Competitive ELISA results from

Other Methods. Marion et al. used a competitive ELISA to measure the specificity of anti-DNA antibodies. Low-affinity, low-avidity antibodies bind to competitor DNAs in solution and thus cannot bind to solid-phase DNA subsequently (M7). A new automatic instrument platform using the immunofluorescence method (EliA ) to detect anti-dsDNA has recently been developed. We found that results from this method correlated well with results from the ELISA method (unpublished). 

Monoclonal antibodies were obtained against atrazine and its metabolite hydroxyatrazine by immunizing mice with atrazine or hydroxyatrazine protein conjugates. By competitive ELISA, we observed that the antibodies raised against hydroxyatrazine cross-reacted mainly with hydroxypropazine. The antibodies raised against atrazine cross-reacted with propa-zine, prometone, prometryne, and to a much lower extent with a few other s-triazines and hydroxy-s-triazines. Atrazine could be detected in water samples down to 50 ppt. Average recoveries measured by ELISA from soil samples fortified with atrazine or hydroxyatrazine were comparable to those measured by GLC or HPLC. Soil samples of unknown atrazine content were analyzed by GLC, GC-MS, and by ELISA. The results show that the ELISA immunoassay represents a valuable detection method for trace amounts of atrazine and hydroxyatrazine in soil. 

Results from competition ELISA experiments suggested that the concentration of heptachlor resulting in a 50% inhibition of the control (i.e. the I50 value) for LLNL-Hept-2 was approximately 10-fold lower than the I50 for LLNL-Hept-1 (I50 for LLNL-Hept-1 = 37 ng and 3.8 ng for LLNL-Hept-2). A 5-fold difference in sensitivity was observed when the competitor was... 

Sandwich and competitive ELISA coupled to the ICP-MS detector yield reproducible and linear responses to antigen concentration. They also enable 96 immunoassays to be run per plate. Primary antibody plates were found to be more sensitive than secondary antibody plates, allowing lower levels of protein to be detected. This is probably due to the restricted well capacity of secondary antibody plates resulting from the steric hindrance of overlying antibodies. Comparative study of three analytes [estriol, a-fetoprotein( AFP) and human chorionic gonadotropin(hCG)] in 81 patient samples has been performed, where it was found that ICP-MS detection reproduced results currently achieved on the same plates with fluorometers (Figure 9.3). 

CE with LIF of this mixture should reveal two resolved peaks corresponding to bound and free Ab (if it still exists in solution). As in the above case, both peaks and their ratio can be used for analyte quantification. Here the higher the amount of Ab in the sample, the more Ab is displaced from the complex, and hence the higher the peak of the tracer and the lower of the complex. Besides CE-related requirements which the analyzed system should meet (see above), special attention must be paid to proper adjustment of the amount of labeled Ag and Ab added to the sample. In both extreme cases of incorrect adjustment, namely, when the amount of either Ag or Ab is too high, the results might be outside the dynamic range of the calibration curve and hence incorrect. These requirements are very typical for any mode of competitive immunoassay, including ELISA-like methods, and are discussed in detail in related books (see, e.g., Ref. 32). 

Since our intent was to use the competitive A-B ELISA to quantify 3-Cys-A adducts formed in biological samples at unknown and perhaps variable levels of protein modification, experiments were conducted to determine the effect of adduct substitution level on quantification. Standards of known substitution level were prepared by derivatizing 9,000 g liver supernatant with various concentrations of [3H]NAPQI. After extensive dialysis to remove noncovalently bound materials, protein concentrations were determined and the substitution level of each standard was determined by scintillation counting. These synthetic standards, which ranged from 0.5 to 30 nmol 3-(cystein-S-yl)[3H]acetaminophen per mg protein, were analyzed in the competitive immunoassay. When the data were plotted with percent inhibition as a function of protein concentration, the results show an ordered family of inhibition curves where the most highly substituted proteins were the... 

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