Ria: Principles and Application
Fuad S. Ashkar, Lelio G. Colombetti in Radiobioassays, 2019
There are two types of enzyme assays. The homogeneous type requires no separation steps, and the heterogeneous type requires at least one separation step. The general reaction is between the test substance (analyte), the same substance labeled with an enzyme, and a specific antibody or binding protein. They all react competitively in the same manner as in the RIA procedure (Figure 13). In the homogeneous assay, an enzyme that is covalently linked to an antigen is inactivated (or activated) by complexing with antibody.90 Competition between enzyme-labeled and nonlabeled antigen allows the enzyme to react with the substrate. A quantitative measurement can then be made spectrophotometrically (EMIT®). EMIT® is probably the most widely used enzymeimmunoassay method. The activity of the enzyme is proportional to the concentration of antigen (analyte) present in the patient’s serum. The EMIT® assay is especially useful for low molecular weight substances that tend to be present in relatively high concentrations.91
Saturation Analysis: Radioimmunoassay and Other Ligand Assays
Joseph Chamberlain in The Analysis of Drugs in Biological Fluids, 2018
The enzyme-multiplied immunoassay technique commercially marketed by Syva Corporation as EMIT kits has proved a very popular method for the semiquantitative analysis of drugs of abuse,1140 and has been extended to quantitative therapeutic drug monitoring. In this procedure, the drug molecule is covalently linked to a stable enzyme such as glucose-6-phosphate dehydrogenase to provide the tracer. When the immunoassay procedure is carried out, the unbound tracer remains active, whereas the bound tracer loses its enzyme activity. Hence, rather than a separation step, the enzyme activity is measured after adding a suitable substrate. The sensitivity of the standard enzyme labeled assays is of the order of μg ml−1 rather than ng ml−1. For enzyme-labeled assays, the special reagents are the drug-enzyme complex and the antiserum; for specific drugs, these and other standards are supplied in kit form. Table 8.4 lists some of the procedures and their evaluation by comparison with other analytical techniques. One great advantage of enzyme-labeled assays is that all the steps of the assay can be performed in the same vessel, making it a homogeneous assay. Thus the technique is very suitable for conversion into ready-made kits for rapid use by nonlaboratory personnel.1147–1149
Detection and Identification of Amphetamine and Related Stimulants
John Caldwell, S. Joseph Mulé in Amphetamines and Related Stimulants: Chemical, Biological, Clinical, and Sociological Aspects, 2019
The procedure for EMIT involves adding to untreated urine known amounts of the enzyme substrate and the reagent containing antibodies to a particular drug. If the urine contains a drug recognized by the antibody, binding occurs. A second reagent containing enzyme-labeled drug is then added, and competes for unfilled antibody binding sites. If some fraction of the binding sites is occupied, the enzyme is not totally inactivated and the enzyme activity is directly releated to the amount of drug present in the urine.
Published population pharmacokinetic models of valproic acid in adult patients: a systematic review and external validation in a Chinese sample of inpatients with bipolar disorder
Published in Expert Review of Clinical Pharmacology, 2022
Yan-Nan Zang, Wei Guo, Fang Dong, An-Ning Li, Jose de Leon, Can-Jun Ruan
The demographic and clinical characteristics of the included patients in the 13 PPK models are summarized in Table 1. Of the 13 PPK models, 6 were from Asian countries, based on the Food and Drug Administration’s definition of Asian ancestry: models A [23], B [24], D [26] and J [30] from Japan, model K [31] from China, and model L [55] from Thailand. Seven were from non-Asian countries, including one from a Middle Eastern country, model M [32] from Saudi Arabia; 4 were from European countries, models F [27], G [28] and H [29] from Serbia and model C [25] from Spain; and 2 were from American countries, model E [54] from the United States and model I [57] from Uruguay. Among the models, 10 were developed in epileptic patients [23–32], 1 in both epileptic and psychiatric patients [54], 1 in manic patients [55], and 1 in healthy subjects [57]. Four were developed with less than 100 individuals [27,29,32,57]. Supplementary Table S1 provides the formulas and parameters included in the PPK model. The analytic methods were: 1) FPIA in 7 studies [23–25,27,29,31,32], 2) enzyme multiplied immunoassay technique (EMIT) in 3 studies [28,30,55], 3) high performance liquid chromatography-ultraviolet-visible (HPLC-UV-Vis) method in 1 study [57], 4) both FPIA and EMIT in 1 study [26] and 5) not reported in another study [54].
Mass spectrometry in emergency toxicology: Current state and future applications
Published in Critical Reviews in Clinical Laboratory Sciences, 2019
Xander M. R. Van Wijk, Robert Goodnough, Jennifer M. Colby
The proposed NACB tier 1 test menu makes use of qualitative screening for drugs-of-abuse (DOA) that can be performed by most hospital laboratories using standard immunoassays. These assays employ antibodies directed against specific drugs or drug classes and results can generally be produced in fewer than 60 min. Because the targets are small molecules, these assays are based on a competition principle, i.e. free drug in urine, or in some cases blood or saliva, competes with a labeled drug for a limited number of antibody binding sites. In the clinical laboratory, these tests are performed on automated chemistry analyzers and the most common methods are cloned enzyme donor immunoassay (CEDIA), enzyme multiplied immunoassay technique (EMIT), and DRI®, all of which produce color or NADH in an enzyme-mediated reaction if the free drug is present. Additional common methods include fluorescence polarization immunoassay (FPIA), where free drug reduces polarized fluorescence, and kinetic interaction of microparticles in solution (KIMS), where free drug reduces aggregation of microparticles.
Psychiatric and non-psychiatric drugs causing false-positive amphetamines urine test in psychiatric patients: a pharmacovigilance analysis using FAERS
Published in Expert Review of Clinical Pharmacology, 2023
Vera Battini, Giovanna Cirnigliaro, Luca Giacovelli, Maria Boscacci, Silvia Massara Manzo, Giulia Mosini, Greta Guarnieri, Michele Gringeri, Beatrice Benatti, Emilio Clementi, Bernardo Dell’Osso, Carla Carnovale
Immunoassay techniques being used for UDS included cloned enzyme donor immunoassay, enzyme-multiplied immunoassay technique (EMIT, a form of enzyme immunoassay), fluorescence polarization immunoassay (FPIA), immunoturbidimetric assay, and radioimmunoassay (RIA). Mass spectrometry (MS) was the most used methodology to verify the results obtained by immunoassay technique. MS is considered the gold standard for confirmatory testing. By definition, all positive results on MS are true positives. The latter is the most accurate, sensitive, and reliable method of testing, able to identify even minimal quantities of substance; however, the test is time-consuming, costly, requires a high level of expertise to perform, and, above all, is limited or nonexistent in many hospital laboratories. Therefore, in clinical practice, positive results from immunoassay are rarely checked with this confirmatory test [53]. Another important limitation of MS is that, once performed, results are generally unavailable for days. Therefore, during the daily practice routine, clinicians are generally prone to trust the result of the immunoassay [39,47,72,73]. Moreover, old GC–MS techniques for confirmation did not have specific isomeric detection for amphetamines and their derivatives. As an example, the l-methamphetamine is marketed as a nasal decongestant, while the other isomer is a drug of abuse: the marketed drug might have percentages of impurity that are detected by mistake. Failures in the identification of the correct methamphetamine stereoisomer in urine might lead to incorrect interpretation of UDS. Nowadays, new techniques are available but still cannot always distinguish between the pharmacologically approved stereoisomer and its impurity [74].
Related Knowledge Centers
- Immunoassay
- Immunosuppressive Drug
- Screening
- Urine
- Serum
- Therapeutic Index
- Drug Test
- Blood Test