Immunoassay employing an enzyme label

A quantitative assay method for the determination of an unknown quantity of a hapten, hereinafter designated hapten X, which method comprises:

• a. preparing a conjugate of the hapten X which conjugate is adapted to ellicit antibody formation, and injecting a mammal with said conjugate so as to result in a specific antibody against said hapten, anti-X;
• b. preparing a conjugate of the hapten X and of another entity Y, which is either another hapten or a larger molecule;
• c. injecting a mammal with the entity Y if it is a larger molecule, or with a conjugate thereof if it is a small molecule which does not by itself ellicit antibody formation, so as to form anti-Y antibodies;
• d. preparing a conjugate of an enzyme and the antibody against Y to form enzyme-labelled anti-Y antibody;
• e. adsorbing the anti-X antibodies on a solid support;
• f. contacting said solid support with a mixture containing the unknown quantity of hapten X and a known quantity of the X-Y conjugate so as to cause all anti-X sites to be occupied;
• g. removing unreacted X and X-Y, leaving all anti-X sites occupied;
• h. contacting the support carrying occupied anti-X sites with enzyme-labelled anti-Y antibody;
• i. contacting the resulting support carrying occupied anti-X-sites and enzyme-labelled anti-Y antibody with a substrate for the enzyme which when acted upon by the enzyme produces a color reaction and measuring the resulting intensity of color so as to determine the quantity of bound enzyme and deducing from calibration curve the quantity of the hapten X.