Analysis of affinity of monoclonal antibody responses by inhibition of plaque-forming cells

It has been suggested that dominant monoclonal responses in mice after transfer of small numbers of spleen cells are the result of strong selection for cell clones producing antibody of high affinity. We have attempted to measure the affinity of anti-DNP (2,4? dinitrophenyl) and anti-NIP (4? hydroxy? 5? iodo? 3? nitrophenacetyl) antibodies produced in monoclonal responses by inhibition of plaque-forming cells (PFC) with free hapten. PFC from individual monoclonal spleen foci and from spleens after adoptive transfer of 5 × 10⁵ to 2 × 10⁶ primed spleen cells were generally inhibited at lower free antigen concentrations than the PFC found in a heterogeneous secondary response. It was possible to compare several individual cell clones, forming monoclonal antibody to DNP, with respect to the free antigen concentration needed for 50 % plaque inhibition. These comparisons did not correlate well with the relative affinities of serum antibody estimated by the Stupp-Farr technique. Published work analyzing PFC from plasma cell tumor MOPC 315 forming antibody to DNP might have predicted that the curve of PFC inhibition by free hapten for a monoclonal PFC population would be steep. Usually this was not the case, and 20–80 % PFC inhibition occurred over 100-fold concentration range of free hapten. Some of the anti-DNP clones formed large plaques and when these PFC were inhibited by free antigen, the plaque diameter decreased progressively as the hapten concentration increased. Large plaques consequently require more free antigen for inhibition than small plaques. The poor agreement between apparent affinity measured by plaque inhibition and that measured in serum antibody for monoclonal antibody responses may well be a result of the observed differences in plaque size between individual clones. We therefore have strong reservations about the use of plaque inhibition by free antigen to estimate antibody affinity at the cellular level.     

Inhibitory effect of analogues of phenylarsonate on purified 19S and 7S antibodies specific for azophenylarsonate

Purified 19S and 7S antibodies specific for protein-conjugated azophenylarsonate were compared with respect to their affinities for the free haptens, phenylarsonate and the o-, m- and p-isomers of mononitrophenylarsonate, as judged from the technique of inhibition of haemagglutination of cells conjugated with the homologous hapten. It was found that the two antibody types were equally effective in discriminating among the isomeric haptens, but that 7S antibody was consistently more readily inhibited than the 19S by all the haptens tested. Various parameters concerning agglutination and its inhibition are discussed with regard to these two antibody forms.     

Dose- and time-dependent changes in the binding capacity of IgM antibody

Past experiments have revealed antigen dose-dependent changes in IgG but not in IgM affinity. This is of importance to the general validity of the receptor concept and led us to investigate IgM affinity as a function of antigen dose and of time after antigen administration. Rabbits were immunized with proteins conjugated with p-azobenzene arsonate and the hapten response was assessed by plaque formation. Dose- and time-dependent changes in binding properties of IgM were determined by inhibition with hapten. The quantity of hapten which led to inhibition of 50% of the plaques, I₅₀, was independent of the amount of hapten antibody in the system. All direct plaques could be inhibited by antibody directed against μ chains, but were not inhibited by antibody against γ chains. The amount of antibody against μ chain which inhibited 50 % of the plaques was the same in the early as in the late plaque-forming response. In the initial stages of antibody response, there was no change in I₅₀, over a considerable range of immunizing doses, including doses sufficient to trigger a plaque-forming response, but insufficient to maintain it. Dose related changes in IgM affinity were observed fifteen days after antigen injection. In general, the avidity of IgM plaque-forming antibody increased with time. However, at a very high dose of antigen, there were no changes in average affinity. After a second administration of antigen, plaque-forming cells were detected earlier than in the primary response, though not in greater numbers. The first plaque forming antibody to be detected was of relatively high affinity; antibody detected subsequently was of the same affinity as the antibody formed in the primary response. Results were discussed in terms of cooperation between receptor binding sites and in terms of interference with this cooperation when antigen antibody complexes trigger antibody formation.     

Anaphylactic hyposensitization of rat mast cells in vitro by antigen

Anaphylactic hyposensitization of rat mast cells in vitro was demonstrated by preincubating the cells with specific antigen in suboptimal concentrations. The reduction of the histamine release was more pronounced when the cells were pre-incubated with increasing concentrations of antigen than with the final concentration. The inhibition remained unchanged for more than 3 hr. Non-specific antigen did not influence the histamine release, whereas a 70% inhibition was demonstrated by specific antigen. In rats sensitized to two different antigens, preincubation with one of these inhibited the subsequent histamine release induced by either of the two antigens. The hyposensitization could not be ascribed to blocking antibodies.     

Radioelectrocomplexing: a counterelectrophoretic method for hapten--antibody interaction.

We have described an application of a radioimmunoassay (RIA)method, known as radioelectrocomplexing (REC), which involves the anodal migration of antigen and the cathodal migration of antibody in agar electrophoresis. The agar is divided into zones of free antigen (DNP125I-HSA) and antigen bound with anti-DNP. Complete assays of anti-DNP can be performed in 2-4 hr since both immune complex formation and separation of free from bound antigen can be accomplished by electrophoresis in 60-90 min. Estimation of the weight of specifically-purified anti-DNP chicken antibodies in the nanogram range by REC is of the same order as the reported sensitivity of other RIA methods. The method was capable of demonstrating the higher avidity of the 17 S than 7 S antibody. Based on hapten inhibition the relative binding constants of DNP derivatives and anti-DNP were of the same order as reported from more definitive methods.     

Quantity and Quality of Anti-Hapten Antibodies in Normal and in T Cell-Deprived Mice Studied at the Cellular Level

The Immune response of normal mice as well as of thymus-deprived (TxB) mice against 2 different hapten-protein conjugates (NNP-BSA and NNP-CG) was tested at various times after immunization at the cellular level. Normal mice developed both direct and indirect plaque-forming cells against the hapten, as determined by a modified local haemolysis in gel assay for detection of anti-hapten antibody-secreting cells. However, TxB mice produced direct plaque-forming cells to the same extent as normal mice, whereas the indirect plaque-forming cell response was greatly impaired. In fact, significantly elevated levels of indirect plaque-forming cells were not detected in such mice. Studies on the affinity of secreted antibodies by means of hapten inhibition revealed that the indirect plaque-forming cells derived from normal immunized mice could be inhibited by decreasing concentrations of free hapten with time after immunization, indicating a gradual increase in the number of cells secreting antibodies of high affinity. No such change was detected in normal or TxB mice with regard to antibodies giving rise to direct lysis, presumably IgM antibodies.     

Direct measurement of antibody affinity distribution by hapten-inhibition enzyme immunoassay

A rapid, simple and reliable technique for determining the affinities of antibody subpopulations in a complex mixture is described. The principle of this method is that antigen conc can be represented as the amount of antigen immobilized on the polystyrene surface of a microwell containing a fixed vol of diluted antibody. Thus, by measuring the proportion of antibody bound to different wells coated with varying amounts of antigen, it is a straightforward matter to calculate an affinity distribution. We have verified that: (1) the amount of antigen bound to a polystyrene plate is proportional to the concn of antigen used for sensitization and follows a typical saturation curve; (2) the antibodies bound to plates sensitized with low concns of antigen are of higher affinity than those bound to plates sensitized with high concns of antigen; (3) an apparent affinity constant (aK) is defined as the reciprocal concn of free hapten required for 50% inhibition of antibody binding to immobilized antigen; (4) the aK determined by this method is in close agreement with the intrinsic affinity constant (K) measured by fluorescence quenching or the Farr assay; and (5) that during the course of immunization in vivo there is a clear shift to higher-affinity antibody subpopulations.     

Activation and hapten inhibition of mast cells sensitized with monoclonal IgE anti-penicillin antibodies: evidence for two-site recognition of the penicillin derived determinant

We utilized an in vitro mast cell activation assay and hapten inhibition of mediator release to characterize the fine specificity of two IgE anti-penicillin monoclonal antibodies (mAb). Cultured mouse mast cells were passively sensitized with IgE mAb anti-benzylpenicillin (BP) or anti-amoxicillin (AX) and challenged with a range of penicillin-human serum albumin (HSA) conjugates. Mast cells sensitized with IgE anti-BP degranulated in response to BP-HSA, but not to AX-HSA or ampicillin(AMP)-HSA, whereas mast cells sensitized with IgE anti-AX responded to AX-HSA but not to BP-HSA or AMP-HSA. Because BP, AX and AMP differ chemically only in the structure of their side chain, these results show that this part of the drug molecule is essential for recognition by IgE antibody. Unexpectedly, although IgE-sensitized mast cells responded to only one penicillin in protein-conjugated form, antigen-induced degranulation was inhibited by the monomeric derivative of more than one penicillin. Furthermore, antigen activation of IgE-sensitized cells was inhibited, although less potently, by haptens representative of the specific penicillin side chain or the binuclear portion of the drug molecule. These patterns of recognition and hapten inhibition were also seen in solid-phase enzyme-linked immunosorbent assay (ELISA), although all haptenic inhibitors were approximately 100 times less potent in the ELISA compared to the mast cell assay. To explain these findings we propose a model in which IgE binding to penicillin-protein antigen is dependent on recognition of two distinct epitopes on the drug molecule: the first comprising the side chain, and the second comprising the binuclear portion plus the proximal region of the side chain. This two-site hypothesis provides a generally applicable model of antibody recognition of penicillins and provides a rational basis for understanding the specificity and cross-reactivity of IgE-mediated allergic reactions to penicillins.     

A study of the heterogeneity of anti-dinitrophenyl (DNP) antibodies using inhibition of bacteriophage neutralization

The dinitrophenyl (DNP) determinant was conjugated to T2 bacteriophage to yield conjugated phage (DNP-phage). Plaque formation by the DNP-phage was neutralized by rabbit anti-DNP antibodies. This neutralization was inhibited by incubating the antibody first with free hapten and subsequently with the DNP-phage. Both DNP-lysine and dinitro-aniline (DN-aniline) inhibited the neutralization of plaque formation. With antibodies obtained 8 days after immunization there was little difference between the inhibitory capacity of DNP-lysine and that of DN-aniline. However, with antibodies obtained 3 weeks after primary immunization, the inhibitory capacity of DNP-lysine was approximately three times greater than that of DN-aniline. The difference between the inhibitory capacity of the two haptens was further increased with respect to antibodies obtained 8 weeks after primary and 4 weeks after secondary immunization. With the latter two preparations of antibodies, DNP-lysine was approximately ten time more effective than DN-aniline. With antibody preparations from all but the earliest bleedings, the difference between the neutralization inhibitory capacity of DNP-lysine and DN-aniline depended on antibody concentration: at lower concentrations of antibodies the difference between the two inhibitors was at a minimum and increased as the antibody concentration increased. This variation in the relative capacity of the two haptens to inhibit neutralization is attributed to the heterogeneity of the anti-DNP antibodies.     

Immunoglobulin diversity in the phylogenetically primitive shark heterodontus francisci: Comparison of fine specificity in hapten binding by antibody to p-azobenzenearsonate

Neutralization and hapten inhibition of neutralization of azobenzenearsonate-(ABA) haptenated bacteriophage by antibody has been used to characterize the response of Heterodontus francisci to immunization with a Brucella abortus—azobenzenearsonate conjugate. Heterodontus appear to have little background antibody to ABA. The liter at 6 months postimmunization is significantly less than that of a rabbit (IgM) anti-ABA; however, the effectiveness of the inhibition of neutralization for a series of hapten analogs is the same for Heterodontus and rabbit antibody. For one hapten pair, the estimated discriminating capacity of Heterodontus antibody was approximately one-tenth that of rabbit antibody. The findings are consistent with the production in Heterodontus of a low affinity antibody population which exhibits limited capacity for specific recognition.     

The Effect of Lipopolysaccharide on the Primary Immune Response to the Hapten NNP

We have studied the effects of lipopolysaccharide (LPS) on the primary in vivo immune response to the hapten (4-hydroxy-3,5-dinitrophenyl)acetyl (NNP), with special reference to the avidity and affinity of the early appearing 19S and antibodies Comparisons were made of the immune response to NNP in groups of mice given either antigen alone. LPS alone, or antigen plus LPS The avidity of antibodies induced by LPS plus antigen was similar to that found after injection of antigen alone, in spite of the fact that the antibodies were more numerous However, when comparing the avidity of antibodies produced in animals given only LPS with those given LPS plus antigen, the latter group was often found to have fewer low-avidity 19S-antibody-producing cells The affinity of 7S antibodies was also similar in the two groups given antigen or antigen plus LPS Kinetic studies of the effect of LPS on the primary immune response to NNP showed that synergy was observed only before or after the peak response in groups given antigen alone It is concluded that LPS under synergy conditions acts preferentially on specific antigen-sensitive cells which are distinct from those that are activated to polyclonal antibody synthesis by LPS alone. Possible mechanisms for the adjuvant effect of LPS are discussed     

Regulation of anti-hapten antibody response by chemically modified carrier antigen preferentially provoking delayed-type hypersensitivity: I. Possible T—T cell interaction in the suppression of antibody response

The i.p. immunization with chemically modified antigen (dodecanoyl-bovine serum albumin, d-BSA) emulsified in Freund's incomplete adjuvant (FIA) of CBA mice provoked delayed-type hypersensitivity (DTH), but not any detectable formation of antibody to the original antigen (BSA). Furthermore, it was found that immunization with d-BSA could generate T cells capable of inhibiting the antibody response to hapten on BSA, and the immunosuppressive effects of these T cells were presumably not due to direct action on hapten-primed and antibody producing B cells. These results were obtained from the following experiments: (1) anti-hapten antibody response to dinitrophenylated-BSA (DNP-BSA) was inhibited when the mice had been primed previously with d-BSA in FIA. This inhibition was regulated by the specificity of the carrier, since the mice treated with d-BSA did not inhibit the anti-DNP antibody response after the immunization with DNP-heterologous carrier, i.e. DNP-keyhole limpet haemocyanin (DNP-KLH). (2) The passive transfer of spleen cells, which had been obtained from donors primed with d-BSA in FIA, inhibited the primary anti-DNP antibody response of syngeneic mice after immunization with DNP BSA. (3) Injection of d-BSA-primed spleen cells suppressed an adoptive anti-DNP antibody response in mice which had been irradiated and had previously had their immunocompetence reconstituted by the cell transfers with both DNP-primed and BSA-primed spleen cells. This in vivo immunosuppressive effect of d-BSA-primed spleen cells did not act on hapten-primed B cells, since d-BSA-primed spleen cells could not suppress the adoptive secondary antibody response reconstituted by DNP-primed cells and bacterial alpha-amylase (BαA)-primed cells. This finding suggests that a T—T cell interaction exists for the suppression of the anti-DNP antibody response to DNP-BSA by d-BSA-primed cells.     

The effect of lipopolysaccharide on the primary immune response to the hapten NNP.

We have studied the effects of lipopolysaccharide (LPS) on the primary in vivo immune response to the hapten (4-hydroxy-3,5-dinitrophenyl)acetyl (NNP), with special reference to the avidity and affinity of the early appearing 19S and 7S antibodies. Comparisons were made of the immune response to NNP in groups of mice given either antigen alone, LPS alone, or antigen plus LPS. The avidity of antibodies induced by LPS plus antigen were similar to that found after injection of antigen alone, in spite of the fact that the antibodies were more numerous. However, when comparing the avidity of antibodies produced in animals given only LPS with those given LPS plus antigen, the latter group was often found to have fewer low-avidity 19S-antibody-producing cells. The affinity of 7S antibodies was also similar in the two groups given antigen or antigen plus LPS. Kinetic studies of the effect of LPS on the primary immune response to NNP showed that synergy was observed only before or after the peak response in groups given antigen alone. It is concluded that LPS under synergy conditions acts preferentially on specific antigen-sensitive cells, which are distinct from those that are activated to polyclonal antibody synthesis by LPS alone. Possible mechanisms for the adjuvant effect of LPS are discussed.     

Affinity maturation of anti-(4-hydroxy-3-nitrophenyl)acetyl antibodies accompanies a modulation of antigen specificity

Anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies bearing λ1 chains are known to possess fine specificity, referred to as heterocliticity, which causes these antibodies to bind to hapten analogues such as (4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NIP) and (4-hydroxy-3,5-dinitrophenyl)acetyl (NNP) with higher affinity than to the autologous hapten, NP. They also show preferential binding to the phenolate form of hapten than to the phenolic form. We address here the question of whether affinity maturation accompanies in the fine specificity of these antibodies by analyzing the interaction between NP₁-, NIP₁-, or NNP₁-hen egg lysozyme and anti-NP antibodies that possess different association constants to NP using a surface plasmon resonance biosensor. We measured interactions at various pH values and found that heterocliticity as well as preferential binding to the phenolate form of hapten were most prominent in a germline antibody having immature affinity and that fine specificity becomes less evident, i.e., anti-NP antibodies become more specific to the immunizing antigen, NP during the process of affinity maturation.     

Avidities of hapten-specific antibodies when the responses are modulated by anti-carrier antibodies.

Either alone or in combination with antibodies having specificity for the carrier erythrocyte, TNP-ORBC were injected i.p. into CBA/J mice. Five days later, their spleens were removed and evaluated for TNP-specific PFC. The spleens from animals receiving 19S antibody (IgM) with carrier specificity displayed 3-4-fold more direct and indirect hapten-specific PFC than control animals receiving the TNP-erythrocyte conjugate only. Animals receiving 7S antibodies (IgG) with carrier specificity displayed very little change in their direct PFC response to the hapten. However, the indirect response to the hapten was suppressed as much as 16-fold by carrier specific IgG. Evaluation by haptenic inhibition of the relative avidity of the antibodies being secreted by these cells revealed the following: the relative avidity of antibodies secreted by indirect PFC in the spleens of animals receiving TNP-ORBC only was approximately 20-fold higher than antibodies secreted by the direct PFC. The 3-4-fold potentiation of the hapten-specific PFC response by carrier-specific IgM antibody did not result in a change in relative avidity of direct or indirect PFC. IgG with carrier specificity did not change the relative avidity of the antibodies secreted by direct PFC having specificity for the hapten. However, evaluation of the remaining PFC in spleens from animals whose indirect hapten-specific PFC response had been suppressed by carrier-specific IgG revealed that the remaining PFC had a lower avidity than indirect PFC from animals receiving TNP-ORBC only. In other words, carrier-specific IgG selectively induced suppression of high avidity hapten-specific IgG antibody secreting cells.     

Determination by ellipsometry of the affinity of monoclonal antibodies

The reaction between monoclonal antibodies and surface-immobilised hapten was studied by ellipsometry, a method allowing absolute measurement of the surface concentration of proteins. Monoclonal antibodies against 2-phenyloxazolone were used and their affinity for the antigen in solution was determined by calculations of the equilibrium constant from data obtained by measuring fluorescence quenching of the hapten due to antibody binding. The binding rate of antibody to surface-immobilised hapten and the dissociation rate of the complex were measured by ellipsometry. The equilibrium constant of the heterogeneous antigen-antibody reaction was determined by a Scatchard plot. The affinity of the antibodies for the antigen was found to be higher in the heterogeneous than in the homogeneous reaction by a factor which varied between different monoclonal antibodies.     

Suppression of reagin synthesis in rabbits by passively administered antibody

The formation of rabbit antibodies, capable of sensitizing homologous skin, (reagins), was completely inhibited by passive administration of serum containing large quantities of 7S antibody 24 hours before or after antigen injection. No evident effect on reagin formation was noted when passive antibody was administered 8 days after antigen injection although some suppression of agglutinating antibody synthesis was observed. In rabbits not treated with passive antibody the injection of haemocyanin resulted in the formation of reagins reaching maximum serum concentrations 1 and 3 weeks following antigen injection. Both the `early' and `late' reagins persisted for a long time in the skin of injected rabbits, they appeared to have similar molecular size and both were devoid of PCA activity when injected into decomplemented rabbits. There was some indication that the `early' reagins may be more heat-labile than the `late' ones. A secondary reagin response was obtained in several animals which had shown a primary reagin response, but not in rabbits with inhibited primary response. The reagins formed in response to secondary antigen stimulation disappeared rapidly from the circulation, simultaneously with the rise in agglutinating antibody titres.

The possible implications of the findings for the immunological treatment of allergic disorders is discussed.

     

Maturation of the antibody response to a protein-coupled form of the organophosphorus toxin soman.

We have analysed the serum antibody response of BALB/c mice to the organophosphorus toxin soman coupled to the protein carrier keyhole limpet haemocyanin (So-KLH) and compared the specificity of the serum antibodies to that of hybridomas described previously. The relative inhibitory capacities of various soman analogues for serum antibodies correlated with those for the monoclonal antibodies. Our results also demonstrate that immune memory to this organophosphorus hapten is stable for greater than 1 year. Interestingly, maturation of the serum antibody response is accompanied by fine specificity changes resulting in increased binding to soman-protein conjugates but not in significant changes in binding to free hapten analogues of soman. This finding suggests that contributions made by the protein carrier or bridge structure, including those made by amino acid side chains involved in the linkage, may play a significant role in the maturation process of antibodies recognizing protein-coupled organophosphorus haptens such as So-KLH. Structurally related but charge-dissimilar organophosphate haptens such as nitrophenylphosphocholine were poorly recognized, even when conjugated to protein with the same diazophenyl linkage used to conjugate soman. This is consistent with maintenance of high specificity in the memory immune response to soman-coupled protein.     

125I-labeled protein A as a general tracer in immunoassay: suitability of goat and sheep antibodies

The immunoassay method in which 125I-labeled staphylococcal Protein A ([125I]PA) serves as a general tracer has been extended to include goat and sheep IgG antibodies. Goat and sheep IgG normally do not react significantly with PA. However, once IgG antibody is bound to immobilized antigen or hapten, binding of [125I]PA is enhanced markedly. Binding efficiencies of [125I]PA to immune complexed goat anti-human IgM, human IgE, methotrexate and sheep anti-IgE were determined and compared quantitatively to rabbit IgG with the corresponding specificity. Immunoassays were developed based on the inhibition of [125I]PA binding as a measure of antibody inhibition by fluid-phase homologous ligand. In terms of sensitivity and specificity, assays using goat and sheep antibodies were comparable to assays developed using rabbit IgG. Goat antibody to the monovalent hapten methotrexate behaved anomalously: for each concentration of IgG tested, there was an optimal amount of methotrexate beads that gave maximum binding of [125I]PA. In the other immune systems, for each antibody concentration maximum binding of tracer was a function only of the amount of immobilized antigen added. In contrast to the results obtained with solid-phase antigen, solutions containing antibody and amounts of antigen ranging from large antigen excess to antibody excess to antibody excess failed to react significantly with PA or [125I]PA.     

Antigenic specificities of delayed hypersensitivity in mice to dinitrophenylated proteins

Humoral antibodies capable of suppressing induction of delayed hypersensitivity to dinitrophenylated proteins in mice were tested for specific absorbability onto chemically insolubilized antigen and for the antigenic determinant specificities of their immunosuppressive (i.e. contrasensitizing) effects. The activity of an antiserum could be completely removed by absorption with homologous antigen, and it could be recovered by dissociating the absorbed antibodies at low pH and high salt concentration. The immunosuppressive antibodies therefore are specific for determinants on the native antigen, and non-antibody serum constituents are non-essential. By selective immunoabsorptions and elutions, antibodies specific for carrier protein, for dinitrophenyl hapten, and for new determinants unique to the hapten—protein complexes were prepared and were compared with unfractionated antiserum for contrasensitizing activity. Nearly all activity could be accounted for by the anti-hapten antibodies, although anti-carrier antibodies also had some. Despite this evidence that immunosuppressiveness was hapten-specific, the delayed hypersensitivity being suppressed by these antibodies was shown to be directed against hapten—carrier complexes or against carrier but not against hapten alone. Hence, humoral antibodies against a portion of an antigen molecule can suppress induction of delayed hypersensitivity specific for other sometimes unrelated parts of this same molecule, and it is possible to induce antibody-mediated tolerance to a multi-determinant antigen with an antibody response against just a part of the antigen. The theoretical and practical implications of these findings are discussed. Notable theoretically is the observation that although determinant specificities of humoral antibody and delayed hypersensitivity responses to one antigen usually differ, the former can regulate the latter for a given species of antigen molecule. Most important practically is the implication that control over development of delayed hypersensitivities and cell-mediated immunologic reactions may be easier than hitherto anticipated, because humoral antibodies to only a portion of an antigen molecule will suffice to control sensitization to the entire molecule.