A Convenient Homogeneous Enzyme Immunoassay for Both Small Molecules and Proteins

The enzyme-multiplied immunoassay technique (EMIT) is a little studied cousin to ELISA that normally is used for small-molecule drug testing.  Unlike ELISA, however, EMIT is a simple, homogeneous assay that also can be performed quickly without expensive instrumentation.  The EMIT approach entails covalent attachment of an antigen-bearing molecule to a reporter enzyme such that when antibody binds to the enzyme-bound antigen, reporter enzyme activity is repressed substantially.  Yet when competing free antigen (the analyte) is present in an analytical sample, antibody binds the analyte and the reporter enzyme is "switched on" to an extent related to the analyte concentration.  We have created an EMIT-based protein assay by conjugating a cysteine-modified HA peptide (from influenza hemagglutinin A) to the reporter enzyme, glucose-6-phosphate dehydrogenase.  The 13-minute assay gave a free HA limit of detection of 10 nM and proved effective for detection of a high molecular weight model protein tagged with HA.  We also have prepared an EMIT assay system for estradiol by covalently binding β-estradiol-6-(O-carboxymethyl)oxime (E2-6-CMO) to glucose-6-phosphate dehydrogenase (G6PDH).  The lower detection limit of the estradiol assay is 1 nM in aqueous solution, and the standard curve is linear on logit-log scale up to 6.7 mM estradiol.  A detection limit of 11.5 nM in estradiol-spiked human serum samples suggests the feasibility of applying this assay to monitoring estradiol levels for the prediction and prevention of ovarian hyperstimulation syndrome.  Toward understanding the antibody-induced inhibition mechanism of EMIT, characterization of morphine-glucose-6-phosphate dehydrogenase (G6PDH) conjugates was pursued.  The conjugates were characterized using a combination of methods including tryptic digestion, immunoprecipitation, matrix-assisted laser desorption/ionization mass spectrometry, and electrospray ionization tandem mass spectrometry.  Twenty-six conjugate sites were identified including some in the vicinity of the enzyme active site.  EMIT assays are convenient, versatile and powerful, and may be developed for a variety of applications including biotoxin and infectious disease detection.