Direct ELISA

Diphenylhydantoin, which has been demonstrated to cause autoimmune phenomena in man (SLE, vasculitis and scleroderma and skin) has also been tested (via drinking water for 6 months) in genetically predisposed mice (C57BL/6-lpr/lpr strain) but the compound depressed rather than increased the levels of ANA (55 and section 4.1, below). In another study [56], a slight shift towards a Th2 response was demonstrated as an increase in the KLH-induced production of IL-4 and IgE (IgE was detected by direct ELISA, which makes these data suspect) in a 4 weeks exposure study. In this same study, proliferative responses of splenocytes to KLH (using spleen cells of KLH-sen-sitized mice), mitogens (ConA, LPS) or anti-CD3 were also reduced, possibly through interference with accessory cell function. 

Although several immunochemical methods have been reported for LAS, few examples of their application to real environmental matrices have appeared. The first immunochemical method for LAS was reported by Fujita et al. [151]. It is a direct ELISA and uses MAbs generated against 5-sulfophenyl valeric acid conjugated to BSA through the carboxylic acid, thus preserving the sulfonic group... 

Immunochemical methods have been reported for both APEs and their metabolites, especially APs. A discussion of the immunochemical methodologies reported to date, the effect of the immunizing haptens employed, and the features of these techniques were recently reviewed [169]. Unfortunately, the detectability achieved is usually far from what is necessary for direct application to environmental samples. Moreover, the selectivity for APs versus APEs is not always satisfactory. Thus, Goda et al. [ 148] developed a direct ELISA for NP with a LOD of 10 pg L 1 and a working range between 70 and 1,000 pg L, but APEs with one to ten ethoxylate units are also well recognized. 

MAbs were also produced by Goda et al. [149], and a direct ELISA has been developed. The LOD accomplished for DBP was 0.2 mg L 1 with a dynamic range of 0.2-4 mg L-1. DEHP was not recognized (CR<1%). BBP showed a cross-reactivity value of 155% and dipropyl phthalate (DPrP) and dipentyl phthalate (DPnP) cross-reacted 60 and 51%, respectively. 

As shown in Fig. 21, in a direct ELISA the unlabeled antigen (a range of standard antigen concentrations or unknown samples) is attached to the solid phase. Enzyme-conjugated (labeled) primary antibody is then added. After incubation and washing of the plate... 

Fig. 21. Principles of ELISA. A In a direct ELISA the unlabeled antigen is attached to the solid phase. Enzyme-conjugated antibody is then added, followed by the enzyme substrate solution and color is allowed to develop. B ELISA with unlabeled antibody attached to the solid support. A variable amount of antigen is then added. A secondary antibody labeled with enzyme, followed by substrate solution, is added to all wells. The amount of color produced is proportional to the amount of antigen present. C Sandwich ELISA assay with the antigen sandwiched between an immobilized, antigen-specific primary antibody and an antigen- or species-specific secondary antibody. An enzyme-labeled tertiary antibody increases the assay specificity and sensitivity...

J. Zeravik, K. Skryjova, Z. Nevorankova, and M. Franek, Development of Direct ELISA for the Determination of 4-Nonylphenol and Octylphenol Anal. Chem. 2004, 76, 1021. 

ELISA. The focus of this chapter, the competitive ELISA, usually is the preferred choice when a DAS ELISA is not available because it provides greater specificity than the direct ELISA, making it more reliable for the diagnostic procedures it is being used for (7,2). 

One week later the animals were bled, and serum was prepared from whole blood and tested in ELISA. (If the sera was negative in the direct ELISA, then the animals would have been reinjected for two additional weeks and then retested with ELISA.)... 

There are two forms of direct ELISA, namely labelled-antibody and labelled-antigen. Both follow a similar protocol, but as the former is arguably the most widespread it is described below (Eigure 10.8). 

Marcobal, A., Polo, M.C., Martin-Alvarez, P.J. Moreno-Arribas, M.V. (2005a). Biogenic amines content of red Spanish wines. Comparison of a Direct ELISA and an HPLC method for the determination of histamine in wines. EoodRes. Int., 38,387-394. 

Table 13.3 Mean and the standard deviation values of the histamine concentrations in 30 commercial wines analyzed by a direct ELISA and an HPLC method, and the results of t test for related samples...

Table 13.3 shows the mean and the standard deviation values of the histamine concentrations in 30 commercial wines analyzed by a direct ELISA and HPLC methods (Marcobal et al. 2005) and the results of the t test obtained with the STATISTICA program (procedure T-Test for dependent samples, in the Basic Statistics and Tables module). The results revealed slightly higher results for ELISA (P < 0.05). 

Cell-Based Cell-based ELISA are indirect or direct ELISA systems that use intact cells... 

Newer techniques have equal sensitivity and greater specificity Enzyme-linked immunosorbent assay (ELISA) tests can identify C. trachomatis, HSV-1 and -2, and adenoviruses through the detection of microbial antigens. In the direct ELISA, an enzyme is covalently linked to an antigen-specific monoclonal or polyclonal antibody. 

Applications of immunoassay to pesticide chemistry have been described which address some difficult problems in analysis by classical methods. These include stereospecific analysis of optically active compounds such as pyrethroids (38), analysis of protein toxins from Bacillus thuringiensis (5,37), and compounds difficult to analyze by existing methods, such as diflubenzuron (35) and maleic hydrazide (15 also Harrison, R.O. Brimfield, A.A. Hunter, K.W.,Jr. Nelson, J.O. J. Agric. Food Chem. submitted). An example of the excellent specificity possible is seen in assays for parathion (10) and its active form paraoxon (3). Some immunoassays can be used directly for analysis without extensive sample extraction or cleanup, dramatically reducing the work needed in typical residue analysis. An example of this is given in Figures 2 and 3, comparing the direct ELISA analysis of molinate in rice paddy water to the extraction required before GC analysis. 

FIGURE 12.2 Noncompetitive immunoassay (A) direct ELISA (B) enhanced assay using a biotin-streptavidin system (C) indirect ELISA. 

Direct ELISA. Antigen is attached to the solid phase by passive adsorption. 

Direct ELISA can be regarded as the simplest fonn of the ELISA, and is illustrated in Fig. 1 and in the following diagram. 

Since a single enzyme-conjugated antibody is used, the system is limited to the specificities and properties inherent in that particular antibody set. This limits the versatility of the testj e.g., each antibody preparation used must be labeled (for different antigens) in the same way as the direct ELISA was limited to single antibody preparations. 

Again there is a requirement to titrate the direct ELISA system, which is then challenged by the addition of test antibodies. The competitive aspect here is between any antibodies in the test sample and the labeled specific antibodies for antigenic sites on the solid-phase bound antigen. The test sample and... 

A. Prc-titration of antigen and conjugate in Direct ELISA Oplimiction of concentrations to be used in competlEion system... 

---