Immunoassay development

Whereas MABs appear to be the choice for use in immunoassays, a majority of immunoassay developers and suppHers use polyclonal antibodies. 

Table 4. Detection Limits of Immunoassays Developed for Mycotoxins and Pesticides...

Alternatively, competitive ELISA can be used to estimate the hapten density if an antibody that specitically recognizes the hapten is available. At first observation this approach seems circular because the immunoassay developed is used to determine hapten density on proteins used for immunization. However, if a small molecule mimic of the protein conjugate is used as a standard, the method can be accurate. For example, a hapten containing a carboxylic acid can be coupled to phenethylamine or tyramine, its structure confirmed and the material used to generate a calibratron curve to estimate hapten density. 

Pesticide immunoassays have been developed for a variety of pesticides and, more recently, GMOs, and have been used for matrices such as surface water, groundwater, runoff water, soil, sediment, crops, milk, meat, eggs, grain, urine and blood. ° Table 9 is a partial list of immunoassays for chemical pesticides developed since 1995 and includes notations on the matrices studied. A fairly comprehensive list of pesticide immunoassays developed prior to 1994 was provided by Gee et al2 ... 

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

Pesticides, including insecticides, herbicides, and fungicides, are widely used in agriculture, and the potential for these residues to accumulate in food has led to concern for human safety. Pesticide residues may enter food animals from environmental sources or from treated or contaminated feeds. Immunoassay development for pesticides has had major impacts for pesticide registrations, analysis of residues in foods, monitoring environmental contamination, determination of occupational exposure, and integration of pest management. 

Table 4 Examples of immunoassays developed for pesticides and their metabolites...

Table 5 Examples of immunoassays developed for anti-infectious agents...

Fluorometers designed for research purposes(31) are typically equipped with a xenon arc lamp, monochromators, one or more photomultiplier tubes, cuvet holders, and a computer interface. Some research level fluorometers, such as the Perkin-Elmer LS50, have optional microtiter plate reading accessories with fiber optic bundles. This is convenient since 96-well microtiter plates are commonly used for immunoassay development, and many commercial immunoassays are based on the use of microtiter plates. Fluorometers designed for commercial immunoassay purposes are generally dedicated instruments with few, if any, data acquisition and reduction parameters that can be manipulated by the user. 

In the past ten years, numerous applications of fluorescence methods for monitoring homogeneous and heterogeneous immunoassays have been reported. Advances in the design of fluorescent labels have prompted the development of various fluorescent immunoassay schemes such as the substrate-labeled fluorescent immunoassay and the fluorescence excitation transfer immunoassay. As sophisticated fluorescence instrumentation for lifetime measurement became available, the phase-resolved and time-resolved fluorescent immunoassays have also developed. With the current emphasis on satellite and physician s office testing, future innovations in fluorescence immunoassay development will be expected to center on the simplification of assay protocol and the development of solid-state miniaturized fluorescence readers for on-site testing. 

Instead of using human serum albumin as a carrier protein, other workers (135) utilized ovalbumin for preparing the diazotized clenbuterol antigen in an enzyme immunoassay developed for screening of clenbuterol residues in bovine urine, liver, and eye. Alkaline phosphatase rather than -galactosidase was also used as an enzyme label in the preparation of the enzyme-clenbuterol conjugate. 

Another area of interest is in the emulsification properties of gum acacia. The protein-rich fractions which are amphiphillic in nature are responsible for stabilization of oil-in-water emulsions by gum arabic. Immunoassays developed against these protein-rich fractions can be used to measure emulsification capability. This was tested out (148) using heated and unheated gum samples. The heated samples exhibit poor emulsification properties versus the unheated and ELISA was able to distinguish these two gums, indicating the viability of using IA for testing the emulsification properties of gum acacia. 

Kishi T, Grass L, Soosaipillai A, Shimizu-Okabe C, Diamandis EP. Human kallikrein 8 Immunoassay development and identification in tissue extracts and biological fluids. Clin Chem 2003 49 87-96. 

Marco, M.P. and B.D. Hammock (1995). Immunochemical techniques for environmental analysis. II. Antibody production and immunoassay development. Trends Anal. Chem., 14 415 125. 

Muldoon, M.T., R-N. Huang, C.J. Hapeman, G.F. Fries, M.C. Ma, and J.O. Nelson (1994). Hapten synthesis and immunoassay development for the analysis of chlorodiamino-s-triazine in treated pesticide waste and rinsate. J. Agric. Food Chem., 42 747-755. 

Homogeneous enzme immunoassay developed by Rubenstein E malate dehydrogenase... 

Fig. 1. Schematic representation of the principle of homogeneous enzyme immunoassay developed by Rubenstein (emit). [Cited and modified from Fig. IV-4, Miyai, K., in Ishikawa et al., eds. (15).]...

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... 

We will first review several of the Immunoassays developed in this laboratory as they illustrate some of the advantages of immunoassay in pesticide residue analysis. Then we will move on to a more detailed discussion of our recent analytical work with the herbicide paraquat. 

Dlflubenzuron. The benzoylphenyl urea Insect growth regulators, for example, pose a formidable residue analysis problem. The compounds are nonvolatile and thus must be derivatized for GC analysis by a rather arduous chemical procedure. The immunoassay developed in this laboratory is much more sensitive and reproducible at a fraction of the cost and can be used to analyze the more difficult matrices such as milk. For instance, a sensitivity of 1 ppb is routinely obtained when milk is added directly to the assay ( .). A series of partition steps can also be added to further clean dlflubenzuron milk extracts yielding a sensitivity in the low ppt range (4). However this increase in sensitivity may not be needed since methods in current use provide a detection limit of only 10-50 ppb. 

Tschmelak JG, Proll J, Riedt J et al (2005) Part I project objectives, basic technology, immunoassay development, software design and networking. Part II intelligent, remote controlled, cost effective, on-line, water monitoring measurement system. Biosens Bioelectron 20 1499-1519... 

Since the immunoassay principle was introduced by Yalow and Berson [2] for the determination of insulin and by Ekins [3] for the determination of thyroxine, there has been an exponential growth in immunoassay development, not only in the range of applications, but also in the number of novel and ingenious designs. This enormous scientific interest is clearly reflected by the number of scientific publications that have appeared in the last four decades in the immunoassay field (Fig. 9.1). 

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