Enzyme-linked immunosorbent assay testing

Field First Aid Remove victim(s) to an area of safety (away from the Hot Zone). Remember patients may contaminate you and/or other emergency responders if you fail to don proper personal protective equipment. Provide victims with emergency medical care as soon as possible. Unless otherwise recommended, remove victim(s) clothing, shoes, and personnel belongings for later return. If the victim was obviously in contact with infectious substance(s), flush skin and eyes for fifteen to twenty minutes. Route victim(s) to hospital for a physician s professional opinion. Ensure that hospital staff is fully aware of the medical situation and the poison or infectious substance that may be involved. An enzyme-linked immunosorbent assay test (ELISA) is now approved for anthrax use in hospital laboratories. 

Naot Y, Desmonts G, Remington JS IgM enzyme linked immunosorbent assay test for the diagnosis of congenital toxoplasma infection. J Pediatr 1981 98 32-36. 

AD, as some patients do not present with such abnormalities. Skin prick tests or enzyme-linked immunosorbent assay tests can be used to aid in identification and exclusion of allergic triggers, but these are not specific or sensitive enough to be diagnostic. 

After more than two decades, advances in IHC have provided a feasible approach to performing immunostain-ing on routinely processed tissues, such that this method is now routine for the performance of IHC special stains in surgical pathology laboratories using EEPE tissues (see Appendix lA). However, demands for improved reproducibility and for quantification have led to a growing recognition that IHC has the potential to be more than just a special stain. If properly controlled in all aspects of its performance, IHC can provide a tissue-based immunoassay with the reproducibility and quantitative characteristics of an ELISA (enzyme-linked immunosorbent assay) test, which not only detects the presence of an analyte (protein or antigen) but also provides an accurate and reliable measure of its relative or real amount (see Quality Control and Standardization section). 

This is directly proportional to the quantity of analyte present. This technique advanced rapidly when monoclonal antibodies became available, since this made it possible to produce unlimited quantities of antibodies with clearly defined characteristics [20]. This second type of assay is also known as a sandwich immunoassay, and is best suited to the assay of proteins rather than haptens. The widespread ELISA (Enzyme Linked Immunosorbent Assay) test belongs to this category [21-24]. 

The ELISA (enzyme-linked immunosorbent assay) test kit for sulfentrazone and SCA residues in groundwater is developed with limit of quantitation (LOQ)... 

Even though stool examination for ova and parasites has remained the major means of diagnosis, other diagnostic tests include enzyme-linked immunosorbent assay (ELISA), which is considered to be between 85% to 98% sensitive and almost 100% specific (ProSpecT, Giardia Microplate Assay, Remel, Lenexa, KS). 

Diagnostic procedures include dark-field microscopy12, non-treponemal exams10 (i.e., the Venereal Disease Laboratory and the rapid plasma reagin test), and treponemal exams (i.e., enzyme immunoassay, the T. pallidum hemagglutination test, the fluorescent treponemal antibody test, and the enzyme-linked immunosorbent assay). 

HIV diagnosis is made either by a positive HIV enzyme-linked immunosorbent assay (ELISA) or rapid test (these tests may be positive as soon as 3 to 6 weeks after infection) and then confirmed by a positive confirmatory test, usually the HIV Western blot (Table 84-1). 

Enzyme-linked immunosorbent assay (ELISA) 3-6 weeks Plasma If nonreactive, no further testing is required, unless acute infection suspected... 

In order to measure the exact amount of a specific protein (analyte) by IHC signal intensity, a critical requirement is the availability of a standard reference material (present in a known amount by weight) that can be used to calibrate the assay (IHC stain). It is then possible to determine the amount of test analyte (protein) by a translation process from the intensity of IHC signals. In this respect it is helpful to consider the IHC stain as a tissue based ELISA assay (Enzyme Linked ImmunoSorbent Assay), noting that ELISA is used in the clinical laboratory as a standard quantitative method for measuring protein by weight in fluids, by reference to a calibrating reference standard. 

Nowadays, antibodies are utilized in numerous immunoanalytical methods. Those widely used in practice, such as radioimmunoassays, fluoroimmunoassays and enzyme-linked immunosorbent assays (ELISA), require labelled reagents. Millions of ELISA tests for diagnostics of various diseases are daily performed in clinical laboratories. The detection of analytes by two-antibody "sandwich" ELISA, is schematically outlined in Figure 3. 

Liposome conjugates may be used in various immunoassay procedures. The lipid vesicle can provide a multivalent surface to accommodate numerous antigen-antibody interactions and thus increase the sensitivity of an assay. At the same time, it can function as a vessel to carry encapsulated detection components needed for the assay system. This type of enzyme-linked immunosorbent assay (ELISA) is called a liposome immunosorbent assay or LISA. One method of using liposomes in an immunoassay is to modify the surface so that it can interact to form biotin-avidin or biotin-streptavidin complexes. The avidin-biotin interaction can be used to increase detectability or sensitivity in immunoassay tests (Chapter 23) (Savage et al., 1992). 

The most commonly used screening method for HIV is an enzyme-linked immunosorbent assay, which detects antibodies against HIV-1 and is both highly sensitive and specific. False positives can occur in multiparous women in recent recipients of hepatitis B, HIV, influenza, or rabies vaccine following multiple blood transfusions and in those with liver disease or renal failure, or undergoing chronic hemodialysis. False negatives may occur if the patient is newly infected and the test is performed before antibody production is adequate. The minimum time to develop antibodies is 3 to 4 weeks from initial exposure. 

Positive enzyme-linked immunosorbent assays are repeated in duplicate and if one or both tests are reactive, a confirmatory test is performed for final diagnosis. Western blot assay is the most commonly used confirmatory test, although an indirect immunofluorescence assay is available. 

Enzyme labels are usually associated with solid-phase antibodies in the technique known as enzyme-linked immunosorbent assay (ELISA). There are several variants of this technique employing both competitive and non-competitive systems. However it is best used in combination with two monoclonal antibodies in the two-site format in which an excess of antibody is bound to a solid phase such as a test-tube or microtitre plate the test antigen is then added and is largely sequestered by the antibody (Figure 7.12). After washing... 

Organ weights (spleen, thymus, all animals) Immunotoxicity tests (1) functional tests (either a splenic plaque-forming cell (PEC) assay or an Enzyme-Linked Immunosorbent Assay (ELISA) to determine the response to antigen administration) (2) enumeration of splenic or peripheral blood total B cells, total T cells, and T-cell subpopulations Detailed clinical observations Functional observations (sensory reactivity to stimuli of different types, grip strength, motor activity, more specialized tests on indication)... 

Currently there are few methods for specific investigation of immunotoxic effects, which are regarded as sufficiently validated for routine use (EC 2003). The plaque forming assay or the equivalent using the ELISA method (Enzyme-linked Immunosorbent Assay) are recommended to identify altered T-cell-dependent humoral responses. Of particular value for hazard assessment are the so-called host resistance models, in which the clinical relevance of immunotoxicity can be evaluated. Other methods may also be of value to provide information on the mode of immunotoxic action, e.g., mitogen stimulation tests and leucocyte phenotyping. However, further work is needed on standardization and validation of these test methods. 

Vos, J.G., Krajnc, E.l. and Beekhof, P. (1982) Use of the enzyme-linked immunosorbent assay (ELISA) in immunotoxicity testing. Environmental Health Perspectives, 43, 115—121. 

The CML is the most characterized AGE and is referred to as a glycoxidation product. The inhibitory effects of C-glycosylflavones on the CML formation were tested by enzyme-linked immunosorbent assay in kidney diabetic subjects. The results showed that the percent inhibition was about 53% for chrysoeriol 6-C-boivinosyl 7-0-glucoside, 64% for chrysoeriol 6-C-boivinosyl, 80%i chrysoeriol 6-C-fucosyl, and only 2% for 4"-OH-3 -methox-ymaysin versus 60%i for the standard glycation inhibitor, aminoguanidine. 

The assessment of DNA adducts may provide a sensitive indicatCH of previous exposure. The enzyme-linked immunosorbent assay (ELISA) has a lower limit of detection of about 0.08 femtomol per microgram of DNA (Perera et oL, 1982). This assay requires (1) the development of an antibody specific for a certain chemical metabolite bound covalently to DNA and (2) the isolation of DNA from some tissue sample, ag., skin biopsy, or lymphocytes of an exposed individual. It is anticipated that further refinement of such immunologic techniques may lower the threshold of sensitivity by one or two orders of magnitude. One such refined test is the ifitrasensitive ymatic radioimmunoassay (USE-RIA), purported to be about five times more sensitive than ELISA (Hsu et oL, 1981 Shamsuddin et oL, 1985 Harris et oL, 1985). Quantification by the development of monoclonal antibodies to aflatoxin Bj metabolites bound to DNA (Groopman et oL, 1982 Sizaret et oL, 1982) has now been reported. 

Antibodies can be combined with enzymes and color reagents or radioactive antigens to produce quantitative testing for drugs. The ELISA or Enzyme-Linked ImmunoSorbant Assay uses antibodies generated against the Ag to be tested for covalently linked to an enzyme which can catalyze a color change reaction such as the NADH to NAD conversion (Xmax at 340 nm). When the Ag-Ab complex is formed the enzyme is activated and the color can be detected. 

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