Enzyme-linked immunosorbent materials

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. 

A. Heginbotham, V. Millay, M. Quick, The use of immunofluorescence microscopy (IFM) and enzyme linked immunosorbent assay (ELISA) as complementary techniques for protein identi fication in artists materials, J. Am. Inst. Cons., 45, 89 105 (2006). 

Enzyme-linked immunosorbent assay (ELISA) is comparable to the immuno-radiometric assay except that an enzyme tag is attached to the antibody instead of a radioactive label. ELISAs have the advantage of nonradioactive materials and produce an end product that can be assessed with a spectrophotometer. The molecule of interest is bound to the enzyme-labeled antibody, and the excess antibody is removed for immunoradiometric assays. After excess antibody has been removed or the second antibody containing the enzyme has been added (two-site assay), the substrate and cofactors necessary are added in order to visualize and record enzyme activity. The level of molecule of interest present is directly related to the level of enzymatic activity. The sensitivity of the ELISAs can be enhanced by increasing the incubation time for producing substrate. 

Wolbers R, Landrey G (1987) The use of direct reactive fluorescent dyes for the characterization of binding media in cross sectional examinations. Preprints of 15th Annual Meeting of the American Institute for Conservation and Artistic Works, Vancouver, 168-202. Heginbotham A, Millay V, Quick M (2006) The use of immunofluorescence microscopy and enzyme-linked immunosorbent assay as complementary techniques for protein identification in artist s materials. J Am Inst Conserv 45 89-105. 

Figure 19-13 Enzyme-linked immunosorbent assay. Anlibody 1, which is specific for the analyte of interest, is bound to a polymer support and treated with unknown. After excess, unbound molecules have been washed away, the analyte remains bound to antibody 1. The bound analyte is then treated with antibody 2. which recognizes a different site on the analyte and to which an enzyme is covalently attached. After unbound material has been washed away, each molecule of analyte is coupled to an enzyme that will be used in Figure 19-14.

Enzyme-linked immunosorbent assay (ELISA) is a very useful technique for the specific and sensitive assay of certain compounds, in which suitable antibodies, monoclonal or polyclonal, to the compounds are available. The technique has found particular application m the monitoring of environmental contaminants and toxins, either studying the primarily contaminated materials, e.g., foodstuffs, or body fluids of potentially exposed humans. The technique has been increasingly applied to monitoring the carcinogenic mycotoxins, the aflatoxins. 

RIA) using 2 I-labeled antibodies and enzyme-linked immunosorbent assay (ELISA) using alkaline phosphatase conjugates, wll be described. It is assumed that in most cases, the second antibodies will be bought either as purified material for radiolabeling or already conjugated to fluorescein, biotin, or the enzyme of choice (seeref. 2 for additional methods). 

Radioactivity, however, is still a very sensitive means of measuring the presence or absence of a given material. Assay methodology has now come full circle, to the development of an ultrasensitive enzyme RIA. In this technique, an antigen is bound to a solid phase. Antibody will bind to the antigen, which could be a drug-protein conjugate, and the presence of bound antibody is detected by means of a second antibody coupled to alkaline phosphatase. So far this is the standard enzyme-linked immunosorbent assay (ELISA). However, if the substrate is tritium-labeled adenosine monophosphate, it is converted by the enzyme to tritium-labeled adenosine, which may be readily separated and measured. The detection limit for this assay for cholera toxin is approximately 600 molecules of the toxin (22). 

To use immunoassays one normally employs a reporter substance or label. This label can be one of many materials including a radiochemical, a heavy metal, or commonly, an enzyme. A widely used format is the enzyme linked immunosorbent assay or ELISA. 

Assay optimization. An optimization step not always taken, but nonetheless important to the success of any immunochemical method of analysis is the selection of materials, such as test tube or plastic plates, which maximize assay performance. An example of this is the selection of 96-well microtiter plates for enzyme-linked immunosorbent assay (ELISA) which give maximum protein binding capacity and minimum interwell variability (28>32 also Harrison, R.O. Nelson, J.O. J. Immunoassay. submitted). The type of microtiter plate may be the most important single determinant of ELISA performance and this selection should not be made carelessly. Significant error may also occur in the reading of assays performed in 96-well microtiter plates, due to alignment errors of automatic plate readers undetected in normal use (Harrison, R.O. Nelson, J.O. J. Immunoassay. submitted Harrison, R.O. Hammock, B.D. J. Assoc. Off. Anal. Chem.. submitted), and a plate reader test should allow further reduction of error. These steps should be taken before a major investment in time, effort, or money is made in an assay system which may later be found to be less than acceptable. 

The separation of trichothecene mycotoxins from biological materials by UV absorption or fluorescence absorption is difficult, but the most suitable analytical methods are gas chromatography mass spectrum analysis. Recently, radioimmunoassay and enzyme linked immunosorbant assay have been developed for T-2 toxin and diacetoxyscirpenol (DAS) and deoxyverrucarol, which are highly sensitive as compared to other biological and chemical methods. 

The levels of creosote in biological materials can be estimated by measuring the PAH content in biological samples. Methods include GC/FID, GC/MS, and HPLC. Synchronous luminescence spectroscopy (SLS), 32P-postlabeling, and immunoassay techniques, i.e., enzyme linked immunosorbent assays (ELISA) and ultrasensitive enzyme radio immunoassay (USERIA), are methods currently being developed to detect and quantify ultratrace levels of PAH adducts bound covalently to macromolecules... 

Heginbotham A, Millay V, Quick M. The use of immunofluorescence microscopy and enzyme-linked immunosorbent assay as complementary technique for proteins identification in artists materials. J Am Inst Conserv 2006 45 89-105. 

A number of commercially available assays are now available for some common laboratory animals (e.g., mouse, rat, and dog). These assays are based on chemiluminescence, enzyme linked immunosorbent (ELISA) colorimetric assays, and radioimmunoassay. For the iodothyronines (e.g., total Tj and TJ, a wide choice of assays designed for human samples is available these can be used across species because the molecule structures are common (Anderson, Nixon, and Akasha 1988 Daminet et al. 1999 Mooney, Shiel, and Dixon 2008). Some assays require adjustments to ensure that the majority of samples are measured within a suitable range because the plasma values vary across the species (Table 10.2.1). When these assays are used, there may be problems associated with calibration materials because external quality assessment schemes continue to show minor and sometimes major differences between assays produced for hormone analyses. With all of these immunoassays, the reagents must be shown to be suitable for the species being studied. Reports of... 

Figure 3. Enzyme linked immunosorbent assay (ELISA) represents a heterogeneous assay in which unreacted materials are washed away between steps. Antibody-antigen binding is visualized by the generation of a colored product.

To date, the commonest type of immunoassay is the enzyme-linked immunosorbent assay (ELISA). The ELISA technique was developed through the pioneering work of Engvall and Perlmann in the 1970s. By immobilizing the reagents to a surface, the facile separation of bound and unbound material is achieved, making ELISA a powerful tool for the measurement of analytes in crude sample preparations.ELISA has become the basic immunoassay on which many of the modern assay formats are based. 

Enzyme-linked immunosorbent assays (for blood or other body fluids)50 or immunohistochemical techniques (for direct analysis of tissues) may be useful in confirming ricin intoxication. However, because ricin is bound very quickly regardless of route of challenge, and metabolized before excretion, identification in body fluids or tissues is difficult. In rats exposed to ricin labeled with iodine 125 by intravenous injection, the radioactive label was found in liver (46%), muscle (13%), and spleen (9%) 30 minutes after intravenous injection.51 Ricin was quickly cleared from the animals, with only 11% remaining after 24 hours 70% was excreted in the urine as low-molecular-weight metabolites. Attempts at identification of the toxin may also include introduction of biological autopsy materials into mice or cultured cells and neutralization through the use of specific antibodies. 

Enzyme-linked immunosorbent assay (ELISA) methods are widely used in medical and food analysis laboratories and it seems appropriate that such methods could be used for the detection of mycotoxins in foods. In the direct ELISA method the antibody is bound to a solid surface, such as a microtiter plate, and it is necessary to prepare a conjugate of the mycotoxin to be analyzed with the enzyme, such as phosphatase or peroxidase, to be used for color development. The sample or standard is mixed with the enzyme-linked mycotoxin and the two allowed to compete for the antibody bound to the surface. After washing away soluble material, the... 

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