Enzyme-Linked Immunosorbent Assay - Lab Report Example

ELISA Clinical Immunology Lab Report 14 December Introduction Immunoassays Immunoassays are biochemical techniques that determine the presence and quantities of macromolecules in solution with the aid of an antibody or antigen. The success of immunoassays is dependent on the ability of an antibody to bind precisely to a given antigen. Therefore, immunoassays are specific due to the specificity of antigen-antibody reactions. Examples of immunoassays include enzyme-linked immunosorbent assays (ELISA), immunofluorescence, immunohistochemistry, immunoprecipitation and Western Blotting. These tests are used in hospital settings to diagnose for certain diseases such as HIV/AIDS, hepatitis and prostate cancer among many others as well as to measure certain hormones such as the human chorionic gonadotrophins. These tests capitalize on the fact that that many viral, bacterial, fungal, and parasitic illnesses generate established antibody reactions. Therefore, the recognition of antibodies specific to infectious agents in patients’ samples aids in the identification of infections such as influenza, HIV and hepatitis (Estridge & Reynolds 2011). In some instances such as syphilis, it is impossible to separate actual disease-causing organisms. Therefore, immunological tests find immense relevance in the diagnosis of these infections. History of Immunoassays Contemporary immunoassays have advanced from the need to identify and measure multifaceted biological molecules in situations where physical and analytical chemical techniques are not feasible (Bonwick & Smith 2004). In the early stages of immunoassays, the workers were restricted to using simple techniques that quantified the precipitation that occurred as a less important event after the binding of a number of antibodies to several antigens. This technique was difficult and had numerous limitations that restricted its use. Advances on improving the method led to the development of enhanced ways of identifying antigen-antibody reactions. The first method was radioimmunoassay by Berson and Yalow, which enabled the identity of the putative molecules by measuring radioactivity (Gan & Patel 2013). However, the problems associated with the disposal of radioactive waste led to the need for safer alternatives. In addition, radioimmunoassay required sophisticated equipment to obtain measurements. ELISA became the most suitable alternative for detecting and quantifying antigens and antibodies ELISA ELISA is among the most common immunological tests in modern use. It entails the addition of an antigen or antibody to a firm surface, which in most instances is usually a polystyrene or polyvinyl surface. The use of a solid support eradicates the need or complicated methods of separation because the unbound substances are eliminated by a simple washing procedure. The test sample is then added to the pre-coated surface and incubated to allow the antibodies or antigen in the sample to attach to the immobilized antigen/antibody. Unbound antigens are eliminated by washing followed by the detection process. A subsequent antibody to which an enzyme is conjugated combines with the first antibody that is attached to the sample. Washing removes the excess conjugate, and the addition of a suitable substrate reveals the existence of the labelled anti-human immunoglobulin. A coloured light-absorbing product shows the evidence of a chemical reaction. The strength of the colour is unequivocally comparative to the amount of antibody attached to the antigen. Preliminary results can be deduced by the naked eye. However, spectrophotometry is used to measure the intensity of colour, which is then related to the precise concentration of the analyte. Some of the enzymes that are used in ELISA tests include horseradish peroxidase (HRP), O-phenylene diamine, alkaline phosphatase, and p-nitrophenyl phosphatase (Wild 2013). ELISA assays are categorized into three main groups depending on the substance that is attached to the surface and the sequence of the assay. These categories include sandwich ELISA, competitive ELISA, direct ELISA and direct ELISA. In sandwich ELISA, a monoclonal antibody is immobilized onto the surface of a microtiter well. The addition of the sample leads to the binding of the target antigen in the sample onto the monoclonal antibody. The addition of a second polyclonal antibody that is attached to an enzyme leads to the binding of the antibody to the antibody that is already bound to the primary monoclonal antibody. Consequently, the antigen is sandwiched between the monoclonal and polyclonal antibodies hence the name. The subsequent addition of substrate elicits a reaction that yields a chromogenic product, which is visualized and quantified by spectrophotometry. For a qualitative assay, for example, a home pregnancy kit, the test results can be determined by the naked eye. However, for quantitative results it is vital for the colour intensities to be measured by spectrophotometry for accurate results. Competitive ELISA is useful for the measurement of small analytes because the procedure requires the binding of one analyte as opposed to two antibodies in the other ELISA formats. The unlabeled primary antibody is incubated with the sample, which contains the target antigen. The antigen-antibody complexes are then added to a well that is already coated with the same antigen. The unbound antibodies are then eliminated via a washing step. Samples containing more antigens will allow few antibodies to bind the antigen in the well, which is the competition process. A second enzyme-linked antibody that binds specifically to the primary antibody is then added followed by a substrate that produces a colour signal. The ultimate signal is indirectly proportional to the concentration of the analyte. This means that a sample with the highest antigen concentration will yield the lowest colour signal. The benefit of competitive ELISA is that it enables the utilization of crude samples and allows selective binding of the target antigen present in the sample. In a direct ELISA, the antigen is immobilized on a solid support instead of the antibody. The primary antibody, which may be the sample, is added followed by a secondary antibody specific for the first antibody. Conversely, in an indirect ELISA the solid support is coated with the primary antibody to which the antigen in the sample binds. Discussion In the above assay, the indirect sandwich ELISA was used to determine the concentration of antigens in the unknown samples X and Y. Capture antibodies were immobilised on a plate after which the analyte was added. The antigens in the analyte attached to the monoclonal mouse anti-rabbit IgG (primary antibody), which was detected by polyclonal antibodies (polyclonal goat anti-rabbit IgG hrp-conjugated antibodies). The intensity of the colour produced when the substrate was added was recorded for the unknown samples and used to determine the concentrations as illustrated in the calculations. The titration of antibodies is vital in the final ELISA steps because it helps determine the optimum concentration of antibodies that will yield the most accurate results without interference and false positive results. However, it is necessary not to over dilute the antibodies as it may yield false negative outcomes. In the above experiment, the optimum dilution of the monoclonal mouse anti-rabbit IgG was determined as 1/2000 for an antibody concentration of 2000 ng/ml whereas the optimum dilution of the polyclonal goat anti-rabbit IgG hrp-conjugated antibodies was 1/2000 for antibody concentration of 2000 ng/ml. The optimum dilution factor for the antibodies was found by establishing the titre that yields the highest optical density because that would be the concentration that would ensure the highest specificity of the assay (Khamehchian et al. 2008). Both titration graphs indicated that the absorbance of the monoclonal mouse anti-rabbit IgG and the polyclonal goat anti-rabbit IgG hrp-conjugated antibodies increased with a decrease in the dilution of the antibodies. This observation implied that high dilution factors would lead to low absorbance values hence reduce the sensitivity of the assay (Badilescu & Packirisamy 2011). During the assay, the known concentrations of rabbit IgG antibodies were used to create the calibration curve shown in figure 3. The relationship exhibited was that the absorbance increased as the concentration of antigen increased. However, the line could not facilitate the direct determination of the concentrations of the unknown antigens. Therefore, it was vital to determine a linear relationship, which was found plotting a logarithmic curve as shown in figure 4. Consequently, the concentrations of the unknown samples were found by extrapolating their absorbance into the linear equation. ELISA has several advantages over other immunological techniques. It gives extremely high sensitivities because it requires only a small amount of the sample. It also attains added objectivity because it utilizes spectrometric reading to sense antigen-antibody reactions. The use of polyclonal antibodies leads to higher amplification of the signal of the primary antibody hence making the say very specific (Badilescu & Packirisamy 2011). In addition, ELISA allows for partial mechanization of the pipetting steps which is helpful in a busy laboratory. However, it is worth noting that ELISA also has its shortcomings. For example, the technique may yield false positive results (Kragstrup et al. 2013). The indeterminate reaction of the enzyme-substrate reaction is responsible for the false positive outcomes (Gan & Patel 2013). In addition, nonspecific binding may occur between the antigens and antibodies further contributing to false positive results. The assay is extremely sensitive to dilution factors, and any error in dilution may mess up the entire assay (Chemicon International n.d.). In addition, the reproducibility of the results from sandwich ELISA relies on the use of pure samples. Therefore, this procedure requires the use of purified samples, which makes it labour intensive. Conclusion Enzyme-linked immunosorbent assay is one of the most reliable methods of identifying the presence and quantifying the amount of antibody or antigen in biological samples. Therefore, this technique finds immense applications in the world of medical diagnostics. The specificity of antigen-antibody reactions makes this technique, particularly the sandwich form, highly specific and lowers the limits of detection of the technique. When a new antibody is discovered or isolated, titration assays are necessary to find the most suitable antibody titre for an assay. Since sandwich ELISA is sensitive to dilution factors, it is vital that the titration is done with precision to obtain accurate results. Reference Badilescu, S. & Packirisamy, M 2011, BioMEMS: science and engineering perspectives, CRC Press, Boca Raton. Bonwick, G. A. & Smith, C. J 2004, “Immunoassays: their history, development and current place in food science and technology,” International Journal of Food Science and Technology, vol. 39, no.2004, pp. 817–827. Chemicon International n.d., Introduction to antibodies (2nd edn), Chemicon International Inc. Temecula, CA. Temecula, CA Estridge, B. H. & Reynolds, A. P 2011, Basic clinical laboratory techniques, 6th edn, Cengage Learning, New York, USA. Gan, S. D & Patel, K. R 2013, “Enzyme immunoassay and enzyme-linked immunosorbent assay,” Journal of Investigative Dermatology, vol.133 no. e12, pp. 1-3. Khamehchian, S., Madani, R., Golchinfar, F., & Taghavian, M 2008, “Development of a sandwich enzyme-linked immunosorbent assay (ELISA) for determining of bovine serum albumin (BSA) in trivalent measles-mump-rubella (MMR) vaccines,” Human Vaccines, vol. 4 no.5, pp. 375-378. Kragstrup, T. W., Vorup-Jensen , T., Deleuran, B., & Hvid, M 2013, “A simple set of validation steps identifies and removes false results in a sandwich enzyme-linked immunosorbent assay caused by antianimal IgG antibodies in plasma from arthritis patients,” SpringerPlus, vol. 2 no.263, pp. 1-20. Wild, D 2013, The immunoassay handbook: theory and applications of ligand binding, ELISA and related techniques, Elsevier, San Diego, CA. Read More