*5.1.3.2 IgM enzyme-linked immunosorbent assay (ELISA)*

Normal ELISA is commonly used to diagnose leptospirosis. Enzyme immunoassay of leptospirosis can be performed using a commercially available kit or antigen obtained internally. Which is commonly used to detect IgM, and sometimes to detect IgG antibodies against leptospiral antigens. The presence of IgM antibodies indicates current or recent leptospirosis. The commercially available *Leptospira* IgM ELISA is used for the serological detection of acute leptospirosis infection in a patient's serum sample. This ELISA is based on the principle that any *Leptospira* IgM antibody present in the patient's serum binds to the *Leptospira* antigen that adheres to the microporous surface of the microwell. Residual serum was removed from these wells by washing with 1% buffer (included in the kit). Peroxidase-conjugated anti-human IgM is presented after adding to the wells, and the plate is reincubated so that the bound antigen-antibody complex binds to the conjugate. The wells are washed again and a colorless substrate system, tetramethylbenzidine hydroperoxide, is added. The substrate is hydrolyzed, and the chromogen is blue. When the reaction is stopped with phosphoric acid, TMB turns yellow. The development of color indicates the presence of visual acuity IgM antibody against *Leptospira* in serum samples [63].
