First Approach to an Ultramicro Enzyme-Linked Immunosorbent Assay for the Detection of Igg Antibodies Associated to Human Leptospirosis

Background: At present, leptospirosis is one of the most widespread zoonosis in the world. Both the microbiological diagnosis and the detection of previous exposition to leptospiras is challenging with the current laboratory tests, because they are consumers of high volumes of samples and time, except rapid tests used for screening.

Objective: To evaluate an Ultra micro enzyme-linked immune sorbent assay (UMELISA) for the detection of IgG antibodies associated to human leptospirosis, proposed for the Faculty of Medical Sciences of Pinar del Rio.

Methods: The UMELISA was evaluated according to the proposed instructions of supplier with a panel of positive and negative human sera to leptospirosis. Different sera dilutions and blockage were assayed. Qualitative performing parameters were calculated, using Micro agglutination test with 2 mercaptoethanol serum treatment as reference.

 Results: The test permitted the differentiation of positive and negative sera, but it did not reach good values of sensitivity (95.45%) and specificity (63.15%); therefore it was carried out a new standardization. It was determined 1:21 as optimal serum dilution instead of 1:3 proposed originally, being proven the cutoff value indicated by the authors for a histogram of frequency of the response of the UMELISA to positive and negative sera confirmed by Micro agglutination test. The blockage of the plates was introduced, obtaining a good precision of the assay for one and three batches of leptospiral antigen. The sensitivity, specificity, positive and negative predictive values were 90.78%, 89.73%, 65.34% and 97.76%, respectively. Conclusion: The proposed UMELISA could be used for the detection of IgG antibodies associated with human leptospirosis according to the modifications described in this study.

Keywords: leptospirosis; UMELISA; diagnosis; Cuba