If these specific antibodies are present in the patients blood sample, they will bind to the antigen. In the subsequent reaction step, an enzyme-conjugated secondary antibody binds to the target antibody, bound to the antigen.
The enzyme transforms an added substrate into a fluorescent product. Comparing the fluorescence signal with that of calibrators of known concentrations enables the antibody concentration in the test sample to be determined.
- The antigen of interest, coated to the solid phase, binds the specific antibodies (e.g. of IgG class) in the patient sample.
- After unbound, non-specific antibodies have been washed away, enzyme-labelled antibodies against the target antibody (e.g. of IgG class) are added to form a complex.
- After incubation, unbound enzyme-labelled antibodies are washed away and the bound complex is then incubated with a developing agent.
- After the reaction has been stopped, the fluorescence of the eluate is measured. The higher the fluorescence, the more specific antibodies (e.g. of IgG class) are present in the sample.