Effect of Heterologous Combination on Competitive Nephelometric Immunoassay. I. Site-Heterologous Combination for Theophylline Immunoassay

A competitive nephelometric immunoassay that is based on the inhibition of immunoprecipitation by a hapten was improved in specificity and in sensitivity for theophylline by the use of a heterologous combination of the assay reagents. Each of two theophylline derivatives, theophylline-7-propionic acid and theophylline-8-butyric acid, was conjugated to bovine serum albumin. These conjugates were injected into rabbits to raise anti-theophylline antiserum. Each derivative was also conjugated to human serum albumin. The competitive nephelometric immunoassay was performed by combined use of this human serum albumin conjugate with the antiserum. This immunoassay system allowed two homologous combinations and two heterologous combinations. Of these four combinations, the heterologous combination of the conjugate of (theophylline-7-propionyl)-(human serum albumin) with the antiserum against (theophylline-8-butyryl)-(bovine serum albumin) gave the most sensitive immunoassay for theophylline with the lowest interference by caffeine.