EVALUATION OF RECOMBINANT ANTIGENS AGAINST LEPTOSPIROSIS SERUM SAMPLES

Rosane de Oliveira (Instituto Butantan); Zenaide Maria de Morais (USP); Eliete Caló Romero (IAL); Silvio Arruda Vasconcellos (USP); Ana Lucia Tabet Oller do Nascimento (Instituto Butantan)

Introduction: Leptospirosis is worldwide zoonotic disease caused by pathogenic spirochaetes of the genus Leptospira. The gold standard reference method for the serological diagnosis of leptospirosis is the microscopic agglutination test (MAT). However MAT presents low sensitivities during the early phase of the disease. In this context, the aim of this project is to study three genes selected from the genome sequences of Leptospira interrogans serovar Copenhageni and to evaluate their immune reactivity in sera from patients diagnosed with leptospirosis. Methods: The genes LIC10258, LIC12880 and LIC12238 were cloned in expression vector pAE and inserted in BL21 SI E. coli strain for proteins expression. The recombinant proteins were purified by using affinity chromatography. The secondary structure content of the purified proteins was evaluated by circular dichroism (CD) spectroscopy; the cellular localization was performed by liquid-phase immunofluorescence assay (L-IFA) and the reactivity of proteins with human sera of patients diagnosed with leptospirosis was analyzed by ELISA. Results and Discussion: All proteins showed secondary structures as assessed by CD spectroscopy. The results obtained with the L-IFA suggest that rLIC12880 is surface exposed. The sensitivity of the proteins against serum from early and convalescent phase of confirmed leptospirosis patient was evaluated by ELISA. All proteins tested were reactive with IgG and IgM antibodies present in convalescent phase, whereas in the early phase only rLIC10258 showed significant frequency of responder against IgG antibodies. The reactivity was detected in 30% of the analyzed samples when undetectable agglutination reaction was achieved by MAT. This is important since in general, the sensitivity of the tests in the first week of illness has been reported to be under 25%. Therefore, further evaluation of the rLIC10258 with larger samples will indicate its appropriateness for diagnosis purposes.