Monoclonal antibodies indirect ELISA

Beyond polyclonal antibodies, monoclonal antibodies to isoxazolyl penicillins were recently produced by immunization of mice with a cloxacillin-human serum albumin conjugate prepared by a mixed anhydride procedure (35). Sensitivity and specificity of these antibodies were tested in an indirect ELISA in which a cloxacillin-glucose oxidase conjugate prepared by an activated ester procedure served as a coating agent. It was found diat die prepared antibodies could be... 

Cell-enzyme-linked immunosorbent assay (cell-ELISA) is an useful technique for the quantitative analysis of cell surface antigen expression that was developed on the basis of enzyme immunohistochemistry (EIH) and ELISA. Since its development, which was made possible by the establishment of monoclonal antibody technology, a wide range of cell types and surface molecules were analyzed by cell-ELISA. Here we show four variants of this method and provide a brief comparison of cell-ELISA with flow cytometry (FACS) and radioimmunobinding assay (BIA), which are other methods for the quantitative detection of cell-surface molecules. We describe step-by-step procedures for both direct and indirect cell-ELISA using either adherent or nonadherent live cells. 

D.L. Brandon, R.G. Binder, A.H. Bates, W.C. Montague, Jr, Comparative ELISA for Thiabendazole Residues in Produce Using Indirect Immobihzed Monoclonal Antibodies , Food Agric. Immunol, 1, 99-108 (1995). 

Lapeyre, C. Janin, F. Kaveri, S. V. Indirect double sandwich ELISA using monoclonal antibodies for detection of staphylococcal enterotoxins A, B, Cl, and D in food samples. Food Microbiol., 5 25-31. 1988. 

Haza, A.I. Morales, P. Martin, R. Garcia, T. Anguita, G. Gonzalez, I. Sanz, B. Hernandez, PJi. Development of monoclonal antibodies against caprine Os2-casein and their potential for detecting the substitution of ovine milk by caprine milk by an indirect ELISA. J. Agric. Food Chem. 1996, 44, 1756-1761. 

Manager B, Solve M, Eriksen H, Brogen CH (1994). Bovine 3-lactoglobulin in hypoallergenic and ordinary infant formulas measured by an indirect competitive ELISA using monoclonal and polyclonal antibodies. Food Agric. Immunol, 6(l) 73-83. 

Many of the early ELISA methods devised for botulism neurotoxin detection, like most of the in vitro tests,suffered from a lack of specificity, due to impurities in the antigen preparation used to produce the antitoxins. More purified toxins are now available for the production of better quality antitoxins. The most sensitive ELISA protocols use an indirect assay sometimes referred to as the sandwich assay. In the basic procedure, a specific antitoxin is first adsorbed to the surface of the wells of a plastic plate. The toxin added to the wells is then bound by these antibodies and detected with a second antitoxin which is conjugated to an enzyme or other labeling molecule. The amount of label is measured by supplying the enzyme substrate, which is converted to a colored product that is measured colorimetrically. Some ELISA protocols use a polyclonal antitoxin on one side of the sandwich and a monoclonal on the other side. Other assays use the same antibody for both sides but label the antibody the second time it is used. A modification of the sandwich assay is the double sandwich ELISA, which employs a third antibody that is conjugated to an enzyme and is directed against the second antitoxin it is an anti-antibody such as rabbit anti-horse IgG. The steps in a typical application of this assay for botulism toxin are shown in Figure 2. 

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