THE DEVELOPMENT OF AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR THE DETECTION OF IgG AND IgM ANTIBODIES TO LEGIONELLA PNEUMOPHILA SEROGROUPS ONE THROUGH SIX

Abstract

An enzyme linked immunosorbent assay (ELISA) was developed for the detection of IgG and IgM antibodies to Legionella pneumophila serogroups 1 through 6. The ELISA compared favorably with the L. pneumophila serogroup 1 through 6 Indirect Fluorescent Antibody (IFA), the current serological method of choice. Two clinical comparisons were conducted to evaluate the performance of the LEGIONELLA ELISA assay relative to the IFA method for detection of IgG and IgM antibodies to L. pneumophila. In the first comparison, a total of 203 clinical specimens were tested in the ELISA and resulted in specificity, sensitivity, and accuracy percentages of 93.0%, 99.4% and 98.0%, respectively, when qualitatively compared to the IFA method. The second clinical comparison was performed by personnel at Maryland Department of Health using the LEGIONELLA ELISA and was based on a collection of samples primarily from the Legionella outbreak in Western Maryland. LEGIONELLA ELISA results indicated 100% sensitivity, 94.115 specificity and an overall accuracy of 95% when qualitatively compared to the IFA method. The specificity of the LEGIONELLA ELISA was further challenged by evaluation of 94 potential cross reactive sera that included other Legionella app., mycoplasma, chlamydia and syphilis specimens. Cross reactivity was observed in the LEGIONELLA ELISA in one L. micdadei specimen with a confirmed negative L. pneumophila IFA titer. A selection of these antisera were additionally evaluated by western immuno-blot analysis in an effort to detect potential cross reactive proteins. Detection of significant increases in antibody against L. pneumophila is critical to diagnosis by the IFA method. Such detection was assessed in the LEGIONELLA ELISA using 131 paired acute and convalescent sera. The ELISA demonstrated a 100% sensitivity, 98.9% specificity and an overall accuracy of 99.2% in detection of significant increases in antibody titer. Paired acute and convalescent sera from four individuals with diagnosed legionellosis were evaluated by western immuno-blot analysis in an effort to define the immune response to L. pneumophila antigens. Differences in the IgG and IgM immune responses were apparent. An IgG only seroconversion was clearly demonstrated in one individual. Monoclonal antibodies specific to the 58 kDa heat shock protein, 24 kDa Mip and common lipopolysaccharide epitopes were used as markers in the western immuno-blots. Periodate oxidation was performed on L. pneumophila serogroup specific antigens to determine the participation of carbohydrate moieties in the serologic reactivity of selected L. pneumophila positive antisera. Monoclonal serogroup specific anti-lipopolysaccharide antibodies were used in the periodate oxidation experiments to verify the effectiveness of various concentrations of periodate on carbohydrate epitopes. Sensitivity to periodate was indicated in the ELISA for all serogroups by percent absorbance reductions in the binding of the monoclonal and polyclonal antisera when compared against the absorbance of the controls. Periodate oxidation was additionally performed on transblotted L. pneumophila serogroup specific antigens in an effort to characterize the effect of periodate on serogroup specific and common epitopes and to possibly identify periodate sensitive antigens. Some sensitivity to periodate was indicated in the western immuno-blot analysis by absence of banding. In overall performance, the LEGIONELLA ELISA compared favorably with the IFA method and will be a rapid and reliable objective alternative for detection of IgG and IgM antibodies to L. pneumophila.