A New Enzyme-Linked Immunosorbent Assay (ELISA) for Spermidine Using Glutaraldehyde Coupling of the Hapten to Carrier-Coated Microtiter Plates

Abstract

An enzyme-linked immunosorbent assay (ELISA) for small molecular haptens needs conjugates of the hapten with larger protein molecules for coating the wells of microtiter plates. The formation of such conjugates is not always reproducible. This makes it difficult to evaluate hapten-protein stoichiometries and to understand the precise orientation of the hapten on the protein. In this paper we describe an assay in which the polyamine spermidine (Spd) is coupled with glutaraldehyde (GA) to carrier poly-L-lysine (PL) or human serum albumin (HSA) coated on polystyrene microtiter wells. Each step of the assay was tested for maximum efficiency. This ELISA detected Spd with excellent reproducibility (coefficient of variation = 5.9%), an EC50 of 4.3 x 10(-6) M and a detection limit of 0.1 x 10(-6) M. The present ELISA was about 100 times more sensitive in detecting Spd than a conventional ELISA for Spd using a Spd-HSA conjugate for coating microtiter wells. The Spd antiserum showed 215% cross-reaction with N1-acetyl-Spd, 30.7% with N8-acetyl-Spd, 10% with spermine, 3.3% with cadaverine, 1.7% with putrescine, and less than 0.43% with 1,3-diaminopropane in the new ELISA system. A much higher degree of hapten binding to the plate occurred with the present method than with the previously reported method using microtiter wells activated directly with GA. The hapten conjugation is simple, reproducible and should provide a general method for developing ELISAs, not only for polyamines but also for amino acid and small peptides.