False-positive immunogenicity responses are caused by CD20+ B cell membrane fragments in an anti-ofatumumab antibody bridging assay.

An electrochemiluminescent (ECL) bridging assay to detect anti-ofatumumab antibodies (ADA) in human serum samples was developed and validated. Using this assay format, clinical samples were first screened to identify potential ADA positive samples, which were then further tested by adding excess drug, confirming the positive signals as drug specific. However, when the method was implemented into clinical studies for ADA testing, a high positive rate was observed in the pre-dose samples collected from patients with chronic lymphocytic leukemia (CLL). Since the positive signals were not associated with ofatumumab (Ofa) treatment, and diminished after treatment, it was suspected that matrix interference might be responsible, resulting in false-positive responses. We performed a series of experimental investigations to identify, characterize, minimize or eliminate the possible false-positive responses. One possible source was identified to be CD20 (the target of Ofa) present on cell membrane fragments (CMFs). The false-positive responses caused by CD20(+) CMFs could be reduced by solid-phase immunodepletion, ultracentrifugation, or inhibited by adding another anti-CD20 antibody (rituximab). As a consequence, the ADA method was modified to minimize the matrix interference caused by CD20(+) CMFs and, then, validated for sample testing.

Enzyme-linked immunosorbent assay for measuring antibodies to pneumococcal polysaccharides for the PNEUMOVAX 23 vaccine: assay operating characteristics and correlation to the WHO international assay.

The Merck pneumococcal (Pn) enzyme-linked immunosorbent assays (ELISAs) for measuring antibodies to 12 serotypes (serotypes 1, 3, 4, 6B, 7F, 8, 9V, 12F, 14, 18C, 19F, and 23F) were validated in 1999. Merck Laboratories developed the Pn assays using 10 microg/ml C polysaccharide, 100 microg/ml Pn polysaccharide (PnPs) 25, and 100 microg/ml PnPs 72 for preadsorption of samples, standards, and controls in order to improve the specificity to the Pn serotypes in the vaccine. The Pn assays utilize postimmunization sera obtained from subjects immunized with PNEUMOVAX 23 as standards for measuring immunoglobulin G concentrations in sera obtained from vaccine clinical trials with adults and infants. This material was calibrated to the Pn reference standard serum, 89SF, subjected to the Merck Pn ELISA adsorbants. Comparisons were made between the Merck Pn assay and the international Pn assay, showing moderate agreement between the two assay formats. This work describes the test procedures and operating characteristics of the Merck Pn assays and the results of experiments performed to compare the Merck Pn ELISAs to the international Pn ELISAs.

Assignment of weight-based immunoglobulin G1 (IgG1) and IgG2 units in antipneumococcal reference serum lot 89-S(F) for pneumococcal polysaccharide serotypes 1, 4, 5, 7F, 9V, and 18C.

Weight-based assignments for immunoglobulin G1 (IgG1) and IgG2 subclass antibodies to Streptococcus pneumoniae capsular polysaccharides (PnPs) in antipneumococcal standard reference serum lot 89-S (lot 89-S), also known as lot 89-SF, have been determined for serotypes 1, 4, 5, 7F, 9V, and 18C. This extends the usefulness of lot 89-S beyond the IgG1 and IgG2 subclass assignments for serotypes 3, 6B, 14, 19F, and 23F made previously (A. Soininen, H. Kayhty, I. Seppala, and T. Wuorimaa, Clin. Diagn. Lab. Immunol. 5:561-566, 1998) to cover 11 major serotypes associated with the highest percentage of pneumococcal disease worldwide. A method of equivalence of absorbances in enzyme immunosorbent assays was used to determine the IgG1 and IgG2 antibody concentrations for the additional serotypes in lot 89-S, based on the subclass values previously assigned for PnPs serotypes 6B, 14, and 23F. This cross-standardization method assures consistency with previous antibody assignments in that reference serum. The newly assigned subclass values for serotype 9V, and previously assigned values for serotype 14, were used to quantitate PnPs antibodies in sera from adult and pediatric subjects immunized with a pneumococcal conjugate vaccine. There was a predominance of IgG1 anti-PnPs antibodies in pediatric sera and IgG2 anti-PnPs antibodies in the adult sera. The IgG1 and IgG2 subclass assignments for the 11 PnPs serotypes in antipneumococcal standard reference serum lot 89-S are useful for quantitating and characterizing immune responses to pneumococcal infection and vaccination regimens.

Effect of antigen coating conditions on enzyme-linked immunosorbent assay for detection of immunoglobulin G antibody to Neisseria meningitidis serogroup Y and W135 capsular polysaccharide antigens in serum.

Human sera collected from 28 consenting adult volunteers were used to define assay conditions for meningococcal vaccine clinical trial serology. Immunoassay parameters were optimized with these test sera and the standard reference serum, CDC1992. Coating conditions for serogroup Y and W135 polysaccharide antigens were found to influence the predicted serum immunoglobulin G (IgG) antibody concentrations. Sera that displayed IgG antibody binding profiles most unlike that of CDC1992 were influenced the most by coating conditions. Our results suggest that presentation of specific epitopes is influenced by antigen-coating concentrations for serogroup Y and W135 polysaccharides.