Effect of polyethylene glycol on the rate of immune complex formation by non-precipitating antibody

A kinetic study formation of large size complexes of non-precipitating pig-Dnp anti-Dnp antibody and multivalent dinitrophenylated serum albumin was performed using light scattering and absorption spectroscopy of the Dnp-group. A very rapid phase of the process resulted in the formation of complexes having molecular weight of about 2 × 10⁶. Further increase of the complex size was much slower. Addition of PEG affected positively the rate of complex growth even in concentrations below 1%. The spectroscopic kinetic curves also showed a rapid and a slow phase, sensitive to the presence of PEG. The character of the kinetic data does not support the simple view that polymers enhance precipitate-formation by the steric exclusion of complexes from the polymer domains. It can be assumed that the interaction of the polymer with the antigen-antibody system consists of a subtle temporary attachment of the polymer to the antibody molecule resulting in a change of the shape and/or flexibility of the antibody molecule, favouring its cross-linking capacity.     

Positive cooperativity in the hapten binding by the VL dimer of protein 315

The hapten binding behavior of the dimer of the variable domains, (V_l)₂, derived from the light chains of protein 315 was investigated by difference absorption spectrophotometric titrations. The stoichiometry of binding of $\text{ε-N-(2,4-}\text{dinitrophenyl)-}\text{l}\text{-lysine}$ (DNPL) and 4-(α-N-alanine)-7-nitrobenz-2-oxa-1,3-diazole is two haptens per (Vl)₂. Positive cooperativity was observed in this binding. Such positive cooperativity has been reported previously for the light chain dimers of protein 315 with their native interchain disulfide bridge preserved or cleaved by reduction and alkylation. The hapten binding was analysed as in the previous cases according to the allosteric model of Monod et al. (1965). This model assumes that the protein exists in two conformations, and the equilibrium between them shifts upon hapten binding. The parameters of the hapten binding and of the allosteric transition of (Vl)₂ are similar to those of L_{2 ncov} showing that the C_l domains play a rather limited role in the behavior of the latter protein. In contrast, these parameters are drastically different from those reported for the L_{2 cov}. This constitutes a further illustration of the importance of the disulfide bond in affecting the protein's conformation and hapten binding.The detailed analysis of the titrations of (V_l)₂ and of L_2nocov with DNPL led to the calculation of the difference absorption spectra between the DNPL complexes formed by each of the two conformers of either protein and the free hapten. The distinct dissimilarity of these two difference spectra illustrates the different environments of the dinitrophenyl ring in the binding sites of each conformer.     

Enhancing effect of C1q on IgG monoclonal antibody binding to hapten

We have previously demonstrated that IgG antibody binding to microfilariae of Dirofilaria immitis increased in the presence of purified C1q. The present study was designed to examine the mechanism of the C1q effect using a system with an antihapten monoclonal antibody (MoAb) and a hapten as an antigen. Microtiter plates were coated with 4-hydroxy-3-nitrophenyl-acetyl (NP)-bovine serum albumin (BSA), and mouse anti-NP MoAb (IgG) was added in the presence of C1q. The amount of IgG which bound to NP-BSA increased with the addition of C1q (p less than 0.01) when the antibody had both specificity to the antigen and ability to fix C1q. The C1q effect, examined using two anti-NP MoAbs with different affinities, was more apparent with the low-affinity antibody (LAMoAb) than with the high-affinity (HAMoAb; percent enhancement of IgG binding was 19 vs. 12%). The C1q effect on LAMoAb binding was doubled when a small amount of HAMoAb was incubated with LAMoAb. The C1q effect on IgG binding might be operative in the early phase of infection, where a small amount of high-affinity antibody and a relatively large amount of low-affinity antibody are produced in the host.     

Chemical basis for diversity in antibody specificity analysed by hapten binding to monoclonal anti-4-hydroxy-3-nitrophenacetyl (NP) immunoglobulins

Anti-4-hydroxy-3-nitrophenacetyl (NP)2 monoclonal antibodies were produced by hybrid cell lines obtained by fusion of NSl myeloma cells with lymphocytes from spleens of C57BL/6 mice immunized with NP conjugated with chicken immunoglobulin (NP-CG). Equilibrium constants of four purified immunoglobulins for N¹²⁵IP-ε-aminocaproate, measured by equilibrium dialysis at 4°C, ranged from 1.0 × 10^?8 to 1.0 × 10^?7M. In order to probe fine-specificity differences, binding constants were measured for a group of structural analogs of NP. This study showed that all four antibodies were heteroclitic and bound the iodo group of NIP-ε-aminocaproate with similar affinities, while the affinity constants for the ε-aminocaproate moiety varied widely. The free energies of binding for iodo group and the side-chain moieties were additive, whereas those for the hydroxy and nitro groups were not. Three of the immunoglobulins bound the aromatic ring system of NIP-ε-aminocaproate similarly, but less effectively than the fourth antibody. The data suggest that anti-NP active sites of diverse specificities were generated on the basis of additive interactions with multiple subsites.     

Effect of glucocorticosteroids on phagosome-lysosome interaction.

The effects of hydrocortisone on fusion of thorotrast-labeled lysosomes with phagosomes containing killed toxoplasmas was studied. Mouse peritoneal macrophages from glucocorticosteroid-treated animals fromed phagolysosomes just as effectively as macrophages from untreated animals (72% thorotrast-positive vacuoles in hydrocortisone-treated animals; 78% in controls). The electron microscopic appearence of macrophages from treated or untreated animals was similar. This observation complements studies which show no change in intracellular microbicidal activity in macrophages treated with glucocorticosteriods.     

Kinetics of antibody binding to solid-phase-immobilised antigen. Effect of diffusion rate limitation and steric interaction

The binding of monoclonal antibodies to surface-immobilised antigen was studied. Antibodies against dinitrophenyl-benzene and O6-ethyl-2'-deoxyguanosine with a known affinity for the antigen were used. The amount of bound antibodies was measured by ellipsometry with an accuracy of +/- 0.15 pmol/cm2, and a sensitivity of 0.11 pmol/cm2. The binding rate of the initial antibody binding could become diffusion rate limited, and the binding rate at surface concentrations above 1 pmol/cm2 was affected by steric interaction between bound antibodies. Bound antibodies did not dissociate when rinsed with saline for up to 20 h, but dissociated in the presence of antigen (0.1 mM). The dissociation rate did not follow any identifiable rate constant. The results are discussed in relation to theoretical models of the kinetics of antigen-antibody reactions at solid-liquid interfaces.     

Effect of macrophage activation on phagocyte-Plasmodium interaction.

We investigated the effect of both immune and normal sera on the binding of free Plasmodium berghei by resident and activated macrophages. Resident macrophages bound plasmodia to a greater extent than did activated macrophages, regardless of treatment. Resident macrophages bound free plasmodia, predominantly trophozoites, in the presence of normal serum by a mechanism inhibited by N-acetylglucosamine and N-acetylmannosamine. Macrophages activated through treatment with Propionibacterium acnes ("Corynebacterium parvum"), on the other hand, did not bind free plasmodia in the presence of normal serum through systems inhibited by N-acetylmannosamine or N-acetylglucosamine. The binding of free plasmodia by activated macrophages was greatest in the presence of immune serum and could be inhibited by immune complexes but not by N-acetylmannosamine or N-acetylglucosamine. These results suggest that a receptor for a carbohydrate component of a normal serum opsonin mediates initial adherence of plasmodial antigen onto resident macrophages, triggering both the immunological cascade and macrophage activation. After activation, the macrophages no longer have the carbohydrate-specific receptor but do have functional Fc receptors which mediate the adherence of immune-serum-opsonized plasmodia.     

Carrier and hapten functions in immune deviation. II. Role of hapten and carrier on the helper functions of the T cells.

The kinetics of haemagglutinating and haemolytic antibody synthesis to the hapten and to the carrier determinants were studied in guinea-pigs injected intravenously with large doses of the carrier, or of the hapten conjugated to an homogous or heterologous protein carrier and subsquently immunized with the hapten-carrier conjugate in Freund's complete adjuvant. The animals treated with the heterologous conjugates exhibited enhanced reactions to the hapten and supressed reactions to the carrier, whereas the animals injected with the homologous conjugate showed depressed reactions to the hapten but unaffected reactions to the carrier. Pretreatment with the carrier alone-seemed to have no effect. These experiments confirm that the hapten determinant must act at the T-cell level but do not exclude the possibility that it could also act at the B-cell level. On the other hand, they allow the dissociation of the different components of the immune response directed against the hapten and carrier determinants of the antigen molecule in immune deviation.     

Carrier and hapten functions in immune deviation. I. Contrary effects of hapten and carrier on cellular and helper functions of T cells.

Carrier and hapten functions have been studied in the immune deviation phenomenon. Delayed hypersensitivity to the carrier and anaphylaxis and Arthus hypersensitivities to the hapten and to the carrier were studied in guinea-pigs injected intravenously with large doses of carrier, homologous and heterologous hapten-carrier conjugates and subsequently immunized with the hapten-carrier conjugate in Freund's complete adjuvant. Pretreatment with DNP-BSA or with HGG were found to modify, in opposite directions, the hypersensitivity reactions induced by DNP-HGG in adjuvant. It is suggested that the hapten and carrier moieties of the antigen molecule might have antagonistic effects on the T cells responsible for cellular immunity as well as on T cells involved in helper functions for B cells.     

Effect of the hapten linking arm on cross-reactivity of sulfonamide-specific antibodies in ELISA

Competitive enzyme-linked immunosorbent assay (ELISA) was used to explore the influence of the linking arm (azo or succinyl) of sulfonamide-protein immunogens on polyclonal antibody (pAb) cross-reactivity to sulfonamide N¹-analogues, N⁴-derivatives, or N⁴-protein conjugates. In addition to the sulfonamide's overall size and N¹-substituent structure, the dominant sulfonamide epitope of the immunogen was defined by the linking arm. Antibodies made from immunizations with azo and succinyl immunogens possessed 12- and 16-fold greater affinity for their N⁴-derivatives, respectively, compared to the underivatized sulfonamide form.     

Mouse monoclonal antibodies at the red cell surface--II. Effect of hapten density on complement fixation and activation

Complement (C)-dependent hemolytic dose-response curves of anti-TNP IgG2b and IgM monoclonal antibodies as a function of TNP density were analyzed: sheep red cells coupled with TNP served as targets. Under conditions when equal numbers of either IgG2b or IgM anti-TNP antibodies were taken up by cells with various TNP densities, both antibodies showed optimal activity at a hapten density of approximately 10(6) TNP/E with regard to C-mediated lysis. These results were similar to those obtained with polyclonal antibodies. The effectiveness of monoclonal antibodies in utilizing guinea pig or mouse C was also investigated. IgM anti-TNP monoclonal antibodies lysed E-TNP in the presence of guinea pig C, but failed to produce lysis in the presence of mouse C. Two monoclonal IgG1 (anti-TNP and anti-SRBC) and an IgG2a anti-TNP antibody failed to produce hemolysis in the presence of guinea pig and mouse C. IgG2b monoclonal antibodies, whether directed against TNP or Forssman antigen, activated guinea pig and, to a lesser extent, mouse C. Finally, monoclonal anti-TNP IgG3 antibodies exhibited a low but measurable hemolytic activity with guinea pig C (10-20 times below that of IgG2b).     

Effect of leukotriene B4 and prostaglandin E2 on the adhesion of lymphocytes to endothelial cells.

The arachidonic acid metabolites leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) may play an important role in inflammation. It is not known whether these mediators influence the binding of lymphocytes to endothelial cells, a process which is important in the extravasation of lymphocytes in inflammatory states. In the present investigation, the effect of LTB4 and PGE2 on the binding interaction between lymphocytes and endothelial cells was examined using a centrifugation cell binding assay. Although LTB4 elicited an aggregation response on human polymorphonuclear leucocytes (PMNL) and enhanced their binding to endothelial cells it had no effect on lymphocyte binding. By contrast, PGE2 caused a dose-dependent inhibition of lymphocyte binding to endothelial cells. The inhibitory effect of PGE2 had a rapid onset but was exhibited only when PGE2 was present continuously during the cell binding assay. Although the mechanism by which PGE2 acts is not clear, it may provide a negative feedback mechanism in regulating the influx of lymphocytes into inflammatory sites.     

Requirement of high-affinity IL-2-IL-2R interaction for T cell anergy induction

Incomplete T cell antigen receptor-mediated signaling induces an unresponsive state known as anergy. Previously, we had shown that anergy can be induced in antigen-primed but not naive T cells. In this report, we found that in vitro primed T cells from IL-2R alpha-deficient mice were resistant to anergy induction in contrast to comparably treated wild-type T cells. This resistance persisted even after proliferation of IL-2R alpha chain-deficient CD4 T cells with high-dose IL-2-IL-2R beta gamma chains interaction. Thus, antigen activation, and/or progression through cell cycle are not sufficient to induce anergy susceptibility in T cells. The high-affinity IL-2-IL-2R interaction appears to play a critical role in this process.     

Studies on the induction of antibody synthesis against sulfanilic acid in rabbits. I. Effect of the number of hapten molecules introduced in homologous protein on antibody synthesis against the hapten and the new antigenic determinants

The present experiments were carried out in order to elucidate further the following three questions:

• (1) Is there any difference in “helper activity” between the diazotized rabbit serum albumin (RSA) molecule and the bovine γ-globulin (BGG) molecule in the induction mechanism of antibody synthesis to sulfanilic acid (Sulf) in rabbits?
• (2) Is there an optimal hapten density on a homologous carrier molecule for induction of and maximum antibody synthesis against sulfanilic acid?
• (3) Does the hapten density affect the expression of, and antibody synthesis against the new antigenic determinants introduced through the haptencoupling reaction?

Carrier antibody titers against diazotized RSA and BGG in the rabbits were significantly different when measured between day 7 and day 30, the BGG titer being higher. Thus, the higher anti-Sulf antibody response observed in rabbits challenged with Sulf₁₀ BGG compared to the anti-Sulf response to Sulf₁₁ RSA can presumably be related to a higher immunogenicity and thereby a more effective “helper” property of the BGG molecule. Anti-Sulf antibody titers of similar magnitude were observed when using Sulf₁₁ -, Sulf₂₃ -, and Sulf₄₀ RSA as immunogens, Sulf₄ RSA elicitating a somewhat lower anti-Sulf response. The anti-Sulf antibodies produced against the different SulfRSA conjugates showed increasing proportions of 19 S antibodies with increasing hapten density. Antibody synthesis against new antigenic determinants was observed when using Sulf_4-, and Sulf₁₁ RSA as immunogens, not observed when using Sulf₂₃, and Sulf₄₀ RSA. This finding was in agreement with the in vitro reactivity of the SulfRSA conjugates against sheep anti-RSA: Sulf₄ and Sulf₁₁ RSA reacted with anti-RSA, Sulf₂₃, and Sulf₄₀ RSA did not. These results are discussed on the basis of the cellular cooperation hypothesis, and multivalent binding of high density conjugates onto antibody-forming precursor cells is proposed as a mechanism alternative to cellular cooperation.