The docking process was achieved using the command prompt within Windows 8. Upon completion of the docking process, the models were exported, viewed and analyzed by PyMOL academic version 1.
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 10 7 8 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 [ 31 ]. Whilst this is not an exact science, care was taken where possible to follow a similar selection strategy for each hapten class increasing stringency though reduction in antigen concentration and the swapping of protein conjugates to minimize the selection of carrier protein specific clones etc.
Typically this was after 3—4 rounds of selection and bio-panning. The isolated scFv phage clones were converted into a scAb single chain antibody format, and the sensitivities of purified soluble antibody fragments to free haptens were determined by competition ELISA Table 2.
An increase in the structural rigidity of the hydrophobic and poly-cyclic second antigen COP did not really improve this moderate IC 50 sensitivity beyond the micromolar range.
The strongest molecular recognitions were seen for HSL molecules, which comprise a hydrophobic tail attached to a hydrophilic lactone ring. Sequences were compared for clones from the pre and post-selection panning and are summarized in Table 1. Sequences were analyzed by examining canonical structures, CDR lengths, and amino acid distribution.
The length of CDRs H1, H2, and L2 were identical for all the post-selection clones isolated from the three libraries, irrespective of binding specificity. In general terms, the selection process has introduced bias into the CDR lengths recovered with CDRs H3, L1, and L3 having lengths in post-selection clones that were represented at low frequency in the pre-selection repertoires.
The pre-selection sequences from all three libraries comprised heavy chain sequences dominated by a 1—1 canonical combination for CDR's H1 and H2 Table 4. There was moderate but not unexpected variability in the classification of lambda light chains within pre-selection sequences.
CDR L1 class 6 represents a 14 amino acid loop length, while class 1 7 amino acids loop is the only identified group for CDR L2 in the literature [ 7 , 41 ]. 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 X 2 IBM SPSS 21 to evaluate whether the canonical class representation was equal within each antibody 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 post-biopanning included only lambda light chains, with a clear antigen specific canonical combination bias seen for each target. The post-selection lambda chains were from canonical classes of CDRs L1 and L3 that were present in low abundance within the pre-selection sequences, and reflected the CDR length trends described previously.
CDR L1 of the highly sensitive post-selection clones was grouped within class 5 or 6. These sensitive clones included CDR H3 with 9 class 4 or 11 class 5 amino acids. 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 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. In sharp contrast to the broad repertoire diversity seen for the pre-selection clones only a relatively small number 3 to 8 different hapten binders were present often repeated several times at the end of each antigen-specific, bio-panning Table 2. 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 Tables 4 and 5.
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.
The number of conserved positions were significantly higher in post-selection sequences positions when compared to their pre-selection counterparts 28 positions Table 5. In addition, FW3 of the heavy and light chains contains the highest levels of conservation, possibly identifying the importance of this region in orientating CDRs 2 and 3 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.
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 H2, H3, and L3 Fig.
The significant influence of CDR H2 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 H3 and L3. These interactions enabled the remaining CDR H2 residues, H58 in particular, to be in direct contact with the different antigens.
For these clones at least, their binding sensitivities might indicate a preference for Phe when establishing interactions with hydrophobic targets like SQA and COP. 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 H58 in clones 2, 3, and 5, which contributed to binding of the polar lactone ring of HSL molecules. In contrast, the H58 Ile of clones 1, 4 and 6 has established hydrophobic interactions with the HSL molecules' aliphatic tail Fig. Consequently, the role and influence of position H58 for antigen binding was confirmed and correlated with each hapten in terms of its chemical structure and polarity.
The structures were viewed by PyMOL 1. Side-chains orientations of potential amino acids within CDR H2. Site-specific interactions of amino acid positions H53, H58, and H Structural, docking, and surface-mapped electrostatic potential of antibodies. Docking analyses were conducted with AutoDock Vina 1. The interaction of H53 and H71 was as predicted because all the post-selection clones contain CDR H2 with a class 1 conformation.
This repositioning appears to partially occlude the hapten binding pocket Fig. 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 Fig.
This positively charged binding pocket might be required to attract HSL molecules that are rich in oxygen molecules. The predicted electrostatic energies of the anti-SQA antibodies ranged from 4. The recorded energies of anti-HSL antibodies all had predicted values around 4.
Antibody fragments scFv are invaluable protein scaffolds that have been extensively used as diagnostics and more recently therapeutics [ 42 ].
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 [ 16 — 18 ].
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 [ 43 ]. This investigation of antibody-hapten interactions has therefore focused on hapten polarity and structural flexibility.
Furthermore, binding sensitivities have been shown both experimentally [ 45 ] and computationally [ 46 ], 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 POR B11, IC 50 nM. This contrasts with the moderate sensitivity to free antigen micromolar range 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 HO 3 SO groups at the carbon position 3 of COP lithocholic acid or glycolithocholic acid sulfate [ 47 , 48 ].
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 eg HSL antigens , super sensitive picomolar range binding interactions can be isolated.
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. Almagro et al. 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 X CDRs L1-L2-L3 having the greatest representation in each of the three libraries.
The high incidence of canonical class X 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.
Whereas the post-selection clones CDRs have shown high level of length conservation to accommodate each hapten. Anti-haptens antibodies with 12 amino acid CDR H3 have been reported previously against arsenate and phosphorylcholine [ 51 ], and it has been postulated that this is the minimum length to enable CDR H3 to form part of a pocket-like binding site. Interestingly, where CDR H3 lengths are 13 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 [ 52 ].
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 Table 5. 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 [ 54 , 55 ]. 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 FW3, 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 Fig. These interactions have enabled the remaining of CDR H2 amino acids, especially position H58, to be oriented toward the target antigens. Position H58 is known from previous work to have an important role in hapten recognition [ 20 , 56 ]. In concert, the residues found at these three positions in CDR H2 H59, H58, and H53 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" [ 58 ], and that this complementarity is important in defining binding site specificity [ 59 ]. 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.
These observations are further supported by the electrostatic optimization of anti-hapten antibodies specific for p-nitrophenyl phosphonate, fluorescein, and N- P-cyanophenyl -N- diphenylmethyl -guanidiniumacetic acid [ 60 — 62 ]. 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. An analysis of the sequences of the variable regions of bence jones proteins and myeloma light chains and their implications for antibody complementarity.
J Exp Med. Padlan EA. Anatomy of the antibody molecule. Mol Immunol. The mechanism and regulation of chromosomal V D J recombination. A hapten-specific chimaeric IgE antibody with human physiological effector function.
Weill J, Reynaud C. The main difference between an antigen and a hapten is that an antigen is a complete molecule that can trigger an immune response by itself whereas a hapten is an incomplete molecule that cannot trigger an immune response by itself. Antigen and hapten are two types of immunogens that can trigger immune responses. What is an Antigen — Definition, Features, Types 2. An antigen is a molecule that can trigger an immune response by acting as an immunogen. It can be either a protein, peptide or polysaccharides.
Lipids and nucleic acids can also serve as antigens when binding to proteins. A particular antigen may contain one or more epitopes, which are the antigen determinants. Antibodies recognize and bind to these epitopes. Furthermore, the immune system produces specific glycoproteins called antibodies in response to epitopes. There are four main types of antigens occur in the body:.
Figure 1: Antigens Directly Bind to an Antibody. A Hapten is an incomplete antigen which is not originally immunogenic. Antigen directly binds to the antibodies produced and initiate an immune reaction. Hapten binds to an antibody but does not have the ability to trigger the host immune system to produce an immune reaction. Haptens conjugate with carrier molecules. Haptens are used in Antibiotics and Anesthetics designing. Antigen reactions are Antigenic and Immunogenic.
Hapten reactions are only Immunogenic. Commonly used Adjuvants 1 Aluminium potassium sulfate Alum First Aluminium salt used as Adjuvant and now completely replaced by aluminium hydroxide and aluminium phosphate for commercial vaccines.
Secreted antibodies or Surface receptors onT-cells. Cross reaction can occur between antigens which bear Stereochemical similarities. Similarly, some substances are Antigenic or Immunogenic in one individual but not in others. Total views 23, On Slideshare 0. From embeds 0. Number of embeds Downloads Shares 0. Comments 0. Likes You just clipped your first slide! Clipping is a handy way to collect important slides you want to go back to later. Now customize the name of a clipboard to store your clips.
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