The isotypic distribution of IgG antibodies was determined in the serum of mice after infection with a panel of RNA and DNA viruses representative of 11 different genera. The antiviral response induced by all these viruses showed a striking preponderance of the IgG2a subclass whatever the strain of mice tested or the time elapsed after infection. Together with the predominance of IgG1 in antiprotein and of IgG3 in anticarbohydrate response, this IgG2a restriction of antiviral antibodies strongly suggests the existence of highly specific mechanisms for the regulation of individual subclasses in the mouse.
Murine IgG antiviral antibodies elicited by infection have recently been shown to belong predominantly to the IgG2a subclass (1). This isotypic distribution ofantiviral antibodies contrasts sharply with that ofantisoluble protein or anticarbohydrate antibodies, which are usually restricted to the IgG1 and IgG3 subclasses, respectively (1-3). Such an isotypic bias of antiviral antibodies could be due to intrinsic biochemical characteristics of viral antigens or, alternatively, to regulatory mechanisms elicited by the infectious process itself. To address this question, we analyzed the effect of infection on the isotypic pattern of antibodies not directed against the viral antigens. Our results indicate that viruses can influence the subclass distribution of IgG antibodies produced in the course of the infection, irrespective of their specificity. Materials and MethodsMice. CBA/Rij and 129/Sv mice, bred at our institute by Dr. G. Warnier, were maintained in specific pathogen-free conditions and used when 6-10 wk old.Viruses. Infections were performed as described in (1). In addition, K virus (a gift ofDr. W. P. Rowe, Bethesda, MD, No. 3134/KS) and EDIM virus (a gift ofDr. T. H. Flewett, Birmingham, UK) were used . For the absorption experiments, pooled sera from infected mice were diluted 100-fold in PBS (120 mM NACI, 5 mM Na2HPO4, 3 mM KH2PO4, pH 7.2) containing 2% FCS and incubated for 2 h at 4°C with serial doses of purified virus or with buffer only. Viral particles were then removed from sera by centrifugation through a 15% sucrose cushion (35,000 rpm for 90 min in an SW41 rotor, Beckman Instruments, Inc., Palo Alto, CA) and remaining Igs were measured in supernatant by ELISA.Immunoglobulin Determinations. Total IgM and IgA levels were determined by RIA as described previously (4) and total IgG subclass levels were measured by ELISA . For the ELISA, polystyrene plates (Greneir, Nurtingen, FRG) were coated overnight with an anti-mouse IgG3 rat mAb (2E .6, American Type Culture Collection, Rockville, MD) (5 Wg/ml in 0.02 M glycine, 0.03 M NaCl, pH 9.2) or with affinity-purified goat antibodies specific for rabbit IgG, followed by rabbit antibodies specific for mouse IgG subclasses (4). After overnight incubation with sera serially diluted in Tris-buffered saline (10 mM Tris, 10 mM merthiolate, 130
After infection with some viruses and intracellular parasites, antibody production is restricted to IgG2a. We first observed that, whereas live viruses such as lactate dehydrogenase-elevating virus (LDV) or mouse adenovirus induced mostly an IgG2a response, a large proportion of antibodies produced against killed viruses were IgG1. This IgG1 antiviral response was suppressed when live virions were added to inactivated viral particles. These results indicate that the IgG2a preponderance is related to the infectious process itself rather than to the type of antigen involved. Since IFN-gamma is known to stimulate IgG2a production by activated B lymphocytes and to be secreted after infection, we examined the role of this cytokine in the antibody isotypic distribution caused by LDV. Most IgG2a responses were relatively unaffected in mice deficient for the IFN-gamma receptor or treated with anti-IFN-gamma antibody. A similar IFN-gamma-independent IgG2a secretion was observed after infection with the parasites Toxoplasma gondii and Trypanosoma cruzi. However, the IFN-gamma-independent IgG2a production triggered by infection still required the presence of functional T(h) lymphocytes. Therefore, signal(s) other than IFN-gamma secretion may explain the T(h)-dependent isotypic bias in antibody secretion triggered by viruses and parasites.
A direct enzyme-linked immunosorbent assay was developed for the measurement of immunoglobulin M (IgM) antibody to cytomegalovirus (CMV). Wells of microtiter plates were coated with anti-human IgM. Each patient's serum was added at a dilution of 1:100, and IgM from the serum was allowed to react with anti-human IgM. The amount of CMV-specific IgM antibody bound was determined by measuring the intensity of color change after the addition of peroxidaselabeled CMV antigen and substrate. Nuclei of infected cells served as an antigen source. CMV IgM could be detected only in IgM fractions of sera from patients with a recent CMV infection. Rheumatoid factor did not cause false-positive results. No cross-reactions were observed when paired sera from 22 patients with herpes simplex or varicella and single sera from 12 patients with suspected infectious mononucleosis were tested by the direct enzyme-linked immunosorbent assay. Each of 17 patients with a seroconversion for CMV antibody showed CMVspecific IgM antibody. In six of these patients the antibody was detected in the initial serum. The direct enzyme-linked immunosorbent assay for CMV IgM is a specific and sensitive test for the diagnosis of recent CMV infections and possesses distinct advantages over indirect tests. 18 to 72 years of age. Four patients were children 3, 4, 9, and 15 years of age, none of whom had been congenitally infected with CMV. In addition, sera were obtained from 34 patients clinically suspected of having herpes simplex or varicella-zoster infection or infectious mononucleosis. Furthermore, 36 sera with 416
A direct enzyme-linked immunosorbent assay (ELISA) is described that uses horseradish peroxidase-labeled antigen for detection of immunoglobulin M (IgM) and IgA antibodies to toxoplasma. In this assay, polystyrene microtiter plates were sensitized with anti-human IgM or IgA antibody to separate IgM or IgA from other classes of antibody. The presence of IgM or IgA antibodies to toxoplasma (Tox-IgM, Tox-IgA) was then detected by sequential addition of soluble horseradish peroxidase-labeled toxoplasma antigen and substrate. As judged by examining sucrose gradient-fractionated sera, the assay was specific for IgM or IgA classes of antibody. In contrast to the indirect immunofluorescence for IgM antibodies to toxoplasma, no inhibition of IgM reactivity by specific IgG antibodies could be detected. Furthermore, rheumatoid factor did not cause false-positive results. Of 80 single sera with high antibody titer to toxoplasma in indirect immunofluorescence and complement fixation, 40 were positive in the direct ELISA for Tox-IgM, 36 were positive in the double-sandwich ELISA, and only 21 were positive in the indirect immunofluorescence for Tox-IgM when whole serum was used. In the indirect immunofluorescence, another 13 sera became positive after sucrose gradient fractionation. The direct ELISA for IgA antibodies to toxoplasma was positive in 43 sera, of which 39 were positive in the direct ELISA for Tox-IgM. High levels of IgM antibodies were found within 3 months after the onset of symptoms, slowly decreasing thereafter. Tox-IgM may persist for more than 1 year after infection.
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