Immunoassay ELISA

This author and coworkers at Beckman Coulter first described the use of a low form 96-well plastic microplate for automated micro-ELISA immunoassays (Matson et al., 2001). The polypropylene plate was first modified by a radiofrequency plasma amination process (Matson et al., 1995) followed by conversion to an acyl fluoride surface chemistry for rapid covalent attachment of biomolecules. Proteins (1 to 2 mg/mL) were prepared in 50 mM carbonate buffer, pH 9, containing 4% sodium sulfate (to improve spot uniformity) and printed using a conventional arrayer system. Approximately 200-pL droplets of monoclonal antibodies (anti-cytokine) were deposited into the bottom of the microwells using a Cartesian PS7200 system equipped... 

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. 

As we learned after the anthrax attacks in 2001, the ability to rapidly detect and to identify a bioterrorism agent is critical. A variety of methods are used for this purpose, including DNA fingerprinting, DNA sequencing, PCR, and ELISA immunoassays. As students read about how these methods are utilized in fighting terrorism, they learn how the methods work. It is even better if they can actually apply at least some of the methods in the laboratory. A number of suppliers provide kits designed for educational use,25 and in most cases, it is possible to use these activities in a way that simulates a bioterrorism scenario. 

The Chief of Naval Research (CNR) should consider Navy funding of technologies for laboratory diagnostics that support a broader technology base for ELISA (immunoassay) and polymerase chain reaction to drive down crossreactivity and false positives and to assess alternative techniques that may not have the inherent shortcomings of these approaches. 

Monoclonal antibodies were obtained against atrazine and its metabolite hydroxyatrazine by immunizing mice with atrazine or hydroxyatrazine protein conjugates. By competitive ELISA, we observed that the antibodies raised against hydroxyatrazine cross-reacted mainly with hydroxypropazine. The antibodies raised against atrazine cross-reacted with propa-zine, prometone, prometryne, and to a much lower extent with a few other s-triazines and hydroxy-s-triazines. Atrazine could be detected in water samples down to 50 ppt. Average recoveries measured by ELISA from soil samples fortified with atrazine or hydroxyatrazine were comparable to those measured by GLC or HPLC. Soil samples of unknown atrazine content were analyzed by GLC, GC-MS, and by ELISA. The results show that the ELISA immunoassay represents a valuable detection method for trace amounts of atrazine and hydroxyatrazine in soil. 

We obtained stabilized hybridoma cell lines secreting MAbs specific for atrazine, a widely used herbicide, and for hydroxyatrazine, an important metabolite. As we observed previously with water samples (15), a good correlation was obtained between the current detection method (HPLC or GLC) and a MAb based immunoassay (ELISA) when fortified soils were analyzed. The limits of detection of atrazine and hydroxyatrazine by both methods were comparable. They corresponded to 50 ppb for soil samples and 0.05 ppb for water samples. However, by evaporating the methanol soil extracts before the ELISA, the limit of detection in soil samples could be reduced down to the ppb level. The analysis of undefined soil samples with respect to their herbicide content showed that for atrazine and hydroxyatrazine, some discrepancies were observed between the two methods due to biased detection during HPLC and GLC measurements. For atrazine this was confirmed by additional GC-MS analysis. Therefore, the ELISA immunoassay represents a valuable detection method for trace amounts of atrazine and hydroxyatrazine in soil, despite its limited specificity due to cross-reacting substances. 

As mentioned above, GC and LC techniques (and GC-MS and LC-MS in particular) represent the major determinative approaches used in current MMRMs. Other, much less widely applicable and applied techniques include (1) capillary electrophoresis and capillary electrochromatography (2) thin-layer chromatography and (3) an array of immunoassays, such as immunosensors and enzyme-linked immunosorbent assay (ELISA). Immunoassays are, however, relatively useful for sensitive and rather rapid and inexpensive screening (followed by a confirmatory method in the case of a positive response) of selected pesticides for which ELISA kits or sensors are available. 

Fig. 20 Scheme of a direct ELISA immunoassay (Reproduced from ref. 115 with permission from The Royal Society of Chemistry). 

Sandwich ELISA is the most popular of the ELISA immunoassays. Here, the measurable antigen is sandwiched between two antibodies (a capture antibody and a detection antibody), which bind to different sites on the antigen or ligand (Fig. 22). 

As in a sandwich ELISA immunoassay a single antigen binds two antibodies, the antigen must have at least two epitopes. The capture and detection antibodies may be ether monoclonal (those that recognize a simple epitope) or polyclonal (those that recognize more than one epitope). 

---