A quantitative ELISA for antigen-specific IgG subclasses using equivalence dilutions of anti-kappa and anti-subclass specific secondary reagents. Application to the study of the murine immune response against the capsular polysaccharide of Neisseria meningitidis serogroup B.
We have developed an enzyme-linked immunosorbent assay (ELISA) to measure murine antigen-specific IgG antibodies of defined subclass using precalibrated equivalence dilutions of anti-kappa (in the standard) and each anti-IgG subclass-specific polyclonal secondary antibody (in the test sample). The calibration of secondary reagents could be carried out easily with a set of monoclonal antibodies (MoAbs) specific for all IgG subclasses. These MoAbs do not require purification or standardization. In addition the MoAbs can be of different antigenic specificity. Once the equivalence dilutions have been determined, they can be applied in a quantitative ELISA using the same antigen in the standard and sample, and using only one IgG subclass standard for the determination of all the IgG subclasses. The method is easy to standardize for many antigenic systems. It is particularly useful when the only standard available is one standardized MoAb of the appropriate specificity, and it could be adapted to use with standard polyclonal antibodies having a known content of total antigen-specific IgG bearing kappa chains but unknown IgG subclass composition. The use of this method to quantitate IgG specific for the capsular polysaccharide of Neisseria meningitidis serogroup B (CpsB) gave highly reproducible measures with an interbatch CV of 5-6% similar for all IgG subclasses and low detection limits ranging from 0.3 ng/well for IgG3 to 0.8 ng/well for IgG2a. The IgG subclass response observed after immunization with live meningococci was mainly IgG2a (74%) and IgG2b (18%). Hyperimmunization modified this IgG distribution to one of mainly IgG3 (62%) and IgG1 (28%) which was maintained in the response to a single immunization 4 weeks later, possibly indicating the generation of resting B cells during continuous stimulation.
Colino
J
J
Immunology Service (C.N.M.V.I.S.), Instituto de Salud Carlos III, Madrid, Spain.
Diez
M
M
Outschoorn
I
I
D000818:Animals; D000888:Antibodies, Anti-Idiotypic; D000907:Antibodies, Bacterial / Q000032:analysis* / Q000145:classification / Q000276:immunology; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000942:Antigens, Bacterial; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D007074:Immunoglobulin G / Q000032:analysis* / Q000145:classification / Q000276:immunology; D007132:Immunoglobulin Isotypes / Q000032:analysis / Q000276:immunology; D007145:Immunoglobulin kappa-Chains / Q000032:analysis / Q000276:immunology; D007202:Indicators and Reagents; D051379:Mice; D008807:Mice, Inbred BALB C; D009345:Neisseria meningitidis / Q000145:classification / Q000276:immunology*; D011135:Polysaccharides, Bacterial / Q000276:immunology*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012703:Serotyping
D000888:Antibodies, Anti-Idiotypic; D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D007074:Immunoglobulin G; D007132:Immunoglobulin Isotypes; D007145:Immunoglobulin kappa-Chains; D007202:Indicators and Reagents; D011135:Polysaccharides, Bacterial