PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODY AGAINST PATHOGENIC Leptospira spp.

Abstract

Leptospirosis is a widespread zoonotic disease in tropical areas caused by pathogenic gram-negative spirochetes Leptospira spp., which affect both human and animals. The disease is transmitted by contacting urine of the infected animals. Leptospira penetrates through mucosa or open wound skin of infected individuals. Symptoms of leptospirosis are extremely broad such as flu-like illness, headache. If patients are not diagnosed or treated in time, symptoms can become severe sepsis with multi-organ failure. This study aimed to generate monoclonal antibodies (MAbs) against pathogenic Leptospira spp. that can be used in a development of immunological based assay for early diagnosis of leptospirosis. Mice were immunized with whole cells of fixed or mixed of fixed and sonicated form of Leptospira interrogans serovar Manilae and the mutant M1352. After the conventional cell hybridization technique, all MAbs were screened by enzyme-linked immunosorbent assay (ELISA) with sonicated cell lysates of various serovars of Leptospira and other bacteria as antigen. The results showed that 14 clones of MAbs were obtained in this study could be divided into 6 groups based on the specificity against bacteria that were tested. MAbs group 1, 2, 3 and 4 were specific to some serovars of pathogenic Leptospira spp. MAbs group 5 and 6 were specific to various serovars of pathogenic leptospires, and also shown cross reactivity with Enterobacter aerogenes. These MAbs could detect L. interrogans serovar Manilae with the half maximal effective concentration (EC50) in the concentration range of 2×106 to 1×107 cells/ml and the limit of detection (LOD) was in the concentration range of 4.7×105 to 3.5×105 cells/ml. All 14 MAbs were isotyped as IgM. By Western blotting, there were some of the obtained MAbs recognizing the protein-like antigen of L. interrogans serovar Manilae with the molecular weight of 41 kDa. Therefore, these preliminary studies indicated that some of the obtained MAbs could be used for application in an immunological detection of pathogenic Leptospira spp.