Selectivity determination immunoassay development

Specificity of an immunoassay is usually measured by determining the extent that compounds that are structurally similar to the test analyte react in the assay. The determination of the assay reactivity of an array of potential cross-reactant is routinely performed in immunoassay development. A panel of suspect cross-reactant, should be selected on the basis of structural similarities to the test analyte and on the expected occurrence along with the test analyte in the sample. Thus, an immunoassay specific for 17/1-estradiol should be tested for reactivity with estrone, estriol, 17a-estraiol and testosterone. Other compound testing might also be indicated. 

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. 

Chen and co-workers have reported a novel, simple, economical, and environmentally friendly, tunable immunosorbent-based immunoassay for sensitive and selective determination of atrazine <2003ABI251>. Hammock and co-workers have developed an antibody for simazine and atrazine that exhibits low cross-reactivity to propazine relative to most atrazine antibodies evaluated heretofore <1996JFA2210>. 

Acrylonitrile metabolites have been measured in blood and urine, but, except for measurement of thiocyanate, these methods have not been developed for routine monitoring of exposed humans. Supercritical fluid extraction/chromatography and immunoassay analysis are two areas of intense current activity from which substantial advances in the determination of acrylonitrile and its metabolites in biological samples can be anticipated. The two techniques are complementary because supercritical fluid extraction is especially promising for the removal of analytes from sample material and immunoassay is very analyte-selective and sensitive (Vanderlaan et al. 1988). 

The issue of which antibody to select for an assay is not a new problem. Certainly anyone involved in the development of an immunoassay has been faced with this choice. Consider attempting to create a multianalyte, microarray-based micro-ELISA of modest density (10 to 100 analytes) and determining which capture antibodies to use based upon their affinities, stabilities, and cross-reactivities. For a sandwich assay, add in the 10 to 100 analyte-specific secondary (reporter) antibodies and determine their levels of cross-reactivity with each other and with the specified antigens and capture antibodies. In other words, achieving high performance for all analytes with a microarray immunoassay is indeed a formidable challenge. 

Ultimately major efforts to develop coupled chromatographic techniques have been performed to alleviate the problem of manual sample pretreatment and to enhance sensitivity and selectivity in the analysis of PAHs in foodstuffs (192) and environmental samples (193-195). Liquid chromatography/MS (196,197), GC/MS (175), HPLC with UV absorbance, fluorescence (177) (see Fig. 4), or electrochemical (ED) detection (179), and ELISA immunoassay (198) have been successfully used for the determination of HAAs. 

In order to decide the lowest possible working concentrations of competitor and antibody, the development of a competitive immunoassay usually starts by performing a two-dimensional dilution experiment, i.e., the antibody concentration, [Ab], is varied in one dimension and the competitor concentration, [Ag ], is varied in the other. In this way the optimal [Ab] and [Ag ] can be determined experimentally and the lowest possible [Ag ] and [Ab] are usually selected, when 30-70% of the tracer is bound to the antibody ([Ab-Ag ]). However, the contradiction in defining immunoassay sensitivity, as mentioned above, has led to different values of the theoretically predicted... 

As reported in the structural determination of BL, CS, DL, and typhasterol, MS is an essential technique for BRs isolated in pure form. However, in most cases, isolation of BRs in pure form is time-consuming and tedious work because of their very low concentration in plant materials. BRs are highly polar and involatile compounds. Therefore, conversion of BRs into volatile derivatives in gas phase makes it easy to characterize BRs in a partially purified bioactive fraction by GC/MS or GC/selected ion monitoring (SIM), which are analytical techniques most frequently used in natural products chemistry. The desired derivatives of BRs are BMBs or MB-TMSs. Another convenient and useful technique is HPLC. HPLC has now been routinely and effectively employed in the purification of natural BRs. Microanalysis of BRs by HPLC has recently been developed, which involves transformation of BRs into derivatives with a fluorophore or an electrophore by use of pre-labeling reagents. Immunoassay techniques to analyze plant hormones have recently advanced and are readily accessible by plant physiologists. RIA for BRs has also been developed. In this section, micro-analytical methods of BRs using GC/MS (SIM), HPLC, and RIA are described. 

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. 

As basic information in plant science increases additional markers will be identified for which immunoassays can be developed. Rapid determination of the nutrient status of the growing crop will allow crop managers to select fertilization that produces maximum return based on the specific needs of the crop as it grows in a specific location (29). Similarly, detection of water stress markers will permit irrigation scheduling based on crop water status rather than on soil moisture levels or other environmental measures. 

---