Immunoassay development requirements

Because standardization of immunoassays requires so many compromises, much work remains to be done, not only to accurately define the analsdical goals for immunoassays, but also to develop the necessary technology to achieve them. The whole process of immunoassay development and standardization is laborious, costly and very time consuming, so that priorities need to be set depending on the values and consequences of the output information obtained. 

At present, the aim is to ensure that a humoral response is generated. For mucosal vaccines, IgA may be more appropriate.21516 T cell-mediated responses may also be considered, and advances in knowledge and the development of different types of vaccines and/or adjuvants suggest that cell-mediated responses may be just as or more important than the humoral response, when considering the selection of species. Adjuvants can selectively switch the T helper cell response, which may influence a protection or pathology dependent response (see Section 19.2.5). However, the current understanding and interpretation of such data are limited in terms of predicitivity, and there are also limitations in the immunoassays developed for species routinely used in nonclinical testing. This is a key area where further development is required. 

Immunoassays are very versatile, and if one could select but a single method, it could be the method of choice. Fortunately we have a variety of techniques available and a good analyst should know when to apply them. Table I provides some general rules for determining how difficult an immunoassay will be. The terms used are relative and possibly other dimensions to the table could be the laboratory s experience with immunoassay and the problems faced. This table does not indicate that good assays cannot be developed for hard compounds it just Indicates that the expense, skill and time required may be greater for those compounds. For instance we have developed successful immunoassays for some lipophilic, small, unstable, volatile compounds. However, such compounds would be a poor choice to use for one s first venture into immunoassay development. 

The list above is not intended to be comprehensive, but it will serve as a general guide to immunoassay development. We must emphasize again the difference between development and implementation. Assays which work in the laboratory generally require modification before they can be used to analyze field samples. Implementation of assays that have been fully validated for field samples may require little additional commitment by the user, other than analyst training. For the near future, there may be considerable pressure to transfer immunoassay methods to the analytical lab as soon as possible after development, and the... 

What distinguishes clinical inununoassays om the assays presented in this book is that clinical assays most frequently quantify polar compounds in the matrices of blood and urine. In contrast, environmental and food applications of immunoassays often require development of antibodies to analytes that are lipophilic and that may occur in such diverse complex matrices as sediments, nuts, and animal fat. 

Assays of ciguatoxin. Determination of ciguatoxin levels in fish was carried out in many laboratories by mouse assays. Enzyme immunoassay to screen inedible fish has been proposed by Hokama (9). No specific chemical assay has been developed, as information on functional groups suitable for fluorescence labeling is not available. Analyses conducted in the authors laboratory on remnant fish retrieved from patients meals indicated that ciguatoxin content as low level as 1 ppb could cause intoxication in adults. An extremely high sensitivity and a sophisticated pretreatment method will be required for designing a fluorometric determination method for the toxin. 

The development of sensitive and inexpensive immunoassays for low molecular weight pesticides has been an important trend in environmental and analytical sciences during the past two decades. 0.27-29 jq design an immunoassay for a pesticide, one can rely on the immunoassay literature for agrochemicals, " but many of the innovations in clinical immunoanalysis are also directly applicable to environmental analysis. - Conversely, the exquisite sensitivity required and difficult matrices present for many environmental immunoassay applications have forced the development of technologies that are also useful in clinical immunoassay applications. In the following discussion we will describe widely accepted procedures for the development of pesticide immunoassays. 

Many groups have developed immunoassays for sulfonamides. These compounds were a problem in the late 1980s when they were commonly found in the milk supplies in the USA. Subsequently, a great deal of effort has been required to develop rapid, reliable assays for the large number of chemical variations of sulfonamides. 

Franek et al developed an immunoassay for sulfadimidine, but they found that milk samples required dilution (1 100), and tissues required dilutions of 1 200 to 1 4000. The LOD was 0.02 pg kg from the buffer calibration curve with the IC50 at 0.15 pgkg The assay measured levels in milk from 10 to 100 pgkg with satisfactory precision. In swine muscle, kidney, and liver samples, levels from 20 to 500 pg kg could be measured when 2 g of tissue were homogenized with 20 mL of buffer and then diluted 1 20. 

Kennedy et al. developed a lasalocid immunoassay for application to residues in chicken meat and liver samples. The antibody was specific and did not cross-react with salinomycin, maduramicin, or monensin. Sample preparation consisted of homogenization in aqueous acetonitrile, removal of fat from an aliquot of the aqueous acetonitrile by hexane extraction, and evaporation of acetonitrile. The sample was then reconstituted with assay buffer. Liver required an additional solid phase extraction step. The LOQ was 0.02 xgkg for muscle and 0.15 agkg for liver. These workers were able to use the system to determine the half-life of lasalocid in the tissues. 

Silverlight and Jackman developed an immunoassay for levamisole in meat and milk. The LOD in both milk and meat samples was 1 xg kg The assay was applied to milk directly, and muscle samples required only homogenization in the presence of 10-fold of buffer prior to analysis. The linear range of the assay was between 5 and 50 pg kg for meat and between 0.2 and 25 qg kg for milk. The linear range of the assay was below the MRL for milk (10 qg kg ) and meat (50 qg kg ). 

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. 

To understand how immunoassay-based analytical methods can be constructed to comply with tolerance enforcement requirements, a brief examination of those requirements is in order. This discussion is not intended to be comprehensive but to highlight aspects of special significance to immunoassay method development. The reader is urged to consult the literature " for further details. 

These requirements have special implications with regard to immunoassay methods. Eirst, the lack of commercial availability of reagents precludes preparing antibody-coated tubes or plates on-site, which may require knowledge of special skills. Commercial availability also ensures the analyst access to a reproducibly manufactured product. Therefore, the method must be based on an immunoassay that is a commercial product. Method developers may choose to introduce an in-house assay to the marketplace by partnering with a manufacturer, although this approach is costly and time-consuming. 

The methylxanthines can be determined in foods and biological systems by the chromatographic methods of TLC, GC, HPLC, or CE. Ultraviolet spectroscopy following a separation procedure can also be used. More recently, immunoassay methods have been developed. There is no single best method the analyst must balance the features of each assay with the final requirements for data precision and reproducibility. 

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