Specificity immunoassay development

The singularity of MAbs and the ease of mass production appeared to be the answer to rapid development of highly specific immunoassays. Companies were formed to produce MAbs and incorporate them into assays. In fact, such assays have been developed and have proved very successful for infectious diseases, hormones, and other clinical analytes. 

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. ... 

A specific immunoassay for measuring two-chain factor VIIa levels in plasma has been developed to identify activation of factor VII in patients with acute coronary syndromes suchs as myocardial infarction and unstable angina (12). Because regulation of factor VIIa is believed to be mediated by tissue factor pathway inhibitor (TFPI), its measurement is also useful in assessing thombotic and cardio-vasular disorders. Because TFPI is released by heparin, its measurement is also useful in assessing the efficacy of heparin and endothelial cell function (93). 

Generation of antibodies that can recognize and bind to specific viruses is straightforward. A sample of live or attenuated virus, or a purified component of the viral caspid, can be injected into animals to stimulate polyclonal antibody production (or to facilitate monoclonal antibody production by hybridoma technology). Harvested antibodies are then employed to develop specific immunoassays that can be used to screen test samples routinely for the presence of that specific virus. Immunoassays capable of detecting a wide range of viruses are available commercially. The sensitivity, ease, speed and relative inexpensiveness of these assays render them particularly attractive. 

Snitkoff et al. [75] reported the development of an EIA for the detection of ciprofloxacin in serum, which was sensitive at picogram per milliliter levels of the antibiotic and no cross-reaction with its metabolites was observed. Gobbo et al. [118] recently described the production of PAb for ciprofloxacin with the aim of detecting fluoroquinolones in Brazilian livestock. On the other hand, Bucknall et al. [77] produced antibodies for quinolones and fluoroquinolones with the aim of developing both generic and specific immunoassays. ELISAs for ciprofloxacin, enrofloxacin, flumequine, and nalidixic acid were developed with sensitivity values around 4 pg kg 1 (on both the generic and specific assays) in bovine milk and ovine kidney. 

Considerable effort is being expended toward the development and understanding of fluorescence-based immunoassay technology. A number of factors can be suggested that tend to drive interest in the pursuit of specific immunoassay methods in general, including sensitivity, accuracy, precision, ease of manufacture, capability for a broad... 

Watanabe, T., G. Shan, D.W. Stoutamire, et al. 2001. Development of a class-specific immunoassay for the type I pyrethroid insecticides. Anal. Chim. Acta 444 119-129. 

Pastor-Navarro, N., E. Gallego-Iglesias, A. Maquieira, et al. 2007. Development of a group-specific immunoassay for sulfonamides Application to bee honey analysis. Talanta 71 923-933. 

CK-MB can be measured in numerous ways. Immunoassays developed in recent years have improved on the analytical and clinical sensitivity and specificity of the earlier immunoinhibition and immunoprecipitation assays. These assays now (1) measure CK-MB directly and provide mass measurements, (2) are easily automated, and (3) provide rapid results (<30 minutes). Mass assays reliably measure low CK-MB concentrations in both samples with low total enzyme activity (<100 U/L) and with high total enzyme activity (>10,000 U/L). Furthermore, no interferences from other proteins have been documented. The majority of commercially available immunoassays that use monoclonal anti-CK-MB antibodies are the same as those listed in Table 5-2 for cardiac troponin assays. Excellent concordance has been shown between mass concentration and activity assays. A primary reference material is commercially available to assist in harmonization. If used for assay standardization, then this material allows... 

The Opus system consists of the Opus analyzer (Fig. 26), assay-specific test modules (Fig. 27) and calibrator sets. This system performs in vitro quantitative and qualitative fluorescent immunoassays developed by PB Diagnostic Systems, a joint venture of the Polaroid Corporation and Behringwerke AG. 

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. 

In the absence of an unambiguous history of ricin exposure, the preferred diagnostic method is specific immunoassay of ricin in serum, respiratory secretions, or other clinical samples associated with poisoning. Most of the methods described for ricin detection are experimental or are under development. The CDC and the Federal Laboratory Response Network have the capability to detect ricin in environmental specimens using validated polymerase chain reaction (PCR) tests and time-resolved immunofluorescence assays, with cell-based bioassays to confirm ricin activity. The U.S. Department of Defense has produced experimental field immunoassays, but commercial distribution of field test kits currently is limited. 

During early immunoassay development, it is important to assess the intended use of the assay. When the intended use is to support pharmacokinetics in either nonclinical PK/TK or clinical bioequivalence (BE) studies, developing a validat-able assay is of utmost importance. A well-thought-out validation plan and fully documented assay methods and results that support the validation are crucial. To withstand the scrutiny of the authorities, and to guarantee the optimal method is applied to your drug development process, the following parameters should be assessed critically, validated to specific criteria, and fully documented. 

The procedures for production of specific antibodies and their application in a competitive inhibition ELISA (Enzyme-Linked Immunosorbent Assay) are discussed in detail in the preceding chapter (Vanderlaan et al., this volume). In addition, other comprehensive overviews of the immunoassay development process in the pesticide field are available in general, a... 

Immunoassays Developments in the production of monoclonal antibodies have led to the production of monoclonal antibodies specific to HbAjc. These antibodies have been used with a variety of immunoassay platforms to produce automated methods. Care must be taken to determine if the epitope of the antibody is... 

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