Immunoassay validation studies

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. 

Brady, J.F., D.P. Tierney, J.E. McFarland, and M.W. Cheung (2001). Inter-laboratory validation study of an atrazine immunoassay. J. Amer. Water Works Assoc., 93 107-114. 

However, in the long term, ELISA is an ephemeral format. Even when streamlined and automated, it has too many steps. Certainly we should realize that it will be replaced by other systems, the most exciting of which will be biosensors. Also, other formats offer a proprietary edge in the market place which will be very important in the maturation of immunoassay systems in the environmental field. Finally, different formats will lend themselves to different environmental problems. We should continually emphasize that the same reagents can be used in many formats. Possibly in small letters we also should caution that certain antibody characteristics may be more important in one format than another, that some formats are more resistant to matrix effects, and that relative cross reactivities of compounds can change as one changes the subtle principles upon which an immunoassay works. For this reason a clear choice of formats should be made before initiating validation studies. 

The 198th ACS meeting certainly is a landmark meeting in the immunoassay field. For the first time at this meeting we have seen reports from a variety of major agricultural chemical companies about the in-house efforts in immunodiagnostics (11-24-29) as well as collaborative validation studies between a biotechnology company and a university (30) and a contract laboratory (31). In addition to the development of polyclonal based systems, there is an increased interest in the development of monoclonal antibodies for environmental chemicals (12,11,32). Deschamps and Hall (32) presented a nice comparison of the relative attributes of mono- vs polyclonal based systems for the herbicide picloram. 

An important extension of our large validation studies involves the use of data bases from field studies in the development of improved statistical methods for a variety of problems in quantitative applications of immunoassays. These problems include the preparation and analysis of calibration curves, treatment of "outliers" and values below detection limits, and the optimization of resource allocation in the analytical procedure. This last area is a difficult one because of the multiple level nested designs frequently used in large studies such as ours (22.). We have developed collaborations with David Rocke and Davis Bunch (statisticians and numerical analysts at Davis) in order to address these problems within the context of working assays. Hopefully we also can address the mathematical basis of using multiple immunoassays as biochemical "tasters" to approach multianalyte situations. 

Implementation of new methods in regulatory work faces several critical difficulties. Among them, lack of certified reference materials, QC/QA protocol application and pending validation studies hinder the transformation of good ideas into official methods for practical regulatory work. For example, Inami et al. recently compared two immunoassays and a 5 h neuroblastoma assay with the MBA for presence/absence detection of saxitoxins in shellfish. The study concluded that a reduction in the number of mouse bioassays in the state of California could be achieved, provided that the alternative assays were applied as a screening tool. 

The versatility and nature of immunoassays allow the detection of virtually any type of compound. The reliability of these detection methods is assessed by validation studies, although they are not always easy to carry out, due to the nature of the analyte and the lack of reference materials. These assays are used by food industry and regulatory agencies for different purposes. Immunoassays can be used by the food industry... 

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. 

It may be important to consider the variability of the matrix due to the physiological nature of the sample. In the case of LC-M/MS-based procedures, appropriate steps should be taken to ensure the lack of matrix effects throughout application of the method, especially if the nature of the matrix changes from the matrix used during method validation. For Microbiological and immunoassay, if separation is used prior to assay for study samples but not for standards, it is important to establish recovery and use it in determining results. In this case, possible approaches to assess efficiency and reproducibility of recovery are ... 

The validity of any statement about the purity of a protein is directly linked to the quality of the analytical method used. The validation of immunoassay systems to detect protein impurities in rDNA pharmaceuticals must be achieved by careful production and characterization of the assay reagents. The studies presented here demonstrate that the blank run approach is reasonable for the isolation of reference materials and that high quality broad spectrum antisera can be produced to these mixtures. Significant improvements in assay sensitivity approaching the ppb level are attainable and should provide the methods to further improve product purity. 

As for any bioanalytical method, the extent of validation for an immunoassay should be related to the intended application of the assay. Thus, if an immunoassay is intended to support rapid screening in discovery R D, the characterization of specificity and the accuracy and precision specifications may be less stringent than if the assay is used to support pre-clinical and clinical development studies. Indeed, an assay for discovery support may be designed to detect active metabolites as well as parent molecule, so that... 

Findlay, J.W.A. Validation in practice-experience with immunoassay. In Bio-International 2. Bioavailability, Bioequivalence and Pharmacokinetic Studies Blume, H.H., Midha, K.K., Eds. Medpharm Scientific Publishers Stuttgart, 1995 361-370. 

Zweig MH, Robertson EA. Clinical validation of immunoassay A well-designed approach to a clinical study. In Chan DW, ed. Immunoassay A practical guide. San Diego Academic Press Inc, 1987 97-128. 

HPLC methods with UV detection for MPA quantification are in common use in clinical pharmacokinetic studies and in clinical laboratories. An immunoassay has been developed and is in routine use in centers outside the United States. Validated HPLC assays with mass spectrometric detection have been developed and are particularly useful for the accurate measurement of free MPA concentration. A new method that uses IMPDH, the natural target receptor of MPA, is in development. Inhibition of IMPDH activity by MPA in an aliquot of patient serum or plasma is the basis for this method. 

Sukovaty, R.L., Lee,J.W., Fox,J., Toney, K., Papac, D.I., Thomas, A., Grover, T.A., and Wells, D.S. (2006) Quantification of recombinant human parathyroid hormone (rhPTH(l 84)) in human plasma by immunoassay commercial kit evaluation and validation to support pharmacokinetic studies. Journal ofPharmaceutical and Biomedical Analysis, 42, 261 271. 

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