Sample immunoassays

Immunoassays are another way to detect foreign substances in urine samples. Immunoassays involve the use of antibodies. 

Besides the methods given in Table 6-1 and Table 6-2, several others are available to determine dinitrophenols in biological and environmental samples. Immunoassay methods with sensitivities comparable to those of the conventional methods given in Tables 6-1 and 6-2 are noteworthy (Bush and Rechnitz 1987 Huang et al. 1992 Kusterbeck et al. 1990 Wannlund and DeLuca 1982). However, methods based on antibody sorption have not yet been validated on samples derived from environmental and biological sources, so it is not clear what methods of clean-up are necessary prior to quantitation of dinitrophenols. Some of the other detection methods with superior sensitivities that can determine dinitrophenols are surface-enhanced resonance Raman spectroscopy (Ni et al. 

Analysis Methods for Liquid Impinger Solutions or Air Filter Samples Immunoassay Methods for Microbial Surface Antigens... 

See also Blood and Plasma. Chemiluminescence Overview. Enzymes Immobilized Enzymes Enzymes in Physiological Samples. Immunoassays, Techniques ... 

In fact, most RIAs and many nonisotopic immunoassays use a competitive binding format (see Fig. 2). In this approach, the analyte in the sample to be measured competes with a known amount of added analyte that has been labeled with an indicator that binds to the immobilized antibody. After reaction, the free analyte—analyte-indicator solution is washed away from the soHd phase. The analyte-indicator on the soHd phase or remaining in the wash solution is then used to quantify the amount of analyte present in the sample as measured against a control assay using only an analyte-indicator. This is done by quantifying the analyte-indicator using the method appropriate for the assay, for example, enzyme activity, fluorescence, radioactivity, etc. 

Latex agglutination immunoassays are easily formatted into simple kits which can provide yes/no and semiquantitative estimates of antigen (or antibody) in a sample. The first such assay was developed in 1957 for rheumatoid factor (15) and assays are on the market for the deterrnination of many species of bacteria, fungi. Mycoplasma, parasites, ckettsia, and vimses, as well as for the deterrnination of autoimmune disease, hormones (qv), dmgs (see Pharmaceuticals), and blood proteins (16). Latex agglutination is also the basis of many home pregnancy tests. 

Enzyme Immunoassay. In EIA, antibody (or antigen) is labeled with (or conjugated to) an enzyme, and this reagent is used to complex and quantify the target antigen (or antibody) in a sample. Conjugation may utilize a variety of chemical methods. 

Radiotracers have also been used extensively for the quantitative rnicrodeterrnination of blood semm levels of hormones (qv), proteins, neurotransmitters, and other physiologically important compounds. Radioimmunoassay, which involves the competition of a known quantity of radiolabeled tracer, usually I or H, with the unknown quantity of semm component for binding to a specific antibody that has been raised against the component to be deterrnined, is used in the rnicro deterrnination of physiologically active materials in biological samples (see Immunoassay). 

Immunoassays. Immunoassays (qv) maybe simply defined as analytical techniques that use antibodies or antibody-related reagents for selective deterrnination of sample components (94). These make up some of the most powerflil and widespread techniques used in clinical chemistry. The main advantages of immunoassays are high selectivity, low limits of detection, and adaptibiUty for use in detecting most compounds of clinical interest. Because of their high selectivity, immunoassays can often be used even for complex samples such as urine or blood, with Httle or no sample preparation. 

A number of soHd-phase automated immunoassay analyzers have been used for performing immunoassays. Table 5 (96) provides usefiil information on maximum tests that can be mn per hour, as well as the maximum number of analytes per sample. A number of immunoassay methods have been found usefiil for environmental analysis (see AUTOMATED INSTRUMENTATION). 

Biopolymers are employed in many immunological techniques, including the analysis of food, clinical samples, pesticides, and in other areas of analytical chemistry. Immunoassays (qv) are specific, sensitive, relatively easy to perform, and usually inexpensive. For repetitive analyses, immunoassays compare very favorably with many conventional methods in terms of both sensitivity and limits of detection. 

Enzyme Immunosensors. Enzyme immunosensors are enzyme immunoassays coupled with electrochemical sensors. These sensors (qv) require multiple steps for analyte determination, and either sandwich assays or competitive binding assays maybe used. Both of these assays use antibodies for the analyte of interest attached to a membrane on the surface of an electrochemical sensor. In the sandwich assay type, the membrane-bound antibody binds the sample antigen, which in turn binds another antibody that is enzyme-labeled. This immunosensor is then placed in a solution containing the substrate for the labeling enzyme and the rate of product formation is measured electrochemically. The rate of the reaction is proportional to the amount of bound enzyme and thus to the amount of the analyte antigen. The sandwich assay can be used only with antigens capable of binding two different antibodies simultaneously (53). 

It is obvious from the provisional risk assessment values for microcystins, and, being of the same order of magnitude of mammalian toxicity, similar values may be calculated for the cyanobacterial neurotoxins, that sensitive detection methods are required to detect these low concentrations of toxins. Of the biological methods of detection discussed earlier, the mouse and invertebrate bioassays are not sensitive enough without concentration of water samples, in that they are only able to detect mg of microcystins per litre. Only the immunoassays (ng-/rg 1 and the protein phosphatase inhibition assays (ng O... 

Example 6-2 The following standard addition plot was obtained for a competitive electrochemical enzyme immunoassay of the pesticide 2,4-D. A ground water sample (diluted 1 20 was subsequently assayed by the same protocol to yield a current signal of 65 nA. Calculate the concentration of 2,4-D in the original sample. 

The sensitivity of enzyme assays can also be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunoassays (ELlSAs) use antibodies covalently finked to a reporter enzyme such as alkafine phosphatase or horseradish peroxidase, enzymes whose products are readily detected. When serum or other samples to be tested are placed in a plastic microtiter plate, the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then blocked by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and these are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody are then determined by adding the substrate for the reporter enzyme. 

The remarkable selectivity that is inherent in the reaction of an antibody with the antigen or hapten against which it was raised is the basis for the extensive use of immunoassay for the rapid analysis of samples in clinical chemistry. Immunochemical reactions offer a means by which the applicability of potentiometric techniques can be broadened. A number of strategies for incorporating immunoassay into the methodology of potentiometry have been explored... 

A homogeneous electrochemical enzyme immunoassay for 2,4-dinitrophenol-aminocaproic acid (DNP-ACA), has been developed based on antibody inhibition of enzyme conversion from the apo- to the holo- form Apoglucose oxidase was used as the enzyme label. This enzyme is inactive until binding of flavin adenine dinucleotide (FAD) to form the holoenzyme which is active. Hydrogen peroxide is the enzymatic product which is detected electrochemically. Because antibody bound apoenzyme cannot bind FAD, the production of HjOj is a measure of the concentration of free DNP-ACA in the sample. 

The main advantage of a homogeneous immunoassay, compared to a heterogeneous immunoassay, is the absence of a separation step. This translates into a simpler procedure and easier automation. However, homogeneous assays are typically less sensitive and more susceptible to sample interferences which are removed in a separation step. 

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