Recombinant LipL32 Protein Developed Using a Synthetic Gene Detects Leptospiraspecific Antibodies in Human Serum Samples

Abstract

Background: Synthetic biology is emerging as a viable alternative for the production of recombinant antigens for diagnostic applications. It offers a safe alternative for the synthesis of antigenic principles derived from organisms that pose a high biological risk.Methods: Here, we describe an enzyme-linked immunosorbent assay (ELISA) using the synthetic recombinant LipL32 (rLipL32) protein expressed in Escherichia coli for the detection of Leptospira-specific antibodies in human serum samples. The rLipL32-based ELISA was compared with a microscopic agglutination test (MAT), which is currently used as the gold standard for the diagnosis of leptospirosis.Results: Our results showed that all the MAT-positive serum samples were positive for Leptospira-specific IgG in an ELISA, while 65% (n = 13) of these samples were also positive for Leptospira-specific IgM. In the MAT-negative serum samples, 80% and 55% of the samples were detected as negative by an ELISA for Leptospira-specific IgM and IgG, respectively.Conclusion: An ELISA using the synthetic rLipL32 antigen was able to distinguish Leptospira-specific IgM (sensitivity 65% and specificity 80%) and IgG (sensitivity 100% and specificity 55%) in human serum samples and has the potential to serve as a rapid diagnostic test for leptospirosis.

Determination of the immunogenic region in the LipL32 protein of Leptospira

“…The study was approved by the Medical Research and Ethical Committee, Ministry of Health Malaysia (study number NMRR-13-677-15713). A detailed protocol for the production of synthetic recombinant LipL32 protein was previously described (Yaacob et al, 2017).…”

“…Determination of the immunodominant region in LipL32 by ELISA. Optimized amount of purified rLipL32 (0.25 μg/50 µl well), NrLipL32 (0.5 μg/50 µl well), CrLipL32 (0.5 μg/50 µl well), and the intermediate LipL32 fragment (0.125 μg/50 µl well) were used to coat 96-well MaxiSorp immunoassay plates (Nunc, Denmark), and the ELISA was performed as previously described (Yaacob et al, 2017). In order to determine the concentrations of antibodies in the serum samples, a standard curve was obtained using human IgM and IgG (25-0.0488 ng/ml).…”