Immunoassays contaminant detection

An enzyme immunoassay technique has been employed for measuring endosulfan and its degradation products (i.e., endosulfan diol, endosulfan sulfate, endosulfan ether, and endosulfan lactone) in water at 3 ppb (Chau and Terry 1972 Musial et al. 1976). However, this technique is not currently in use in environmental residue analysis. Further research into this technique could produce a rapid, rehable, and sensitive method for identifying contaminated areas posing a risk to human health. No additional methods for detecting endosulfan in environmental media appear to be necessary at this time. However, methods for the determination of endosulfan degradation products are needed. 

A prerequisite to pharmacokinetic/pharmacodynamic studies is the availability of a sufficiently selective and sensitive assay. The assay must be capable of detecting and accurately quantifying the therapeutic protein in the presence of a complex soup of contaminant molecules characteristic of tissue extracts/body fluids. As described in Chapter 7, specific proteins are usually detected and quantified either via immunoassay or bioassay. Additional analytical approaches occasionally used include liquid chromatography (e.g. HPLC) or the use of radioactively labelled protein. 

Hoffman advises, Relying solely on a process-specific assay is ill advised and can result in failure to detect atypical process contaminants. In cases with a defined, persistent, and problematic host cell protein impurity, a down-stream process-specific assay may be justified. It is critical that the immunoassay be capable of detecting every possible host cell protein contaminant. 13... 

If the product is an antibody, then it is essential to distinguish the immunoglobulin product, e.g., mouse IgG, from any media immunoglobulin components, e.g., bovine IgG. Lucas et al.16 developed an immunoassay to measure nanogram quantities of bovine IgG in the presence of a large excess of a structurally homologous protein, mouse MAb. The bovine IgG was a contaminant that copurified with the product from a protein A column. For the bovine IgG assay, whole IgG and protein A-purified IgG reacted differently in the assay. It is important to evaluate these types of assays for cross-reactivity. For other media components, such as chemicals or antibiotics, ELISA is probably not the most appropriate method due to the low immunogenicity of chemicals. Techniques such as HPLC would be better to detect these chemical components. 

Cell components or metabolites capable of recognizing individual and specific molecules can be used as the sensory elements in molecular sensors [11]. The sensors may be enzymes, sequences of nucleic acids (RNA or DNA), antibodies, polysaccharides, or other reporter molecules. Antibodies, specific for a microorganism used in the biotreatment, can be coupled to fluorochromes to increase sensitivity of detection. Such antibodies are useful in monitoring the fate of bacteria released into the environment for the treatment of a polluted site. Fluorescent or enzyme-linked immunoassays have been derived and can be used for a variety of contaminants, including pesticides and chlorinated polycyclic hydrocarbons. Enzymes specific for pollutants and attached to matrices detecting interactions between enzyme and pollutant are used in online biosensors of water and gas biotreatment [20,21]. 

Allen RL, Manning W, McKenzie KD, et al. 1992a. Development of a monoclonal antibody immunoassay for the detection of gasoline and diesel fuel in the environment. Assoc Am Railroads Contaminated Soils-Diesel Fuel Contamination Research Triangle Park NC. 

Most recombinant biopharmaceuticals are produced in microbial or mammalian cell lines. Thus, although the product is derived from a human gene, all product-unrelated contaminants will be derived from the producer organism. These non-self-proteins are likely to be highly immunogenic in humans, rendering their removal from the product stream especially important. Immunoassays may be conveniently used to detect and quantify non-product-related impurities... 

Endotoxicity results from the interaction of a bacterial cell envelope component (e.g., LPS or PG with a cell surface receptor constituting part of the nonspecific immune system, (i.e., a toll-like receptor on white blood cells). This results in the production of cytokines [e.g., interleukin 1 (IL-1) or tumor necrosis factor (TNF)] as part of an intracellular enzyme cascade which can cause severe tissue injury. Bioassays or immunoassays can be used to detect such reactions respectively. As noted above the most widely used bioassay is the LAL assay. A lysate of amoebo-cytes of the horseshoe crab (Limulus) contains an enzymatic clotting cascade which is activated by extremely low levels of LPS (nanogram levels or lower). There are variants of this assay that can detect PG, but they are not as widely used. As noted above, other bioassays employ cultured cell lines that respond to LPS or PG, respectively. Unfortunately bioassays are highly amenable to false positives (from the presence of cross-reactive substances) or false negatives from inhibition (by contaminants present in the sample) [10]. A detailed discussion of these assays is beyond the scope of this chapter and has been reviewed elsewhere [1]. 

ELISA s (54, 55) are one of the most commonly used immunoassays in the food industry for detection of a wide variety of substances including contaminants, toxins, adulterants, herbicides and carcinogens. They use an enzyme as a label and visualization is achieved via conversion of the substrate to a colored product. There are different types of ELISA s i.e., competitive (Fig. 7), non-competitive (Fig. 8), sandwich (Fig. 9) and homogeneous enzyme immunoassay (48). 

Food products are prone to accidental or deliberate abuse anywhere in the food processing/storage/distribution/consumption cycle, starting from raw materials to finished products. Faced with this problem a food analyst needs rapid, sensitive, reliable and cost effective techniques. Immunoassays are ideally suited for this type of tasks and their role in food diagnostics is expanding due to the numerous analytes like adulterants/additives, allergens and contaminants/toxins that it can detect. For more detailed information on applications of immunoassays one can refer to some of the recent review articles and books (11-13, 38, 44). 

Salmonella and Listeria are a source of numerous food borne illness. As a result, a lot of attention has been focused on these microorganisms. The effort in the Salmonella area focused on obtaining antibodies which can detect several serotypes (14-16, 61,64, 90) and shortening the assay time from 3-4 days to 1-2 days by use of more sensitive formats and enrichment protocols (74, 75, 91,92). Numerous immunoassay kits for Salmonella were developed, such as the Salmonella-Tek (74, 75), Tecra Salmonella (93) and Bio-Enza Bead (94), to name a few. More information on kits is given in section 2.9. Additional information is available for Salmonella, Listeria and other microbiological contaminants and toxins (see Table 1) in review articles and books (5, 7,11-13, 88,95-99). 

The test kit is based on immunoassay techniques and the method takes about 10 minutes to provide an analysis. A unique tag, permanently attached to the polymer, changes the color of special test strips exposed to ppm levels of polymer. The strips indicate the amount of tagged polymer that is present, without interference from other additives and contaminants. This technology is not currently available for continuous inhibitor detection, but given the importance of AH Organic Programs, there is little doubt that further developments will take place. The Water Additives Division of Great Lakes Chemical Corp. have also recently introduced a similar simple and accurate immunoassay test for the detection of Belclene 200 antiscalent. 

The development of immunoassays for the detection of food components and contaminants has progressed rapidly in the last few years [7]. Antibodies against almost all the important food residues compounds are currently available. Classical immunochemical methods such as immunodiffusion and agglutination methods for food analyses generally involve no labeled antigen or antibody. Concentration of the antigen-antibody complex is estimated from the secondary reaction that leads to precipitation or agglutination. These methods are not sensitive, are subject to... 

Detection limits in the range of low parts per billion (ppb) can be achieved by immunoassay testing for certain parameters in aqueous samples. For soil samples, detection limits of <10 ppm can be achieved for many contaminants... 

PCBs can be conveniently determined by most of the common analytical techniques which include GC-ECD, GC-HECD, GC-FID, GC/MS, HPLC, NMR, and enzyme immunoassay. Among these, GC-ECD and GC/MS are by far the most widely used techniques for the determination of PCBs in the environmental samples at a very low level of detection. While the former can detect the PCBs at subnanogram range, the mass selective detector (GC/MS) identifies the components relatively at a higher detection range, 10 to 50 times higher than the ECD detection level. GC/MS, however, is the best confirmatory method to positively confirm the presence of PCBs, especially in heavily contaminated samples. Aqueous and nonaqueous samples must be extracted into a suitable solvent prior to their analysis. 

Gruessner, B. and M.C. Watzin (1995). Patterns of herbicide contamination in selected Vermont streams detected by enzyme immunoassay and gas chromatography/mass spectrometry. Environ. Sci.Technol., 29 2806-2813. 

As part of SW-846, the EPA has validated and approved many immunoassay and colorimetric screening methods for a wide range of contaminants, such as petroleum fuels, pesticides, herbicides, PCBs, and explosives. Immunoassay technology uses the property of antibodies to bind to specific classes of environmental pollutants allowing fast and sensitive semiquantitative or qualitative detection. Colorimetric kits are based on the use of chemical reactions that indicate the presence of target analytes by a change in color. Table 3.9 presents a summary of EPA-approved screening methods and their detection capabilities. 

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