Evaluation of a western blot method for the detection of Yersinia antibodies: evidence of serological cross-reactivity between Yersinia outer membrane proteins and Borrelia burgdorferi

Abstract

Yersinia enterocolitica and Yersinia pseudotuberculosis have been identified as causative organisms of reactive arthritis in humans. We evaluated a Western blot assay which uses Yersinia outer membrane proteins as antigens for the detection of Yersinia antibodies as a replacement for the complement fixation (CF) assay. Clinical agreement, sensitivity, and specificity were determined by testing 19 positive and 21 negative serum samples by the CF assay, Western blot assay, and enzyme-linked immunosorbent assay (ELISA). The CF assay and ELISA were compared to the Western blot assay, which was the reference method used in this study. Sera with antibodies that could potentially cross-react with Yersinia were also tested by the Western blot assay. The agreement, sensitivity, and specificity of the CF method were 61%, 26%, and 95%, respectively; and those for the ELISA were 89%, 95%, and 82%, respectively. The prevalences of Yersinia antibodies in 50 healthy donors were 6% for immunoglobulin G (IgG), 2% for IgA, and 2% for IgM. Sera positive for Bartonella henselae, Brucella, Chlamydia pneumoniae, and Rickettsia rickettsii antibodies showed cross-reactivity by the Western blot assay. The highest cross-reactivity was observed with Borrelia burgdorferi; 5 of 11 (45%) specimens were cross-reactive by the IgM-specific assay. Overall, the Western blot assay performs acceptably and is more sensitive than the CF assay, warranting replacement of the CF assay in the laboratory. Due to the evidence of cross-reactivity, particularly with B. burgdorferi, which can cause an oligoarthritis similar to reactive arthritis, the diagnosis of reactive arthritis should be based on clinical findings and complete serologic analysis of the potential causative infectious pathogens.

FIG. 1.

Western blot nitrocellulose strips reacted with the individual manufacturer's negative, cutoff, and positive control sera. Each strip contains a control section and an analytical section (the separation is indicated by an arrow). The control section contains the strip number; a serum control band (SC); and an IgG, IgA, or IgM conjugate control band (CC). The analytical section of each strip contains Yop antigens of different sizes (in kDa) that develop as dark bands if antibodies against the specific Yop antigens are present in the sera: YopH (51 kDa), YopM (44 kDa), YopB (41 kDa), LcrV (37 kDa), YopD (35 kDa), YopN (33 kDa), and YopE (23 kDa). (A) Negative control showing the appearance of no positive bands in the analytical section of the strip; (B) cutoff control, showing the appearance of the 35-kDa band in the analytical section of the strip; (C) positive control, showing the appearance of 51-, 44-, 41-, 37-, 33-, and 23-kDa bands in the analytical section of the strip.