Enzyme-linked immuno-sorbent assay ELISA

Fig. 1. Comparison of enzyme-linked immuno sorbent assay (ELISA, left) and immuno-polymerase chain reaction (IPCR, right). During ELISA, an antibody-enzyme conjugate is bound to the target antigen. The enzyme converts a substrate in solution to a detectable product. In IPCR, the antibody-enzyme conjugate is replaced by an antibody-DNA conjugate. The subsequent addition of a DNA polymerase enzyme (e.g., Taq), nucleotides and a specific primer pair uses the antibody-linked DNA marker sequence as a template for amplification of the DNA. The PCR product is finally detected as an indicator of the initial amount of antigen.

Fig. 3. Comparison of different enzyme-linked immuno sorbent assay (ELISA) methods adapted for immuno-polymerase chain reaction (IPCR). Dependent on the purification grade of the sample to be analyzed and the availability of specific and functionalized antibodies, several typical ELISA protocols were adapted to IPCR. In the direct approach (A), the pure antigen is immobilized to the microplate surface and subsequently detected by a labeled specific antibody. If no labeled antibody is available (e.g., because of unpurified ascites fluid containing the antibody or loss in activity following labeling), a standardized labeled secondary species-specific antibody is used for detection of the primary antigen-specific antibody (B). For the detection of the antigen from matrices such as serum, plasma, tissue homogenate, and so on, a capture antibody immobilized to the microplate surface was used either in a direct (C) or indirect (D) sandwich approach, with the latter one additionally including a secondary species-specific detection antibody. For different methods of coupling antibody and DNA, abbreviated by in this figure, compare Fig. 2. Note that protein A chimeras (Fig. 2A) are not compatible with capture antibodies (Fig. 3C, D).

As E. coli 0157 H7 is the most implicated STEC serotype in human cases, numerous immuno-based methods for the detection of this specific serotype have been documented. They include conventional Enzyme Linked Immuno Sorbent Assays (ELISA) in microplates, one-step immuno-detection systems, fully automated systems and various non enzymatic immunological based systems like Rapid Plate Latex Agglutination (RPLA), Immuno-Magnetic Separation (IMS) or immuno-chromatography (Table 3). 

Screening test kits - enzyme linked immuno-sorbent assay (ELISA)... 

Gasc6n J, Oubina A, Barcelb D. (1997). Detection of endocrine-disrupting pesticides by enzyme-linked immuno-sorbent assay (ELISA) Application to atrazine. Trends in Analytical Chemistry 16 554-562. 

ELISA enzyme linked immuno sorbent assay... 

It is necessary to give some background information to understand the principle underlying immunochemical tests such as ELISA (Enzyme Linked Immuno Sorbent Assay). Such information is not always available to chemists, since chemistry and immunology have traditionally been separate disciplines. 

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The basic tool for detecting antibodies binding to a specific antigen is the ELISA, or Enzyme Linked Immuno-Sorbent Assay [3]. In this assay, antigen-specific antibodies are detected by antigen-mediated attachment to a solid support and secondary detection with an Fc-specific enzyme-labeled secondary antibody (Fig. 2). For hapten immunization, ELISA is performed using a carrier protein, typically BSA (bovine serum albumin), different from the carrier protein used for immunization. In this manner only antibodies with binding specificity to the hapten are revealed by the assay. 

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