BACKGROUND
the vertebrate immune system is a highly specific defensive barrier to infection that can develop antibodies with the remarkable capability to recognize different antigen classes such as proteins, carbohydrates, and lipids.
the antibody molecule contains two light chains and two heavy chains.
every light chain is composed of one variable  and one constant  domain, and each heavy chain of these antibodies  folds into four domains: one variable , and three constant .
the v regions of each of these chains comprise three complementarity determining regions  separating four more conserved framework  regions  <cit> .
v domains are produced by random rearrangement of germline variable , diversity , and joining  gene segments at the heavy chain loci, and of vl and jl gene segments at the light chain locus  <cit> .
the primary repertoire of antibodies is generated through the assembly of these genes by somatic dna recombination, and its size depends on the diversity of these genes.
during the assembly process, further junctional diversity is introduced at the gene segment junctions by palindromic and nontemplated nucleotides addition through the activity of vj recombinase  <cit> .
additionally, the primary repertoire diversity is enhanced by combinatorial linkage of heavy and light chains.
this second phase of diversification is antigen dependent, occurs in the activated b-cells, and arises from three mechanisms: somatic mutations, gene conversion, and class-switching .
despite the potential to generate an almost incalculable variability in final domain sequences and the lengths of the hypervariable loops, these regions have been shown to exhibit a much smaller number of core main chain conformations, which are usually referred to as "canonical structures"  <cit> .
the conformation of a specific canonical structure is determined by the loop length and the presence of specific structural residues at key sites along the domain  <cit> .
apart from the particularly variable cdr h <dig> loop, different canonical classes have been assigned to cdr h <dig> and h <dig> of the heavy chain  <cit> , and the three cdrs of the kappa and lambda light chains  <cit> .
although numerous canonical combinations  are theoretically possible, domination of relatively few combinations is typically observed for antibodies specific for protein, carbohydrate, or hapten antigens  <cit> .
haptens are ubiquitously distributed in our environment as pollutants, and widely utilized as pharmaceuticals, hormones, and antibiotics.
these small molecular weight molecules  cannot be efficiently recognized by the mammalian immune system unless conjugated to larger immunogenic carrier molecules  <cit> .
“tricking” the immune system in this way has allowed anti-hapten binders with exquisite specificity and sensitivity to be successfully generated from a number of sources  <cit> .
antibody based immunosorbent assays have been developed as efficient and inexpensive tools for hapten detection  <cit> .
antibodies developed against various antigen classes have been shown to display different binding surfaces that tend to accommodate the shape of the target antigen  <cit> .
this phenomenon can be summarized as deep binding pockets for anti-hapten antibodies, and binding grooves for linear peptide antibodies, or antibodies that bind proteins possessing relatively flat binding sites  <cit> .
the development of cavity-like binding sites that recognize haptens has been reported for a number of species including: human  <cit> , mouse  <cit> , llama  <cit> , and rabbit  <cit> .
several studies have attempted to analyze in greater detail the relationship between antigen specificity and binding site structure.
each study has tended to focus on the application, in isolation, of a number of different approaches including: somatic hypermutation  <cit> , continuum electrostatic methods  <cit> , cdr length and sequence composition  <cit> , and conformational changes on binding  <cit> .
however, no previous research has tried to use these strategies collectively to analyze antibodies developed against different hapten classes.
in fact, the main focus of research within the anti-hapten antibody field has been typically limited to the characterization of anti-hapten binding sensitivity and cross-reactivity in relation to their targets; rather than considering how this remarkable ability of antibodies to recognize a small, but diverse class of molecules is achieved at the molecular level.
consequently, we describe the development and characterization of panels of antibody binders to four very different haptens: sqa, por, cop, and hsl, all derived from a series of immunized sheep phage display libraries.
sqa represented the minimum signature in terms of its simplicity, hydrophobicity, and flexibility of chemical structure.
an increase in structural rigidity was offered through the selection of the haptens cop and por.
however, the former lacks the presence of any polar group, when compared to por, which possess four nitrogen atoms within its core.
finally, hsl molecules were chosen as they combine molecular flexibility with the presence of potential polar groups.
here our characterization approach has focused on the antibody-hapten interaction and the complementarities of the binding counterparts in terms of their shape, size, and electrostatic energy.
furthermore, these factors were correlated with the canonical structures, cdr lengths, amino acid distribution, and antigen sensitivities of these anti-hapten binders to generate detailed and predicted models of anti-hapten antibody binding sites.
methods
sheep immunization and sera characterisation
three conjugates were prepared  following standard protocols  <cit> , and characterized by the aberdeen proteomics facility using a maldi-ms system.
in collaboration with ig-innovations ltd , the sheep were immunized with the desired hapten-conjugates .
animals were immunized with 100 μg/ml initial concentration, and four subsequent 50 μg/ml boosts, which were administered subcutaneously atsix sites.
freund's adjuvant was used in the immunization process, as a complete form  in the initial dose, and as an incomplete form  in the four successive boosts.
bleeds were taken from the jugular vein two weeks after each boost.
all animal work/procedures carried by ig innovations ltd, are performed under a rigorous home office  licence.
the project licence covers antibody production in large animals and describes the various techniques and procedures that were used as part of this study.
lymphocytes were isolated from the blood using histopaque®- <dig>  and accuspin tubes , and the isolated lymphocytes were stored in rna stabilization buffer .
sera characterization was performed using binding and competition elisa following protocols that were described previously  <cit> .
the elisa plates were coated with hapten-carrier protein conjugates at 1 μg/ml, and donkey anti-sheep  hrp antibody  was utilised as the secondary antibody to generate a binding signal.
phage display library construction
phage display library construction followed standard protocols  <cit> .
briefly, rna was isolated from whole blood lymphocytes using rneasy® midi kit: .
cdna was then synthesized by pcr using three sheep forward primers .
the pcr reaction included 25 μl of dnase treated rna, 3 μl of  <dig> pmol forward primers, 8 μl of 5x first strand buffer, 4 μl of  <dig>  m dtt, 1 μl of 10 mm dntps mixture, and 1 μl  superscript® iii reverse transcriptase .
sequence amplification rescuing of the heavy and light chains was conducted by pcr utilizing sheep gene-specific primers  <cit> .
pcr reactions comprising 1 μl  each of ovvh back primers and combination of ovjh for primers, 1 μl cdna, 25 μl 2x hf phusion® mix , and 22 μl sterile water.
the λ and k light chains were amplified as above but using  back/ovjλ for) or  back/ovjk for) primer combinations.
the amplified heavy and light chains were digested with asci  and mlui  restriction enzymes, respectively.
the complementary sites created by digestions with asci and mlui permitted ligation of the heavy and light chains and the formation of a  <dig> amino acids linker  between and joining these domains.
the linked dna material was amplified by pull-through pcr using ovvh backsfi primer and ovjλ fornot or ovjk fornot primers.
the linked vh and vl  dna  was purified and digested overnight at 37 °c with  ncoi  and noti  restriction enzymes.
the digested dna was then purified and ligated  into a phen <dig> phagemid vector.
this step was followed by ethanol precipitation of the ligated dna, and transformation of the vector into electrocompetent e.coli tg <dig> cells  using an electroporator .
library panning and screening
phage display library selections were performed as detailed previously  <cit> .
in brief, maxisorp tubes  were coated with the desired antigen-conjugates, and incubated overnight at 4 °c.
the tubes were then washed and phage particles  added in 4 ml 2 % marvel-pbs.
the bound phage were eluted by the addition of 1 ml of 100 mm triethylamine  for 10 min, then neutralized by the addition of  <dig>  ml of 1 m tris buffer .
the eluted phage were used to infect e.coli  cells at their exponential growth phase for 30 min in a water bath, and for another 30 min at 37 °c in a shaking  incubator.
the cells were then plated on tye agar containing 1 % glucose and 100 μg/ml ampicillin, and incubated overnight at 30 °c, before being colony scraped and stored at −80 °c.
to rescue the library, m13k <dig> helper phage  were used to infect the library and inoculated into the culture at 1: <dig> ratio .
phage clones were isolated and characterized by elisa according to established protocols  <cit> .
expression and purification of scab proteins
the plasmid dna of the anti-hapten scfv were digested with ncoi and noti restriction enzymes, and cloned into a similarly digested soluble expression vector pims <dig>  <cit> .
the ligated product was transformed into e. coli  cells  by electroporation.
expressions of scabs in transformed e. coli xl- <dig> blue bacterial cells were carried out in terrific broth according to standard protocols  <cit> .
the expressed scabs were purified via the hexa-histidine tag using immobilized metal ion chelate affinity chromatography .
the sensitivity of expressed scab proteins for their target antigen was examined using a scab binding elisa  <cit>  and/or an indirect competition elisa  <cit> .
all elisa plates were coated with hapten-carrier protein conjugates at 1 μg/ml.
determination of cdr length, canonical structures, and amino acid distribution
the cdrs lengths of the heavy and light chains were defined throughout this article following the well-established kabat numbering system.
to analyze amino acid distribution and conservation throughout the entire antibody, amino acids were classified into seven groups using a microsoft excel visual basic macro sheets  <cit> .
these sheets were downloaded from the aaaaa server  <cit> .
here again, each amino acid, within the analyzed sequences, was numbered according to the kabat scheme  <cit> .
in addition, the canonical classifications of the loops were determined according to a chothia sdr template  <cit> .
these numbering and classification steps were further aided by reference to dr. andrew c.r.
martin's group website .
antibody modelling and electrostatic potential measurements
the variable domains of anti-hapten antibodies were modelled using the project mode in the swiss-model website  <cit> .
the heavy and light chains templates were identified using the swiss-model workspace template identification portal.
templates with the highest sequence identities, and similar canonical classes, were then selected.
the homology models of the heavy and light chains were generated by the swiss-model workspace automated modelling portal with appropriate specification for each selected template.
the generated model qualities were then examined following standard protocols  <cit> .
finally, the heavy and light chains were linked by swiss-pdbviewer  <dig> .
<dig>  and visualized by pymol .
electrostatic energy of the selected antibodies was calculated using adaptive poisson-boltzmann solver  version  <dig> .
<dig> in the python molecule viewer  version  <dig> .
<dig>  the produced energy was mapped to the surface with medium surface quality and at a 1 Å distance.
the map colour was coded as white:  <dig> kt/e, blue:  <dig>  kt/e, red: − <dig>  kt/e.
docking analysis
automated docking was used to predict suitable binding orientations and conformations of the various haptens positioned at/within their corresponding binding sites.
the antibody-antigen docking analyses were conducted using the molecular docking and visual screening program autodock vina  <cit> .
polar hydrogen atoms were added, and the generated models were saved as pdbqt files.
the ligand  rotatable bonds were examined and saved in similar pdbqt format.
a potential option within autodock vina is the ability to determine the docking site of the antibody by setting the dimensions of the docking grid box.
this was achieved by setting the x, y, and z axes of the grid box to cover the binding sites of the antibody.
the docking process was achieved using the command prompt within windows  <dig>  the command script included , , .
upon completion of the docking process, the models were exported, viewed and analyzed by pymol .
RESULTS
library construction, bio-panning, and characterization
for each immunized sheep, lymphocytes were used as the genetic source for scfv.
sheep sera samples were routinely examined during the immunization process, and the selection of material for library construction made based on the best measured responses.
sequences of the different antibody fragment libraries confirmed a full diversity within the cdr loops.
each of the libraries was considered highly diverse, containing at least 107- <dig> unique clones.
the panning strategies employed to select phage-binders included a series of steps to encourage the enrichment and selection of the more sensitive and specific clones  <cit> .
whilst this is not an exact science, care was taken where possible to follow a similar selection strategy for each hapten class .
the selection process was stopped when clones of the same sequence began to appear at a level of great than 30 % of positive clones analyzed.
typically this was after 3– <dig> rounds of selection and bio-panning.
binding and competition elisas of monoclonal phage were used to identify unique clonal panels specific for sqa, por, hsl, or cop .
the isolated scfv phage clones were converted into a scab  format, and the sensitivities of purified soluble antibody fragments to free haptens were determined by competition elisa .
the “naïve” or simple chemical structure of sqa was recognized with only micromolar sensitivity .
an increase in the structural rigidity of the hydrophobic and poly-cyclic second antigen cop did not really improve this moderate ic <dig> sensitivity beyond the micromolar range.
however, clone por b <dig> was able to detect the rigid but polar por with an ic <dig> value of  <dig> nm.
the strongest molecular recognitions were seen for hsl molecules, which comprise a hydrophobic tail attached to a hydrophilic lactone ring.
several anti-hsl antibodies displayed super-sensitive interactions with clone hsl  <dig> having an ic <dig> in the picomolar range .table  <dig> summary of the sequence analysis strategy
the analyzed sequences were obtained from three libraries.
the pre and post-selection sequences included heavy  and lambda light  chains.
pre-selection clones were randomly selected sequences during library construction process
summary of the binding sensitivities of the analyzed clones together with the chemical structures of their corresponding antigens.
#: ic <dig> values were not successfully detected
sequences analysis
sequences were compared for clones from the pre and post-selection  and are summarized in table  <dig>  sequences were analyzed by examining canonical structures, cdr lengths, and amino acid distribution.
cdr length and canonical structures
cdr lengths determination of each individual library has revealed clear conservation in cdrs h <dig>  h <dig>  and l <dig> .
in addition, there was moderate diversity in the lengths of cdr l <dig>  and high variability in cdrs h <dig> and l <dig>  the length of cdrs h <dig>  h <dig>  and l <dig> were identical for all the post-selection clones isolated from the three libraries, irrespective of binding specificity.
in contrast, cdr h <dig> and l <dig>  lengths could be clustered into two groups:  antibodies that were developed against cop  having generally shorter cdr h <dig> and l <dig> when compared to  the antibodies developed against sqa, por, and hsl .
in general terms, the selection process has introduced bias into the cdr lengths recovered with cdrs h <dig>  l <dig>  and l <dig> having lengths in post-selection clones that were represented at low frequency in the pre-selection repertoires.table  <dig> cdr length distribution of the pre and post-selection sequences
the most prevalent cdrs lengths  within each library in  pre-selection clones and  post-selection clones.
the percentage of each specific cdr length within  the entire library population or  post-selection clones is in parenthesis
canonical classification was performed utilizing a strict chothia sdr template, on all the cdrs except cdr h <dig>  the pre-selection sequences from all three libraries comprised heavy chain sequences dominated by a 1– <dig> canonical combination for cdr's h <dig> and h <dig> .
typically, these classes corresponded to cdr lengths of  <dig>  and  <dig>  amino acids.
there was moderate but not unexpected variability in the classification of lambda light chains within pre-selection sequences.
a significant contribution to the overall library variability was from a canonical class combination 6-1-x for cdrs l1-l2-l <dig>  cdr l <dig> class  <dig> represents a  <dig> amino acid loop length, while class  <dig>  is the only identified group for cdr l <dig> in the literature  <cit> .
class x is used here to indicate that no canonical class has been reported previously with a similar loop length.
the three sampled antibody libraries were analyzed statistically using a chi square goodness of fit test   to evaluate whether the canonical class representation was equal within each antibody library.
however, the null hypothesis was rejected , and therefore, there was domination of specific canonical classes within each library.
it is impossible to conclude whether this bias was present as a result of the different immunizations or as an artifact from the library cloning process.
clones  included only lambda light chains, with a clear antigen specific canonical combination bias seen for each target.
in contrast, the heavy chains cdrs h1-h <dig> were all classified as 1– <dig> ; a canonical class combination that dominated all the three libraries’ pre-selection sequences.
the post-selection lambda chains were from canonical classes of cdrs l <dig> and l <dig> that were present in low abundance within the pre-selection sequences, and reflected the cdr length trends described previously.
cdr l <dig> of the highly sensitive post-selection clones was grouped within class  <dig> or  <dig>  these sensitive clones included cdr h <dig> with  <dig>  or  <dig>  amino acids.
in addition, cdr l <dig> class x was evident in post-selection clones that have not shown high binding sensitivities, like sqa  and cop clones .table  <dig> canonical combinations of the pre and post-selection sequences
the canonical classes were determined using strict chothia sdr template  <cit> .
data presented represent the most prevalent canonical combinations within each library in  pre-selection clones and  post-selection clones.
x: indicates no currently recognized canonical class with the same loop length.
the percentage of specific canonical combination within the entire library population of the analyzed pre  and post -selection sequences are in parenthesis
amino acid distribution
analyses of the pre-selection sequences confirmed the high level of amino acid site conservation in the fw regions and remarkable variability within the cdr loops .
the majority  of the fw positions were well conserved .
comparison between the sheep heavy and light chain's fw regions indicated that the heavy chain fw positions are 30 % more conserved than the light chain.
as anticipated, the most variable region was cdr h <dig>  of which >80 % of the positions showed >50 % variability.
the most diverse positions within the heavy chains were h32- <dig>  and h50- <dig> .
positions l28- <dig>  and l50- <dig>  were highly diverse within the light chains.fig.
<dig> amino acid variability within various regions of the isolated sequences.
variability within a vh, b vl regions of the pre-selection sequences.
diversity at each amino acid position was classified into five groups.
the total cdr or fw region variability was determined as a percentage of the overall diversity of positions within the selected regions
in sharp contrast to the broad repertoire diversity seen for the pre-selection clones only a relatively small number  different hapten binders were present  at the end of each antigen-specific, bio-panning .
this extreme contraction of repertoire diversity is independent of the hapten type and is further reflected in an extreme narrowing of the canonical structures represented within the hapten binding clones .
it must be concluded, therefore, that only a tiny percentage of the original and highly diverse sheep repertoires, even following bias through immunization and boosts, have a paratope-shape that is pre-disposed to bind haptens, as a class.
all post-selection sequences were collectively “searched” for signs of possible hapten-driven similarities or differences.
the number of conserved positions were significantly higher in post-selection sequences  when compared to their pre-selection counterparts  .
in addition, fw <dig> of the heavy and light chains contains the highest levels of conservation, possibly identifying the importance of this region in orientating cdrs  <dig> and  <dig> required to form a pocket for recognizing haptens.
not unexpectedly, the cdr regions themselves contain a significantly lower level of conservation, as each of these antibody panels was selected against different hapten antigens.
taken together, the concentration of site conservation within the fw regions, but not in the cdrs, appears to suggest an important structural role for the fw regions in enabling cdrs to be displayed in the required orientation to accommodate haptens without compromising antigen affinity.table  <dig> comparison of the conserved amino acid positions following antigen selection
analysis was conducted on the pre and post-selection sequences.
a conserved site was defined as: a position  in the post-selection clones where an amino acid remained unchanged or had similar bio-physical characteristics to the original pre-selection residues
surface analysis of post-selection clones
homology models of post-selection clones were constructed utilizing swiss-model workplace.
examination of the binding site topographies revealed pocket like surfaces for all of the modelled anti-hapten antibodies.
the sizes and shapes of these pockets were influenced by the antigens they were selected against, with the main contributions to antigen binding coming from cdrs h <dig>  h <dig>  and l <dig> .
the significant influence of cdr h <dig> on antigen binding was via direct interaction with antigen and also via the less obvious indirect impact on the orientation and corresponding pocket shape and size delivered by the positioning of cdrs h <dig> and l <dig>  in particular, two positions in cdr h <dig>  greatly influenced antigen binding by controlling the pocket shape between cdr h <dig> and cdr l <dig> .
in all cases, position h <dig> was occupied with phe, tyr, ile, or leu, and these amino acids play a key role within a network or web of interactions with amino acids at positions h <dig>  h <dig>  h <dig>  h <dig>  l95a, and l95b .
these interactions enabled the remaining cdr h <dig> residues, h <dig> in particular, to be in direct contact with the different antigens.
analyses of position h <dig> revealed the presence of phe in anti-sqa  clones , whilst tyr was observed in anti-sqa  and all the anti-cop clones .
for these clones at least, their binding sensitivities might indicate a preference for phe when establishing interactions with hydrophobic targets like sqa and cop.
in contrast, the presence of tyr at this position appears to be important for the high binding sensitivity of clone por b <dig>  when compared to clones por a <dig> and por d <dig> that contained a h <dig> phe .
the four polar nitrogen atoms at the core of the por structure should interact readily with the hydroxyl group of tyr.
for the anti-hsl antibodies, there was an arg at position h <dig> in clones  <dig>   <dig>  and  <dig>  which contributed to binding of the polar lactone ring of hsl molecules.
in contrast, the h <dig> ile of clones  <dig>   <dig> and  <dig> has established hydrophobic interactions with the hsl molecules' aliphatic tail .
consequently, the role and influence of position h <dig> for antigen binding was confirmed and correlated with each hapten in terms of its chemical structure and polarity.fig.
<dig> the effect of amino acids within cdr h <dig> on the predicted positioning of cdrs h <dig> and l <dig>  amino acid positions h <dig> , h <dig> , and h <dig>  in clones a hsl <dig>  b hsl <dig>  c sqa a <dig>  d sqa b <dig>  e por b <dig>  f por a <dig>  g cop h <dig>  and h cop a <dig>  position h <dig> has a profound effect on the orientation of cdr h <dig> , whilst positions h <dig> and h <dig> influence cdr l <dig>  orientation.
these homology images of the post-selection clones were constructed utilizing swiss-model workplace.
the structures were viewed by pymol  <dig>
fig.
<dig> side-chains orientations of potential amino acids within cdr h <dig>  site-specific interactions of amino acid positions h <dig>  h <dig>  and h <dig>  a sqa a <dig> , sqa b <dig> , and sqa f <dig> .
b por a <dig> , por d <dig> , and por b <dig> .
c cop e <dig> , cop h <dig> , and cop a <dig> .
d hsl  <dig> , hsl  <dig> , and hsl  <dig> .
these homology images of the post-selection clones were constructed utilizing swiss-model workplace.
the structures were viewed by pymol  <dig>
fig.
<dig> structural, docking, and surface-mapped electrostatic potential of antibodies.
the homology structures of the post-selection clones were determined by swiss-model workplace for clones a hsl <dig>  b sqa a <dig>  c por b <dig>  d cop h <dig>  docking analyses were conducted with autodock vina  <dig> .
<dig>  the structures were viewed by pymol  <dig>  .
measurements of electrostatic energies were calculated using python molecule viewer  version  <dig> .
<dig>  the produced energy was mapped with medium surface quality and at a 1 Å distance from the surface.
the map color was coded as white:  <dig> kt/e, blue:  <dig>  kt/e, red: − <dig>  kt/e
the surface topography of the area between cdrs h <dig> and h <dig> was also greatly influenced by a single position at h <dig> within cdr h <dig>  which can establish interactions with h <dig>  h <dig>  and h <dig>  the interaction of h <dig> and h <dig> was as predicted because all the post-selection clones contain cdr h <dig> with a class  <dig> conformation.
for the less antigen-sensitive clones such as sqa f <dig>  por d <dig> and a <dig> , position h <dig> was occupied by an arg.
when the same position was represented by a smaller amino acid such as ser , met , or thr , the clones all showed increased sensitivity to their respective target hapten.
in general terms, this structural analysis suggested that the presence of relatively bulky amino acids  at position h <dig> might cause cdr h <dig> to be oriented further away from cdr h <dig> and towards cdr l <dig>  this repositioning appears to partially occlude the hapten binding pocket .
furthermore, we were able to explore this hypothesis further and show, using computer generated binding site “mutants” and their subsequent modelling, that changes in amino acid composition at position  <dig> was predicted to cause significant and functionally profound re-positioning of the cdr <dig> loop .
electrostatic potential measurements of the post-selection clones
the surface-mapped electrostatic energy was examined across the entire population of post-selection clones.
here, four antibodies, one for each target, have been selected for illustration purposes .
sqa e <dig>  por b <dig>  and cop h <dig> antibodies generally have relatively uncharged binding pockets ideally suited to their hydrophobic targets, whilst in stark contrast, the hsl antibodies have a positively charged binding pocket, as typified by the hsl  <dig> antibody.
this positively charged binding pocket might be required to attract hsl molecules that are rich in oxygen molecules.
these observations were supported by measurements of total electrostatic potential utilizing adaptive poisson-boltzmann solver  version  <dig> .
<dig> in python molecule viewer   <dig> .
<dig>  the predicted electrostatic energies of the anti-sqa antibodies ranged from  <dig>  e +  <dig>  to < <dig>  e + 04 kj/mol.
low electrostatic energy  was also predicted for the three por clones.
in contrast, most of the anti-cop antibodies had a higher electrostatic energy .
the recorded energies of anti-hsl antibodies all had predicted values around  <dig>  e + 04 kj/mol.
the total electrostatic potential of these antibodies complements the hydrophobic/hydrophilic nature of these haptens .
discussion
antibody fragments  are invaluable protein scaffolds that have been extensively used as diagnostics and more recently therapeutics  <cit> .
they can recognize and bind to a diverse range of antigens including polypeptides, carbohydrates, lipids, nucleic acids, or even small hapten molecules.
haptens are not inherently immunogenic due to their small size but can elicit anti-hapten responses when coupled to a suitable immunogenic carrier protein prior to immunization.
sheep immunization followed by library construction and characterization by phage display technology has proved to be a successful strategy to develop highly sensitive anti-hapten antibodies .
the binding of antibody to antigen is principally generated by complementarities of the binding surfaces, which allows various interactions between the two molecules including the formation of hydrogen bonds, salt bridges, and van der waals interactions  <cit> .
this investigation of antibody-hapten interactions has therefore focused on hapten polarity and structural flexibility.
the isolated sqa antibodies recognized the free sqa antigen in the micromolar  range.
a possible reason for the moderate sensitivity to sqa might be attributed to its pronounced hydrophobic nature preventing/limiting the establishment of specific binding interactions, as has been reported for other hydrophobic targets  <cit> .
furthermore, binding sensitivities have been shown both experimentally  <cit>  and computationally  <cit> , to be enhanced to more rigid hapten structures.
whilst structural flexibility is clearly important, the por used in this study has an unmetalated core, yet still generated antibodies with sub-micromolar sensitivity .
this contrasts with the moderate sensitivity to free antigen  observed for the cyclic aliphatic and nonpolar cop.
improvement in binding sensitivities have been reported in other studies following the chemical addition of polar oh or ho3so groups at the carbon position  <dig> of cop   <cit> .
it would appear therefore, that the presence of polar groups within the por structure was more important in the generation of higher sensitivity binders than structural rigidity alone.
when moderate flexibility and high polarity are combined , super sensitive  binding interactions can be isolated.
here again polarity at the carbon  <dig> position  was a key determinant in overall sensitivity.
one clear conclusion from this work is that the combination of relative structural rigidity with the presence of several polar groups is predictive of an enhanced hapten-antibody interaction.
this proposed influence of hapten chemistry on antibody binding might also be evident when examining amino acid distribution, cdrs lengths, and canonical classifications.
canonical combination 1– <dig> for cdr h1-h <dig> dominated both the pre and post-selection heavy chain sequences .
structurally, cdr h <dig> bridges the two β-sheets and packs across the top of the vh domain.
while class  <dig> of cdr h <dig> represents the shortest observed, and most commonly seen, loop for this cdr  <cit> .
almagro et al.
<cit>  have reported the domination of these canonical classes within sheep heavy chain populations.
in contrast to the restricted canonical classes of the sheep heavy chains, several canonical classes were observed in the pre-selection lambda chain sequences, with the class combination 6-1-x  having the greatest representation in each of the three libraries.
this newly proposed class  for cdr l <dig>  was found in 60-80 % of the pre-selection sequences, and interestingly in the less sensitive post-selection clones.
the high incidence of canonical class  within the pre-selection clones, but not in the highly sensitive post-selection sequences, suggest this class is not pre-disposed to binding haptens as a group.
cdr length comparisons revealed high conservation in cdrs h <dig>  h <dig>  and l <dig>  but much more variability in cdrs l <dig>  h <dig>  and l <dig> .
whereas the post-selection clones cdrs have shown high level of length conservation to accommodate each hapten.
a cdr h <dig> with  <dig> amino acids was observed with sqa, por, and hsl post-selection clones .
anti-haptens antibodies with  <dig> amino acid cdr h <dig> have been reported previously against arsenate and phosphorylcholine  <cit> , and it has been postulated that this is the minimum length to enable cdr h <dig> to form part of a pocket-like binding site.
interestingly, where cdr h <dig> lengths are  <dig> amino acids or greater, it has also been postulated that they become increasingly exposed to solvent, resulting in a more unstable structure and less defined binding site  <cit> .
antibody-antigen sensitivity is of course greatly influenced by the type of amino acids in contact with the different hapten targets.
analyses of the pre and post-selection sequences revealed more conserved sites in the post-selection sequences, when compared to their pre-selection counterparts, especially within the fw regions .
this type of conservation phenomenon has been reported previously but to a lesser extent , following examination of the  <dig> common core residues of ~ <dig> kabat sequences of antibodies to a full range of different antigen classes  <cit> .
one possible reason for the very high level of conservation we have seen is as an artefact of the antibody isolation process.
phage enrichment is known to be significantly influenced by toxicity and expressibility of the displayed antibody binding sites  <cit> .
however, the conserved positions seen here are consistent even across clones isolated from different libraries, or from structurally dissimilar haptens or different panning strategies.
the only shared factor here is the overall low molecular weight of the haptens and the need to form a binding site shape able to accommodate them.
these structural and topographical perquisites could drive high conservation in the fw regions, especially fw <dig>  and result in the selection of clones from only a small subset of the full repertoire diversity.
whilst all the homology-modelled sheep antibodies possess pocket-like binding sites with various shapes and sizes , the development of more compact binding pockets has been attributed to closure of the vl-vh interface of the anti-hapten antibodies and improved binding sensitivities  <cit> .
here, the shape and overall size of the binding pockets was greatly influenced by a small number of amino acid positions in cdr h <dig>  that appeared to control the orientation of cdrs h <dig> and l <dig>  position h <dig> was occupied with phe, tyr, ile, or leu, and was involved in a network of interactions with positions h <dig>  h <dig>  h <dig>  l95a, and l95b.
these interactions have enabled the remaining of cdr h <dig> amino acids, especially position h <dig>  to be oriented toward the target antigens.
position h <dig> is known from previous work to have an important role in hapten recognition  <cit> .
the average relative side chain solvent-accessible surface of this position was estimated at 50-75 %  <cit> .
this study is able to correlate the amino acids found at this site with antigen polarity, and with phe, tyr, and arg pairing up with sqa and cop, por, and hsl, respectively.
position h <dig> appears to impart a second and key structural influence on cdr h <dig> .
this residue is at the tip of cdr h <dig> and is believed to pack against residues at positions h <dig> and h <dig>  <cit> .
from our models, the presence of arg at this position causes steric hindrance with cdr h <dig> pushing this important cdr away from cdr h <dig> and towards cdr l <dig> .
in concert, the residues found at these three positions in cdr h <dig>  appear to exert a significant influence over the size and shape of the hapten binding pockets.
previous studies have concluded that antibody-protein interactions are based on "charge complementarity" and "electrostatic complementarity"  <cit> , and that this complementarity is important in defining binding site specificity  <cit> .
therefore, it was interesting to examine whether this proposed electrostatic complementarty could be expanded to hapten antigens.
here, hapten complementarity is not limited to shape and size but extends also to surface electrostatic potential.
sqa, por, and cop binding pockets have hydrophobic surfaces, in complete contrast to the positively charged surfaces of anti-hsl antibodies .
these observations are further supported by the electrostatic optimization of anti-hapten antibodies specific for p-nitrophenyl phosphonate, fluorescein, and n--n--guanidiniumacetic acid .
consequently, we can predict that antibodies with high binding sensitivity show an improved capacity to recognize haptens by establishing electrostatically complementary binding pockets.
CONCLUSIONS
various structural and molecular factors appear to profoundly influence the successful binding of antibodies to hapten molecules.
haptens possessing a relatively rigid chemical backbone, together with the presence of polar groups, are much more likely to be recognized by antibodies with high sensitivity.
these highly sensitive antibodies tend to show an improved capacity to recognize their antigens by establishing complementary binding pockets.
these complementarities are influenced by amino acid composition and control the pocket size, shape, and electrostatic potential.
abbreviations
cdrcomplementarity determining regions
copcoprostane
dadalton
fwframework
hslhomoserine lactone
poretioporphyrin
scabsingle chain antibody
scfvsingle-chain variable fragments
sqasqualane
competing interests
no potential conflicts of interest were disclosed.
authors' contributions
ma: performed experiments, participated in the design of the study, analyzed data, and drafted the manuscript.
sp: performed experiments, participated in the design of the study, analyzed data.
gb: participated in the design of the study, analyzed data.
dc: provided material, participated in the design of the study, and analyzed data.
kc: participated in the design of the study, and analyzed data ap: participated in the design of the study, analyzed data, and drafted the manuscript.
all authors read and approved the final manuscript.
the authors would like to thank the scottish universities life sciences alliance  for their support.

 the vertebrate immune system is a highly specific defensive barrier to infection that can develop antibodies with the remarkable capability to recognize different antigen classes such as proteins, carbohydrates, and lipids. the antibody molecule contains two light chains and two heavy chains. every light chain is composed of one variable and one constant domain, and each heavy chain of these antibodies folds into four domains: one variable, and three constant. the v regions of each of these chains comprise three complementarity determining regions separating four more conserved framework regions. v domains are produced by random rearrangement of germline variable, diversity, and joining gene segments at the heavy chain loci, and of vl and jl gene segments at the light chain locus. the primary repertoire of antibodies is generated through the assembly of these genes by somatic dna recombination, and its size depends on the diversity of these genes. during the assembly process, further junctional diversity is introduced at the gene segment junctions by palindromic and nontemplated nucleotides addition through the activity of vj recombinase. additionally, the primary repertoire diversity is enhanced by combinatorial linkage of heavy and light chains. this second phase of diversification is antigen dependent, occurs in the activated b-cells, and arises from three mechanisms: somatic mutations, gene conversion, and class-switching. despite the potential to generate an almost incalculable variability in final domain sequences and the lengths of the hypervariable loops, these regions have been shown to exhibit a much smaller number of core main chain conformations, which are usually referred to as "canonical structures". the conformation of a specific canonical structure is determined by the loop length and the presence of specific structural residues at key sites along the domain. apart from the particularly variable cdr h loop, different canonical classes have been assigned to cdr h and h of the heavy chain, and the three cdrs of the kappa and lambda light chains. although numerous canonical combinations are theoretically possible, domination of relatively few combinations is typically observed for antibodies specific for protein, carbohydrate, or hapten antigens. haptens are ubiquitously distributed in our environment as pollutants, and widely utilized as pharmaceuticals, hormones, and antibiotics. these small molecular weight molecules cannot be efficiently recognized by the mammalian immune system unless conjugated to larger immunogenic carrier molecules. “tricking” the immune system in this way has allowed anti-hapten binders with exquisite specificity and sensitivity to be successfully generated from a number of sources. antibody based immunosorbent assays have been developed as efficient and inexpensive tools for hapten detection. antibodies developed against various antigen classes have been shown to display different binding surfaces that tend to accommodate the shape of the target antigen. this phenomenon can be summarized as deep binding pockets for anti-hapten antibodies, and binding grooves for linear peptide antibodies, or antibodies that bind proteins possessing relatively flat binding sites. the development of cavity-like binding sites that recognize haptens has been reported for a number of species including: human, mouse, llama, and rabbit. several studies have attempted to analyze in greater detail the relationship between antigen specificity and binding site structure. each study has tended to focus on the application, in isolation, of a number of different approaches including: somatic hypermutation, continuum electrostatic methods, cdr length and sequence composition, and conformational changes on binding. however, no previous research has tried to use these strategies collectively to analyze antibodies developed against different hapten classes. in fact, the main focus of research within the anti-hapten antibody field has been typically limited to the characterization of anti-hapten binding sensitivity and cross-reactivity in relation to their targets; rather than considering how this remarkable ability of antibodies to recognize a small, but diverse class of molecules is achieved at the molecular level. consequently, we describe the development and characterization of panels of antibody binders to four very different haptens: sqa, por, cop, and hsl, all derived from a series of immunized sheep phage display libraries. sqa represented the minimum signature in terms of its simplicity, hydrophobicity, and flexibility of chemical structure. an increase in structural rigidity was offered through the selection of the haptens cop and por. however, the former lacks the presence of any polar group, when compared to por, which possess four nitrogen atoms within its core. finally, hsl molecules were chosen as they combine molecular flexibility with the presence of potential polar groups. here our characterization approach has focused on the antibody-hapten interaction and the complementarities of the binding counterparts in terms of their shape, size, and electrostatic energy. furthermore, these factors were correlated with the canonical structures, cdr lengths, amino acid distribution, and antigen sensitivities of these anti-hapten binders to generate detailed and predicted models of anti-hapten antibody binding sites. methods sheep immunization and sera characterisation three conjugates were prepared following standard protocols, and characterized by the aberdeen proteomics facility using a maldi-ms system. in collaboration with ig-innovations ltd, the sheep were immunized with the desired hapten-conjugates. animals were immunized with 100 μg/ml initial concentration, and four subsequent 50 μg/ml boosts, which were administered subcutaneously atsix sites. freund's adjuvant was used in the immunization process, as a complete form in the initial dose, and as an incomplete form in the four successive boosts. bleeds were taken from the jugular vein two weeks after each boost. all animal work/procedures carried by ig innovations ltd, are performed under a rigorous home office licence. the project licence covers antibody production in large animals and describes the various techniques and procedures that were used as part of this study. lymphocytes were isolated from the blood using histopaque®- and accuspin tubes, and the isolated lymphocytes were stored in rna stabilization buffer. sera characterization was performed using binding and competition elisa following protocols that were described previously. the elisa plates were coated with hapten-carrier protein conjugates at 1 μg/ml, and donkey anti-sheep hrp antibody was utilised as the secondary antibody to generate a binding signal. phage display library construction phage display library construction followed standard protocols. briefly, rna was isolated from whole blood lymphocytes using rneasy® midi kit:. cdna was then synthesized by pcr using three sheep forward primers. the pcr reaction included 25 μl of dnase treated rna, 3 μl of pmol forward primers, 8 μl of 5x first strand buffer, 4 μl of  m dtt, 1 μl of 10 mm dntps mixture, and 1 μl superscript® iii reverse transcriptase. sequence amplification rescuing of the heavy and light chains was conducted by pcr utilizing sheep gene-specific primers. pcr reactions comprising 1 μl each of ovvh back primers and combination of ovjh for primers, 1 μl cdna, 25 μl 2x hf phusion® mix, and 22 μl sterile water. the λ and k light chains were amplified as above but using back/ovjλ for) or back/ovjk for) primer combinations. the amplified heavy and light chains were digested with asci and mlui restriction enzymes, respectively. the complementary sites created by digestions with asci and mlui permitted ligation of the heavy and light chains and the formation of a amino acids linker between and joining these domains. the linked dna material was amplified by pull-through pcr using ovvh backsfi primer and ovjλ fornot or ovjk fornot primers. the linked vh and vl dna was purified and digested overnight at 37 °c with ncoi and noti restriction enzymes. the digested dna was then purified and ligated into a phen phagemid vector. this step was followed by ethanol precipitation of the ligated dna, and transformation of the vector into electrocompetent e.coli tg cells using an electroporator. library panning and screening phage display library selections were performed as detailed previously. in brief, maxisorp tubes were coated with the desired antigen-conjugates, and incubated overnight at 4 °c. the tubes were then washed and phage particles added in 4 ml 2 % marvel-pbs. the bound phage were eluted by the addition of 1 ml of 100 mm triethylamine for 10 min, then neutralized by the addition of  ml of 1 m tris buffer. the eluted phage were used to infect e.coli cells at their exponential growth phase for 30 min in a water bath, and for another 30 min at 37 °c in a shaking incubator. the cells were then plated on tye agar containing 1 % glucose and 100 μg/ml ampicillin, and incubated overnight at 30 °c, before being colony scraped and stored at −80 °c. to rescue the library, m13k helper phage were used to infect the library and inoculated into the culture at 1: ratio. phage clones were isolated and characterized by elisa according to established protocols. expression and purification of scab proteins the plasmid dna of the anti-hapten scfv were digested with ncoi and noti restriction enzymes, and cloned into a similarly digested soluble expression vector pims. the ligated product was transformed into e. coli cells by electroporation. expressions of scabs in transformed e. coli xl- blue bacterial cells were carried out in terrific broth according to standard protocols. the expressed scabs were purified via the hexa-histidine tag using immobilized metal ion chelate affinity chromatography. the sensitivity of expressed scab proteins for their target antigen was examined using a scab binding elisa and/or an indirect competition elisa. all elisa plates were coated with hapten-carrier protein conjugates at 1 μg/ml. determination of cdr length, canonical structures, and amino acid distribution the cdrs lengths of the heavy and light chains were defined throughout this article following the well-established kabat numbering system. to analyze amino acid distribution and conservation throughout the entire antibody, amino acids were classified into seven groups using a microsoft excel visual basic macro sheets. these sheets were downloaded from the aaaaa server. here again, each amino acid, within the analyzed sequences, was numbered according to the kabat scheme. in addition, the canonical classifications of the loops were determined according to a chothia sdr template. these numbering and classification steps were further aided by reference to dr. andrew c.r. martin's group website. antibody modelling and electrostatic potential measurements the variable domains of anti-hapten antibodies were modelled using the project mode in the swiss-model website. the heavy and light chains templates were identified using the swiss-model workspace template identification portal. templates with the highest sequence identities, and similar canonical classes, were then selected. the homology models of the heavy and light chains were generated by the swiss-model workspace automated modelling portal with appropriate specification for each selected template. the generated model qualities were then examined following standard protocols. finally, the heavy and light chains were linked by swiss-pdbviewer. and visualized by pymol. electrostatic energy of the selected antibodies was calculated using adaptive poisson-boltzmann solver version. in the python molecule viewer version. the produced energy was mapped to the surface with medium surface quality and at a 1 Å distance. the map colour was coded as white: kt/e, blue: kt/e, red: − kt/e. docking analysis automated docking was used to predict suitable binding orientations and conformations of the various haptens positioned at/within their corresponding binding sites. the antibody-antigen docking analyses were conducted using the molecular docking and visual screening program autodock vina. polar hydrogen atoms were added, and the generated models were saved as pdbqt files. the ligand rotatable bonds were examined and saved in similar pdbqt format. a potential option within autodock vina is the ability to determine the docking site of the antibody by setting the dimensions of the docking grid box. this was achieved by setting the x, y, and z axes of the grid box to cover the binding sites of the antibody. the docking process was achieved using the command prompt within windows the command script included,,. upon completion of the docking process, the models were exported, viewed and analyzed by pymol.  library construction, bio-panning, and characterization for each immunized sheep, lymphocytes were used as the genetic source for scfv. sheep sera samples were routinely examined during the immunization process, and the selection of material for library construction made based on the best measured responses. sequences of the different antibody fragment libraries confirmed a full diversity within the cdr loops. each of the libraries was considered highly diverse, containing at least 107- unique clones. the panning strategies employed to select phage-binders included a series of steps to encourage the enrichment and selection of the more sensitive and specific clones. whilst this is not an exact science, care was taken where possible to follow a similar selection strategy for each hapten class. the selection process was stopped when clones of the same sequence began to appear at a level of great than 30 % of positive clones analyzed. typically this was after 3– rounds of selection and bio-panning. binding and competition elisas of monoclonal phage were used to identify unique clonal panels specific for sqa, por, hsl, or cop. the isolated scfv phage clones were converted into a scab format, and the sensitivities of purified soluble antibody fragments to free haptens were determined by competition elisa. the “naïve” or simple chemical structure of sqa was recognized with only micromolar sensitivity. an increase in the structural rigidity of the hydrophobic and poly-cyclic second antigen cop did not really improve this moderate ic sensitivity beyond the micromolar range. however, clone por b was able to detect the rigid but polar por with an ic value of nm. the strongest molecular recognitions were seen for hsl molecules, which comprise a hydrophobic tail attached to a hydrophilic lactone ring. several anti-hsl antibodies displayed super-sensitive interactions with clone hsl having an ic in the picomolar range.table summary of the sequence analysis strategy the analyzed sequences were obtained from three libraries. the pre and post-selection sequences included heavy and lambda light chains. pre-selection clones were randomly selected sequences during library construction process summary of the binding sensitivities of the analyzed clones together with the chemical structures of their corresponding antigens. #: ic values were not successfully detected sequences analysis sequences were compared for clones from the pre and post-selection and are summarized in table  sequences were analyzed by examining canonical structures, cdr lengths, and amino acid distribution. cdr length and canonical structures cdr lengths determination of each individual library has revealed clear conservation in cdrs h h and l. in addition, there was moderate diversity in the lengths of cdr l and high variability in cdrs h and l the length of cdrs h h and l were identical for all the post-selection clones isolated from the three libraries, irrespective of binding specificity. in contrast, cdr h and l lengths could be clustered into two groups: antibodies that were developed against cop having generally shorter cdr h and l when compared to the antibodies developed against sqa, por, and hsl. in general terms, the selection process has introduced bias into the cdr lengths recovered with cdrs h l and l having lengths in post-selection clones that were represented at low frequency in the pre-selection repertoires.table cdr length distribution of the pre and post-selection sequences the most prevalent cdrs lengths within each library in pre-selection clones and post-selection clones. the percentage of each specific cdr length within the entire library population or post-selection clones is in parenthesis canonical classification was performed utilizing a strict chothia sdr template, on all the cdrs except cdr h the pre-selection sequences from all three libraries comprised heavy chain sequences dominated by a 1– canonical combination for cdr's h and h. typically, these classes corresponded to cdr lengths of and amino acids. there was moderate but not unexpected variability in the classification of lambda light chains within pre-selection sequences. a significant contribution to the overall library variability was from a canonical class combination 6-1-x for cdrs l1-l2-l cdr l class represents a amino acid loop length, while class is the only identified group for cdr l in the literature. class x is used here to indicate that no canonical class has been reported previously with a similar loop length. the three sampled antibody libraries were analyzed statistically using a chi square goodness of fit test to evaluate whether the canonical class representation was equal within each antibody library. however, the null hypothesis was rejected, and therefore, there was domination of specific canonical classes within each library. it is impossible to conclude whether this bias was present as a result of the different immunizations or as an artifact from the library cloning process. clones included only lambda light chains, with a clear antigen specific canonical combination bias seen for each target. in contrast, the heavy chains cdrs h1-h were all classified as 1–; a canonical class combination that dominated all the three libraries’ pre-selection sequences. the post-selection lambda chains were from canonical classes of cdrs l and l that were present in low abundance within the pre-selection sequences, and reflected the cdr length trends described previously. cdr l of the highly sensitive post-selection clones was grouped within class or these sensitive clones included cdr h with or amino acids. in addition, cdr l class x was evident in post-selection clones that have not shown high binding sensitivities, like sqa and cop clones.table canonical combinations of the pre and post-selection sequences the canonical classes were determined using strict chothia sdr template. data presented represent the most prevalent canonical combinations within each library in pre-selection clones and post-selection clones. x: indicates no currently recognized canonical class with the same loop length. the percentage of specific canonical combination within the entire library population of the analyzed pre and post -selection sequences are in parenthesis amino acid distribution analyses of the pre-selection sequences confirmed the high level of amino acid site conservation in the fw regions and remarkable variability within the cdr loops. the majority of the fw positions were well conserved. comparison between the sheep heavy and light chain's fw regions indicated that the heavy chain fw positions are 30 % more conserved than the light chain. as anticipated, the most variable region was cdr h of which >80 % of the positions showed >50 % variability. the most diverse positions within the heavy chains were h32- and h50-. positions l28- and l50- were highly diverse within the light chains.fig. amino acid variability within various regions of the isolated sequences. variability within a vh, b vl regions of the pre-selection sequences. diversity at each amino acid position was classified into five groups. the total cdr or fw region variability was determined as a percentage of the overall diversity of positions within the selected regions in sharp contrast to the broad repertoire diversity seen for the pre-selection clones only a relatively small number different hapten binders were present at the end of each antigen-specific, bio-panning. this extreme contraction of repertoire diversity is independent of the hapten type and is further reflected in an extreme narrowing of the canonical structures represented within the hapten binding clones. it must be concluded, therefore, that only a tiny percentage of the original and highly diverse sheep repertoires, even following bias through immunization and boosts, have a paratope-shape that is pre-disposed to bind haptens, as a class. all post-selection sequences were collectively “searched” for signs of possible hapten-driven similarities or differences. the number of conserved positions were significantly higher in post-selection sequences when compared to their pre-selection counterparts. in addition, fw of the heavy and light chains contains the highest levels of conservation, possibly identifying the importance of this region in orientating cdrs and required to form a pocket for recognizing haptens. not unexpectedly, the cdr regions themselves contain a significantly lower level of conservation, as each of these antibody panels was selected against different hapten antigens. taken together, the concentration of site conservation within the fw regions, but not in the cdrs, appears to suggest an important structural role for the fw regions in enabling cdrs to be displayed in the required orientation to accommodate haptens without compromising antigen affinity.table comparison of the conserved amino acid positions following antigen selection analysis was conducted on the pre and post-selection sequences. a conserved site was defined as: a position in the post-selection clones where an amino acid remained unchanged or had similar bio-physical characteristics to the original pre-selection residues surface analysis of post-selection clones homology models of post-selection clones were constructed utilizing swiss-model workplace. examination of the binding site topographies revealed pocket like surfaces for all of the modelled anti-hapten antibodies. the sizes and shapes of these pockets were influenced by the antigens they were selected against, with the main contributions to antigen binding coming from cdrs h h and l. the significant influence of cdr h on antigen binding was via direct interaction with antigen and also via the less obvious indirect impact on the orientation and corresponding pocket shape and size delivered by the positioning of cdrs h and l in particular, two positions in cdr h greatly influenced antigen binding by controlling the pocket shape between cdr h and cdr l. in all cases, position h was occupied with phe, tyr, ile, or leu, and these amino acids play a key role within a network or web of interactions with amino acids at positions h h h h l95a, and l95b. these interactions enabled the remaining cdr h residues, h in particular, to be in direct contact with the different antigens. analyses of position h revealed the presence of phe in anti-sqa clones, whilst tyr was observed in anti-sqa and all the anti-cop clones. for these clones at least, their binding sensitivities might indicate a preference for phe when establishing interactions with hydrophobic targets like sqa and cop. in contrast, the presence of tyr at this position appears to be important for the high binding sensitivity of clone por b when compared to clones por a and por d that contained a h phe. the four polar nitrogen atoms at the core of the por structure should interact readily with the hydroxyl group of tyr. for the anti-hsl antibodies, there was an arg at position h in clones and which contributed to binding of the polar lactone ring of hsl molecules. in contrast, the h ile of clones and has established hydrophobic interactions with the hsl molecules' aliphatic tail. consequently, the role and influence of position h for antigen binding was confirmed and correlated with each hapten in terms of its chemical structure and polarity.fig. the effect of amino acids within cdr h on the predicted positioning of cdrs h and l amino acid positions h, h, and h in clones a hsl b hsl c sqa a d sqa b e por b f por a g cop h and h cop a position h has a profound effect on the orientation of cdr h, whilst positions h and h influence cdr l orientation. these homology images of the post-selection clones were constructed utilizing swiss-model workplace. the structures were viewed by pymol fig. side-chains orientations of potential amino acids within cdr h site-specific interactions of amino acid positions h h and h a sqa a, sqa b, and sqa f. b por a, por d, and por b. c cop e, cop h, and cop a. d hsl, hsl, and hsl. these homology images of the post-selection clones were constructed utilizing swiss-model workplace. the structures were viewed by pymol fig. structural, docking, and surface-mapped electrostatic potential of antibodies. the homology structures of the post-selection clones were determined by swiss-model workplace for clones a hsl b sqa a c por b d cop h docking analyses were conducted with autodock vina. the structures were viewed by pymol. measurements of electrostatic energies were calculated using python molecule viewer version. the produced energy was mapped with medium surface quality and at a 1 Å distance from the surface. the map color was coded as white: kt/e, blue: kt/e, red: − kt/e the surface topography of the area between cdrs h and h was also greatly influenced by a single position at h within cdr h which can establish interactions with h h and h the interaction of h and h was as predicted because all the post-selection clones contain cdr h with a class conformation. for the less antigen-sensitive clones such as sqa f por d and a, position h was occupied by an arg. when the same position was represented by a smaller amino acid such as ser, met, or thr, the clones all showed increased sensitivity to their respective target hapten. in general terms, this structural analysis suggested that the presence of relatively bulky amino acids at position h might cause cdr h to be oriented further away from cdr h and towards cdr l this repositioning appears to partially occlude the hapten binding pocket. furthermore, we were able to explore this hypothesis further and show, using computer generated binding site “mutants” and their subsequent modelling, that changes in amino acid composition at position was predicted to cause significant and functionally profound re-positioning of the cdr loop. electrostatic potential measurements of the post-selection clones the surface-mapped electrostatic energy was examined across the entire population of post-selection clones. here, four antibodies, one for each target, have been selected for illustration purposes. sqa e por b and cop h antibodies generally have relatively uncharged binding pockets ideally suited to their hydrophobic targets, whilst in stark contrast, the hsl antibodies have a positively charged binding pocket, as typified by the hsl antibody. this positively charged binding pocket might be required to attract hsl molecules that are rich in oxygen molecules. these observations were supported by measurements of total electrostatic potential utilizing adaptive poisson-boltzmann solver version. in python molecule viewer. the predicted electrostatic energies of the anti-sqa antibodies ranged from e +  to < e + 04 kj/mol. low electrostatic energy was also predicted for the three por clones. in contrast, most of the anti-cop antibodies had a higher electrostatic energy. the recorded energies of anti-hsl antibodies all had predicted values around e + 04 kj/mol. the total electrostatic potential of these antibodies complements the hydrophobic/hydrophilic nature of these haptens. discussion antibody fragments are invaluable protein scaffolds that have been extensively used as diagnostics and more recently therapeutics. they can recognize and bind to a diverse range of antigens including polypeptides, carbohydrates, lipids, nucleic acids, or even small hapten molecules. haptens are not inherently immunogenic due to their small size but can elicit anti-hapten responses when coupled to a suitable immunogenic carrier protein prior to immunization. sheep immunization followed by library construction and characterization by phage display technology has proved to be a successful strategy to develop highly sensitive anti-hapten antibodies. the binding of antibody to antigen is principally generated by complementarities of the binding surfaces, which allows various interactions between the two molecules including the formation of hydrogen bonds, salt bridges, and van der waals interactions. this investigation of antibody-hapten interactions has therefore focused on hapten polarity and structural flexibility. the isolated sqa antibodies recognized the free sqa antigen in the micromolar range. a possible reason for the moderate sensitivity to sqa might be attributed to its pronounced hydrophobic nature preventing/limiting the establishment of specific binding interactions, as has been reported for other hydrophobic targets. furthermore, binding sensitivities have been shown both experimentally and computationally, to be enhanced to more rigid hapten structures. whilst structural flexibility is clearly important, the por used in this study has an unmetalated core, yet still generated antibodies with sub-micromolar sensitivity. this contrasts with the moderate sensitivity to free antigen observed for the cyclic aliphatic and nonpolar cop. improvement in binding sensitivities have been reported in other studies following the chemical addition of polar oh or ho3so groups at the carbon position of cop. it would appear therefore, that the presence of polar groups within the por structure was more important in the generation of higher sensitivity binders than structural rigidity alone. when moderate flexibility and high polarity are combined, super sensitive binding interactions can be isolated. here again polarity at the carbon position was a key determinant in overall sensitivity. one clear conclusion from this work is that the combination of relative structural rigidity with the presence of several polar groups is predictive of an enhanced hapten-antibody interaction. this proposed influence of hapten chemistry on antibody binding might also be evident when examining amino acid distribution, cdrs lengths, and canonical classifications. canonical combination 1– for cdr h1-h dominated both the pre and post-selection heavy chain sequences. structurally, cdr h bridges the two β-sheets and packs across the top of the vh domain. while class of cdr h represents the shortest observed, and most commonly seen, loop for this cdr. almagro et al. have reported the domination of these canonical classes within sheep heavy chain populations. in contrast to the restricted canonical classes of the sheep heavy chains, several canonical classes were observed in the pre-selection lambda chain sequences, with the class combination 6-1-x having the greatest representation in each of the three libraries. this newly proposed class for cdr l was found in 60-80 % of the pre-selection sequences, and interestingly in the less sensitive post-selection clones. the high incidence of canonical class within the pre-selection clones, but not in the highly sensitive post-selection sequences, suggest this class is not pre-disposed to binding haptens as a group. cdr length comparisons revealed high conservation in cdrs h h and l but much more variability in cdrs l h and l. whereas the post-selection clones cdrs have shown high level of length conservation to accommodate each hapten. a cdr h with amino acids was observed with sqa, por, and hsl post-selection clones. anti-haptens antibodies with amino acid cdr h have been reported previously against arsenate and phosphorylcholine, and it has been postulated that this is the minimum length to enable cdr h to form part of a pocket-like binding site. interestingly, where cdr h lengths are amino acids or greater, it has also been postulated that they become increasingly exposed to solvent, resulting in a more unstable structure and less defined binding site. antibody-antigen sensitivity is of course greatly influenced by the type of amino acids in contact with the different hapten targets. analyses of the pre and post-selection sequences revealed more conserved sites in the post-selection sequences, when compared to their pre-selection counterparts, especially within the fw regions. this type of conservation phenomenon has been reported previously but to a lesser extent, following examination of the common core residues of ~ kabat sequences of antibodies to a full range of different antigen classes. one possible reason for the very high level of conservation we have seen is as an artefact of the antibody isolation process. phage enrichment is known to be significantly influenced by toxicity and expressibility of the displayed antibody binding sites. however, the conserved positions seen here are consistent even across clones isolated from different libraries, or from structurally dissimilar haptens or different panning strategies. the only shared factor here is the overall low molecular weight of the haptens and the need to form a binding site shape able to accommodate them. these structural and topographical perquisites could drive high conservation in the fw regions, especially fw and result in the selection of clones from only a small subset of the full repertoire diversity. whilst all the homology-modelled sheep antibodies possess pocket-like binding sites with various shapes and sizes, the development of more compact binding pockets has been attributed to closure of the vl-vh interface of the anti-hapten antibodies and improved binding sensitivities. here, the shape and overall size of the binding pockets was greatly influenced by a small number of amino acid positions in cdr h that appeared to control the orientation of cdrs h and l position h was occupied with phe, tyr, ile, or leu, and was involved in a network of interactions with positions h h h l95a, and l95b. these interactions have enabled the remaining of cdr h amino acids, especially position h to be oriented toward the target antigens. position h is known from previous work to have an important role in hapten recognition. the average relative side chain solvent-accessible surface of this position was estimated at 50-75 %. this study is able to correlate the amino acids found at this site with antigen polarity, and with phe, tyr, and arg pairing up with sqa and cop, por, and hsl, respectively. position h appears to impart a second and key structural influence on cdr h. this residue is at the tip of cdr h and is believed to pack against residues at positions h and h. from our models, the presence of arg at this position causes steric hindrance with cdr h pushing this important cdr away from cdr h and towards cdr l. in concert, the residues found at these three positions in cdr h appear to exert a significant influence over the size and shape of the hapten binding pockets. previous studies have concluded that antibody-protein interactions are based on "charge complementarity" and "electrostatic complementarity", and that this complementarity is important in defining binding site specificity. therefore, it was interesting to examine whether this proposed electrostatic complementarty could be expanded to hapten antigens. here, hapten complementarity is not limited to shape and size but extends also to surface electrostatic potential. sqa, por, and cop binding pockets have hydrophobic surfaces, in complete contrast to the positively charged surfaces of anti-hsl antibodies. these observations are further supported by the electrostatic optimization of anti-hapten antibodies specific for p-nitrophenyl phosphonate, fluorescein, and n--n--guanidiniumacetic acid. consequently, we can predict that antibodies with high binding sensitivity show an improved capacity to recognize haptens by establishing electrostatically complementary binding pockets.  various structural and molecular factors appear to profoundly influence the successful binding of antibodies to hapten molecules. haptens possessing a relatively rigid chemical backbone, together with the presence of polar groups, are much more likely to be recognized by antibodies with high sensitivity. these highly sensitive antibodies tend to show an improved capacity to recognize their antigens by establishing complementary binding pockets. these complementarities are influenced by amino acid composition and control the pocket size, shape, and electrostatic potential. abbreviations cdrcomplementarity determining regions copcoprostane dadalton fwframework hslhomoserine lactone poretioporphyrin scabsingle chain antibody scfvsingle-chain variable fragments sqasqualane competing interests no potential conflicts of interest were disclosed. authors' contributions ma: performed experiments, participated in the design of the study, analyzed data, and drafted the manuscript. sp: performed experiments, participated in the design of the study, analyzed data. gb: participated in the design of the study, analyzed data. dc: provided material, participated in the design of the study, and analyzed data. kc: participated in the design of the study, and analyzed data ap: participated in the design of the study, analyzed data, and drafted the manuscript. all authors read and approved the final manuscript. the authors would like to thank the scottish universities life sciences alliance for their support.
analyses of the pre and post- selection sequences revealed more conserved sites within the post-selection sequences, when compared to their pre-selection counterparts. the isolated antibodies were analysed in term of cdr length, canonical structure, and binding site shape and electrostatic potential. to better understand this range of antibody-hapten interactions, analyses were extended to examine the binding loop canonical structures and cdr lengths of a series of anti-hapten clones. haptenphage displayantibody modelinghapten dockingcanonical structurecdr lengthelectrostatic potentialissue-copyright-statement© the author 2015 low molecular weight haptens cannot be recognized by the immune system unless conjugated to larger carrier molecules. an increase in structural rigidity of the hydrophobic and cyclic coprostane did not improve this binding sensitivity beyond the micromolar range, whilst the polar etioporphyrin was detected with nanomolar sensitivity. the simple, chemically naïve structure of squalane was recognized with micromolar sensitivity. three antibody libraries were constructed and used as potential pools to isolate specific antibodies to each target. different hapten targets with a broad range of structural flexibility and polarity were conjugated to carrier proteins, and utilized in sheep immunization. the best binding antibodies have shown improved capacity to recognize these haptens by establishing complementary binding pockets in terms of size, shape, and electrostatic potential. a detailed understanding at the molecular level of this incredible ability of antibodies to recognize haptens, is still limited compared to other antigen classes. homoserine lactone molecules, which combine molecular flexibility and polarity, generated super-sensitive interactions. the strong selection pressure, generated by panning against these haptens resulted in the isolation of antibodies with significant sequence conservation in the fw regions, and suitable binding site cavities, representing only a relatively small subset of the available full repertoire sequence and structural diversity. as part of this process, the important influence of cdr h on antigen binding was observed through its direct interaction with individual antigens and indirect impact on the orientation and the pocket shape, when combined with cdrs h and l the binding pockets also displayed electrostatic surfaces that were complementary to the hydrophobic nature of cop, sqa, and por, and the negatively charged hsl.
 low molecular weight haptens cannot be recognized by the immune system unless conjugated to larger carrier molecules. antibodies to these exceptionally small antigens can still be generated with exquisite sensitivity. a detailed understanding at the molecular level of this incredible ability of antibodies to recognize haptens, is still limited compared to other antigen classes. methods different hapten targets with a broad range of structural flexibility and polarity were conjugated to carrier proteins, and utilized in sheep immunization. three antibody libraries were constructed and used as potential pools to isolate specific antibodies to each target. the isolated antibodies were analysed in term of cdr length, canonical structure, and binding site shape and electrostatic potential.  the simple, chemically naïve structure of squalane was recognized with micromolar sensitivity. an increase in structural rigidity of the hydrophobic and cyclic coprostane did not improve this binding sensitivity beyond the micromolar range, whilst the polar etioporphyrin was detected with nanomolar sensitivity. homoserine lactone molecules, which combine molecular flexibility and polarity, generated super-sensitive interactions. to better understand this range of antibody-hapten interactions, analyses were extended to examine the binding loop canonical structures and cdr lengths of a series of anti-hapten clones. analyses of the pre and post- selection sequences revealed more conserved sites within the post-selection sequences, when compared to their pre-selection counterparts. the strong selection pressure, generated by panning against these haptens resulted in the isolation of antibodies with significant sequence conservation in the fw regions, and suitable binding site cavities, representing only a relatively small subset of the available full repertoire sequence and structural diversity. as part of this process, the important influence of cdr h on antigen binding was observed through its direct interaction with individual antigens and indirect impact on the orientation and the pocket shape, when combined with cdrs h and l the binding pockets also displayed electrostatic surfaces that were complementary to the hydrophobic nature of cop, sqa, and por, and the negatively charged hsl.  the best binding antibodies have shown improved capacity to recognize these haptens by establishing complementary binding pockets in terms of size, shape, and electrostatic potential. keywords haptenphage displayantibody modelinghapten dockingcanonical structurecdr lengthelectrostatic potentialissue-copyright-statement© the author 2015 