Double antibody sandwich

Valdivieso-Garcia, A. Riche, E. Abubakar, O. Waddell, T. E. Brooks, B. W. A double antibody sandwich enzyme-linked immunosorbent assay for the detection of Salmonella using biotinylated monoclonal antibodies. J. Food Prot. 2001, 64,1166-1171. 

Double-antibody sandwich. Antibody is coated on or adsorbed to the plastic plate. The sample to be assayed containing the antigen (antibiotic) is added followed by a second antibody that is conjugated to the enz)rme (horseradish peroxidase, alkaline phosphatase, or 3 galactosidase). The substrate is added and the intensity of the color produced is directly proportional to the antigen in the test sample. 

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. 

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

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-... 

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.

Crook and Payne (1980) compared the direct (Section 14.2.1.1), indirect (Section 14.2.1.2) and the double-antibody sandwich methods (variant A) for their ability to detect and discriminate between several granulosis viruses. The indirect method was the most sensitive (1 ng/ml), the direct method was the least sensitive (15 ng/ml) and method A had an intermediate detectability (10 ng/ml). 

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) ... 

Fig. 3 Double-antibody sandwich technique. The capturing antibody (Abl) is immobilized onto the solid phase. The analyte from the sample is captured by forming Abl-Ag immunocomplex. After washing off the extraneous materials from the sample, the reporting antibody (Ab2, which has an enzyme label in this illustration) is introduced. The double-antibody sandwich is formed Abl-Ag-Ab2E. Compounds not recognized by both Abl and Ab2 will be washed away. A chromogenic substrate is added to produce the product for detection. The occupied Ab2 by the Ag is measured this is an excess-reagent assay. As the sample analyte concentration increases, the signal responses increases proportionally.

The double-antibody sandwich technique is applicable to large molecules. Small analytes have difficulty forming the double-antibody sandwich immunocomplex. The smallest analytes reported that have been used in a double sandwich assay are peptides of around 10 amino acid residues (158). The double-antibody sandwich technique measures the occupied antibodies using an excess-reagent assay protocol. A limited-reagent assay protocol can also be designed, as shown in Fig. 4. In this format, the antibodies are immobilized onto the solid phase,... 

Scheme 2. Double antibody sandwich reaction (noncompetitive).

Kerschbaumer R J, Hirschl S, Kaufmann A, et al. (1997). Single-chain Fv fusion proteins suitable as coating and detecting reagents in a double antibody sandwich enzyme-linked immunosorbent assay. Anal. Biochem. 249 219-227. 

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). 

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