An affinity-amplified immunoassay for juvenile hormone esterase

András Székács, Shirley J. Gee, Freia Jung, Bill F. McCutchen, Bruce D. Hammock

Research output: Contribution to journalArticle

2 Scopus citations

Abstract

A method is described for increasing the specificity of an immunoassay for catalytically active enzymes and is specificically illustrated with a sensitive assay for an important regulatory enzyme from insects. Trifluoromethyl ketone haptens, potent inhibitors of insect juvenile hormone esterase, were bound to proteins such as hemocyanin (keyhole limpet) and conalbumin (chicken embryo). Haptens containing a thiol group were conjugated using heterobifunctional coupling reagents, and haptens with a carboxylic acid moiety were conjugated by the mixed anhydride method. The trifluoromethyl ketone-protein conjugates, shown to retain their inhibitory activity against juvenile hormone esterase, were used as coating antigens in several solid-phase enzyme-linked immunosorbent assay formats along with specific antibodies raised in rabbits against purified juvenile hormone esterase. The previously unreported format, termed affinity-amplified immunoassay (AAIA), was successfully used for quantitative monitoring of low levels of the esterase in dilute hemolymph and egg homogenates from various lepidopteran insect species, as well as for detection of the native and mutant forms of the enzyme obtained in a recombinant baculovirus expression system. The AAIA format was more sensitive for the target esterase and detected only the catalytically active form of the enzyme.

Original languageEnglish (US)
Pages (from-to)291-297
Number of pages7
JournalAnalytical Biochemistry
Volume207
Issue number2
DOIs
StatePublished - 1992

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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    Székács, A., Gee, S. J., Jung, F., McCutchen, B. F., & Hammock, B. D. (1992). An affinity-amplified immunoassay for juvenile hormone esterase. Analytical Biochemistry, 207(2), 291-297. https://doi.org/10.1016/0003-2697(92)90014-X