ELISPOT assay for chlamydia-specific, antibody-producing cells correlated with conventional complement fixation and microimmunofluorescence

Abstract

Chlamydia antigens cross-reactive with pneumoniae (TWAR), psittaci, and trachomatis strains were used to evaluate the ELISPOT assay for detecting antigen-specific, antibody-secreting cells (ASC). Human blood specimens from healthy and hospitalized persons were randomly collected and tested by coating the nitrocellulose membrane at the base of microtiter wells. Ficoll- separated mononuclear cells from blood specimens collected in EDTA were incubated in the wells with Iscove's growth medium in CO2 atmosphere at 37°C. An IgG-specific conjugate labeled with biotin was used in an avidin- peroxidase chromogen system for indicating the areas (spots) of immunologically committed lymphocytes. Positive specimens had median levels of ASC above 8 per 106 cells (range 15-23 ASC/106 cells). Evidence that the ELISPOT is reliable, sensitive, and specific includes the following: (1) immunized animal and clinical human specimens in control experiments were selectively reactive in the presence of antigen, but negative without antigen, (2) serologically characterized reference sera demonstrated homologous rather than heterologous reactions with the antigens, (3) conventional complement fixation and microimmunofluorescence on serum fractions of clinical specimens correlated well (P<0.02) with ELISPOT results that were both TWAR- and psittaci-positive, and (4) the array of specimens (from healthy donors, community hospitalized, and pulmonary service patients) selected for their increasing likelihood in that order for being positive due to illness was then confirmed and supported by their respectively increasing positivity rates (6, 15, and 25%) for TWAR/psittaci combined. The incidence of positive specimens for either TWAR or psittaci was greatest (23/54, 43%) in specimens from the hospitalized patients and least (8/33, 24%) in specimens from healthy individuals. These findings suggest that ELISPOT detects chlamydial antibody production at the cellular level. ELISPOT positivity thus indicates previous exposure and would favor earlier detection.