Microplates are coated with Tp immunodominant synthetic antigens (recombinant p47, p17 and TmpA).In the 1st incubation, the solid phase is treated with diluted samples and anti-Tp IgM are captured, if present, by the antigens.
After washing out all the other components of the sample, in the 2nd incubation bound anti-Tp IgM are detected by the addition of anti hIgM antibody, labeled with peroxidase (HRP). The enzyme captured on the solid phase, acting on the substrate/chromogen mixture, generates an optical signal that is proportional to the amount of anti-Tp IgM antibodies present in the sample.
The presence of IgM in the sample may therefore be determined by means of a cut-off value able to discriminate between negative and positive samples.
Neutralization of IgG anti-Tp, carried out directly in the well, is performed in the assay in order to block interferences due to this class of antibodies in the determination of IgM.