Indirect ELISA procedure

Indirect ELISA Procedure Using Antigen-Coated Plates... 

Several variations of indirect ELISA procedures have been described.25-38... 

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... 

A partially purified Bacillus thurlnglensis var. israelensls (Bti) 6-endotoxin was used to Immunize rabbits. The antisera obtained have an improved specificity towards the mosquito larvacidal activity of the toxin, as opposed to antiserum raised when the whole crystal was used as immunogen. Using a two step/indirect ELISA (enzyme linked immunosorbent assay) procedure developed in our laboratory, fourteen experimental formulations were tested, and the results were compared with bioassays. An average of 69.1 international units 20% c.v. was found to associate with each ug of toxin detected by the ELISA. Our data indicate that when toxin specific antisera are available, Immunoassays can be used to predict the biological activity of Bti samples with reasonable accuracy. 

There are several modifications to the procedure that allow the target to be identified and quantified. Since the topic is immunocytochemistry, the procedures described deal with ELISAs performed on intact cells fixed onto the wells of 96-well plates. There are two forms of the procedure direct and indirect. The direct method calls for linking the enzyme directly to the antibody of interest. Depending on the availability of the antibody and its activity, direct conjugation of the enzyme to the antibody may interfere with specificity or success of binding with the target. The preferred method is the indirect ELISA. The antigen or cell of interest is immobilized onto the well. The primary antibody (often a... 

Wash the plates. (From now on we are performing a procedure similar to the indirect ELISA.)... 

The substrate is cleaved by the enzyme-labeled hapten in the immunocomplex at the surface. The fluorescence can be measured in solution or at the surface. For the determination of high molecular mass antigens a two-site sandwich assay can be applied. A variation represents the double antibody assay, whereby the second antibody, which is directed against the hapten-specific antibody, is enzyme-labeled. Reagent-excess based assays are mainly used for the determination of antibodies in a noncompetitive format. In Scheme 6, a procedure for an indirect ELISA for the determination of antibodies is described. 

ELISA is the indirect method for measuring antibody concentration. This procedure employs immobilized antigen and enzyme-labeled second antibody against IgG of the species in which the test antibody has been elicited. This method has been used to measure antibodies to a variety of anti-... 

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. 

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. 

---