Assignment of Weight-Based Antibody Units to a Human Antipneumococcal Standard Reference Serum, Lot 89-S

Transcription

1 CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Sept. 1995, p Vol. 2, No X/95/$ Copyright 1995, American Society for Microbiology Assignment of Weight-Based Antibody Units to a Human Antipneumococcal Standard Reference Serum, Lot 89-S SALLY A. QUATAERT,* CAROL S. KIRCH, LAURIE J. QUACKENBUSH WIEDL, DONNA C. PHIPPS, SUSAN STROHMEYER, CAROLYN O. CIMINO, JANICE SKUSE, AND DACE V. MADORE Lederle-Praxis Biologicals, West Henrietta, New York Received 23 January 1995/Returned for modification 14 April 1995/Accepted 2 June 1995 A human reference serum pool, lot 89-S, has been developed for use in quantitating concentrations of antibody to Streptococcus pneumoniae. Weight-based units have been assigned to antibodies to 11 pneumococcal polysaccharide (PnPs) serotypes (1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F) by using enzyme-linked immunosorbent assay methodology and a human standard reference serum, USNRP IS The experimentally derived assignments for anti-pnps antibodies of the immunoglobulin G (IgG), IgM, and IgA isotypes in lot 89-S correlate well to the separately determined immunoglobulin assignment. These assignments for this antipneumococcal standard serum were used to quantitate IgG, IgM, and IgA isotype levels and the total immunoglobulin level in pediatric samples from a pneumococcal conjugate vaccine trial. The data indicate that these assignments may be used to assess levels of antibody to PnPs serotypes in human serum. Antibody responses to the serotype-specific capsular polysaccharides of Streptococcus pneumoniae, a major human pathogen causing pneumonia, bacteremia, and meningitis, as well as otitis media, are known to be protective (1). Several pneumococcal (Pn) polysaccharide (PnPs) vaccines containing 23 serotypes are licensed but have limited use in children 2 years of age. Young children are susceptible to S. pneumoniae disease because their immune systems are not mature enough to respond to PnPs antigens. However, they may respond to new conjugate vaccines which are presented to the immune system as T-cell-dependent antigens (13). This new conjugate vaccine technology is being applied to PnPs serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F, which are prevalent in causing disease in children and immunocompromised individuals. To evaluate these new vaccines, it is necessary to accurately quantitate antibodies in clinical trial samples. Recently, enzyme-linked immunosorbent assays (ELISAs) have been used to detect serotype-specific PnPs antibodies (3, 5, 7). Their usefulness in comparative vaccine trials is dependent on the availability of standardized reagents (15). The availability of human reference serum with assigned anti-pnps antibody values will allow comparative assignment of antibody levels between laboratories and methods. We have prepared a human reference standard from pooled serum specimens from 17 adults immunized with a 23-valent PnPs vaccine. This reference standard was quantitated for total immunoglobulin (Ig) as well as for levels of IgG, IgM, and IgA isotypes to 11 S. pneumoniae serotypes by a modified Zollinger technique (17). A similar approach to quantitation was undertaken by Rudolph and Parkinson for assignment of IgG isotype values to reference standard PSAB90 (14). The sum of the IgG, IgM, and IgA assignments for antibodies specific for each of the serotypes in the new reference standard serum is consistent with the independently determined total Ig assignment for that serotype. In this study, pediatric sera were quantitated by using a standard reference serum, lot 89-S. Good agreement between isotype and total antibody levels quantitated in the * Corresponding author. Mailing address: Lederle-Praxis Biologicals, 211 Bailey Rd., West Henrietta, NY Phone: (716) Fax: (716) pediatric sera was observed. The new standard reference preparation can be used for quantitation of serotype-specific antibody to S. pneumoniae in both adult and pediatric sera. This anti-pn standard serum is available as U.S. standard reference serum lot 89-SF from the Center for Biological Evaluation and Review (CBER), U.S. Food and Drug Administration, Bethesda, Md., for use in evaluating immune status and responses to immunization. MATERIALS AND METHODS Sera. Human anti-pn standard serum lot 89-S was prepared by pooling 17 high-titered serum specimens as described previously (9). Briefly, plasma units were obtained from 68 adult human donors following immunization with a 23-valent PnPs vaccine (PNU-IMUNE; Lederle), a meningococcal polysaccharide vaccine (MENOMUNE; Connaught), and a Haemophilus conjugate vaccine (ProHIBIT; Connaught). Relative titers of antibody to 12 specific PnPs serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F, and 23F) were determined by ELISA. Plasma samples from the 17 individuals showing the highest titers of antibody to a majority of the serotype-specific PnPs antigens were pooled, defibrinated, and filtered. This pooled plasma is stored frozen at 20 C and is also available in lyophilized aliquots (U.S. standard reference serum lot 89-SF) from Carl Frasch, CBER, U.S. Food and Drug Administration. Pediatric sera were obtained from infants (ages, 2 to 7 months) pre- and postimmunization with an experimental pentavalent (6B, 14, 18C, 19F, and 23F) pneumoconjugate vaccine (Lederle-Praxis). ELISA quantitation method for assigning antibody values. The method for quantitating antibodies is based on equivalence of absorbances between a reference ELISA and the anti-pnps ELISA performed in parallel under identical assay conditions, including buffers, enzyme conjugate dilutions, and incubation times and temperatures (Fig. 1) (17). The reference ELISA is an antibody capture ELISA in which the Ig molecules in the human reference serum preparation USNRP IS 1644 (U.S. National Reference Preparation of human serum proteins, Center for Infectious Disease, Centers for Disease Control and Prevention, Atlanta, Ga.) are captured by goat anti-human light-chain-specific reagents. The reference preparation USNRP IS 1644 has been assigned mg of IgG per ml, 2.15 mg of IgA per ml, and 1.40 mg of IgM per ml (12). In the anti-pnps ELISA, which is adapted from a method described by Koskela (4), microtiter plates are coated separately with each serotype-specific PnPs for direct binding of serum antibodies. The endpoint dilutions at 0.3 absorbance units which under the same assay conditions for both the reference ELISA and the anti-pn ELISA represent equivalent amounts of antibodies are determined from a logarithmic regression of the reciprocal of dilution versus absorbance. The known concentration (in micrograms per milliliter) of the reference preparation at the endpoint dilution is used to assign antibody concentrations to the endpoint dilution of the standard serum, lot 89-S, in the anti-pn ELISA. Reference ELISA for the quantitation method. An antibody capture ELISA was performed with microtiter plates (Nunc Polysorp; catalog no ) coated for 90 min at 37 C with 100 l of goat antibodies to human kappa and lambda light chains (Tago, Burlingame, Calif.; catalog no and 4308, re- 590

2 VOL. 2, 1995 HUMAN ANTIPNEUMOCOCCAL STANDARD REFERENCE SERUM 591 FIG. 1. Principle of equivalence of absorbances obtained with known concentrations of reference serum antibody in a reference ELISA and serotype-specific antibody in an anti-pnps ELISA. Std, standard; 2, secondary;, enzyme linked. spectively) per well, each at a final concentration of 0.8 g/ml in sterile 0.01 M phosphate-buffered saline (PBS) 0.02% azide (ph 7.2 to 7.4). After each step, the plates were washed with 250 l of 0.01 M PBS 0.1% Tween 20 per well by using an automated enzyme immunoassay plate washer programmed for a 30-s soak and five wash cycles (Biotek, Winooski, Vt.). Eight twofold serial dilutions of human reference serum preparation USNRP IS 1644 in PBS 0.05% Tween 0.02% azide were added to duplicate wells at 50 l per well, and the wells were incubated at room temperature for 2 h. The plates were washed as described above, and 50 l of either alkaline phosphatase-labeled conjugate goat antihuman IgG, IgM, or IgA (Tago; catalog no. 2490, 2492, or 2491, respectively) or goat anti-human Ig (IgG, IgM, or IgA, heavy chain specific) (Caltag; catalog no ) was added per well, and the mixtures were incubated for 2 h at room temperature. The goat anti-human Ig enzyme conjugate reagent was selected for balanced reactivity against IgG, IgM, and IgA and no reactivity with light chains as tested in a direct-binding ELISA. The isotype-specific conjugates selected have 5% cross-reactivity with the other isotypes and have no reactivity with light chains. In addition, the selected anti-igg and anti-ig reagents have equal sensitivities for IgG subclasses, with no bias for light-chain usage by the IgG subclass. The plates were washed a final time, 100 l ofp-nitrophenylphosphate (Sigma Chemicals, St. Louis, Mo.) per well at 1 mg/ml in diethanolamine buffer with MgCl 2 (ph 9.8) was added, and the mixture was incubated for 1 h. The reaction was stopped by adding 50 l of 3 N NaOH per well. A 405 values were determined with an enzyme immunoassay reader (Biotek) with a 690-nm reference wavelength. Anti-PnPs ELISA. The anti-pnps ELISA, starting with the primary antibody incubation step, was performed with the same assay conditions as the reference ELISA. The antigens, PnPs serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F, were purchased from American Type Culture Collection (Rockville, Md.), diluted in sterile water for irrigation (Kendall-McGraw Laboratories, Inc., Irvine, Calif.), and stored at 20 C as 1-mg/ml stocks. The optimal coating concentrations for the antigens diluted in sterile PBS on Nunc Microwell plates (catalog no ) were 0.5 g/ml for PnPs serotypes 4 and 9V; 1 g/ml for PnPs serotypes 7F, 14, and 18C; 2 g/ml for PnPs serotype 1; 5 g/ml for PnPs serotype 5; and 10 g/ml for PnPs serotypes 3, 6B, 19F, and 23F. All sera were preabsorbed at a 1:50 dilution with 2.5 g of Pn ELISA absorbent, lot B, per ml to minimize any non-pnps-specific reactivity to the PnPs antigen coatings (4, 9). Pn ELISA absorbent is a preparation of soluble capsular components including C polysaccharide (C-Ps) made by Lederle-Praxis Biologicals from a serotype-specific capsule-negative variant (CSR-II) of S. pneumoniae (6, 8, 16). A separate microtiter plate was coated with each PnPs serotype, 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, or 23F, at 100 l per well for 5hat37 C. Fifty microliters of twofold serial dilutions of preabsorbed sera per well was added in duplicate to the antigen-coated plates, which were then incubated for 2 h at room temperature. The anti-pnps ELISA was completed according to the procedure described for the reference ELISA. Quantitative precipitin method. Anti-PnPs antibodies to PnPs serotypes 6B, 14, and 18C were quantitated by precipitating 100 l of the standard reference serum lot 89-S with 100 l of each antigen separately. The PnPs concentrations ranged from 1.25 to 20 g/ml in 0.01 M PBS. The mixture was incubated in glass tubes for 1hat37 C and then for 1 to 4 days at 4 C. The precipitate was centrifuged at 900 g for1hat4 C and washed twice with PBS. The precipitate was dissolved in 0.02 ml of 0.1 N NaOH. Antibody protein concentrations were then determined with a bicinchoninic acid protein determination kit (Pierce, Rockford, Ill.; catalog no X) with human IgG (Pierce; catalog no ) as the protein reference standard. RESULTS Reference preparation USNRP IS 1644 was run in the reference ELISA concurrently with the anti-pn standard reference serum, lot 89-S, in the anti-pnps ELISAs for total Ig, IgG, IgM, and IgA antibody determinations. The average concentrations of antibody (in nanograms per milliliter) detected in the reference ELISA at an absorbance of 0.3 were for total Ig, for IgG, for IgM, and for IgA. The slopes of the regression lines for the log of the absorbance versus the log of Ig concentration for USNRP IS 1644 typically range from 0.9 to 1.0 for total Ig, 0.85 to 1.0 for IgG and IgA, and 0.95 to 1.1 for IgM. The anti-pn standard serum, lot 89-S, was run on three or four separate days in each of the 11 anti-pnps ELISAs for total Ig, IgG, IgM, and IgA antibody determinations. The same primary and secondary antibodies, substrate incubation times and temperatures, buffers, and enzyme conjugate dilutions were used for the reference and the anti-pnps ELISAs. The titration of anti-pn standard serum lot 89-S for Ig, IgG, IgM, and IgA binding to PnPs serotype 19F shown in Fig. 2 is typical of that seen with the other 10 PnPs serotypes. The slopes of the lines typically run in the range of 0.9 to 1.0 for total Ig and IgG, 0.85 to 0.95 for IgA, and 0.7 to 0.8 for IgM in the anti-pnps ELISA for lot 89-S as well as for adult and pediatric sera. Antibodies of the IgG, IgM, and IgA isotypes in the reference preparation USNRP IS 1644 and the anti-pn standard serum reach similar equilibrium binding in the respective assays. The titrated USNRP IS 1644 and anti-pn standard serum samples were transferred after the primary antibody binding on a capture plate or a PnPs serotype 19F antigen-coated plate to a second capture or 19F antigen-coated plate. For USNRP IS 1644, 83, 71, and 80% of IgG, IgM, and IgA, respectively, were captured on the first reference ELISA plate. For the anti-pn standard serum, 79, 54, and 63% of the IgG, IgM, and IgA antibodies, respectively, were captured on the first PnPs serotype 19F antigen-coated plates. With these similar binding kinetics, the IgG assignments for anti-pn antibodies will reflect the same numbers of micrograms per milliliter as the reference

3 592 QUATAERT ET AL. CLIN. DIAGN. LAB. IMMUNOL. FIG. 2. Titration of anti-pn standard serum lot 89-S in an ELISA for Ig (j), IgG (}), IgM (º), and IgA ( ) antibodies to PnPs serotype 19F. standard, USNRP IS 1644, and the IgM and IgA assignments will be within 75 to 80% of the actual amount present in the reference standard. Anti-PnPs antibody levels were assigned to the anti-pn standard serum, lot 89-S, by assigning the concentration of known total Ig or isotype giving an absorbance of 0.3 in the concurrently run reference ELISA to the dilution of the anti-pn standard yielding an absorbance of 0.3 in the corresponding anti-pnps-specific assay for total Ig or isotype. The quantitated values for total Ig, IgG, IgM, and IgA antibodies to the 11 PnPs serotypes present in anti-pn standard serum lot 89-S are shown in Table 1. The overall coefficients of variation (CV) when all 11 PnPs serotype determinations are combined are 22.2% for total Ig, 16.2% for IgG, 9.8% for IgM, and 15.4% for IgA, indicating very good reproducibility in assignments. It is important to recognize that the CV described here and in Table 1 reflect the variation in the method, which depends on two separate ELISAs. The sum of the three independently determined values for anti-pnps antibodies of the IgG, IgM, and IgA isotypes agrees well with the independently determined value for total Ig of the same PnPs serotype. The sum of the values for IgG, IgM, and IgA anti-pnps antibodies against the 11 PnPs serotypes ranged from 83 to 113% of the total Ig anti-pnps value determined in the respective serotype assay. The overall average for the sum of IgG, IgM, and IgA with all 11 serotypes compared with the total Ig was 96%, showing good agreement among the independent determinations. This suggests that the Antigen (PnPs serotype) TABLE 1. Quantitation of anti-pnps antibody in anti-pn standard serum lot 89-S g of anti-pnps antibody/ml (CV [%]) a Total Ig (n 3) IgG (n 4) IgM (n 4) IgA (n 4) Sum (IgG IgM IgA) (19.2) (13.4) (7.0) (13.9) (20.1) (12.5) (4.6) (7.8) (12.9) (11.5) (8.5) (6.9) (22.6) (6.3) (18.7) (14.3) B (19.0) (16.4) (14.1) (7.7) F (21.0) (23.9) (6.5) (19.0) V (17.6) (26.7) (5.9) (18.9) (20.4) (13.2) (3.4) (16.8) C (25.3) (21.0) (5.9) (26.0) F (20.5) (20.6) (14.6) (17.2) F (46.1) (13.1) (18.5) (20.8) Overall CV (%) a Antibody levels shown are means standard deviations. n, number of determinations. b Ratios of sums of IgG, IgM, and IgA to total Ig, expressed as percentages (overall average, 96%). Sum/Ig (%) b

4 VOL. 2, 1995 HUMAN ANTIPNEUMOCOCCAL STANDARD REFERENCE SERUM 593 TABLE 2. Assignment of anti-pnps antibody levels to human anti-pn standard serum lot 89-S PnPs serotype Anti-PnPs antibody level ( g/ml) Total Ig IgG IgM IgA B F V C F F assignments to each of the four independently determined values did not show any assay bias and that the values are consistently assigned. In consideration of assay variability and ease of use, the antibody levels assigned to anti-pn standard serum lot 89-S for the 11 PnPs serotypes were rounded to the nearest 10th of a microgram per milliliter. The assignments generated by this comprehensive study are listed in Table 2. Similar quantitative relationships were seen for total Ig and IgG isotype with antibodies in the anti-pn standard serum. PnPs serotype-specific antibodies ranked 14 6B 19F 23F 1, 5, 9V, and 7F 3, 4, and 18C. IgG antibodies were the predominant isotype against the majority of PnPs serotypes, with a geometric mean value for IgG antibody fivefold greater than that for the next dominant isotype, IgM. IgG assignments ranged from 2.7- to 23-fold higher than IgM assignments. Notable exceptions were the preponderance in lot 89-S of IgA isotype antibodies against PnPs serotype 3 and an equal distribution of IgG and IgM antibodies against PnPs serotype 5 (IgG/IgM ratio, 1.38). The levels of IgA antibody against PnPs serotype 3 were two to five times higher than the levels of IgA antibody to the other PnPs serotypes tested. The anti-pn standard serum is a pool of sera from adults who were immunized with a 23-valent PnPs vaccine and were selected for having the highest titers of anti-pnps antibodies against the greatest number of the 12 PnPs serotypes tested. The relationships among the anti-pnps antibodies described for the anti-pn standard serum may not apply to nonimmunized populations or to low responders. Pediatric serum samples from infants immunized at 2, 4, and 6 months with a pentavalent Pn conjugate vaccine were evaluated in anti-pnps serotype 6B, 14, 18C, 19F, and 23F ELISAs for total Ig, IgG, IgM, and IgA antibodies, using the anti-pn standard serum as a reference standard with the values assigned by the ELISA quantitation method (shown in Table 1). Titration of pediatric sera yielded regression lines parallel to that for anti-pn standard serum lot 89-S, as evidenced by similar ranges for slopes in total Ig, IgG, IgA, and IgM determinations. For each pediatric sample and for each PnPs serotype, the sum of the IgG, IgM, and IgA was compared with the total Ig by linear regression analysis (Fig. 3 through 7). The slope of the linear regression was close to 1 for all serotypes except for PnPs serotype 19F, for which the slope of the regression line was Although the slope of the line comparing the sum of the IgG, IgM, and IgA with the total Ig determined for all 35 pediatric serum specimens is 0.7, a slope of about 0.9 can be obtained if values at the lowest sensitivity are removed from the datum set. This suggests that the slightly greater variation FIG. 3. Comparison of sum of IgG, IgM, and IgA with total Ig antibodies to PnPs serotype 6B as determined by ELISA for pediatric sera. Pre- and postimmunization pediatric serum samples (n 37) were quantitated as described in Materials and Methods for total Ig, IgG, IgM, and IgA specific to PnPs serotype 6B. The antibody titers (in micrograms per milliliter) are shown after log transformation.

5 594 QUATAERT ET AL. CLIN. DIAGN. LAB. IMMUNOL. FIG. 4. Comparison of sum of IgG, IgM, and IgA with total Ig antibodies to PnPs serotype 14 as determined by ELISA for pediatric sera. Pre- and postimmunization pediatric serum samples (n 33) were quantitated as described in Materials and Methods for total Ig, IgG, IgM, and IgA specific to PnPs serotype 14. The antibody titers (in micrograms per milliliter) are shown after log transformation. of the anti-pnps serotype 19F ELISA seen at the lowest sensitivity (data not shown) is influencing the relationship and that the values assigned to lot 89-S are valid for pediatric sera. These slopes indicate a good correlation for antibody values covering a broad range of titers based on the anti-pnps assignments in lot 89-S. To further corroborate that these anti-pnps Ig assignments are consistent, an independent quantitative method was employed. Three PnPs serotypes (6B, 14, and 18C) were chosen for quantitative precipitation of antibodies against these serotypes in anti-pn standard serum lot 89-S. Results are shown in Table 3. The anti-pn standard serum was not preabsorbed with soluble Pn absorbent because of the difficulty of optimizing the precipitin reaction for multiple antigens found in the Pn absorbent material. Seventy to 85% of the anti-pn absorbent activity as determined by ELISA was retained in the anti-pn standard serum after precipitation with the optimal precipitating concentration of either PnPs serotype 6B, 14, or 18C. Protein quantitated by the precipitin reaction should reflect predominantly antibodies specific to the PnPs serotype used. With PnPs serotypes 6B and 14, there was good agreement between the reference ELISA and the quantitative precipitin methods in assigning micrograms of Ig per milliliter to the anti-pn standard serum. The quantitative precipitin method yielded 50% more Ig antibody than the reference ELISA method for PnPs serotype 18C. Since the PnPs serotypes may contain C-Ps, some anti-c-ps antibodies may be present in the precipitate. DISCUSSION In a previous report on preparation of anti-pn standard serum lot 89-S (9), tentative antibody values were assigned to lot 89-S (11). While these tentative assignments were derived by the ELISA quantitation method described here, total Ig and isotype values were determined in separate experiments at different times. In addition, a variety of microtiter plate types were used in determining the tentative assignments. These and other experimental variables may have contributed to a bias, with the tentative total Ig assignment for serotypes 1, 5, 6B, 18C, and 23F being 1.5- to 2.6-fold greater than the sum of the tentative isotype values. The total Ig and isotype assignments reported here were derived concurrently and show no bias in assignment with the sum of the isotype values, varying by 15% of the total Ig value for all serotypes. Assurance that the ELISA quantitation method provides consistent values hinges on several factors. The reference ELISA for capturing a known amount of Ig (total or isotype specific) employs anti-kappa and anti-lambda antibodies for capture of the Ig near the antibody binding site in the same orientation as the binding of anti-pnps antibody, with the Fc portion outward, to the PnPs antigen-coated plate. The enzyme conjugates used to detect antibody are able to label exposed epitopes on the Fc portion of the primary antibodies similarly in the reference and the antigen-specific ELISAs. In addition, equivalent microtiter plates need to be used for the solid phase in reference and specific ELISAs for consistent presentation of antigen or capture antibody (10). The equilibrium of the antigen-antibody binding reaction must be equivalent or close to equivalent in the reference and specific ELISAs (2). If different proportions of the reference preparation antibodies and the specific antibodies to be detected bind, the reference ELISA value will not be able to be used to accurately assign antibody levels to the unknown sample. If the equilibrium reactions are different, the actual assignment may be either an overestimate or an underestimate of antibody

6 VOL. 2, 1995 HUMAN ANTIPNEUMOCOCCAL STANDARD REFERENCE SERUM 595 FIG. 5. Comparison of sum of IgG, IgM, and IgA with total Ig antibodies to PnPs serotype 18C as determined by ELISA for pediatric sera. Pre- and postimmunization pediatric serum samples (n 30) were quantitated as described in Materials and Methods for total Ig, IgG, IgM, and IgA specific to PnPs serotype 18C. The antibody titers (in micrograms per milliliter) are shown after log transformation. levels. In the case of lot 89-S, the same proportion of IgG antibodies bound under similar assay conditions in the reference and the anti-pnps ELISAs. This was important for the best assignment of antibody values, since IgG antibodies predominated the antibody repertoire in lot 89-S for all PnPs serotypes tested except serotype 3, for which the level of IgA antibodies was almost twofold higher than that of IgG. For the most part, IgM and IgA constituted a minor portion of the anti-pnps antibodies present and were adequately assessed by the method. To avoid antibody affinity differences between the assays influencing the quantitation, both the reference preparation and the anti-pn standard serum were titrated over a FIG. 6. Comparison of sum of IgG, IgM, and IgA with total Ig antibodies to PnPs serotype 19F as determined by ELISA for pediatric sera. Pre- and postimmunization pediatric serum samples (n 35) were quantitated as described in Materials and Methods for total Ig, IgG, IgM, and IgA specific to PnPs serotype 19F. The antibody titers (in micrograms per milliliter) are shown after log transformation.

7 596 QUATAERT ET AL. CLIN. DIAGN. LAB. IMMUNOL. FIG. 7. Comparison of sum of IgG, IgM, and IgA versus total Ig antibodies to PnPs serotype 19F as determined by ELISA for pediatric sera. Pre- and postimmunization pediatric serum samples (n 32) were quantitated as described in Materials and Methods for total Ig, IgG, IgM, and IgA specific to PnPs serotype 23F. The antibody titers (in micrograms per milliliter) are shown after log transformation. wide range. Analysis was done with linear regression of absorbance values of 1.5 to ensure that capture antibody or PnPs antigen would be in excess to antibody, thus minimizing any effect of antibody affinity (18). Parallelism in titration of USNRP IS 1644 in each reference ELISA compared with the serotype-specific anti-pnps ELISA for total Ig, IgG, IgA, and IgM is important for the precision of the assignment. The slopes for total Ig, IgG, and IgA in the anti-pnps ELISA (range, 0.85 to 1.0) are parallel to those for USNRP IS 1644 in the reference ELISA. The slopes of the IgM assays are slightly different, 0.7 to 0.8 for the anti-pnps ELISA and 0.95 to 1.1 for the reference ELISA with USNRP IS 1644, and may result in a slight overestimation of IgM in lot 89-S. However, no bias appears to occur, as the sum of the IgM, IgG, and IgA approximates the total Ig value for serotype-specific antibodies. The ELISA quantitation method is also complicated by the specificity and balanced reactivity, or lack thereof, of the enzyme conjugates used to detect antibody binding. We were particularly attentive to evaluating the anti-human Ig (total and isotype) enzyme conjugate reagents used to detect antibodies for quantitating the anti-pn standard serum. The anti-ig reagent detects IgG, IgM, and IgA equally and does not react with light chains. Therefore, values for total Ig in serum will not be overestimated. Similarly, the IgG reagent was balanced to equally detect IgG1, -2, -3, and -4 subclasses of either lambda or kappa light-chain specificity. Excellent agreement of the sum of the IgG, IgM, and IgA values for each serotype with the total Ig value assigned to that serotype is evidence that the enzyme conjugates met our stringent criteria for detecting antibodies. The secondary reagents we used were polyclonal reagents, which may have an advantage when antibody responses to carbohydrates are assessed, since they are directed to multiple epitopes on the antibody molecule and may not be as susceptible to allotypic or idiotypic restrictions that potentially may affect detection by monoclonal reagents. The quantitative precipitin results for PnPs serotypes 6B and 14 verify the antibody values assigned by the ELISA quantitation method. Although the precipitin value is not as close for 18C as for 6B and 14, the results are only about twofold different. The variability in other components, such as C-Ps, which cannot be removed and may be part of the PnPs antigen itself, compromises the specificity of the precipitin reaction. Our analysis in an ELISA using a monoclonal antibody specific for phosphorylcholine (the major C-Ps sugar determinant) suggests that the PnPs serotype 18C lot used may contain more C-Ps than the other two types tested (data not shown). The precision with which the sum of the values assigned to the anti-pn standard serum agrees with the total value assigned for each of the 11 serotypes tested substantiates that consistency is achieved by this quantitation method. Of critical importance, the use of these values for anti-pn standard serum lot 89-S as a reference for pediatric samples from vaccine trials has been shown. Antibody reference preparations should reflect antibody composition and properties similar to those PnPs serotype TABLE 3. Antibody values in anti-pn standard serum lot 89-S determined by quantitative precipitin n g of antibody/ml (mean SD) Ratio a 6B C a Ratio of precipitin value to anti-pn standard serum Ig value assigned by total Ig reference ELISA.

8 VOL. 2, 1995 HUMAN ANTIPNEUMOCOCCAL STANDARD REFERENCE SERUM 597 present in the sample population. Anti-Pn standard serum lot 89-S is a pool from PnPs-immunized adults whose response to PnPs vaccination is mainly IgG2. The aim of multiple-vaccine trials in progress is to induce IgG1 responses to PnPs in infants by immunizing them with S. pneumoniae polysaccharide conjugate vaccines. This adult reference standard can be used to quantitate pediatric samples, as evidenced by the very good correlation of total Ig to the sum of the IgG, IgM, and IgA anti-pnps antibodies quantitated in pediatric trial samples using anti-pn standard serum lot 89-S. This indicates that although lot 89-S is an adult reference preparation, the values assigned to lot 89-S work well in the ELISA as a reference standard for pediatric samples. Considering that each of the four independent assays has an assay CV of about 25%, the linearity of the regression line which approaches 1.0 for the comparison of the sum of the IgG, IgM, and IgA versus the total Ig for PnPs serotypes 6B, 14, 18C, and 23F is remarkable. These results suggest that under the assay conditions used in the anti-pn ELISA, differences in antibody type and affinity between the pediatric samples and anti-pn standard serum lot 89-S are not influencing the assignment of antibody values. The excellent agreement of the sum of the IgG, IgM, and IgA with the total Ig anti-pnps values for both pediatric samples and anti-pn standard serum lot 89-S verifies the consistency of the values assigned, independent of isotype or donor age. The anti-pn standard serum is available as U.S. standard reference serum lot 89-SF from the CBER, U.S. Food and Drug Administration, for use in evaluating immune status and responses to immunization. The ELISA quantitation method will be useful for assigning antibody levels in lot 89-S to other PnPs serotypes. The anti-pnps ELISA for any of the 11 serotypes can be used as the reference assay in the ELISA quantitation method. This will provide a convenient method for assigning further values for antibodies in anti-pn standard serum lot 89-S to additional serotypes in a manner similar to that described here. This consistent method has been applied to the assignment of values to anti-pnps antibodies to 11 serotypes of S. pneumoniae in lot 89-S. These assignments show no bias in assignment of antibody between total Ig and isotype values and replace the previous assignments made by Phipps et al. (9). A consistent method for antibody assignment is important for reference preparations such as anti-pn standard serum lot 89-S, since this preparation is used to evaluate responses to multivalent PnPs vaccines. When consistently assigned, the microgram values for antibodies to each PnPs serotype not only yield absolute anti-pnps antibody values, but also allow comparison of responses to the serotypes. Importantly, consistent antibody assignments to the reference standard will permit assessment of relative protective antibody levels necessary to protect against disease caused by different S. pneumoniae serotypes. REFERENCES 1. Committee on Issues and Priorities for New Vaccine Development New vaccine development establishing priorities, vol. II, appendix D-17, p National Academy Press, Washington, D.C. 2. Griswold, W. R., and R. R. Chalquest Theoretical analysis of the accuracy of calibrated immunoassays for measuring antibody concentration. Mol. Immunol. 28: Konradsen, H. B., U. B. S. Sorensen, and J. Henrichsen A modified enzyme-linked immunosorbent assay for measuring type-specific anti-pneumococcal capsular polysaccharide antibodies. J. Immunol. Methods 164: Koskela, M Serum antibodies to pneumococcal C polysaccharide in children: response to acute pneumococcal otitis media or to vaccination. Pediatr. Infect. Dis. J. 6: Koskela, M., and M. Leinonen Comparison of ELISA and RIA for measurement of pneumococcal antibodies before and after vaccination with 14-valent pneumococcal capsular polysaccharide vaccine. J. Clin. Pathol. 34: Liu, T.-Y., and E. C. Gotschlich The chemical composition of pneumococcal C-polysaccharide. J. Biol. Chem. 238: Musher, D. M., M. Luchi, D. A. Watson, R. Hamilton, and R. E. Baughn Pneumococcal polysaccharide vaccine in young adults and older bronchitics: determination of IgG responses by ELISA and effect of absorption of serum with non-type specific cell wall polysaccharide. J. Infect. Dis. 161: Pedersen, F. K., J. Henrichsen, U. B. S. Sorensen, and J. L. Nielsen Anti-C-carbohydrate antibodies after pneumococcal vaccination. Acta Pathol. Microbiol. Scand. Sect. C 90: Phipps, D. C., S. Strohmeyer, S. A. Quataert, G. Siber, and D. V. Madore Standardization of ELISA for the quantitation of antibodies to S. pneumoniae capsular polysaccharides (PnPs). Pediatr. Res. 27:179A. 10. Pruslin, F. H., S. E. To, R. Winston, and T. C. Rodman Caveats and suggestions for the ELISA. J. Immunol. Methods 137: Quataert, S. A., L. Quackenbush Wiedl, D. C. Phipps, and D. V. Madore Quantitation of anti-polysaccharide (PnPs) antibodies in a new human reference serum for streptococcal pneumonia. Pediatr. Res. 33:179A. 12. Reimer, C. B., S. J. Smith, T. W. Wells, N. K. Nakamura, P. W. Keitges, R. F. Ritchie, G. W. Williams, D. J. Hanson, and D. B. Dorsey Collaborative calibration of the U.S. National and the College of American Pathologists reference preparations for specific serum proteins. Am. J. Clin. Pathol. 77: Robbins, J. B Vaccines for the prevention of encapsulated bacterial diseases: current status, problems and prospects for the future. Immunochemistry 15: Rudolph, K. M., and A. J. Parkinson Measurement of pneumococcal capsular polysaccharide serotype-specific immunoglobulin G in human serum, a method for assigning weight-based units to proposed reference sera. Clin. Diagn. Lab. Immunol. 1: Siber, G. R., C. Priehs, and D. V. Madore Standardization of antibody assays for measuring the response to pneumococcal infection and immunization. Pediatr. Infect. Dis. J. 8:S84 S Sorensen, U. B. S., and J. Henricksen C-polysaccharide in a pneumococcal vaccine. Acta Pathol. Microbiol. Immunol. Scand. Sect. C 92: Zollinger, W. D., and J. W. Boslego A general approach to standardization of the solid phase radioimmunoassay for quantitation of class-specific antibodies. J. Immunol. Methods 46: Zollinger, W. D., J. M. Dalrymple, and M. S. Artenstein Analysis of parameters affecting the solid phase radioimmunoassay quantitation of antibody to meningococcal antigens. J. Immunol. 117: