Optimizing ELISA

Recently, a method for optimizing ELISA procedures has been demonstrated (13). This method attempts to compare the net effects of different conditions using an experimental design called the Taguchi method. The method attempts to reduce the effects of the interactions of optimized variables, making it possible to access the optimal conditions even in cases in which there are large... 

Lopez-Avila and Benedicto combined SEE with ELISA to determine sulfamethazine in powdered milk. Various conditions were tested in order to achieve quantitative extraction of sulfamethazine. Variations in extraction pressure, temperature, extraction period, and the presence of organic modifier resulted in extraction efficiencies of 0-92%. Once optimal extraction conditions had been developed, a commercially available ELISA was utilized to determine sulfamethazine concentrations. The LOD was 2.5 pgkg and satisfactory recoveries were obtained at levels from 5 to 15 pgkg-i. 

Ramandeep, Dikshit KL, Raje M. Optimization of immunogold labeling TEM an ELISA-based method for rapid and convenient simulation of processing conditions for quantitative detection of antigen. J. Histochem. Cytochem. 2001 49 355-367. 

Myc A, Anderson MJ, Baker JRJ. Optimization of in situ cellular ELISA performed on influenza A virus-infected monolayers for screening of antiviral agents. /. Virol. Methods 1999 77 165-177. 

An enzyme-linked immunosorbent assay (ELISA) has two major components. The first is the immunological reaction that occurs between an antigen and antibody. This reaction is crucial and needs careful optimization. The second compo-... 

Several qualitative and quantitative immunochemical methods for CAP analysis in biological matrices of animal origin have been described [101,102, 104,105] (see Table 3). Van de Water et al. [ 102] described an ELISA that detected CAP in swine muscle tissue with an IC50 value of 3 ng mL1. This immunoassay was improved and subsequently optimized incorporating the streptavidin-biotin amplification system. There are also several commercially available test kits (see Table 4). RIDASCREEN is a competitive enzyme immunoassay for the quantitative analysis of CAP residues in milk, eggs, and meat in a microtiter plate. The measurement is made photometrically, obtaining a LOD of 100 ng L 1 in meat and eggs and 150 ng L 1 in milk. The test has been also applied to the analysis of tetracyclines. 

Once the reagents are available, the ELISA method is fast and can be modified to accommodate high throughput. But the time to obtain the appropriate reagents must be considered. It may take considerable time to generate the appropriate antibodies by immunization, as discussed previously. Also considerable time must be allowed to develop and optimize the assays. 

There are several important advantages RPMAs have over antibody arrays and other proteomic techniques such as immunohis-tochemistry or tissue arrays. Antibody arrays usually require a second specific antibody, made in a different species, for each captured protein to be visualized in a manner analogous to enzyme-linked immunosorbent assays (ELISA). Therefore, it becomes difficult to simultaneously optimize the antibody-antigen hybridization conditions for so many antibodies at once present on antibody arrays while minimizing nonspecific cross-reactivity and ensuring that proteins over a wide range of concentrations can be quantitated in a linear fashion (14). Antibody arrays also consume or require much higher inputs of protein than reverse phase arrays. With antibody arrays. 

The additional optimization of other ELISA factors may be necessary after determining the best concentration of antigen/enzyme conjugate to use (see Note 8). Three parameters may be varied to make improvement. Each one should be adjusted independently and an optimum dilution factor or value determined for routine use. Molecules of interest may be masked by components of the test fluid leading to unexpectedly low signals. This may be remedied by mixing the sample with PBS/Tween supplemented with 10% fetal bovine serum (see Note 9). 

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