In the secondary reaction an anti immunoglobulin (anti-Ig; an antibody that reacts with antibodies) is added into the vessel and allowed to react with the Ag-Ab complex already formed; the anti-Ig binds to the antibody component of the Ag-Ab complex. The anti-Ig is linked or conjugated to an appropriate enzyme molecule (i.e., is labelled with an enzyme molecule) in such a way that its anti-Ig activity is not impaired.

The unreacted anti-Ig is washed away and, finally, substrate of the enzyme is added along with the necessary reagents to develop colour due to the enzyme activity. The intensity of colour is proportional to the enzyme concentration; therefore, colour intensity is used to determine the quantity of antigen or antibody or simply to detect their presence.

For an easy and a rapid assay, a computerised ELISA reader may be used. The sensitivity of ELISA is in the range of nanograms (10-9 g)/ml. This method of ELISA is often called direct antigen coating (DAC) ELISA. This approach is useful in estimating the amount of specific antibody present in the antiserum used for the primary reaction.

The protein A may be first immobilized in the microtiter well, to which the unlabelled antigen specific antibody is added; the antibody becomes immobilized due to the binding of protein A in the Fc region. The rest of the procedure is the same as that for DAS ELISA described in the preceding paragraph. The enzymes used for labelling of antibodies are horseradish peroxidase (most commonly used), alkaline phosphatase, β-galactosidase, lacto­ peroxidase, etc.

In case of peroxidase, the substrate hydrogen peroxide (H₂O₂) is converted into water and O₂ in the presence of electron donors like diaminobenzidine or 4-chloronaphthol which are themselves oxidized in the reaction. Oxidation of diaminobenzidine produces dark brown colour, while that of 4-chloronaphthol yields purple colour, which is the basis of the ELISA.