Affinity of T and B lymphocyte receptors for hapten determinants

The affinity of anti-hapten antibodies, produced by single cells, was studied by hapten inhibition of plaque-forming cells using the local hemolysis in gel assay. The shift in affinity of anti-hapten antibodies, that occurs with time after immunization, was parallelled by an analogous shift, at the cellular level, with regard to indirect plaque-forming cells (PFC). Thus, there was a gradual decrease of the hapten concentration which was needed to suppress indirect plaque formation with time after immunization, indicating a gradual increase in the number of high affinity cells. No such shift was observed with cells causing direct plaque formation. Analogous studies were performed with hapten-specific antigen-binding cells of T and B origin by using hapten-inhibition of rosette-forming cells. Hapten-specific antigen-binding cells were present in both cell populations. Lymphocyte binding of haptenated red cells could be specifically inhibited by free hapten. It was found that antigen-binding B lymphoid cells became gradually more susceptible to hapten inhibition with time after immunization, whereas no such change was observed in antigen-binding T lymphoid cells. Possible reasons for this discrepancy are discussed, as well as the implications for these findings regarding the specificity and structure of the T cell receptors.     

Affinity labeling of the Fc receptor on human monocytes using bifunctional cross-linking agents

To affinity label the Fc receptor on human monocytes, Fc fragments of monoclonal human IgG1 radiolabeled with iodine 125 were covalently bound to the surface of intact monocytes using a variety of bifunctional cross-linking agents including ethylene glycol bis(succinimidyl succinate), dithio-bis-(succinimidyl proprionate), maleimidobenzoyl N-hydroxysuccinimide, glutaraldehyde and dimethyl suberimidate. After cross-linking, cells were solubilized and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by radioautography. Each of these cross-linkers caused a portion of cell-bound Fc fragments to form a covalent complex with a monocyte membrane component. This complex migrated on electrophoresis with an apparent molecular weight of 120,000. Deducting the molecular weight of Fc fragments alone (53,000) the molecular weight of the second component of the complex therefore was about 67,000. A similar estimate of receptor size also was obtained after reduction with dithiothreitol. Complex formation was potently inhibited by unlabeled Fc fragments, IgG1 or IgG3, all of which would be expected to compete with Fc fragments for IgG Fc receptor on human monocytes, but was not inhibited by Fab fragments, IgG2 or IgG4, which do not bind avidly to this receptor. By quantitating the amount of complex formed in the presence of varying concentrations of labeled ligand, it could be demonstrated that complex formation was saturable, and that Fc fragments formed complexes with avidity comparable to that with which Fc fragments bound to receptors on intact monocytes. The findings establish the feasibility of using radiolabeled Fc fragments to affinity label the IgG Fc receptors on human leukocytes. Potential advantages of this approach to studying receptor structure are discussed.     

Affinity maturation of recombinant antibodies using E. coli mutator cells

Background: Phage libraries can display repertoires of antibodies which are greater in number than the mammalian immune response. However, the selected antibodies often have low binding affinity to their target antigen or hapten (K_D below 10⁻⁶ M), which is characteristic of the primary immune repertoire. There is a need for procedures to mimic somatic hypermutation through antigen driven affinity maturation, thereby increasing the affinity of selected immunoglobulins. Objective: To investigate the effectiveness of mutation and affinity selection of recombinant antibody genes with mutator E. coli cells, incorporating phage-display strategies. Study design: Unique human scFvs were selected from a naive Fd-phage library. These genes were mutated by propagation in mutD5 mutator E. coli cells (mutD5-FIT) which were competent for Fd (M13) based phagemid transfections and generated point mutations (transversions and transitions) in the scFv genes. Individual phage-displayed scFvs were affinity selected from the mutation library and were assayed as soluble scFvs by ELISA and BIAcore for binding to antigen. Results: The in vivo mutation of phage-displayed scFvs in E. coli mutD5-FIT, combined with affinity selection against antigen, produced scFv molecules with improved binding activity. The point mutations which resulted in single amino acid substitutions frequently produced ten fold increases in apparent binding affinity. Structural comparisons revealed that these point mutations were in framework regions (adjacent to the CDRs) and within the CDRs. In one case the apparent affinity of an antiglycophorin scFv after mutation in the VL framework region close to CDR3 increased by 10³. However, this increase in apparent affinity was accompanied by an increased propensity to dimerise and form aggregates. Conclusions: A strategy for the rapid affinity maturation of scFv and Fab antibody fragments has been developed which utilises mutator strains of E. coli and incorporates phage display of antibody repertoires (libraries).     

Production and utilisation of antibodies directed against oestrone sulphate using specific hapten synthesis.

Oestrone-3-sulphate (E13S) is an important metabolite of oestrone. Studies in cattle had previously shown that it is synthesised in the gravid uterus by the foetus, but not by the corpus luteum. Progesterone measurement in milk by radioimmunoassay (RIA) is routinely carried out in some laboratories as a pregnancy test for cattle. The major drawback of progesterone measurement in milk, by RIA, as a pregnancy test was the failure to detect the lack of conceptus in those cows where early embryonic death had occurred but the corpus luteum still persisted thereby giving false positive results. We have developed a direct RIA for E13S by raising antibodies to an immunogen prepared from a specific hapten synthesised by an unambiguous chemical synthesis. The sensitivity of the RIA in milk was found to be 0.368 nmol/l. The levels of E13S in non-pregnant cows are undetectable but during a viable pregnancy, the levels are elevated to greater than 0.40 nmol/l by day 100. There was no cross-reactions in the assay with any free oestrogens. The measurement of this metabolite of oestrone promises to provide an accurate marker for the detection of a viable conceptus in cattle.     

Spin-label studies of hapten combining sites in anti-p-azobenzoate antibodies

The structural nature of the hapten combining site for anti-p-azobenzoate from four rabbits has been studied using spin-labeled benzoate haptens in which, (a) the spin-label (nitroxide free-radical) was attached directly to the p-aminobenzoate or with a spacer residue, viz., glycyl, β-alanyl or γ-aminobutyryl and (b), the spin-label was attached directly to m-aminobenzoate, o-aminobenzoate or to o-(methylamino) benzoate. Studies using the haptens under (a) showed that the rotational-freedom of the bound spin-label for the four preparations was increased with increased spacing and that although, for two of the preparations, the binding constant was decreased slightly by the introduction of the glycyl spacer, it increased with increasing spacer length. Of the haptens under (b), the o-(methylamino) benzoate derivative was not bound, presumably because the carboxylate group was displaced out of the plane of the benzene ring. On the other hand, the o-aminobenzoate derivative was measurably bound, presumably because the carboxylate group forms an intramolecular hydrogen bond to the amino group and is coplanar with the benzene ring. The meta-derivative was more strongly bound than the ortho-derivative but both the ortho- and meta-derivatives were bound less than the unspaced-p-aminobenzoate derivative. In the cases of both ortho-and meta-derivatives the spin-label was strongly immobilized indicating a tight fit for p-azobenzoate around these positions as was also indicated by the low binding of these compounds.     

The effect of homologous and heterologous carriers on the immunogenicity of the galactocerebroside hapten

Glycolipid haptens are capable of eliciting an immune response when complexed with a protein carrier. These experiments demonstrate that an immunogenic (foreign) carrier is required.

Antibody to galactocerebroside can be produced in rats by injecting the hapten associated with heterologous (bovine serum albumin) but not homologous (rat serum albumin) protein. It appears after primary immunization in animals given pertussis vaccine or after repeated injections of the antigen emulsified in Freund's incomplete adjuvant. However, if Mycobacterium tuberculosis is added to the emulsion a protein carrier is unnecessary. In this case the bacilli may serve a carrier function in addition to their usual adjuvant action.

Galactocerebroside, therefore, behaves in much the same way as conventional, covalently bound, non-lipid haptens. Antibody is only produced when the hapten is bound to an immunogenic carrier or in the presence of an adjuvant, capable of providing a stimulus to replace the carrier specific `helper' function of the T cell.

Since galactocerebroside is a normal body constituent, the findings also define some of the conditions for the failure of tolerance to endogenous glycolipids in the central nervous system.

     

An improved method for the production of antibodies to lipophilic carboxylic hapten using small amount of hapten-carrier conjugate

A rapid, simple and low cost procedure for preparing minute amount of hapten-protein conjugates was developed using 4-acetyl benzoic acid (ABA) and two other closely related small chromophoric haptens. The amide bond-generating mixed anhydride method of Erlanger was modified to promote conjugation to various proteins (bovine serum albumin, ovalbumin, casein and hemocyanin) or to a synthetic homopolymer (Poly- -lysine). The key process in this synthesis is the use of a reversed micellar medium allowing strong carrier haptenization as determined by spectrophotometric measurement at characteristic hapten absorption peaks. This coupling procedure is applicable to as little hapten material as 0.2 μmol and is disclosed to be most valuable for other rare lipid haptens which pose analytical problem in biological fluids and matrices. Specific mice polyclonal antibodies were produced following multiple intraperitoneal injections of (ABA)₂₃–BSA conjugate as revealed by indirect and competitive ELISA. Calculated K_D for the interaction of the antibodies with free ABA was found to be 5×10⁻⁵ M.     

Generation of llama single-domain antibodies against methotrexate, a prototypical hapten

Single-domain antibodies specific to methotrexate (MTX) were obtained after immunization of one llama (Llama glama). Specific VHH domains (V-D-J-REGION) were selected by panning from an immune-llama library using phage display technology. The antibody fragments specific to MTX were purified from Escherichia coli (C41 strain) periplasm by immobilized metal affinity chromatography with an expression level of around 10mg/L. A single band around 16,000Da corresponding to VHH fragments was found after analysis by SDS-PAGE and Western blotting, while competition ELISA demonstrated selective binding to soluble MTX. Surface plasmon resonance (SPR) analysis showed that anti-MTX VHH domains had affinities in the nanomolar range (29-515nM) to MTX-serum albumin conjugates. The genes encoding anti-MTX VHH were found by IMGT/V-QUEST to be similar to the previously reported llama and human IGHV germline genes. The V-D and D-J junction rearrangements in the seven anti-MTX CDR3 sequences indicate that they were originated from three distinct progenitor B cells. Our results demonstrate that camelid single-domain antibodies are capable of high affinity binding to low molecular weight hydrosoluble haptens. Furthermore, these anti-MTX VHH give new insights on how the antigen binding repertoire of llama single-domain antibody can provide combining sites to haptens in the absence of a VL. This type of single-domain antibodies offers advantages compared to murine recombinant antibodies in terms of production rate and sequence similarity to the human IGHV3 subgroup genes.     

Combining site specificity of monoclonal antibodies to the organophosphate hapten soman

The combining site specificities of eight monoclonal antibodies raised against the organophosphorous-containing hapten Soman are compared to monoclonal antibodies specific for a naturally occurring organophosphorous compound, phosphocholine (PC). Although these haptens share some structural and spatial features, differences in their chemical structures, most notably the presence or absence of a positive charge, appear to prevent significant cross-reactivity between antibodies specific for each. The murine memory response to PC-KLH has been shown previously to be characterized by the presence of two major groups of antibodies differentiated on the basis of their specificity for free PC and for the nitrophenyl derivative of PC, nitrophenylphosphocholine (NPPC). Interestingly, two groups of hybridoma antibodies were detected in the immune response to Soma-KLH which possess differential specificity for Soman and for a nitrophenyl derivative of Soman.     

A simple and rapid method for quantification of hapten specific antibodies

A sensitive and simple method for quantitation of antibodies against small molecules is described using DNP-lysozyme as the enzyme conjugate. The anti-DNP antiserum was raised against DNP-bovine serum albumin conjugate. Anti-DNP antibody or its monovalent fragment (Fab) reduced the enzyme activity of DNP-lysozyme conjugate in a concentration-dependent manner. The inhibition of enzyme activity is a specific measure of the antibody and Fab content of the sample. The specificity of the reaction was assessed by reduction of antibody-induced inhibition by DNP-lysine. The ability of DNP-lysine to reduce the antibody-induced inhibition of DNP-lysozyme activity also makes possible a sensitive assay for DNP-lysine.     

Affinity ranking of antibodies using flow cytometry: application in antibody phage display-based target discovery

Application of antibody phage display to the identification of cell surface antigens with restricted expression patterns is often complicated by the inability to demonstrate specific binding to a certain cell type. The specificity of an antibody can only be properly assessed when the antibody is of sufficient high affinity to detect low-density antigens on cell surfaces. Therefore, a robust and simple assay for the prediction of relative antibody affinities was developed and compared to data obtained using surface plasmon resonance (SPR) technology. A panel of eight anti-CD46 antibody fragments with different affinities was selected from phage display libraries and reformatted into complete human IgG1 molecules. SPR was used to determine K(D) values for these antibodies. The association and dissociation of the antibodies for binding to CD46 expressed on cell surfaces were analysed using FACS-based assays. We show that ranking of the antibodies based on FACS data correlates well with ranking based on K(D) values as measured by SPR and can therefore be used to discriminate between high- and low-affinity antibodies. Finally, we show that a low-affinity antibody may only detect high expression levels of a surface marker while failing to detect lower expression levels of this molecule, which may lead to a false interpretation of antibody specificity.