William P. Vann

A luminol-assisted, competitive-binding immunoassay of human immunoglobulin G

Publisher Summary This chapter discusses the different aspects of a luminol-assisted competitive binding immunoassay of human immunoglobulin G. The use of luminol as a chemiluminescent label for immunoassays has several advantages. Luminol is readily available and inexpensive, can be coupled to large and small molecules, and is stable. The separation of reacted and unreacted analyte is accomplished by a precipitin reaction. The precipitate is solubilized and tested for chemiluminescent activity (CA). This approach may be extended to the nonprecipitating immune complexes, by the use of either a second antibody or a separating agent, such as polyethylene glycol. The CA was monitored, using a forced flow system, together with inexpensive light-monitoring equipment. The luminol-IgG label was synthesized, using a periodate oxidation of IgG 2 , and subsequent Schiffs base formation with luminol. Increasing the initial mole ratio of luminol to IgG from 100- to 1000-fold only increased the final effective level of conjugation to 4 luminol molecules per molecule of IgG. The oxidation of IgG with periodate was performed in the presence of excess luminol in order to prevent copolymerization of IgG. The immunological activity of the luminol-IgG label was also determined in a precipitin reaction with rabbit anti-IgG.