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

Abstract

Leptospira immunoglobulin (Ig)-like (Lig) proteins are a novel family of surface-associated proteins in which the N-terminal 630 amino acids are conserved. In this study, we truncated the LigA conserved region into 7 fragments comprising the 1st to 3rd (LigACon1-3), 4th to 7.5th (LigACon4-7.5), 4th (LigACon4), 4.5th to 5.5th (LigACon4.5-5.5), 5.5th to 6.5th (LigACon5.5-6.5), 4th to 5th (LigACon4-5), and 6th to 7.5th (LigACon6-7.5) repeat domains. All 7 recombinant Lig proteins were screened using a slot-shaped dot blot assay for the diagnosis of equine leptospirosis. Our results showed that LigACon4-7.5 is the best candidate diagnostic antigen in a slot-shaped dot blot assay. LigACon4-7.5 was further evaluated as an indirect enzyme-linked immunosorbent assay (ELISA) antigen for the detection of Leptospira antibodies in equine sera. This assay was evaluated with equine sera (n = 60) that were microscopic agglutination test (MAT) negative and sera (n = 220) that were MAT positive to the 5 serovars that most commonly cause equine leptospirosis. The indirect ELISA results showed that at a single serum dilution of 1:250, the sensitivity and specificity of ELISA were 80.0% and 87.2%, respectively, compared to those of MAT. In conclusion, an indirect ELISA was developed utilizing a recombinant LigA fragment comprising the 4th to 7.5th repeat domain (LigACon4-7.5) as a diagnostic antigen for equine leptospirosis. This ELISA was found to be sensitive and specific, and it yielded results that concurred with those of the standard MAT.

Fig 1

A schematic diagram showing the structure of LigA proteins and the truncated LigA proteins used in this study.

Fig 2

Expression of truncated LigA proteins. Shown are stained SDS-PAGE gels. Analysis of affinity chromatography-purified recombinant fragments by Coomassie brilliant blue-stained SDS-PAGE. Lane M, molecular mass (kDa)marker; lane 1, LigACon1-3; lane 2, LigACon4-7.5; lane 3, LigACon4-5; lane 4, LigACon4.5-5.5; lane 5, LigACon5.5-6.5; lane 6, LigACon6-7.5; lane 7, LigACon4.

Fig 3

Reactivity of different truncated LigA proteins to Leptospira-positive serum in slot blot assays. (A) Analyzed results using Phoretix 1D software. Data are plotted as pixel intensity versus pixel location (the distance from left to right on the membrane). Lane 1, LigACon1-3; lane 2, LigACon4-7.5; lane 3, LigACon6-7.5; lane 4, LigACon4.5–5.5; lane 5, LigACon4-5; lane 6, LigACon5.5–6.5; lane 7, LigACon4. (B) Scanned slot blot assay image.

Fig 4

Kinetic curves of horse antibodies to rLigACon4-7.5 tested by ELISA at different days postinfection. The graph shows the OD readings of rLigACon4-7.5 ELISA using sera from three horses experimentally infected with Leptospira interrogans serovar Kennewicki at different days postinfection. Antibody was detected as early as day 3 after infection, increased over time, and reached a peak after day 14.

Fig 5

Graph of the ELISA samples showing the IgG ELISA reactivity of 220 equine sera. The x axis indicates the MAT titers of the tested sera. The y axis indicates the ELISA reading of OD₄₅₀.

Fig 6

Western blot results of sera that were MAT negative but ELISA positive. Lanes 1 through 12, sera that are MAT negative and ELISA positive; lane 13, MAT positive and ELISA positive (positive control); lane 14, MAT negative and ELISA negative (negative control).