assays are serological tests that examine for the presence or adsence of antibody in human serum directed against the pathogen being tested. ELISA is the first test done in assessment of a patient with suspected HIV infection and positive ELISA results are confirmed by western blotting.The most frequently used form of ELISA is the indirect form where the test detects antibody in the patient's serum directed against a known antigen. The specific steps of this assay are outlined below.
1.Known antigen,such as antigen from the HIV virus,is fixed to the bottom of a well.
2.Patient serum is then added to these wells containing known antigen. If present, patient antibody specific for the known antigen will bind the antigen and remain fixed to the well.
3.The plate is washed to remove unbond patient antibody. After this wash, only the patient antibody-known antigen complexes(Patient anti-HIV antibody complexed to HIV antigen ) remain attached to the well.
4.Next a second antibody is added to the wells. This antibody is an anti-human IgG antibody, and as such it will bind to the patient immunoglobulin that was bound to the fixed antigen in the previous steps. These second antibodies are coupled to the substrate-modifying enzyme (''enzyme linked'')
5.Wash the wells so that excess unbound enzyme-linked antibodies are removed.
6.Apply a substrate, such as chromogen, which is converted by the enzyme to elicit a chromogenic or fluorescent signal.
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