Enzyme multiplied immunoassay technique
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|Enzyme multiplied immunoassay technique|
Enzyme multiplied immunoassay technique (EMIT) is a common method for qualitative and quantitative determination of drugs and certain proteins in serum and urine. First introduced by Syva Company in 1973, it is the first "homogeneous immunoassay" to be widely used commercially.
Homogeneous immunoassays can be carried out using exceptionally simple and rapid mix and read protocols. The most widely used applications for EMIT are for therapeutic drug monitoring (serum) and as a primary screen for abused drugs and their metabolites in (urine). The original US patents covering major aspects of the method, 3,817,837 and 3,875,011, have expired although US patent 6,455,288 that covers a major improvement is still valid. While still sold by Siemens Healthcare under its original trade name, EMIT, assay kits with different names that employ the same technology are supplied by other companies.
Determination of drug levels in serum is particularly important when the difference in the concentrations needed to produce a therapeutic effect and adverse side reactions is small, the therapeutic window. EMIT therapeutic drug monitoring tests provide accurate information about the concentration of such drugs such as digoxin and other cardioactive drugs, theophylline, anti-epileptic drugs, and antibiotics. EMIT urine assays for drugs such as cannabinoids, morphine, and amphetamine are designed to detect the drug itself or a metabolite of the drug present in a concentration above a pre-specified minimum detection cutoff limit. In the U.S., the cutoff limits must be set in accordance with Mandatory Guidelines for Federal Workplace Drug Testing Programs that were developed by SAMHSA (The Substance Abuse and Mental Health Services Administration is a branch of the U.S. Department of Health and Human Services). The setting of reasonable cutoff limits help reduce false positive results that occur from assay limitations. Because of the social and legal consequences, a positive test result must be confirmed by an alternative method, usually Gas Chromatography/Mass spectrometry. As an example the SAMHSA cutoffs for cannabinoids are 50 ng/ml for the immunoassay and 15 ng/ml as confirmed by GC/MS. Immunoassays that do not conform with SAMHSA, featuring a cutoff of 20 ng/ml, have been shown to produce false positives from passive inhalation of marijuana smoke.
Like all immunoassays, EMIT uses antibodies that are specifically designed to bind the molecule(s) of interest (analyte) without binding to other substances in the sample. Its unique feature is the ability to detect this binding without resorting to a cumbersome separation of the bound component. This is accomplished by including (in the mixture of antibodies and sample) an enzyme that is attached to the analyte being tested for. Antibodies that do not become bound to the sample drug bind instead to this analyte-bound enzyme. The analyte-bound enzyme is designed so that when antibodies bind to its analyte portion, the enzyme is deactivated. Two general examples are here given:
- If a high amount of sample analyte is present, this sample analyte will bind a large portion of antibodies leaving a large portion of the analyte-bound enzymes free in solution. Much substrate will be converted by high concentration of free enzyme.
- If a low concentration of sample analyte is present, this small concentration of sample analyte will bind only a small portion of the antibodies, leaving a large portion of the antibodies to bind the analyte-bound enzymes and deactivate them. In this case, much of the substrate will not be converted.
- Combine a sample suspected of containing the drug of interest with a solution containing a known concentration of antibody and the enzyme substrate.
- After a short period (usually less than a minute) to allow binding, add a known concentration of the conjugate.
- Measure the rate of appearance of color or fluorescence.
- Determine the drug concentration by comparing the observed rate to the rates produced by known concentrations of the drug.
- http://www.gpo.gov/fdsys/pkg/FR-2010-08-16/pdf/2010-20095.pdf[full citation needed]
- Cone, E. J.; Bigelow, G. E.; Herrmann, E. S.; Mitchell, J. M.; Lodico, C.; Flegel, R.; Vandrey, R. (2014). "Non-Smoker Exposure to Secondhand Cannabis Smoke. I. Urine Screening and Confirmation Results". Journal of Analytical Toxicology 39 (1): 1–12. doi:10.1093/jat/bku116. PMID 25326203.
- "The Immunoassay Handbook", 4th Edition, David Wild, Ed., Elsevier,2013, p 76