(804a) 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.