THE DEVELOPMENT OF A WESTERN BLOT (IMMUNOBLOT) ASSAY FOR THE DETECTION OF IgG AND IgM ANTIBODIES TO TREPONEMA PALLIDUM IN HUMAN SERUM

Abstract

A Western Immunoblot assay was developed to detect IgG and IgM antibodies to Treponema pallidum, the causative agent of Syphilis, in human serum. A total of 220 human serum samples and 3 cerebral spinal fluid samples were run on the Syphilis Western ImmunoBlot (SWIB) IgG assay and the Fluorescent Treponemal Antibody Absorption (FTA-ABS) assay to determine relative sensitivity, specificity and accuracy. High specificity for the 14, 15 and 48 kDa antigens was demonstrated with FTA-ABS positive samples versus FTA-ABS negative samples. The development of only one of these three bands in the SWIB IgG assay was indicative of a positive sample. Sensitivity, specificity and accuracy were checked further by running 30 human serum samples from a well-defined CDC syphilis panel and 14 potentially cross- reactive autoimmune samples on the SWIB IgG assay. The sensitivity, specificity and accuracy for all 267 samples were 97.4% (111/114), 98% (150/153) and 97.8% (261/267), respectively. A total of 151 human serum samples were tested on the SWIB IgM assay and a commercially available Syphilis IgM Capture ELISA to determine relative sensitivity, specificity and accuracy. A lack of specificity with the 37 kDa antigen was demonstrated. Overall, very few bands were visualized on IgM blots and no definitive positive cut-off was evident. Three samples were equivocal by ELISA and were not included in the calculations. The sensitivity, specificity and accuracy of the SWIB IgM assay were checked further by running 14 potentially cross-reactive autoimmune samples. With the appearance of one band other than the 37 kDa antigen band as indicative of a positive, the sensitivity, specificity and accuracy for 162 samples tested were 46.7% (14/30), 84.8% (112/132) and 77.8% (126/162), respectively. Immunochemical analysis of separated Treponema pallidum antigens immobilized on nitrocellulose strips was conducted using mild periodate oxidation to identify any carbohydrate moieties. The 31, 34 and 41 kDa antigen bands lost reactivity with mild periodate oxidation compared with control strips. These antigens are possible glycoproteins of Treponema pallidum.