Evaluation of Leptospiral Recombinant Antigens MPL17 and MPL21 for Serological Diagnosis of Leptospirosis by Enzyme-Linked Immunosorbent Assays

Abstract

Leptospirosis is a zoonosis of multisystem involvement caused by pathogenic strains of the genus Leptospira.In the last few years, intensive studies aimed at the development of a vaccine have provided important knowledge about the nature of the immunological mechanisms of the host. The purpose of this study was to analyze the immune responses to two recombinant proteins, MPL17 and MPL21 (encoded by the genes LIC10765 and LIC13131, respectively) of Leptospira interrogans serovar Copenhageni in individuals during infection. The recombinant proteins were expressed in Escherichia coli as six-His tag fusion proteins and were purified from the soluble bacterial fraction by affinity chromatography with Ni 2؉ -charged resin. The recombinant proteins were used to evaluate their ability to bind to immunoglobulin G (IgG) (and IgG subclass) or IgM antibodies in serum samples from patients in the early and convalescent phases of leptospirosis (n ‫؍‬ 52) by enzyme-linked immunosorbent assays. The prevalences of total IgG antibodies against MPL17 and MPL21 were 38.5% and 21.2%, respectively. The titers achieved with MPL17 were statistically significantly higher than those obtained by the reference microscopic agglutination test. The specificity of the assay was estimated to be 95.5% for MPL17 and 80.6% for MPL21 when serum samples from individuals with unrelated febrile diseases and control healthy donors were tested. The proteins are conserved among Leptospira strains that cause human and animal diseases. MPL17 and MPL21 are most likely new surface proteins of leptospires, as revealed by liquid-phase immunofluorescence assays with living organisms. Our results demonstrate that these recombinant proteins are highly immunogenic and, when they are used together, might be useful as a means of diagnosing leptospirosis.

Development of an Enzyme-Linked Immunosorbent Assay Using a Recombinant LigA Fragment Comprising Repeat Domains 4 to 7.5 as an Antigen for Diagnosis of Equine Leptospirosis

“…Most state animal diagnostic laboratories in the United States use MAT for the serologic diagnosis of animal leptospirosis. Because of the serious drawbacks of the MAT assay, numerous attempts have been made to develop a serodiagnostic ELISA (10)(11)(12)(13)(14)(15)(16)(17)(18) or to develop a dual-path platform (DPP) assay (19). We previously used the LigACon protein for diagnosis of equine and canine leptospirosis (26,27).…”

“…Previously, several research groups have attempted to utilize different recombinant proteins from Leptospira spp. as antigens in an indirect enzyme-linked immunosorbent assay (ELISA) as a serodiagnostic test (10)(11)(12)(13)(14)(15)(16)(17)(18)(19). However, as far as we know, no one has applied them to equine leptospirosis diagnosis.…”

OmpL1 Is an Extracellular Matrix- and Plasminogen-Interacting Protein of Leptospira spp

“…Human and hamster IgG antibodies against OmpL1 were detected by an ELISA, as previously described (50). In brief, MAT-negative and -positive serum samples from 32 patients with confirmed leptospirosis and 44 experimentally infected hamsters were diluted (1:100) and evaluated for total IgG by using HRP-conjugated anti-human IgG antibodies (1:5,000) (Sigma) and HRP-conjugated anti-hamster IgG antibody (1:5,000).…”

Characterization of Three Novel Adhesins of Leptospira interrogans

“…Human IgG against Lsa26, Lsa23, and Lsa36 was detected by using an enzyme-linked immunosorbent assay (ELISA) as described. 32 In brief, positive MAT serum samples from 16 confirmed-leptospirosis patients were diluted 1:200 and evaluated for total IgG by using horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibodies (1:5,000) (Sigma-Aldrich). Data were analyzed by using Graph Pad Prism software version 3.0 (Graphpad, San Diego, CA).…”