Chapter 25

Panel Discussion

The speakers on regulatory and acceptance issues, P. Schuda, J. Brady, J. Rittenburg, S. Berberich, S. Coates, and M. Trucksess were panelists for a discussion which was moderated by R. B. Wong. The major points of discussion centered upon the data analysis and interpretation issues. Other questions were related to the AOCA Research Institute validation process, EPA's acceptance of immunoassay data, and problems with cross-reactivity as exemplified by the validation of aflatoxin immunoassay kits. The following is a summary of the panel discussion.

According to Dr. Brady, an immunoassay method is an analytical method and so it should satisfy the same criteria as conventional analytical methods must meet. As such, the analyst must provide evidence the limit of detection (LOD) satisfies some statistical criteria to distinguish its response from the response of the zero dose standard in addition to the LOD merely being the smallest standard (one approach to this statistical problem is to use a modified form of Rodbard's work that employs Student's t distribution for a one-sided test at 95% probability). The limit of quantitation (LOQ), by contrast, is determined by achieving satisfactory recoveries in fortified samples of each matrix the assay is intended to be used for. The lowest level at which those recoveries are achieved becomes the LOQ. The LOD thus establishes the lower limit of the standard curve but the LOQ sets the concentration at which sample residues may be quantified. The LOQ is consequently a practical, utilitarian level whereas the LOD is merely the lower end of the standard curve. Depending upon the complexity of the matrix, these levels may not necessarily be the same. A method with an LOD of 0.05 ppb, for example, may achieve satisfactory recoveries at 0.05 ppb in water but fail to do so in soil. Recoveries in soil may be that an LOQ is set at a higher level such as 0.10 ppb.

There were differences in opinion about the logit-log transformation of immunoassay data. Brady took a conservative view that the only useful portion of the sigmodial dose-response curve is the central linear dose-response region. A response corresponding to a sigmodial tail can be extrapolated back to multiple doses on the x-axis, in contrast to

0097-6156/95/0586-0354$12.00/0 © 1995 American Chemical Society

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25. Panel Discussion 355

the unique one-to-one dose-response relationships found in the linear central portion. To make the tail appear to have a one-to-one dose response relationships is merely a mathematical manipulation that is unsupported by physical reality. Others in the audience consider logit-log transformation as valid, citing a long history of clinical application and also referenced Baud et al., (1) showing logit-log as the best and most accurate among the methods compared for eight different immunoassays. However, it was also acknowledged that any method of transformation or curve fitting presupposes that the data conform to a particular mathematical model. While data for microplate ELISAs and some other formats are well approximated by the four-parameter logistic equation or the logit-log transformation, data from other formats may be better fitted by other models. The most conservative view is that with any assay format it is necessary to obtain enough data to justify the use of a particular mathematical model and accurately establish the variance and confidence limits at different analyte concentrations.

The AO AC Research Institute (AO AC RI) validation procedure was clarified by Dr. Coates. The validation protocol requires that the kit manufacturer provide all the data in support of the claims. After reviewing the package, the AO AC RI will test the claims and provide a certification if validation is successful. The cost of the procedure is $7,500 per application plus billing to the sponsor for independent testing (cost range from $5,000 to $10,000). Each test kit can include any number of matrices as long as the data for such matrices are supplied at the time of application. Additional fee will be charged if data for additional matrices are provided at a later date. The AOACRI routinely check the performance of the certified kits. Adverse Advisory Statements are sent to the sponsor for unsatisfactory performance and the manufacturer has thirty days for corrective action. The manufacturer is required to send a copy of the package insert annually to the Institute and revise any changes in format, reagents or formulation. The annual maintenance fee for certification is $1,000.

The EPA-Office of Pesticide Programs views immunoassay as a positive step. This office is different in that its methods are performance based and not prescriptive. Registrants using immunoassay methods should approach the agency on an individual basis since different reviewers and their supervisors have different perspectives about the technology and no defined mechanism for immunoassay implementation is available at this time. With the work of the Analytical Environmental Immunochemical Consortium (AEIC) and the Summit Meetings sponsored by the Environmental Monitoring Systems Laboratory in Las Vegas (EMSL-LV), some of the key issues required to accept immunoassay are being resolved. Another important point Dr. Schuda brought up was a training program. While it is useful for reviewers in the agency to attend meetings such as the ACS, it is extremely difficult because of limited travel budget. Some training program will have to be devised which can accomplish the goal of introducing this technology. In response to the question of assay kit certification, the agency views certification of assay kits by recognized groups as an added measure of credibility. The idea of environmental laboratory certification adds a measure of confidence and helps to provide the agency with a "level of comfort" in accepting immunoassay methods and data.

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356 IMMUNOANALYSIS OF AGROCHEMICALS

A concern was raised about cross-reactivity in immunoassays, observation by Dr. Trucksess that four aflatoxins cross-reacted in tests that she and her colleagues evaluated. Dr. Trucksess pointed out that first of all, the user must make an effort to know or determine the cross-reactivity of the kit. This information is usually provided by the kit manufacturer. Also, there are different populations of aflatoxins in various commodities. For example, in corn, Bl and B2 are most commonly found and Bl is often much greater than B2. In peanuts, all four aflatoxins can be found and in some rare instances, the amount of G 1 level can be greater than that of Β1. Therefore, as a rule, when immunoassay shows a positive for total aflatoxin, a second method of analysis will be needed to quantify individual aflatoxins.

Dr. Rubio elaborated on Lawruk et al.'s (2) work which was cited in Dr. Brady's presentation. In addition to the information provided by the publication that the graph (figure 1, page 1427) represented the mean of 68 determinations, Dr. Rubio stated that the error bars represented an average of 68 runs by different analysts using different lots of reagents in a three months period.

1. Baud,M, Mercier, M., and Chatelain, F. J. Clin Lab Invest .51, (Suppl 205), 1991,120-130. 2. Lawruk, T. S.; Lachman, C. E.; Jourdan, S. W.; Fleeker, J. R.; Herzog, D. P.; Rubio, F. M. J. Agric. Food Chem. 1993, 41, 1426-1431.

RECEIVED December 7, 1994

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