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Enzyme Multiplied Immunoassay (EMIT) for qualitative and quantitative determination of proteins (CAT#: STEM-MB-0172-WXH)

Introduction

Enzyme multiplied immunoassay technique (EMIT) is a common method for qualitative and quantitative determination of therapeutic and recreational drugs and certain proteins in serum and urine.<br />It is an immunoassay in which a drug or metabolite in the sample competes with a drug/metabolite labelled with an enzyme, to bind to an antibody. The more drug there is in the sample, the more free enzyme there will be, and the increased enzyme activity causes a change in color.<br />In ELISA, the enzyme is a passive passenger through the actual immunoassay. In EMIT, the enzyme plays a key role throughout the assay process. ELISA requires very little knowledge of enzyme technology, whereas enzymology is the key to success in EMIT.




Principle

The EMIT assay is based on competition between drug in the specimen and drug tagged to the enzyme glucose-6-phosphate dehydrogenase for antibody binding sites. Enzyme activity decreases upon binding of the drug to the antibody. The enzyme converts glucose-6-phosphate into glucose-6-phosphate gluconate. The reaction is coupled to the conversion of NAD to NADH. Enzyme activity is determined by spectrophotometric measurement of the absorbance of the NADH. An increase in absorbance indicates the presence of the drug in the analyzed sample.

Applications

• Qualitative and quantitative determination of therapeutic and recreational drugs and certain proteins in serum and urine.
• Used to presumptively identify several drugs or drug metabolites and/or drug classes as being present in biological specimens.

Procedure

1. Mix sample containing drug with fixed quantity of enzyme bound drug, and antibody
2. Add substrate
3. Measure absorbance at 15 and 45 seconds after substrate addition
4. Quantitate by measuring enzyme-substrate reaction (by UV - visible spectroscopy)
5. Determine standard curve