Enzyme-linked Immunosorbent Assay - Term Paper Example

?Introduction There are several environmental problems today which threaten the normal processes in our entire world. The continuous globalization and excessive or inappropriate use of natural resources has transformed local problems into international issues. One of the major environmental problems today includes environmental toxicants. The environmental toxicants may be in the form of pesticides, solvents or heavy metals. These environmental toxicants are usually released as chemical waste into the atmosphere, water bodies and soil. Most of the time, this waste is hazardous and may cause detrimental effect to any living organisms, including human (Freedman, 3-9). One of the most common environmental toxicant includes the polychlorinated biphenyls or PCBs. PCBs are man-made organic chemicals belonging to a broad family of hydrocarbons. It was used in many industrial and commercial applications because of its good chemical properties like being non-flammable, high boiling point, chemical stability and good electrical insulating properties. These compounds are widely used for many applicants such as transportation and coolants. PCBs are also used as plasticizers in paints, plastics, and rubber products. It was first manufacture during 1929 but their use had been banned in 1979 upon determining its dangerous effects to both the environment and human health (Davis and Wade, 2-6). PCBs have a range of toxicity and vary in consistency from thin, light-colored liquids to yellow or black waxy solids. These toxic compounds are considered to be persistent organic pollutant for it do not degrade easily and may remain for a long time in the environment. PCBs can contaminate some parts of plants and food crops. PCBs may also be taken up into the bodies of small organisms in that lake including fishes. These toxic compounds may bioaccumulate in these food sources and ingestion may have many adverse health effects in human with symptoms such as skin condition or changes in the blood which may lead to liver damage. Some food which contains PCBs can cause poisoning to the consumers. Moreover, some studies report the health effect of PCBs in children of mothers who were exposed to PCBs (ATSDR, 285-290). Hence, it is important to detect this compound in the environment to prevent its hazardous effect. One of these methods of detection includes the use of Enzyme-Linked Immunosorbent Assay or ELISA Analytical Technique Enzyme-linked immunosorbent assay (ELISA) is a biochemical technique which allows rapid screening and quantification of an antigen in a sample. It is used mainly to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and as detection or measuring tool of environmental toxicants. In ELISA, an antigen is affixed to a surface and a specific antibody is applied over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal, which is most commonly a color change in a chemical substrate (Lequin, 2415-2416). Enzyme-linked immunosorbent assay or (ELISA) analysis biochemical technique is used to measurement of organic contaminants such as polychlorinated biphenyls in an environmental sample. Performing an ELISA includes at least one or more antibody with specificity for an antigen. The sample, which has unknown amount of antigen, is immobilized on a solid support either non-specifically or specifically. The immobilization process occurs in two ways. One process is by the adsorption to the surface wherein a sample containing an antigen is adsorbed into an inert surface, usually a 96-well polystyrene well. Another process involves capturing another antibody specific to the same antigen, as in a "sandwich" ELISA (Lequin, 2417). The detection antibody is added after the antigen is immobilized, forming a complex with the antibody. This is usually done by washing the surface with a solution of a non-specific protein to block proteins introduced in the subsequent steps from adsorbing to these surfaces. Then the surface is treated with a solution containing the primary antibody or the antibody against the protein of interest (Van Weemen & Schuurs, 233). The detection antibody can be connected to an enzyme, or can itself be detected by secondary antibody, which is connected with an enzyme through bioconjugation. Between each step, the plate is typically washed with a mild cleaner solution to remove any proteins or antibodies that are not specifically bound. Finally, after the last wash step, the plate is developed by adding an enzymatic substrate to produce a visible signal that indicates the quantity of antigen in the sample which is usually a color reaction. Product formation, which is monitored as color intensity, is proportional to the concentration of the protein of interest in the sample (Van Weemen & Schuurs, 235). ELISA is one of the immunoassay technologies that have been used in environmental toxicology for the detection of toxic substances in soil, water and even biological samples. It has shown potential for providing rapid, low-cost methods for determination of environmental pollutants including PCBs. There are many semi-quantitative field methods using ELISA that determine levels of PCBs whether it is above or below a certain threshold. Many studies have indicated the use of ELISA in the detection and Quantitative ELISA has been used for the determination of PCBs in environmental soil and sediment samples (Johnson and Van Emon, 162). These studies use a similar conventional principle in performing ELISA from sample collection, preparation, incubation, detection and preservation. In a study, ELISA was performed to determine quantitatively levels of Aroclors 1242, 1254, 1260 and 1248 in soil and sediment samples compared with that of Gas Chromatography (GC). The study develop a quantitative ELISA based on antibodies which preferentially allow determination of environmentally important PCBs (Aroclors 1242, 1248, 1254, 1260). It also characterize the assay accuracy, precision, working range, and susceptibility to interferences from structurally similar compounds. In the study, two sets of environmental soil samples were analyzed using the quantitative PCB ELISA. These two sets include one hundred forty-eight (148) environmental soils, sediment, and paper pulp samples. These samples were obtained from two EPA listed Superfund sites. The assay in the study was based on the indirect competitive ELISA format. Initial manipulations containing free PCBS were carried out in glass vessels to avoid adsorption of PCBs onto plastic surfaces. Optimizations of antibody and antigen dilutions were done using checkerboard titration. For the ELISA, Polystyrene 96-place microwell plates were used. It was treated with solution of coating antigen in carbonate coating buffer. The plates were then covered with acetate plate sealing tape and stored overnight at 4 °C. Rabbit anti-PCB anti-serum was used as the antibody. It was diluted in PBST (Phosphate-buffered saline with Tween 20) and a part of the resulting solution (around 1 mL) was added to the glass tubes. The solution was added into either the calibration standard, the one with the soil sample extract or to the reference soil extract. Then each glass tubes were added with methanol solution. Upon incubation, the antibody was allowed to react with the PCBs. After almost one day of incubation, the plates were washed with PBST to remove antigens which did not bind to the antibody. This was done several times with shaking. On the final time that it was washed, a solution of nitrophenyl phosphate in diethanolamine buffer was added to each well. This was done to have a color reaction after 30 minutes. The colorimetric measurements were done at 405-650 nm with a Vmax plate reader. The PCB levels in the soil samples were calculated based on a standard curve that was derived from a fit of optical density versus the logarithm of concentration. Extraction of all samples was done by shaking with methanol. The subsequent solutions were then more rigorously extracted by using Soxhlet or supercritical fluid extraction. ELISA analysis of the methanolic shake extracts frequently gave results which were not in close agreement with values obtained using conventional GC-based methodologies. Based on the analysis of results, it showed that the ELISA determination of PCBs in Soxhlet or supercritical fluid soil extracts were essentially identical with results obtained using conventional GC-based methodologies. This suggests that the ELISA can be used as the determinative step in analyzing PCB. Moreover, comparing results from ELISA analyss with GC analyses using the same sample extracts will help decrease the uncertainty resulting from two separate analyses of separate samples. In addition, the current results raise concern with the use of the abbreviated “quick shake” extraction procedures typically found in the commercially available semiquantitative PCB ELISA kits. Results from the current study suggest the possibility that many of the false negative and positive results encountered in application of the kit methods may in fact result not from the ELISA itself but rather from coupling poor extraction procedures to the ELISA After many analytical processes, the study demonstrated that the ELISA determination of PCBs in soil extracts recorded quantitative results that were similar to those which were given by GC-based methodologies. While the benefits of such low-cost, field-portable, immunoassay based methods are evident for use in site characterization and monitoring of site remediation, these methods can be considered as being complementary but not comparable with current GC-based methodologies. Consequently, there exists a large performance gap between these two analytical methods. In the most general sense, this study was undertaken with the goal of narrowing this gap. This study has indicated the use of ELISA in determining levels of PCB in soil samples. It have been showed that There are advantages and disadvantages of using ELISA in the detection of toxic substances in environmental samples. One of the advantages, as indicated, is that immuno-chemical methods like ELISA provide a rapid, sensitive, and cost effective analysis for a variety of environmental contaminants. Being cost-effective is very important since large number of samples are usually collected and analyzed in environmental toxicology studies for it to be valid. Expensive assays or techniques for detection of environmental toxicants might limit the number of samples to be analyzed. ELISA was found to be inexpensive when compared to traditional GC/MS (Gas chromatography-mass spectrometry) and liquid chromatography (TechData Sheet, 2). An enzyme immunoassay laboratory using ELISA can be established for as low as $1000, with a per-sample cost of as low as $10. In contrast to this, establishment of a laboratory which uses conventional analytical techniques like GC/MS can cost for as low as $150,000, with a per-sample cost of as low as $200 (TechData Sheet, 3). It can be clearly seen here an almost 20-fold increase in cost of using GC/MS compared to ELISA. Moreover, ELISA does not require a multi-step cleanup process like many traditional methods. However, it should be noted that a specific ELISA may be used for detection of one specific chemical. This should be a consideration when choosing ELISA because there is a point of diminishing returns in terms of cost and time when compared to GC/MS systems which can determine 70 or more chemicals from a single sample run. Hence, there is a disadvantage of using ELISA in relation to time duration when the study aims to determine several kinds of chemicals in an environmental sample (TechData Sheet, 2-3). Another advantage of using ELISA is that it elicits high sensitivity to specific antibodies, and provides analytical systems which can detect chemicals even at very low concentrations. Hence, there is no pressure in getting huge amounts of samples for detection of environmental toxicants, in this case, the PCBs. The high sensitivity may be attributed to the very strong catalytic capacity of enzymes used in the assays. These methods are also highly selective due to the extraordinary discriminatory capabilities of antibodies (Aiyagari & La Breche). ELISA is also fast and relatively easy compared to conventional techniques like GC/MS and HPLC. GC/MS may need 1 hour for analysis of one sample while in ELISA, 30 samples can already be analyzed for 1 hour. This might be because ELISA has fewer and less complicated instructions. Moreover, ELISA kits are usually portable and can be brought at the site of sample collection conveniently (Aiyagari & La Breche). Immunoassays are also considered to be a very efficient screening tool. There is a low detection limits for environmental samples. Preprocessing of the samples is also not required. There is no need to stabilize samples using other chemicals. Retrieval of analyte from spiked samples is also more effective with ELISA compare to conventional methods (TechData, 2). However, no method of determining environmental toxicants like PCBs is perfect. Inspite of its numerous advantages in helping make significant progress in research, ELISA cannot be independently used as the detection protocol of environmental toxicants and replace conventional methods altogether. ELISA is usually best used in combination with these conventional methods. One of the reasons is that some ELISA is not highly selective. It has a different manner of effect to certain compounds. It may respond to certain kinds of chemicals in pesticides such as cyclodienes, and not to other chemicals. This leads to the presence of more than one compound in the environmental sample. During this condition, it is better to use conventional GC/MS or HPLC methods to identify the analytes rather than ELISA (Baker et al, 306). Because of the design of the immunoassay, sample contaminants that might interfere with the antigen-antibody reaction can produce positive readings when samples are indeed negative (called false positive results). This design eliminates the possibility of obtaining false negatives due to interfering materials. Conventional methods can then have be used as a backup test for positive samples to be sure sample contamination is not skewing your results. Lower molecular weight molecules often lack specific antigens, and sometimes there are crosslinking problems (Baker et al, 302). Application to Environmental Issue ELISA has been a very useful technique for the preliminary assessment of PCB distribution in environmental field screening applications. The immunoassay technique is very useful for preliminary assessments of contaminant distribution as in environmental field screening applications (Belton, 12). In evaluating desktop trackdown methods and innovative fields methods , another study in New Jersey has used PCB ELISA to detect PCB from environmental samples such as water and soil. The use of ELISA for this study was validated for use by NJDEP (New Jersey Department of Environmental Protection) and USEPA (United States Environmental Protection Agency) for it is known to be quick, inexpensive and accurate. This approach was done to document suspected PCB source in sewer water systems using water environmental samples. It also identifies readily accessible regulatory datasets and uses street soil samples at storm drains in front of suspected PCB sources. ELISA was also applied during various phases of the Ecological Risk Assessment process, depending on the specific objectives being addressed. Combining screening data with a select number of laboratory analysis resulted in more efficient sampling and analysis to characterizing the nature and extent of contamination at a site. ELISA is found to be not applicable to sites with unknown site conditions and contaminants. Sites with a single contaminant, or only one type of chemical class of contamination, are the sites most suited for ELISA. ELISA may not also be applicable to sites contaminated with complex mixtures of chemicals because interference may arise. However, most studies have found that the immunoassay screening data for PCBs using ELISA in sediments have been successfully validated against standard laboratory methods. Reports have indicated the presence of hazardous waste such as PCBs at high concentrations in soils that significant levels were found in indoor residential air at certain areas. The waste site was used to illustrate the complexities in exposure assessment and toxicity assessment of PCBs (Davis & Wade, 2-3). Aroclors are common commercial mixtures of different arrays of the 209 individual PCB types. In reality, what was seen at given site would be a complex mixture of PCB chemicals. The exact composition of the PCB depends on the components of the original material released at the site, fate of the environmental sample and its transport to the environment (Davis & Wade, 3). Individual PCB chemical analysis has supported the reports on Aroclors. However, long-term toxicity studies which includes the criteria for toxicity, are available exclusively to Aroclors as a whole rather than individually. The PCB mixtures at a waste site may be quite different than the mixture on which toxicity criteria are based (Davis & Wade, 5-6). There are many health hazard of PCB. The US Environmental Protection Agency (EPA) currently provides upper bound cancer slope factors of 2.0 for High Risk and Persistence PCBs, 0.4 for Low Risk and Persistence PCBs, and 0.07 for Lowest Risk and Persistence PCBs, based on bioassays of Aroclors 1260, 1254, 1242, and 1016. Another consideration for toxicity assessment is the dioxin-like activity of co-planer PCBs. However, toxicity assessment of PCB at relatively low dose levels indicated the absence of cancer incidence. This has implied certain levels of PCB to be non-toxic (Davis & Wade, 4). Exposure assessment of PCBs is complicated by its bioaccumulative properties. This necessitates consideration of the potential for indirect exposure to PCBs from the waste site through food pathways, including fish, poultry and livestock, and breast milk. Another exposure assessment consideration is the near ubiquitous ambient low level presence of PCBs in environmental media including air, soil, and food. Hence, detection of PCB in environmental samples is important. This is to prevent health hazards in human and prevents its toxic effect from spreading. Rapid and cheap detection, through immunoassay techniques like ELISA may help in doing these measures of detection. This should be done in conjunction with conventional analytical techniques to further verify its presence in the environment. This would provide assurance to the safety from the threats of contamination to PCB of basic human resources such as water and plant products. References: Agency for Toxic Substances and Disease Registry (ATSDR). “Toxicological Profile for Polychlorinated Biphenyl”. U.S. Department of Health and Human Services: Public Health Service. 2000. pp. 285-370 Aiyagari, N & La Breche, T. “Immunoassay”. Environmental Sampling and Monitoring Primer. 1997 Retrieved March 20, 2011 http://www.cee.vt.edu/ewr/environmental/teach/smprimer/immuno/immuno.html Baker G.B., Dunn, S., Lajtha A. and Holt A. “Handbook of neurochemistry and molecular biology”. Springer Publication. 2007. pp 301-306. Belton, T., Botts, J., Lippincott, L. & Stevenson, E. Pollution Minimization Plans and PCB Source Trackdown in Camden City. PMP/PCB Report. 2008. pp. 10-21 Davis, B. & Wade, M. Risk assessment of Polychlorinated Biphenyls at hazardous waste sites. Department of Toxic Substances Control: Sacramento, California. 2003. pp 2-6. Freedman, B. Environmental Ecology: The Impacts of Pollution and Other Stresses on Ecosystem Structure and Function. Academic Press: San Diego. 1989. pp.3-9 Johnson, J & Van Emon, J. Quantitative Enzyme-Linked Immunosorbent Assay for Determination of Polychlorinated Biphenyls in Environmental Soil and Sediment Samples. Anal. Chem. 1996. 68: pp. 162-169. Lequin, R. "Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA).". Clin. Chem. 2005. 51(12): pp. 2415–8. TechData Sheet. 2001. Rapid Sediment Characterization of PCBs With ELISA: An Immunoassay Technique; A Rapid Sediment Characterizaton (RSC) Tool. TDS-2086-ENV. Naval Facilities Engineering Service Center. Port Hueneme, California. pp. 1-3 Van Weemen BK, Schuurs AH. "Immunoassay using antigen-enzyme conjugates". FEBS Letters. 1971. 15 (3): 232–6. Read More