Binding of a mouse monoclonal IgE (anti-DNP) antibody to radio-derivatized polystyrene-DNP complexes

Polystyrene (PS) labware products (microtiter plates, test tubes, etc.) show radiation (gamma) dose-dependent carbodiimide (EDC)-mediated uptake of carboxyl-containing molecules such as DNP-[3H]Gly. The presence of water during irradiation increases, and oxygen decreases the coupling capacity of irradiated PS. The EDC-mediated attachment of DNP-Gly to PS is stable because it resists prolonged exposure to acids at low or high salt concentrations; the bond is sensitive to bases and oxidizing agents. Commercially available radiation-sterilized PS labware products also show elevated EDC-mediated uptake of DNP-[3H]Gly. Nonirradiated PS products or non-PS materials in these studies were inactive. Commercially irradiated PS microtiter plates were coated with DNP-amino acids in an EDC-mediated reaction, and the binding of a radiolabeled mouse monoclonal IgE (aDNP) to DNP-coated plates was studied. 1) The minimal ligand (DNP-Gly) and reagent (EDC) concentrations for plate coating to reach saturating antibody binding were found to be 0.2 mM and 0.2 mg/ml, respectively. High coating densities were suboptimal for antibody binding; 2) five to 60 min of coupling times yielded optimal coating densities; 3) The binding of antibody to DNP-Gly-coated plates reached half-maximal levels in approximately 40 min; 4) Dissociation of antibody from DNP-Gly and DNP-Ser-coated plates was very slow. Intra- and interassay coefficients of variation of antibody binding to plates coated with DNP-Gly under optimal conditions were generally below 3%. We conclude that the PS-bound DNP produced by this method is recognizable by anti-DNP antibodies. The optical quality of PS is not affected by radio-derivatization, and the ligand-coated plates obtained by these methods are suitable for colorimetric assays. All brands of heavily irradiated PS examined were suitable carriers for EDC-mediated coupling of DNP-aminoacids.     

Numbers and avidity of anti-DNP antibody plaques in different inbred mouse strains

Spleen cells of mice from eight inbred strains and three F₁ hybrids, undergoing a secondary immune response to dinitrophenylated keyhole limpet hemocyanin (DNP-KLH), were examined for numbers of indirect DNP-specific plaque forming cells (PFC) as well as avidity of anti-DNP antibodies. The results indicated that the magnitude of the immune response is under genetic control. Differences in average avidity and heterogeneity of avidity were found among different mouse strains, suggesting genetic control of these parameters. However, no simple pattern of inheritance for these characteristics emerged from the study.     

Immune responses to hapten--lipopolysaccharide conjugates. 1. Modulation of in vivo antibody responses to the polysaccharide

The immune responses of mice to various lipopolysaccharides (LPS) and hapten-LPS conjugates were compared. We found that some strains of mice ($\text{A}\text{J}$ and BALB/c) produced equivalent amounts of anti-LPS antibody after the injection of either LPS or hapten-LPS conjugates. In contrast, however, other strains of mice (C57BL/6J, C3H/St, DBA/1J, DBA/2J, and Swiss) produced fewer anti-LPS-antibody-secreting cells after stimulation with hapten-LPS conjugates than did mice injected with unsubstituted LPS. The covalent coupling of hapten to LPS changed neither the mitogenic capacity nor the antigenicity of the LPS. The differences in the magnitude of antibody responses to hapten-LPS and LPS in these latter strains of mice occurred in the absence of mature T lymphocytes and was restricted to the primary immune response. Furthermore, these differential responder mice (C57BL/6J) did produce anti-LPS antibody when primed with LPS before challenge with the hapten-LPS conjugate. These data are discussed with respect to both the modulatory capacity of the hapten-LPS in the regulation of the primary immune response to LPS and the biochemical and structural requirements of the hapten-LPS conjugate for immunogenicity.     

Thymus-dependent network responses to a monoclonal cross-reactive antiidiotypic antibody.

BALB/c mice were inoculated i.p. with a cross-reactive anti-Idiotypic mAb (designated FD5-1) in the absence of Ag or adjuvants. Injection with unmodified FD5-1 resulted in the induction of serum antibodies reactive with both FD5-1 (Ab3) and the hapten DNP (Ab1'). Endpoint titers of the Ab3 response showed an increase in serum IgM, which was dose-responsive to both the number of injections and the amount of FD5-1 antibody injected. The serum IgM Ab3 response was found to be thymus dependent and idiotypically specific for FD5-1. Athymic mice injected with FD5-1 were unable to produce a serum IgM Ab3 response, whereas their euthymic littermates produced strong Ab3 responses. Serum Ab3 responses and Ab1' were detectable only in the IgM isotype; no specific IgG responses were observed. Indeed, IgG recognized by FD5-1 appeared to be suppressed by FD5-1. Injection of mice with FD5-1 modulated serum IgM responses to DNP, (4-hydroxy-3-nitrophenyl)acetyl (NP), 4-ethoxymethylene-2-phenyloxazol-5-one (OX), phosphorylcholine (PC), and alpha 1,3-dextran (DEX) in a thymus-dependent manner. FD5-1 injection induced IgM responses against DNP, (4-hydroxy-3-nitrophenyl)acetyl (NP), 4-ethoxymethylene-2-phenyloxazol-5-one, and DEX but decreased IgM binding to PC. No detectable Ab1' responses to any of the aforementioned molecules were found when the same sera were probed for IgG. The specificity of serum Ab1' from FD5-1-injected mice was evaluated by antigenic inhibition. Binding of serum Ab1' to DNP-BSA was inhibitable by DNP-lysine, whereas equivalent concentrations of lysine alone had no inhibitory effect. The antigenic specificity of IgM from normal serum binding to PC-BSA was demonstrated by inhibition with free PC, and the binding of Ab1' from FD5-1-injected mice to DEX-coated plates was shown to be inhibitable by DEX. We have described in vivo network perturbation in adult BALB/c mice injected with anti-Id antibody in the absence of Ag or adjuvants. Our findings show that injection of the cross-reactive anti-Id FD5-1 can induce thymus-dependent Ag-specific responses. In two systems where FD5-1 functions as an anti-anti-anti-Id antibody (PC and DEX), thymus-dependent responses were also observed. FD5-1 injection suppressed antibodies binding to PC, whereas DEX-specific responses were induced.     

Sexual-stage antibody responses to P. falciparum in endemic populations

Gametocytes and sporogonic stages are responsible for the spread of disease and drug resistance in the population. Sexual stage immunity affects the infectiousness of gametocytes to mosquitoes. Specific antibodies including anti-Pfs48/45 and anti-Pfs230 antibodies are found in individuals with limited prior exposure to malaria. Sexual stage antibodies are rapidly acquired after infection and are relatively prevalent in gametocytaemic individuals. Functional transmission reducing activity (TRA) is found after primary infections and in young children and appears to depend on recent rather than cumulative exposure to gametocytes. Exposure to gametocytes decreases with age most likely as a consequence of the acquisition of asexual-stage immunity that controls asexual parasite density and consequently gametocytaemia. This results in lower exposure to the antigenic load of gametocytes in semi-immune individuals. Since sexual stage immunity is probably short-lived in the absence of gametocytes, we hypothesize that sexual stage immunity will wane, resulting in low antibody and TRA prevalences in clinically semi-immune carriers.     

Thymus-independent Step in the Immune Response to Sheep Erythrocytes

THE nature of the T and B cell interaction in the response to erythrocyte antigens¹ has been an area of intense interest recently. Perhaps because of the influences of molecular biology, there has been a tendency to invoke specific mechanisms such as informational RNA and thymus specific immunoglobulins as mediators of this synergism. But once it was demonstrated that antigen specific receptors of immunoglobulin nature were on the surface of immune competent cells², it was possible to approach the problem more simply; that is from the viewpoint of control of protein synthesis and/or release of proteins from the cell surface. This communication outlines recent evidence concerning the role of non-specific mitogens in the control of antibody precursor (B cells) and antibody secreting cells.     

Serodiagnostic antibody responses to Psoroptes sp. infestations in bighorn sheep

The antibody responses of bighorn sheep (Ovis canadensis) infected with Psoroptes sp. mites were investigated by enzyme linked immunosorbent assay on western blots of P. cuniculi antigens. Serum from 20 Psoroptes sp.-infested bighorn sheep (O. canadensis mexicana, O. canadensis nelsoni, O. canadensis canadensis) from New Mexico, Nevada, California, and Idaho reacted strongly with mite antigens ranging from 12 to 34 kd. Serum from 35 Psoroptes sp.-free bighorn sheep of unknown tick infestation status and from three Psoroptes sp.-free bighorn sheep infested with Dermacentor hunteri ticks did not react with these antigens. Psoroptes sp.-specific antibody responses were present throughout a 16 mo period in one infected bighorn sheep, but were not detectable 8 mo following successful treatment. These results demonstrate that specific serodiagnosis of Psoroptes sp. infestation is feasible in bighorn sheep and suggest that antibody responses are indicative of current or recent infestation.     

Subtype cross-reactive, infection-enhancing antibody responses to influenza A viruses.

Antibody-dependent enhancement of the uptake of influenza A virus by Fc receptor-bearing cells was analyzed by using virus strains of the three human influenza A virus subtypes, A/PR/8/34 (H1N1), A/Japan/305/57 (H2N2), and A/Port Chalmers/1/73 (H3N2). Immune sera obtained from mice following primary infection with an H1N1, H2N2, or H3N2 subtype virus neutralized only virus of the same subtype; however, immune sera augmented the uptake of virus across subtypes. Immune sera from H1N1-infected mice augmented uptake of the homologous (H1N1) and H2N2 viruses. Antisera to the H2N2 virus augmented the uptake of virus of all subtypes (H1N1, H2N2, or H3N2). Antisera to the H3N2 virus augmented the uptake of the homologous (H3N2) and H2N2 viruses. These results show that subtype cross-reactive, nonneutralizing antibodies augment the uptake of influenza A virus strains of different subtypes. Antibodies to neuraminidase may contribute to the enhanced uptake of viruses of a different subtype, because N2-specific monoclonal antibodies augmented the uptake of both A/Japan/305/57 (H2N2) and A/Port Chalmers/1/73 (H3N2) viruses.     

Human antibody responses to Brugia malayi antigens in brugian filariasis.

Human antibody responses to Brugia malayi antigens were studied with sera from a Brugia endemic area in South India. Patients with clinical filariasis had significantly higher IgE and lower IgG4 levels to adult worm antigens than people with asymptomatic microfilaraemia. Intermediate antibody levels were observed in endemic normals. A majority of sera from each clinical group contained IgG antibodies to surface antigens of infective larvae (L3) by IFAT. IgG immunoblot studies did not reveal group differences in L3 antigen recognition. IgE antibodies bound to a subset of antigens bound by IgG. IgE antibodies in sera from clinical filariasis patients preferentially bound to L3 antigens at 200, 97, 68 and 58 kDa compared with sera from microfilaria carriers. These results are consistent with prior studies of antibody responses in filariasis and add new information on the targets of IgG and IgE antibodies to L3 antigens in brugian filariasis.     

Neutralizing antibody responses to HIV-1 infection

Neutralizing antibodies represent an important component of immune control in many viral infections. In HIV-1 infection, almost all individuals develop antibodies capable of neutralizing autologous viruses in vitro; however, the role of these antibodies in vivo still remains unclear. Their absence during the acute phase of infection, when the viral levels are brought under control, suggests they play a minor role in immune control and that cellular immune responses are more critical during this time. However, during chronic infection these antibodies may be important in preventing cell-to-cell spread and they still represent our best hope of providing sterilizing immunity (i.e., prevention of infection) by vaccination. Significant advances over the last few years in understanding the structure of the envelope glycoproteins have renewed interest in the role of neutralizing antibodies and the possibility that immunogens capable of stimulating a neutralizing antibody response can be developed.     

Macrophage-mediated suppression of immune responses in Toxoplasma-infected mice. III. Suppression of antibody responses to parasite itself

In acute Toxoplasma infection, anti-sheep erythrocytes (SRBC) antibody responses were strongly suppressed in the infected C57BL/6 mice, and the mice produced low titers of only 2-mercaptoethanol (2-ME)-sensitive antibodies but not 2-ME-resistant antibodies. By contrast, the infected BALB/c mice produced much higher titers of both 2-ME-sensitive and -resistant anti-SRBC antibodies than the infected C57BL/6 mice. In anti-Toxoplasma antibody responses, the 2-ME-resistant antibody titers were significantly lower in the infected C57BL/6 mice than in the BALB/c mice in the early phase of infection, suggesting that the suppressive effect of Toxoplasma infection affects antibody responses to Toxoplasma itself as well as to the unrelated antigen, SRBC. A histological study revealed that in the infected C57BL/6 mice, a large number of acid phosphatase-positive, macrophage-like cells infiltrated into the follicles of their spleens, and an involution of follicles occurred in the acute phase of infection. This histological change was not observed in the infected BALB/c mice. The infected C57BL/6 mice, which had the suppressed anti-Toxoplasma antibody responses, made five times as many as cysts in their brains as compared with the BALB/c mice at the fifth week of infection.     

Thymus-independent induction and antigen-dependent recovery of idiotype-specific suppression

BALB/c nude (nu/nu) mice and euthymic (nu/+) littermates were treated as neonates with anti-T15 antibody and challenged at various ages with either a thymus-independent, PC-Brucella abortus (PC-BA), or thymus-dependent, PC-keyhole limpet hemocyanin (PC-KLH), form of phosphorylcholine (PC). Nu/nu mice challenged with PC-KLH received KLH-primed splenic T cells prior to immunization. Neither neonatally anti-idiotype-treated nu/+ nor nu/nu mice responded with the production of T15-positive anti-PC antibodies after challenge with either form of PC antigen. It is concluded that neither induction nor maintenance of a state of T15-specific suppression requires thymus-matured T cells. Recovery of anti-PC responsiveness in suppressed nu/+ or nu/nu mice was similar and was found to be related to the form of antigen used to elicit the response. Immunization with PC-KLH revealed a long-lasting unresponsiveness (up to 16 weeks). In contrast, immunization with PC-BA elicited a full anti-PC response as early as at 6.5 weeks of age.     

Maternal antibody blocks humoral but not T cell responses to BVDV.

Bovine viral diarrhoea virus (BVDV) contributes significantly to health-related economic losses in the beef and dairy industry. Antibodies of maternal origin can be protective against BVDV infection, however, calves with low titres of maternal antibody or that do not receive colostrum may be at risk for acute BVDV infection. Interference by high titres of maternal antibodies prevents the development of an antibody response following vaccination with either a killed or attenuated BVDV vaccine. However, the T cell mediated immune response to BVDV may be generated in the absence of a detectable serum neutralizing antibody response. Two trials were conducted to evaluate the potential to elicit T cell mediated immune responses to BVDV in calves with circulating maternal antibody to BVDV. In the first trial, calves with high levels of circulating maternal antibody to BVDV 1 and BVDV 2 were experimentally infected with BVDV 2 (strain 1373) at two to five weeks of age. The T-cell mediated immune responses of the experimentally infected calves and non-infected calves were monitored monthly until circulating maternal antibody was no longer detectable in either treatment group. Calves experimentally infected with BVDV developed BVDV specific CD4(+), CD8(+), and delta T cell responses while high levels of maternal antibody were circulating. A second challenge with BVDV 2 (strain 1373) was performed in the experimentally infected and control calves once maternal antibody could no longer be detected. Previous exposure to BVDV in the presence of maternal antibody protected calves from clinical signs of acute BVDV infection compared to the control calves. In the second trial, three groups of calves with circulating maternal antibody to BVDV were given either a modified live vaccine (MLV) containing BVDV 1 and BVDV 2, a killed vaccine containing BVDV 1 and BVDV 2, or no vaccine, at seven weeks of age. Serum neutralizing antibody levels and antigen specific T cell responses were monitored for 14 weeks following vaccination. Calves vaccinated with MLV BVDV developed BVDV 1 and BVDV 2 specific CD4(+)T cell responses, and BVDV 2 specific gammadelta T cell responses, in the presence of maternal antibody. Vaccination with killed BVDV did not result in the generation of measurable antigen specific T cell immune responses. In this trial, a second vaccination was performed at 14 weeks to determine whether an anamnestic antibody response could be generated when calves were vaccinated in the presence of maternal antibody. Calves vaccinated with either a MLV or killed BVDV vaccine while they had maternal antibody developed an anamnestic antibody response to BVDV 2 upon subsequent vaccination. The results of these trials indicate that vaccinating young calves against BVD while maternal antibody is present may generate BVDV specific memory T and B cells. The data also demonstrated that seronegative calves with memory T and B cells specific for BVDV may be immune to challenge with virulent BVDV.     

Short synthetic glycopeptides successfully induce antibody responses to carcinoma-associated Tn antigen.

Glycopeptides containing a tumor-associated carbohydrate antigen (mono-, tri- or hexa-Tn antigen) as a B-cell epitope and a CD4+ T-cell epitope (PV: poliovirus or TT: tetanus toxin) were prepared for immunological studies. Several Tn antigen residues [FmocSer/Thr (alpha-GalNAc)-OH] were successively incorporated into the peptide sequence with unprotected carbohydrate groups. The tri- and hexa-Tn glycopeptides were recognized by MLS128, a Tn-specific monoclonal antibody. The position of the tri-Tn motif in the peptide sequence and the peptide backbone itself do not alter its antigenicity. As demonstrated by both ELISA and FACS analysis, the glycopeptides induced high titers of anti-Tn antibodies in mice, in the absence of a carrier molecule. In addition, the generated antibodies recognized the native Tn antigen on cancer cells. The antibody response obtained with a D-(Tn3)-PV glycopeptide containing three alpha-GalNAc-D-serine residues is similar that obtained with the Tn6-PV glycopeptide. These results demonstrate that short synthetic glycopeptides are able to induce anticancer antibody responses.