Article Figures & Data
Figures
- 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).
Tables
- Table 1.
Optimal PS and mHSA concentrations for coating of Immulon I ELISA plates by the comix method
Pneumococcal type PS concn (μg/ml) mHSA concn (μg/ml) 1 5.0 2.0 2 2.0 1.0 3 5.0 1.0 4 2.5 1.0 5 5.0 5.0 6B 2.0 5.0 7F 5.0 5.0 9V 2.5 2.5 14 1.0 0.5 18C 5.0 5.0 19F 5.0 5.0 23F 5.0 5.0 - Table 2.
Estimation of concentrations of antibody to individual pneumococcal types in reference serum lot 89SF by cross-standardization by the mHSA comix methoda
Type used as reference standard Concn (μg/ml) of IgG antibody in serum 89SF against the following type PSs: 1 3 4 5 6B 7F 9V 14 18C 19F 23F 1 7.6 5.8 3.5 14.8 5.3 8.3 22.9 6.8 6.7 10.6 3 2.0 1.7 1.1 4.4 1.7 2.6 6.9 2.0 2.1 3.5 4 4.7 5.6 2.6 10.0 4.1 6.1 16.7 4.8 4.9 8.8 5 11.0 13.0 10.1 23.8 9.8 13.8 40.0 11.3 11.7 17.9 6B 8.0 9.7 7.2 4.4 6.9 10.5 28.4 8.2 8.5 13.9 7F 6.2 7.8 5.4 3.5 13.2 8.9 20.9 6.2 6.4 10.6 9V 5.6 7.2 4.9 3.2 12.0 4.7 18.9 5.7 5.8 9.6 14 8.2 10.4 7.2 6.3 17.7 6.9 10.3 8.3 8.5 14.1 18C 5.1 6.6 4.3 3.0 10.9 4.1 7.7 16.5 5.2 8.4 19F 7.3 11.9 7.7 5.1 22.7 8.4 13.6 35.4 11.0 18.8 23F 5.3 7.0 4.5 3.1 10.8 4.3 8.1 17.3 5.4 5.5 Meanb 6.2 7.7 5.5 3.7 12.8 5.2 8.5 20.2 6.5 6.4 10.8 ↵a Data are for one of six assays to estimate IgG antibodies. Similar assays were done to estimate levels of total antibody to each type. Reference antibody values to a given pneumococcal type were used.
↵b Arithmetic means of antibody estimates obtained with individual types with their previously assigned IgG antibody concentrations (14) to generate the reference curve. Estimates obtained with types 3, 5, and 19F as standards were excluded from the mean calculations.
- Table 3.
Comparison of antibody values assigned to the 89SF reference serum with those assigned by cross-standardization by mHSA ELISA
Pneumococcal type Total antibody concn (μg/ml) IgG specific-antibody concn (μg/ml) Assigneda mHSAb Assigneda mHSAb 1 10.7 8.4 6.3 6.2 3 7.9 7.0 2.4 4.0c 4 7.0 8.4 4.1 5.5 5 10.0 4.1c 5.8 3.7 6B 24.3 22.0 16.9 12.8 7F 7.3 6.1 5.2 5.2 9V 10.2 8.0 6.9 8.5 14 37.0 43.2 27.8 20.2 18C 6.7 8.5 4.5 6.5 19F 18.8 8.4c 13.0 6.4c 23F 11.9 9.2 8.1 10.8 ↵a Values assigned to 89SF serum by Quataert et al. (14) and included in the circular sent to other laboratories with serum 89SF.
↵b Values were obtained by comix mHSA and cross-standardization (see Table 2 for IgG values), except for type 3, for which the mHSA precoat method was used.
↵c For this value, the limits of a ±20% interval do not overlap for both the assigned and the mHSA estimates.
- Table 4.
Estimation of concentrations of antibody to individual pneumococcal types in reference serum lot 89SF by cross-standardization by the mHSA comix method
Type used as reference Concn (μg/ml) in serum 89SF of IgG antibody against the following type PSs with reference antibody values: 2 9N 19A 4 4.5 7.4 10.3 6B 7.6 7.6 11.4 7F 5.7 6.6 9.1 9V 5.2 9.6 13.4 23F 4.8 8.3 12.5 Meana 5.6 7.9 11.3 ↵a Arithmetic mean of antibody estimates obtained by using individual types with their previously assigned IgG antibody concentrations (14) to generate the reference curve.
