Evaluation of previously assigned antibody concentrations in pneumococcal polysaccharide reference serum 89SF by the method of cross-standardization

Abstract

An enzyme-linked immunosorbent assay (ELISA) and the antibody concentrations assigned to different pneumococcal capsular polysaccharide types were used to estimate concentrations of antibody to additional pneumococcal types in reference serum 89SF and to confirm assigned antibody values. This was possible because the slopes of curves of antibody binding to all polysaccharide types evaluated (1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F) were similar. The point estimates for total anti-pneumococcal antibody and immunoglobulin G (IgG) antibody determined by cross-standardization by an ELISA based on use of methylated human serum albumin (mHSA) to improve the efficiency of polysaccharide binding to the ELISA plate differed by less than 40% from those reported by Quataert et al. (Clin. Diagn. Lab. Immunol. 2:590-597, 1995) for types 1, 4, 6B, 7F, 9V, 14, 18C, and 23F. However, large differences were found between the assigned values and those obtained by our mHSA ELISA for types 3 and 19F. The mHSA ELISA and the direct polysaccharide coat ELISA may not measure antibodies to the same epitopes on polysaccharides of types 3 and 19F. The functional importance of these different antibody specificities is being investigated. We have thus confirmed the assigned IgG antibody values for most types by a different method and have extended antibody assignments to several additional types.

FIG. 1

Optimization of ELISA plate coating conditions by checkerboard titration of PS and mHSA concentrations for pneumococcal types 19F and 23F. Concentrations of 0, 1.0, 2.5, and 5.0 μg of mHSA per ml comixed with each of four different PS concentrations were used. Serum 89SF was used at a dilution of 1:400. Absorbance values were read 20 to 40 min after addition of the substrate and were normalized to the values that would have occurred at 100 min.

FIG. 2

Comparability of slopes for IgG antibodies with those for PS types used for cross-standardization. Reference serum 89SF was diluted beginning with 1:200 for each of the eight polysaccharides and used in the mHSA comix method.

FIG. 3

Comparison of two methods for blocking of C PS antibodies. Reference serum 89SF was used against type 19F PS without C PS absorption (solid line), preadsorbed with 50 μg of Danish C PS per ml (dotted line), without preabsorption but with 1 μg of C PS per ml added to the serum dilution buffer (dashed line), or both with preadsorption and with C PS added to the serum dilution buffer (dashed and dotted line).

FIG. 4

Specificity of the mHSA comix method for measurement of type 18C antibodies in reference serum 89SF.

FIG. 5

Effect of PS coating conditions on the specificity of IgG antibodies to the type 3 pneumococcal PS in reference serum 89SF. The serum was used at a dilution of 1:200 and was examined for type specificity following attachment of type 3 PS to the plate by one of the following three methods: precoating of the ELISA plate with 5 μg of mHSA per ml for 6 h and then addition of 5 μg of PS per ml (A), comixing of 1 μg of mHSA per ml with 5 μg of PS per ml (B), and direct coating of 5 μg of PS per ml without mHSA (C). In each method, the antibodies were competitively inhibited by type 3 PS (solid line), type 1 PS (dotted line), type 2 PS (dashed and dotted line), or type 4 PS (dashed line).