 
UV-absorption of the fractions reached the background level, the column 
was washed further with PBS for several fractions, then the buffer was 
switched to 0.1M citrate buffer (50 mM citric acid, 50 mM Na.sub.2 
HPO.sub.4, pH 3.6), and the protein-A bound IgG was eluted. The fractions 
were collected until the absorbance reached the background, then the 
fractions containing significant amounts of IgG were pooled and dialyzed 
against PBS at 4.degree. C. overnight. 
The column was washed with excess citrate buffer and PBS, then the 
anti-serum obtained after 45 days from the beginning of the immunization 
was purified the same way. 
After completing the dialysis, the pre-immune IgG was passed through the 
KLH column 5 times, then the column was washed with PBS. The 
IgG-containing fractions were collected and pooled. Then the column was 
washed with citrate buffer, but no significant amount of protein was 
eluted. 
In the next step, the IgG obtained from the immune-serum was passed through 
the KLH column five times, PBS fractions were collected, and all these IgG 
fractions were pooled. Then a large amount of IgG was eluted from the 
column with 0.1 citrate buffer (pH 3.6), and further with 0.1M citric acid 
(pH 2.2). The acidic IgG fractions were dialyzed against PBS. 
FIG. 8 demonstrates that the non-IgG protein levels were similar before and 
after the immunization, while the IgG fraction was substantially higher in 
the immune serum. 
4.5 Testing the purified IgG molecules. Nunc-Immuno Plate I F was coated 
with 1.0 .mu.g/ml 5'-3' CP or 1.0 .mu.g/ml KLH, 100 .mu.l/well, 4.degree. 
C., 16 hr. The wells were washed 5 times, then the non-specific binding 
sites were blocked. Anti 5'-3' CP or antiKLH IgG molecules were added at 
various concentrations to the wells, then the reaction was evaluated as 
described above. As the Nunc plate has substantially higher 
protein-binding capacity then the Dynatech Immulon-1 plate, the reaction 
became so intensive that the optical density exceeded the range of the 
reader. Thus, 100 .mu.l reaction product was transferred from each well to 
another plate and diluted with 100 .mu.l distilled water. The absorbances 
are plotted in FIG. 9. 
As indicated in FIG. 9, the pre-immune IgG had no significant binding 
capacity to either the 5'-3' CP or KLH. The anti 5'-3' CP IgG was bound 
equally to the solid phase 5'-3' CP throughout the whole dilution range 
tested, hence it obviously has high affinity to the antigen. The antiKLH 
IgG also exhibited binding to the 5'-3' CP, though the affinity was 
significantly lower. The anti 5'-3' CP IgG fraction demonstrated a 
moderate, but significant binding to the KLH, while the anti-KLH IgG 
showed higher binding capacity to its antigen. 
EXAMPLE 5 
Effect on GH Secretion in vitro 
5.1 Preparation and culture of pituitary cells. Pituitary glands were 
collected from adult male Holtzman rats in Medium 199 (M199) with 3 g/l 
BSA, 10 mM HEPES (pH 7.4), penicillin (100 U/ml) and streptomycin (0.1 
g/l). The tissue was minced and digested with 0.5% solution of the 1:250 
Trypsin (Difco) followed by DNAase treatment according to the procedure of 
Loumaye & Catt (1983). The cell yield was ca. 1.3.times.10.sup.6 
cells/gland. The viability as determined by trypan blue exclusion was over 
95%. The cells were cultured in Falcon 24-well plates of a density of 
2.5.times.10.sup.5 cells/well in 500 .mu.l M199 containing 5% fetal calf 
serum, 5 nM dexamethasone, 10 mM HEPES, 100 U/ml penicillin, 0.1 g/l 
streptomycin at 37.degree. C. in humidified 95% O.sub.2 /5% CO.sub.2. 
After 4 days of culture, the cells were washed twice with M199 containing 
0.3% BSA 5 mM dexamethasone, 10 mM HEPES (pH 7.4), penicillin (100 U/ml) 
and streptomycin (0.1 g/9) and were incubated with the same culture medium 
to obtain basal secretion rate, or with growth hormone releasing hormone 
(GHRH, 5'-3' CP, 3'-5' CP, anti 5'-3' CP IgG or combination of GHRH and 
anti 5'-3' IgG), at various concentrations for 18 hrs at 37.degree. C. in 
95% O.sub.2 /5% CO.sub.2. Each treatment was carried out in triplicate. 
The culture media from each well were collected at the end of the 
incubation and stored frozen until assayed for GH by RIA. 
5.2 Growth Hormone Radioimmunoassay (GH RIA). In the GH RIA, the NIADDK rat 
GH kit was used. GH was labelled with .sup.125 I using the chloramine-T 
method, and the tracer was purified on Sephadex G-75 column. 16,000 cpm 
tracer in 100 .mu.l volume was added to each tube. The final dilution of 
the antibody was 1:30,000, resulting in about 50% specific binding. The 
detection limit was &lt;25 pg/tube. Precipitation was obtained with IgG-sorb 
(The Enzyme Center, Inc.). 
5.3 GH secretion in vitro: effect of various compounds. A small aliquot was 
pooled from each control culture to establish the parallelism in the 
displacement between a standard reference preparation of GH (GH RP-2), 
courtesy of Dr. A. F. Parlow, NIADDK, NIH, and the culture media 
containing GH secreted in vitro as obtained in paragraph 5.1 above. Based 
on this experiment, we determined the dilution needed to measure GH 
levels, and we chose that part of the displacement curve, where the 
parallelism was the best. See FIG. 10. 
The baseline secretion was established in five triplicates assigned between 
the treatment wells. Each treatment was carried out in triplicate. See 
FIG. 11. GHRH stimulated GH secretion in all doses studied (10.sup.-9 
-10.sup.-7 M). The 3'-5' CP had no effect at the doses of 10.sup.-5 and 
10.sup.7, but markedly stimulated GH secretion at the doses of 10.sup.-6 
and 10.sup.-8 M. The 5'-3' CP itself had no substantial effect on GH 
secretion (10.sup.-8 -10.sup.-5 M). On the other hand, the anti 5'-3' CP 
IgG demonstrated GH increasing activity in all doses studied (0.1 
.mu.g/ml-1.0 mg/ml). This stimulating activity is not attributed to the 
salts being present in the IgG preparation, since the lyophilized IgG was 
diluted and dialyzed against the culture medium before use. The 0.1 
.mu.g/ml dose of the IgG preparation contains less than 10.sup.-9 M IgG, 
hence its action is based on high-affinity binding. Co-incubation of the 
IgG with 10.sup.-8 GHRH was synergistic only at the highest dose used (1.0 
mg/ml IgG). 
It is understood that the examples and embodiments described herein are for 
illustrative purposes only, and that various modifications or changes in 
light thereof that will be suggested to persons skilled in the art are to 
be included in the spirit and purview of this application and the scope of 
the approved claims.