Immunoassay assay development

However, if a class-selective assay is desirable (for multi-analyte assays), the handle should be located at or near a position that differentiates members of the class and exposes features common to the class. Using the pyrethroid example, an ideal immunogen should retain the phenoxybenzyl moiety and link the protein from the distal acid end (Figure 9). Using such an immunogen hapten, a class-specific immunoassay was developed that was highly cross-reactive with the type I pyrethroids permethrin, phenothrin, resmethrin and bioresmethrin. ... 

An immunoassay was developed to determine the penicillinase stable isoxazolyl penicillins cloxacillin and dicloxacillin in milk by Usleber et alJ The assay detected lOpgkg" of cloxacillin and 30pgkg of dicloxacillin with recoveries of 102% and 84%, respectively. The calibration curve was prepared by fortifying skimmed milk powder (lOOgL ) with standards. Fortified samples were prepared in pasteurized milk and analyzed directly after decreaming by centrifugation. This immunoassay was performed with minimal sample preparation, probably because the extensive water solubility of the penicillins prevents problems associated with more lipid-soluble analytes. 

The DELFIA assay, the first effective lanthanide-based immunoassay, was developed and commercialized by the early 1980s.108-112 DELFIA (Dissociation Enhanced Lanthanide Fluoro-ImmunoAssay) is a heterogeneous assay which uses a lanthanide complex based on aminocarboxylate ligands such as EDTA, EGTA, or DTPA, linked to the antibody by reaction of appended isothiocyanate groups (e.g., complex (45)) with nucleophilic residues, particularly amines, on the protein surface (Figure 11). 

The expense of an analytical procedure depends upon much more than the cost of the final analysis. Much of the expense of an assay is related to sample preparation, and for many applications immunoassays have tremendously reduced the time needed for sample preparation. Another consideration is the amount of time needed for the development of an assay. The additional expertise which must be developed in an analytical laboratory before immunoassays can be used with confidence may seem formidable, and waiting for an animal to develop antibodies may lead to unacceptable delays in assay development. On the other hand, once a usable antibody titer is obtained, the development of a workable assay is usually straightforward. It is also likely, if immunoassays become accepted for some aspects of pesticide analysis, immunoassay kits or at least critical reagents will become commercially available. Such kits already exist for many pharmaceutical products and hormones, and numerous companies will supply antibodies to a user supplied hapten on a contract basis (83). 

The TRH radio-immunoassay was developed as a double antibody assay, with modifications mainly depending on the antiserum used, and on the conditions of incubation. Addition of an enzyme inhibitor to the assay tubes is essential to avoid degradation of TRH in samples and of radioiodinated TRH. 

A cell culture/ELISA assay has recently been developed to detect anticoagulant rodenticides in treated grain (Lawley et al, 2006). A prior immunoassay was developed to detect diphacinone and chlorophacionone (Mount et al, 1988). Enantiomers of warfarin, coumachlor, and coumafuryl can be separated chromatographically (Armstrong et al, 1993). 

Immunoglobulin Levels in Serum.Similar immunoassays were developed for human IgM and IgE. Levels of these Igs were determined in the sera of normal individuals and are shown in Table VI. The IgE analyses were performed on samples that were absorbed with PA-Sepharose as described above to remove components responsible for nonspecific (lipids) and specific (IgG) interference in the assay. The IgE levels were comparable to values obtained for the same samples by double-antibody RIA. The concentration of IgG in these sera (Table VI) was determined by the procedure described above [Eq. (1)]. 

To understand how the Department has selected target pesticides for immunoassay development, it should be understood that the EM PM Branch monitors the environment only for residues of certain pesticides. Since we have regulatory authority only over those pesticides in current use, we do not monitor the environment for pesticides such as EOT and related chemicals, which are no longer in use in the State. Therefore, although they are important environmental contaminants, they are not included in our assay development program. 

In order to achieve maximum value to the Department, the immunoassays we develop will be validated through testing to determine within- and between-laboratory variability. At present, we plan to defer validation by the Association of Official Analytical Chemists (AQAC) until we have an assay which is more specific than the current atrazine assay, vrtuch is 40% cross-reactive to simazine. 

The differential equations describing the reactions involved in AM-type immunoassays are rather complicated for regression analysis. They were modified by the simplification procedures of Naus et al. (1977) and Wellington (1980). These rather complex mathematical procedures serve mostly as an aid in assay development. 

This section elucidates some of the practical aspects that should be considered while developing an immunoassay. Although written with an emphasis on the sandwich assay format, and more specifically ELIS As over RIAs and ECL assays, it also applies to the competitive assay format. A systematic approach for immunoassay method development may consist of the steps shown in Fig. 3.3. 

Affinity and Avidity Antibody strength of binding and specificity are the main determinants of the sensitivity and ruggedness of any immunoassay. Strength of binding reflects both affinity and avidity, which are described in more detail below and summarized in Table 3.3. Understanding these antibody characteristics will help in the appropriate selection of antibodies for assay development. A variety of methods for determining the affinity constant [13] and avidity index [14,15] have been reported. 

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