Monoclonal antibodies direct ELISA

Geysen et al. [2] first successfully applied this approach in the multipin system in an on-pin ELISA assay to define a mimotope for a monoclonal antibody directed against a discontinuous epitope on the foot-and-mouth disease virus particle. Later, Houghten et... 

Both circulating OxLDL and MDA-LDL have been measured in plasma by ELISA. Most commonly, the capture antibody is a monoclonal antibody against OxLDL or MDA-LDL and the detection antibody, a polyclonal or monoclonal antibody directed against apo B (El, S12, Tl). Usually, the capture antibody is developed against chemically modified MDA-LDL or OxLDL but antibody has been developed against homogenate of human arteriosclerotic plaque. This antibody reacted with oxidized phosphatidylcholines but not native LDL, or MDA-LDL (S12). 

Fig. 2. Biochemical classification of the nia mutants, mutated domains and intragenic complementation. A collection of Nicotiana plumbaginifolia nia mutants (defective for NR apoenzyme) was tested for NR protein and activities (Cherel et al., 1990) and checked for in vivo and in vitro intragenic complementation (Pelsy Gonneau, 1991). NR protein amounts were measured by a sandwich ELISA test using as first antibody the anti-corn NR monoclonal antibody 96.9.25 (Ch6rel eta/., 1985) shown to be directed against the haem domain (M. Kavanagh, personal communication Meyer etal., 1991). The result is shown here as + or when a positive or negative ELISA test, respectively, was observed. Many class 4 mutants, most probably frameshift and deletion mutants, complement class 3 but not class 2 mutants.

In sum, EMPs have emerged as a preferred direct method for assessing EC injury in different disorders. EMP analysis could provide insight into the actual status of the endothelium in vivo by a simple blood analysis. However, there is a need for refinement and standardization of the assay method. Overall, most groups have relied on flow cytometry for the measurement of EMPs nevertheless, other methods such as ELISA are available and may be an option in the future. The main challenge remains in the selection of specific and sensitive monoclonal antibodies that may yield consistent results between different laboratories. In addition, the protocols for sample handling and storage need to be clearly delineated. The assay is still a... 

Robertson, D.M., Stephenson, T., Cahir, N. et al Development of an inhibin snbnnit ELISA with broad specificity. Mol. Cell. Endocrinol. 180,79-86, 2001 Robertson, D.M., Stephenson, T., Prnysers, E. et al., Inhibins/activins as diagnostic markers for ovarian cancers. Mol. Cell. Endocrinol. 191, 97-103, 2002 Khosravi, J., Krishna, R.G., Khaja, N., Bodani, U., and Diamandi, A., Enzyme-linked immnnosorbent assay of total inhibin direct determination based on inhibin subunit-specific monoclonal antibodies, Clin. Biochem. 37, 370-376, 2004 Cook, R.W., Thompson, T.B., Jardtzky, T.S., and Woodruff, T.K., Molecular biology of inhibin action, Semin. Reprod. Med. 22, 269-276, 2004. 

Newer techniques have equal sensitivity and greater specificity Enzyme-linked immunosorbent assay (ELISA) tests can identify C. trachomatis, HSV-1 and -2, and adenoviruses through the detection of microbial antigens. In the direct ELISA, an enzyme is covalently linked to an antigen-specific monoclonal or polyclonal antibody. 

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. 

Nayeri et al. [27] presented an SPR (Biacore 2000)-based direct qualitative detection of hepatocyte growth factor (HGF), which is an angiogenic growth factor related to breast cancer, in reconstituted fecal samples from patients with infectious gastroenteritis (n = 20) and normal controls (n = 10) (dissolved in distilled water at a dilution rate of 1 6). Mouse anti-human HGF monoclonal antibodies and recombinant hmnan HGF receptor were immobilized on a dextran surface. The proportion of antibody-positive patient samples detected by SPR correlated well with results obtained using ELISA. 

This is directly proportional to the quantity of analyte present. This technique advanced rapidly when monoclonal antibodies became available, since this made it possible to produce unlimited quantities of antibodies with clearly defined characteristics [20]. This second type of assay is also known as a sandwich immunoassay, and is best suited to the assay of proteins rather than haptens. The widespread ELISA (Enzyme Linked Immunosorbent Assay) test belongs to this category [21-24]. 

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