Enzyme-linked immunosorbent assay for titration of Haemophilus influenzae capsular and O antigen antibodies

Abstract

The enzyme-linked immunosorbent assay (ELISA) was elaborated for the detection of immunoglobulin M (IgM) and IgG antibodies against capsular and O antigens of Haemophilus influenzae. Purified capsular polysaccharide and lipopolysaccharide were used as antigens, with optimal coating concentrations being about 50 and 100 micrograms/ml, respectively. The antibody content was expressed as the highest serum dilution (-log10) showing an absorbance of 0.2 above the background level. The titers of hyperimmune sera (reference sera) ranged between 5 and 7 -log10. The sensitivity of the method was about 80 ng/ml with regard to anticapsular antibodies and 3 to 5 ng/ml with regard to anti-lipopolysaccharide antibodies. For detection of antibodies against capsular polysaccharide in sera obtained after primary immunization, ELISA was about 100-fold more sensitive than the indirect hemagglutination assay, whereas in hyperimmune sera, ELISA was about 10-fold more sensitive than the indirect hemagglutination assay. The sensitivity of ELISA for detecting anticapsular antibodies after primary and booster immunizations was 50-fold higher than that of the bactericidal assay using capsulated bacteria, whereas the sensitivity of the two methods was the same when hyperimmune sera were tested. ELISA performed with lipopolysaccharide as the antigen was about 50- and 150-fold more sensitive than the complement fixation and bactericidal assays tested with noncapsulated variants after primary injection and hyperimmunization, respectively.