Validation immunoassays

Spiehler, V. Fay, J. Rogerson, R. Schorenforfer, D. Nied Bala, R.S. Enzyme immunoassay validation for qualitative detection of cocaine in sweat. Clin. Chem. 1996, 42, 34-38. 

Immunoassay validation with emphasis on applications for environmental monitoring... 

Lee N, Beasley HE, Silbum M, et al. 1997b. Validation and application of immunoassays to studies of the environmental fate of endosulfan. 213th National Meeting Of The American Chemical Society, San Francisco, California, USA, April 13-17, 1997. Abstracts of Papers American Chemical Society. Abstract 117. 

The use of immunoassay methodology for residue trial analysis is in principle just as acceptable as for enforcement methods, provided that the method has been adequately validated. Because the validation of such methods requires a different approach, as opposed to chromatographic and spectrometric methods, some important points to be aware of in the use are explained in SANCO/3029/99. The authors do not go into detail on this subject here, since on the one hand very few methods have been submitted up to the present, and on the other it would go beyond the scope of this article. 

Consistent with other analytical methods, immunoassays must be validated to ensure that assay results are accurate. Initial validation involves an evaluation of the sensitivity and specificity of the immunoassay, while later validation includes comparison with a reference method. Because a goal of immunoassays is to minimize sample preparation, validation also includes testing the effects of sample matrices and(or) sample cleanup methods on results. The final steps in validation involve testing a limited number of samples containing incurred residues to determine if the method provides reliable data. 

For pesticide residue immunoassays, matrices may include surface or groundwater, soil, sediment and plant or animal tissue or fluids. Aqueous samples may not require preparation prior to analysis, other than concentration. For other matrices, extractions or other cleanup steps are needed and these steps require the integration of the extracting solvent with the immunoassay. When solvent extraction is required, solvent effects on the assay are determined during assay optimization. Another option is to extract in the desired solvent, then conduct a solvent exchange into a more miscible solvent. Immunoassays perform best with water-miscible solvents when solvent concentrations are below 20%. Our experience has been that nearly every matrix requires a complete validation. Various soil types and even urine samples from different animals within a species may cause enough variation that validation in only a few samples is not sufficient. 

Technology providers use quantitative immunoassays to determine expression data of field material for regulatory submissions. Regulatory authorities require that expression levels of introduced proteins in various plant parts be determined by quantitative, validated methods. Immunoassays are also used to generate product characterization data, to assess food, feed and environmental characteristics, to calculate concentrations for toxicology studies and to obtain tolerance exemption or establish tolerances for pesticidal proteins. 

Several considerations influence the suitability of the immunoassay as a qualitative or quantitative tool for the determination of tissue residues. These include the assay format, the end user (on-farm or laboratory use), effects of sample matrix on the analysis, cross-reactivity considerations, detection levels required of the assay, target tissues to be used in the assay, and the use of incurred or fortified tissues for validation of the immunoassay against accepted instrumental methods. Although these variables are often interrelated, each topic will be discussed in further detail below. 

The use of immunoassays for the determination of pesticides and veterinary medicines in food animals has increased since the early 1990s. The advantages of simple analysis, quick results, and high throughput make immunoassays a powerful technique for problematic matrices commonly encountered in animal agriculture. Careful development and validation are required to obtain accurate results, however. This review has demonstrated that most immunochemical techniques have been designed for use with milk samples, but a number of applications have also been developed for liver and muscle samples. The development of immunoassay techniques for residue analysis in eggs has clearly not been pursued to the extent of other edible tissues. 

E5. Ekins, R., Validity of analog free thyroxin immunoassays. Clin. Chem. 33,2137-2144 (1987). 

I.A. Darwish and D.A. Blake, One-step competitive immunoassay for cadmium ions development and validation for environmental water samples. Anal. Chem. 73, 1889-1895 (2001). 

Geng, D., Shankar, G., Schantz, A., Rajadhyaksha, M., Davis, H., and Wagner, C. 2005. Validation of immunoassays used to assess immunogenicity to therapeutic monoclonal antibodies. Journal of Pharmaceutical and Biomedical Analysis 39, 364-375. 

There are some other firms that also commercialize immunoassay kits for PCBs based on different formats (see Table 3). They have been mainly validated for soil and wipe matrices, although the manufacturer can provide optional protocols for testing sediment or water samples. 

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