Immunoassay cross-reacting metabolites

Shelver and Smittf confirmed fhaf commercial clenbuterol immunoassays cross-react with some, but not all, clenbuterol metabolites. As a result, quantitative clenbuterol immunoassays may differ from determinative methods if substantial concentrations of metabolites are present. For clenbuterol, the parent clenbuterol level is... 

Structurally related compounds may cross-react with the antibody, yielding inaccurate results. In screening for the herbicide alachlor in well water by immunoassay, a number of false positives were reported when compared with gas chromatography (GC) analysis. A metabolite of alachlor was found to be present in the samples and it was subsequently determined that the cross-reactivity by this metabolite accounted for the false-positive results. On the other hand, cross-reactivity by certain structural analogs may not be an issue. For example, in an assay for the herbicide atrazine, cross-reactivity by propazine is 196% because of atrazine and propazine field use... 

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. 

It follows that an immunoassay for quantitative determination of atrazine, based on the use of MAb 4063-21-1, would not be fully specific the ELISA values representing a compound response to all cross-reacting substances. One way to improve the specificity would be to combine the use of MAb 4063-21-1 with that of the anti-hydroxyatrazine MAb 4009-85-3, allowing a clear distinction between the response due to s-triazines and that of cross-reacting hydroxylated metabolites. However, the immunoassay would still not discriminate between atrazine and a few other triazines. 

We obtained stabilized hybridoma cell lines secreting MAbs specific for atrazine, a widely used herbicide, and for hydroxyatrazine, an important metabolite. As we observed previously with water samples (15), a good correlation was obtained between the current detection method (HPLC or GLC) and a MAb based immunoassay (ELISA) when fortified soils were analyzed. The limits of detection of atrazine and hydroxyatrazine by both methods were comparable. They corresponded to 50 ppb for soil samples and 0.05 ppb for water samples. However, by evaporating the methanol soil extracts before the ELISA, the limit of detection in soil samples could be reduced down to the ppb level. The analysis of undefined soil samples with respect to their herbicide content showed that for atrazine and hydroxyatrazine, some discrepancies were observed between the two methods due to biased detection during HPLC and GLC measurements. For atrazine this was confirmed by additional GC-MS analysis. Therefore, the ELISA immunoassay represents a valuable detection method for trace amounts of atrazine and hydroxyatrazine in soil, despite its limited specificity due to cross-reacting substances. 

Currently available immunoassays designed to detect and quantify TCAs provide a Total concentration of cross-reacting substances. This total concentration may be adequate for drugs and equipotent metabolites that exhibit comparable cross-reactivity with the detection antibody upon which the immunoassay is based, especially when the total concentrations are consistent with clinical expectations. Immunoassay-based results are less useful for evaluating compounds of differential cross-reactivity and potency, wherein falsely elevated or falsely low concentrations may be reflected by a total. Falsely elevated immunoassay-based results are likely when non-TCA... 

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