Double antibody sandwich ELISA

Double antibody sandwich ELISA along with their modifications (Hefle et al., 1994) (Figure 3.1.1)... 

We have also applied ELISA to several biological pesticides including the endotoxin of Bacillus thurineiensis kurstaki (Btk). In this application to a macromolecular analyte, we have used a double antibody sandwich ELISA for Btk to measure the amount of ELISA reactive material in formulations of the pesticide. Figure 7 shows the use of an ELISA standard curve of gel purified Btk endotoxin to measure the immunoreactive material in dilutions of two Btk formulations. It has been demonstrated that ELISA can serve as a quick quality control check for formulations of Bacillus thurineiensis lsraelensis (44). Such examples indicate that immunoassays will be increasingly important as biologicals and products of recombinant DNA research impact our field (M) ... 

Figure 7. Standard curve of gel purified 60 kd protein endotoxin of Btk was generated using a double antibody sandwich ELISA. The arrows indicate dilutions of two Btk formulations absorbance values were used to determine the endotoxin concentrations of the formulations, based on the standard curve. The formulation dilutions gave curves that were virtually superimposable on the standard curve. Such similarity in shape and slope indicate that the antibody is likely binding to a specific determinant common to the purified Btk and the Btk in the formulation. In general immunoassays for biopolymers are much easier to develop than assays for small molecules. However, only recently has an interest in trace analysis of such materials begun to develop in the environmental field. Thus, sample cleanup and handling is not as sophisticated as with small molecules.

Since its introduction in medical research in 1971, various types of enzyme immunoassays have been developed (21) The double antibody sandwich ELISA (DAS-ELISA) introduced in plant pathology in 1976 (23) has become the major test system for plant indexing in the Netherlands This technique can be applied to a wide range of plant pathogens Figure 1 shows the principle for the complex antigen situation for detecting bacteria (23) ... 

Double-antibody (sandwich) ELISA using specific anti-soy trypsin inhibitor and other soy protein antibodies coated onto microwells... 

The Soy Protein Residue Assay is a double-antibody (sandwich) ELISA using specific anti-soy tripsyn inhibitor and other soy protein antibodies coated onto microwells. After addition of the sample, the enzyme conjugate, and the TMB substrate, a positive reaction (indicating the presence of soy protein), produces a blue color. Addition of the stop solution ends the assay and turns blue to yellow. The result may be read visually (in the qualitative method) or with an ELISA reader (in the qualitative or quantitative method). Quantification can be obtained by mnning positive control standards (2.5-5-10-25 ppm) together with the samples. A standard curve is then plotted using the optical density (OD) values of the control standards (OD vs. concentration). 

It is not always possible to use exactly the same format for screening as will be used in the final assay but it is advisable to attempt to ensure that the position that the monoclonal antibodies (MAb) will occupy in the final format is the position that is used for screening. For example MAbs which will eventually be conjugated to enzymes and used in Double Antibody Sandwich (DAS) ELISA should be screened for using TAS ELISA. This ensures that the epitope-binding portion of the antibody is in the same position as it will be in the final test format with the analyte bound to the coating antibody. 

In addition to ELISA and Western blots for detecting host-cell protein impurities there is a third immunoassay now available to the bioanalyst for this purpose the immunoligand assay (ILA). Like the ELISA, it is configured as a double-antibody sandwich. The anti-host-cell protein antibodies are separately conjugated to biotin and to fluorescein. A tripartite immune complex is formed between host-cell protein impurities and these two anti-... 

The most popular form of this technique is solid-phase heterogeneous ELISA, and the inherent use of passive adsorption facilitates flexibility in assay design. ELISA is generally classified as direct, indirect, sandwich (double-antibody) or competitive. Principles of each of these formats are briefly discussed below. 

Figure 10.10 Diagrammatic overview of sandwich (double-antibody) ELISA.

The principle of a double sandwich assay is also used for the sandwich ELISA, which avoids the use of radioactivity. The second antibody contains an enzyme (e.g., horseradish peroxidase), which serves as catalyst for a color reaction of a suitable substrate. Quantification is performed using UV-spectrophotometry. This type of ELISA may be used for trace impurity analyses. Immunization for such a multiantigen assay requires the representative preparation of all host cell proteins, but this must be completely free from the product protein. 

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. 

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. 

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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