Indirect competitive ELISA

Figure 1 Schematic sequence of the direct and indirect competitive ELISA. The principle difference is that for direct competitive immunoassay, the well is coated with primary antibody directly, and for indirect competitive immunoassay, the well is coated with antigen. Primary antibody (Y), blocking protein (Y), analyte (T), analyte-tracer ( ), enzyme labeled secondary antibody ), color development ( J)...

Other attempts have been made to detect BPA at a low concentration range. Thus Kodaira et al. [274] analyzed BPA in urine samples with an assay that showed a working range between 0.5 and 5 pg L The assay was validated by HPLC. DeMeulenaer et al. [275] developed an indirect competitive ELISA using PAbs obtained from chicken egg yolk, but the assay achieved an IC50 value of only 570 pg L-1. 

An indirect competitive ELISA has been also developed for the determination of streptomycin and dihydrosticptomyciri in milk (24). Prior to the analysis, the milk sample was skimmed and treated with oxalic acid. The antiserum was raised in rabbits using streptomycin linked to a bacterial protein as the antigen. To perform the test, microtiter plates were coated with streptomycin, and antiserum and milk samples were mixed to be added in the wells where they were incubated for 1 h. Depending on the amount of residues in the sample, more or less antibody remained available for binding to the streptomycin coat. A pig antirabbit antibody-enzyme conjugate was subsequently added and incubated for 90 min. Using a suitable substrate, streptomycin and dihydrostreptomycin could be detected down to 1.6 ppb, whereas quantification could be made possible up to 100 ppb when samples were used undiluted. 

This method is further divided into two major types. One type is competitive ELISA, which can be used for the analysis of both hapten and macromolecule the other is noncompetitive sandwich-type ELISA, which is only used for divalent and multivalent antigens. Two major types, i.e., direct competitive ELISA (dC-ELISA) and indirect competitive ELISA (inC-ELISA), are used most commonly in food analysis. 

Duan, J. and Z. Yuan. 2001. Development of an indirect competitive ELISA for ciprofloxacin residues in food animal edible tissues. J. Agric. Food Chem. 49 1087-1089. 

Fig. 9.10. Matrix eflFects in immunoassay are generally revealed as a decrease in absolute signal at any level of spiked anal5d e concentration. This effect decreases with the dilution of the sample. Example presented an indirect competitive ELISA for 4-nitrophenol in milk at different dilutions. A 4-nitrophenol derivative coupled to bovine serum albumin was used as the competitor, an anti-4-nitrophenol (polyclonal, from rabbit) as the antibody and goat anti-rabbit immunoglobulin G coupled to peroxidase as the detection system [43]. (Reprinted with permission.)...

Manager B, Solve M, Eriksen H, Brogen CH (1994). Bovine 3-lactoglobulin in hypoallergenic and ordinary infant formulas measured by an indirect competitive ELISA using monoclonal and polyclonal antibodies. Food Agric. Immunol, 6(l) 73-83. 

Wu JE, Chang C, Ding WP, He DP, Determination of florfenicol amine residues in animal edible tissues by an indirect competitive ELISA, J. Agric. Food Chem. 2008 56 8261-8267. 

Plate Data for Indirect Competition ELISA to Measure Antigen... 

Indirect competitive ELISA (or double antibody ELISA) In the indirect ELISA, Instead of using a mycotoxin-enzyme conjugate, a mycotoxin-protein (or polypeptide) conjugate is first prepared and then coated to the microplate (1-13). The plate is then Incubated with specific antibody against the mycotoxin in the presence or absence of the homologous mycotoxin. The amount of antibody bound to the plate coated with mycotoxin-protein conjugate is then determined by reaction with a second antibody-enzyme complex such... 

The most commonly used detection for HPLC of MC is UV at 238 nm, usually performed with photodiode-array detectors, which is also employed as reference method, e.g., for MC determination using a novel indirect competitive ELISA method " or for a time-resolved fluoroinununometric assay. The detection limit of MC in HPLC system without any preconcentration or clean-up is too high for practical applications... 

Ivanov A, Evtugyn A, Budnikov H, Girotti S, Ghini S, Feiri E, Montoya A, Mtncader JV (2008) Amperometric immunoassay of azinphos-methyl in water and htmeybees based on indirect competitive ELISA. Anal Lett 41 392-405... 

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