Measurement of Human Antibodies to Type III Group B Streptococcus

We disagree with the conclusions reached by Bhushan et al. (5) in their study comparing different methods of coating enzyme-linked immunosorbent assay (ELISA) plates for estimation of group B Streptococcus type III (GBS III) polysaccharide-specific antibody concentrations in human serum. We believe that interpretation of the data as suggested in their paper could lead to serious errors in the use of serum antibody concentrations as surrogates of vaccine efficacy. We propose four alternate conclusions that are supported by the data given by Bhushan et al. (5).

(i) ELISAs that used free GBS III polysaccharide or polysaccharide mixed with methylated human serum albumin (mHSA) as a coating antigen failed to detect all of the antibodies reactive with type III polysaccharide in a quantitative precipitin assay. Bhushan et al. found that ELISA methods using GBS III polysaccharide alone or type III polysaccharide mixed with mHSA as coating antigens measured substantially lower concentrations of specific immunoglobulin G (IgG) in a reference serum than did methods using polysaccharide covalently conjugated to HSA or to biotin. However, the specific IgG concentration detected by the conjugated polysaccharide methods in ELISAs agreed very closely with the concentration of specific antibodies in the reference serum (standard human reference serum III [SHRS III]) determined by quantitative precipitin analysis with the purified type III polysaccharide as antigen, while the ELISA methods using unconjugated polysaccharide underestimated the antibody concentration by more than 50%. The data provided in the paper are inadequate to permit the assessment of the relative sensitivities of the free versus conjugated polysaccharide methods, but experiments in our laboratory have shown that assays using free polysaccharide as the coating antigen are less sensitive than those using polysaccharide conjugated to HSA (III-HSA) (6).

(ii) Antibody measurements in the III-HSA assay are highly correlated with antibody measurements in the RABA. The radioactive antigen binding assay (RABA) is an assay in which soluble polysaccharide (unencumbered by attachment to a plastic surface) is allowed to interact in solution with antibodies; the antibody concentration is determined by quantifying the amount of polysaccharide complexed to specific antibodies. The RABA is the gold standard assay because it is the only immunoassay shown to give results that correlate with neonatal susceptibility to GBS III infection (1–3).

To test the validity of the unconjugated polysaccharide ELISA, III-specific IgG in 16 serum specimens from healthy adults was measured by ELISA independently in the laboratory of Bhushan et al. at the Food and Drug Administration (FDA) and by RABA in the laboratory of one of us (C.J.B.). Both the FDA and the Baylor laboratories also measured specific IgG concentrations by the III-HSA ELISA method. Antibody concentrations determined by the RABA and the III-HSA ELISA (irrespective of the laboratory in which the III-HSA ELISA was performed) were in better agreement than those determined by RABA and the free polysaccharide plus mHSA method (unpublished data). These results support the previously published excellent correlation (r = 0.92) between antibody concentrations determined by the III-HSA ELISA and by the RABA (6). Of particular concern is the overestimation of specific antibodies by the free polysaccharide plus mHSA method in the eight serum samples that contained <1.0 μg of specific antibodies per ml according to RABA. In those eight samples, the range of specific IgG concentrations determined by III-HSA ELISA was <0.1 to 0.5 μg/ml at the FDA and <0.05 to 0.47 μg/ml at Baylor. By contrast, the free polysaccharide plus mHSA method measured >1.0 μg of specific IgG per ml (range, 1.0 to 4.2 μg/ml) in each of the samples. Overestimates by the free polysaccharide plus mHSA method are of serious concern because Bhushan et al. propose to use this ELISA to establish the minimum level of maternal antibody that is protective against neonatal infection. Overestimation of specific antibody levels would yield a falsely elevated value for the minimum protective level in such studies.

(iii) The III-HSA ELISA measures antibodies that cross-react with PN-14 polysaccharide but that also bind to GBS III polysaccharide. Bhushan et al. suggest that conjugation of the type III polysaccharide to a protein alters the antigenic specificity of the polysaccharide. Because the type III ELISA methods using conjugated polysaccharide also detected antibodies cross-reactive with the structurally related type 14 pneumococcal (PN-14) polysaccharide, the investigators conclude that the conjugated polysaccharide ELISAs are less specific than assays using free polysaccharide. Antibody cross-reactions between GBS III and PN-14 polysaccharides have been recognized for many years (4, 6–8). We have reported that in some subjects immunization with the purified type III polysaccharide evokes antibodies that cross-react with PN-14 polysaccharide (9). Of importance is that these are truly cross-reacting antibody populations: the GBS III-HSA ELISA measured no increase in specific antibodies in the postimmunization sera of four patients who responded to vaccination with PN-14 polysaccharide (6). Thus, the III-HSA ELISA detects antibodies that bind to type III polysaccharide and cross-react with PN-14, but it does not detect all antibodies that recognize PN-14. The lower sensitivity of the free polysaccharide method accounts for the inability of this method to detect lower-affinity antibodies such as those that cross-react with PN-14 polysaccharide.

(iv) The III-HSA ELISA, but not the free polysaccharide ELISA, has been shown to correlate directly with opsonic activity of serum against GBS III. In addition to the problem of assay sensitivity, it is critical to determine which assay system gives results that most closely reflect the concentration of functionally active and protective antibodies. Specific IgG concentrations measured by the III-HSA method have been shown to correlate with opsonophagocytic killing activity of the antisera to GBS III in sera from subjects immunized either with type III polysaccharide or with polysaccharide conjugated to tetanus toxoid (10). No such relationship with functional activity has been demonstrated for antibody detection by the free polysaccharide method.

On the basis of these data and the results presented in the article by Bhushan et al. (5), neither the free polysaccharide method nor the polysaccharide plus mHSA ELISA method can be considered a valid technique for measurement of naturally acquired GBS III polysaccharide-specific IgG in human serum.