Specificity of antibody

Commercial use of cell and tissue culture continues to expand. Improvement of organisms through recombinant nucleic acid techniques has become commonplace. Formerly, a few laboratories were well ahead of most others, but now the methods have been perfected for routine use. Another technique that is widely practiced is culturing of cells that excrete high concentrations of just one antibody protein. The specificity of antibodies and antigens is exploited in medical testing procedures using these pure monoclonal antibodies. 

Baldo BA, Fisher MM, Pham NH On the origin and specificity of antibodies to neuromuscular blocking (muscle relaxant) drugs an immunochemical perspective. Clin Exp Allergy 2009 39 325. 

While much care has to be used in performing competitive protein binding assays, most well-equipped and staffed clinical laboratories should have no serious problem in undertaking such assays. The biggest problem that may be encountered is the selection of a dependable and reliable manufacturer for reagents. Problems that may arise are non-purity of standards and label non-specificity of antibodies or the inability to maintain any of these characteristics from lot to lot. It therefore is a good practice to evaulate a few manufacturers before selecting one for routine use. 

Differences in the relative proportion of f-PSA and PSA-ACT can affect the result obtained for t-PSA because of the differences in the nature of calibration and the molar response, sensitivity, and specificity of antibodies used in various immunoassays. The efficiency of these immunoassays has been evaluated by several investigators. Because the proportion of free and complexed PSA varies in benign and malignant diseases, these immunoassays measure one form or the other, giving rise to different results for different patient groups. It is very important that data from clinical studies support the proposed intended uses of these assays, since as many as 5 percent of men with a negative free PSA test (free PSA values >25 percent) will have cancer and not be recommended for biopsy. Therefore, a goal for standardization is to detect total and free PSA accurately in equimolar fractions. 

The limitations of ELISA methods include the specificity of antibodies, the concentrations of primary antibody and antigen, and the type of reaction solution. Nonspecific binding of either of the antibodies to related antigens, unrelated proteins of other bacteria, or even the microtiter plate may lead to false positive reactions.49,52 57 Use of a monoclonal antibody may decrease crossreactivity with other antigens. For detection of low numbers of bacteria, as in drinking water, the sample may be filtered to concentrate the cells or cultured in a selective broth until it reaches the minimum detection limit for ELISA.49,58 Commercial test kits using dipsticks, immunoblots, and sandwich ELISA methods have been developed for the identification of pathogenic bacteria.58,59... 

A common application for (strept)avidin-biotin chemistry is in immunoassays. The specificity of antibody molecules provides the targeting capability to recognize and bind particular antigen molecules. If there are biotin labels on the antibody, it creates multiple sites for the binding of (strept)avidin. If (strept)avidin is in turn labeled with an enzyme, fluorophore, etc., then a very sensitive antigen detection system is created. The potential for more than one labeled (strept)avidin to become attached to each antibody through its multiple biotinylation sites is the key to dramatic increases in assay sensitivity over that obtained through the use of antibodies directly labeled with a detectable tag. 

Immunoassays employ monoclonal or polyclonal antibody preparations (Chapter 13) to detect and quantify the product (Box 7.1). The specificity of antibody-antigen interaction ensures good assay precision. The use of conjugated radiolabels (RIA) or enzymes (EIA) to allow detection of antigen-antibody binding renders such assays very sensitive. Furthermore, when compared with... 

The Agilent multiple affinity removal system utilizes the specificity of antibody-antigen recognition for 14 highly abundant proteins from human serum samples. The affinity column achieves reproducible and specific depletion from human serum and plasma to eliminate 94% of interfering proteins. It allows identification of proteins down to nanograms per milliliter level as reported by Agilent. 

Specificity of Antibody binding of Chlordiazepoxide A good number of benzodiazepines are tested for their ability to complete with labelled chlordiazepoxide for the respective antibody binding site. The various competitors are adequately tested at a concentration of 200 ng i.e.., 10-times the concentration of chlordiazepoxide required to produce a 50% inhibition of binding as shown in Table 32.2. 

The specificity of antibodies can be exploited in order to probe the in situ organization of cells and tissues. Cellular antigens can be identified both in viable cells and in frozen or fixed tissue sections. Antibodies are used to identify the appropriate antigen in the section and then the position of this primary antibody may itself be detected either directly if it was initially labelled or indirectly using another secondary antibody or molecule to attach to the antibody (Figure 7.8). Samples need to be carefully washed after addition of the primary or labelled antibody in order to prevent any non-specific reactions. Labels that have been successfully linked to antibodies include the following ... 

In the immunoglobulin heavy and light chain loci (ensuring that one B cell makes only one specificity of antibody)... 

The classical methods of demonstrating specificity of antibody reactions with antigens, such as immunodiffusion plates, are obsolete. Immunofluorescence is a very sensitive method that requires controls for specificity of equal sensitivity. The most important controls are not just those that demonstrate the presence of an antibody that reacts with the antigen of interest, but more importantly, the absence of antibodies that react with other things. 

Precise controls for the specificity of antibody reactions reside with other techniques, such as immunoprecipitation or immunoblotting. Controls for the labeling... 

V4. Van Vunakis, H., Bradvica, H., Benda, P., and Levine, L., Production and specificity of antibodies directed toward 3,4,5-trimethoxyphenylethylamine, 3,4-dimethoxyphenylethylamine and 2,5-dimethoxy-4-methylamphetamine. Biochem. Pharmacol. 18, 393-404 (1969). 

A final caveat in the RPAIA field that limits biomarker discovery as well as other uses in drug development is the somewhat limited panel of antibodies that have been validated to date for the platform. Many antibodies that work on western blots have the potential to work with RPMAs as well, because in both platforms proteins are in a denatured state. However, because the molecular weight of analytes detected by RPMA cannot be known, it is imperative to demonstrate the specificity of antibodies, usually measured by the absence of nonspecific reactive bands on western blots developed under similar blocking conditions. A second requirement for antibody validation is proof of principle that an antibody detects quantitative changes in the expected target on an... 

Immunoaffinity chromatography is one of the most popular techniques of affinity derivatived method and it enables to produce ligands in case the ligand required is not available [7]. In this technique, stationary phase comprises of an antibody or antibody-related agent [1]. It is possible to isolate variable subtances using this technique due to high specifity of antibodies [1]. It is reported that immunoaffinity chromatography may be used for natural food contaminants such as aflatoxins, fumonisins and ochratoxins [11]. 

The development of hybridoma technology by Milstein and Kohler in 1975 revolutionized the antibody field and radically increased the purity and specificity of antibodies used in the clinic and for diagnostic tests in the laboratory. Hybridomas consist of antibody-forming cells fused to immortal plasmacytoma cells. Hybrid cells that are stable and produce the required antibody can be subcloned for mass culture for antibody production. Large-scale fermentation facilities are now used for this purpose in the pharmaceutical industry. 

The specificity of antibodies has practical uses. A selected antibody can be covalently attached to a resin and used in a chromatography column of the type shown in Figure 3-18c. When a mixture of proteins is added to... 

The specificity of antibody-antigen interactions is a result of a structural fit between the antibody binding site and the antigenic determinant. An understanding of the mechanism of the antigen-antibody interactions has revealed the existence of hypervariable and conserved regions. The... 

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