HEV IgM is an indirect solid-phase enzyme assay based on micro wells coated with recombinant antigens representing ORF2 and mosaic artificial antigen representing immunodominant regions of ORF2 and ORF3. The conjugate is a mixture of HRP-labeled anti-human IgM antibodies. Serum or plasma samples are added to the wells and if antibodies specific for HEV ORF2 or/and ORF3 immunodominant regions are present in a sample, they form stable complexes with the HEV antigens immobilized on the wells. Then the antigen-antibody complexes are identified by the addition of HRP antibody conjugate. The unbound components are removed by washing. After addition of the solution containing TMB and hydrogen peroxide, the wells with bound conjugate develop a blue colour which is converted to yellow after the reaction has been stopped with sulphuric acid. The colour intensity is directly proportional to the concentration of HEV antibodies in the specimen and can be read at 450 nm or 450/620 nm.
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