Estimation of group B streptococcus type III polysaccharide-specific antibody concentrations in human sera is antigen dependent

Abstract

The presence of immunoglobulin G (IgG) antibodies against group B streptococcus (GBS) type III polysaccharide (PS) has been correlated with protection against GBS disease. The GBS type III PS is structurally similar to the pneumococcal type 14 PS, differing only in the presence of sialic acid residues. Four different preparations of GBS type III PS were evaluated for their specificity in enzyme-linked immunosorbent assay (ELISA): free PS, free PS mixed with methylated human serum albumin (mHSA), PS conjugated to biotin and PS conjugated to human serum albumin. Three groups of human sera were used to evaluate these PS preparations: sera from recipients of a GBS PS vaccine, sera from women receiving a GBS type III PS-tetanus toxoid conjugate vaccine, and sera from nonimmunized healthy women of childbearing age. Estimated antibody concentrations were different depending on the PS preparation used. Using any of the four preparations, we were able to measure </=0.05 micrograms of IgG antibody to the GBS type III PS per ml. The specificity of the assay was determined by competitive inhibition with homologous and heterologous PS. The pneumococcal type 14 PS did not inhibit binding of antibody to the native GBS type III PS in sera from adults receiving the GBS PS vaccine or in sera from nonimmunized adults (except serum G9). The pneumococcal type 14 PS inhibited 50% in sera from recipients of GBS type III conjugate vaccine and in serum G9 when GBS type III PS conjugated to biotin or to HSA was used as antigen in ELISA. These data show that free GBS type III PS or PS mixed with mHSA is a sensitive and specific antigen for ELISA and that conjugation can alter the antigenic specificity of a PS.

FIG. 1

Structures of GBS type III PS and PN-14 PS repeating units.

FIG. 2

Relative sensitivities of ELISAs with four GBS type III PS preparations as antigens. Symbols: ⧫, free GBS type III PS; ■, GBS type III PS mixed with mHSA; ▴, GBS type III PS conjugated to HSA; ×, GBS type III PS conjugated to biotin. The assays were performed with reference serum 19 (A) and SHRS-III serum (B) based on their previously assigned IgG antibody concentrations.

FIG. 3

Competitive inhibition with reference serum 19. Fivefold-increased PS concentrations from 0.04 to 5 μg/ml were used. Percent inhibition was determined by comparing the absorbances in the presence and absence of the PS inhibitor. III, II, Ia, Ib, and V, PSs from GBS types III, II, Ia, Ib, and V; B, GBS group B antigen; 14, PN-14 PS.

FIG. 4

Competitive inhibition with G9 and SHRS-III sera. A serum dilution corresponding to the upper portion of the linear range of a dilution curve was mixed with 5 μg of GBS type III PS per ml (A) or 5 μg of PN-14 PS per ml (B) and then preincubated at 37°C for 2 h. The rest of the GBS type III ELISA was performed as described in the text. Symbols: ■, free GBS type III PS; ▧, GBS type III PS mixed with mHSA; □, GBS type III PS conjugated to HSA; ░⃞, GBS type III PS conjugated to biotin.

FIG. 5

Results of ELISA with a rabbit PN-14 typing antiserum. Free and conjugate GBS type III PSs and Hib PS were used for coating microtiter plates. The ELISA was performed by using twofold dilutions of the PN-14 antiserum, and the absorbance was extrapolated to 100 min. Symbols: ⧫, free GBS type III PS; ■, GBS type III PS mixed with mHSA; ▴, GBS type III PS conjugated to HSA; ×, GBS type III PS conjugated to biotin; ✻ Hib PS.