The Principles and Types of Enzyme-Linked Immunosorbent Assay - Essay Example

? Health sciences and medicine, Essay By of [Word Count] Introduction Enzyme linked immunosorbent assay (ELISA) is perhaps the most available immunoassay used in biological experiments and trials due to its greater sensitivity, rapidity and specificity. The reason for which ELISA is favoured foe biological tests is that its reactions can be measured both qualitatively and quantitatively. Consequent to this advantage, ELISA is used in biological and medical researches and in clinical setting for tests and diagnoses (Icon Group International, 2011). Immunoassays or examinations in which ELISA is widely used refer to examinations in which the presences of anti-bodies or antigens in a given sample are detected. To understand the importance of ELISA in modern clinical and educational fields, it is necessary that one understands what antibodies and antigens are. It is this knowledge of antigens and antibodies that makes one appreciate the need to detect them in biological samples (Fedarko et al., 2008). Antigens refer to the proteinacious humoral substances found on the surfaces of body cells and tissues with each organism having a set of biochemical identities by which form its antigens. It is due to the presence of the different antigens on the cell or tissue surfaces of organisms that a foreign substance or organism is detected (Fedarko et al., 2008). It is after this identification of foreign antigens that the invaded body rejects or destroys the invading molecule through the production of antibodies, which function to recognize the intruding antigens. This paper explores the principles and types of ELISA tests. The Principles and Types of ELISA Immunoassays are important in the detection and recognition of antigens that invade the system of an organism, thus identifying the cause of diseases in the organism. ELISA is thus one of the tests used to detect the presence of foreign antigens in biological fluids and tissues in patients to help in achieving accurate diagnoses and effective treatment. ELISA, like many other biological laboratory techniques, is based on certain principles on which its processes are based. The most basic of ELISA’s principles is that antibodies or antigens stick on a polyvinyl plate after which the plate is washed and the antigens or antibodies are separated. To fix the antigen adhered to the plate; a corresponding second antibody to which an enzyme is tagged is added. When a suitable substrate is added to this mixture of antibody/antigen and an enzyme, a colour measurable to the quantity or function of the antigens or antibodies present in a given biological sample is produced. There are different broad categories of ELISA namely direct, indirect, heterogeneous and homogeneous ELISA. In heterogeneous Elisa, the antigens added first are solid in nature and are thus fixated on the plate. The second antibody or antigen added is often in liquid phase. On the other hand, in homogeneous Elisa, the first antigen to be added is always a liquid and the second antibody added is also in liquid form. In direct ELISA, only a set of antigens and a set of antibodies mixed to react. That is, antibodies tagged to an enzyme are made to react with sample antigens fixated on Elisa plates. In other terms, direct Elisa involves the direct application or addition or enzyme-linked antibody to the antigen under study to produce a given colour with an ELISA reagent/substrate. The reaction below summarizes the direct ELISA reaction. Antigen or Antibody + Antibody- or Antigen-(Enzyme) > Reaction colour On the other hand, the principle of the indirect ELISA reaction involves the addition of one more additional antibody to an ELISA reaction. That is, an extra antibody is added to the antigen fixated to the ELISA plate. To this reaction, another secondary enzyme-linked antigen is added since in some patients, the antigen of the intruder pathogen may not be available in the system but a corresponding one could be present in the sample. In these instances, it is the corresponding antigen that is traced (Frank et al., 2005). Below is the reaction for the indirect Elisa reaction. Antigen or Antibody + Antibody or Antigen +Antigen- or Antibody-(Enzyme) > Reaction colour An example of an indirect ELISA test is the Sandwich ELISA in which there is an antigen between two antibodies. The bottom antibody is fixed to the plate surface and an antigen is fixed over it. On this antigen, one more antibody junction is attached and it is on this junction that an enzyme-linked antibody, which gives the colour reaction when substrate is added, is present (Frank et al., 2005). Antibody + Antigen + Antibody-(Enzyme) > Reaction colour A slightly different category to the direct, indirect and sandwich ELISA is the competitive ELISA that has some modifications on the above types. In competitive ELISA, a biotinated substrate complete with antibody and antigen to bind are added to the already added antigen and antibody in an Elisa reaction. The main function of the competitor biotinated substance is to stop the unwanted binding of antigen and antibody, only allowing binding to occur due to the affinity between the antigen and the antibody (Perlman & Engvall, 1971). That is, the mere addition of the reagents should not be the reason they bind to each other, rather, they should bind due to their high affinity for each other. Limitations of ELISA As stated earlier, ELISA is a rather straightforward and routine procedure that professionals and students apply in laboratories to detect the presence of an antibody in a biological sample. However, there are several limitations to the ELISA procedure, more so with regards to the variables involved. In fact, these variables must always be checked to ensure that errors are prevented, minimised and controlled. Among these variables in ELISA are time, volume, temperature and reagent selection, all of which must be monitored and adjusted so that all the ELISA steps and outcomes are accurate and reproducible. The second limitation of ELISA is that a positive result that confirms the detection and presence of an antibody does not necessarily imply that an individual is suffering from a given condition. That is, people’s systems may continue to produce antibodies against diseases suffered earlier and from which an individual may have recovered. On the other hand, some people are poor producers of antibodies since there could be substances in people’s bodies that interfere with the production of antibodies, thus hindering the synthesis of antibodies (Deshpande, 1996). Hence, the antibodies produced could be undetectable, thus giving an inaccurate outcome. A non-specific reaction between an unrelated antibody and an antigen could also give a positive outcome, implying that this reaction, though positive, is considered false since the specific antibody is not detected. To distinguish between true and false results, it would thus require workers to carry out more tests. The use of duplicate or even triplicate samples in running tests is thus recommended (Deshpande, 1996). The other limitation of the ELISA test is its cost. Although not quite cheap, ELISA is reliable and cost effective in terms of its results. Luckily, some ELISA kits are affordable, costing between US $200 to $ 300. Nonetheless, some ELISA kits are rather expensive and cost a few thousand dollars. These costs however vary from kit type and manufacturers. Conclusion ELISA is a widely available and used immunoassay by professional and students in biological experiments for its greater sensitivity, rapidity and specificity. Direct and indirect tests are the two most common categories of ELISA. In addition to these types is the sandwich ELISA, which has an antigen sandwiched between antibodies. In spite of its specificity, rapidity and sensitivity, ELISA is quite expensive and might encounter certain false positive results or may even fail to detect antibodies in patients that cannot produce detectable quantities. References Deshpande, S. S. (1996) Enzyme immunoassays: from concept to product development, first edition. Springer. Fedarko, G. K., Leng, J., McElhaney, J. Walston, D., and Xie, N. (2008). Elisa and Multiplex Technologies for Cytokine Measurement in Inflammation and Aging Research. Journal of Gerontology 63 (8): 879. Frank, J. Griffin, T. Spittle, E., Rodgers, R., Liggett, S., Cooper, M., Bakker, D., and Bannantine, J. P. (2005). Immunoglobulin G1 Enzyme-Linked Immunosorbent Assay for Diagnosis of John’s disease in Red Deer. Clinical Diagnosis Laboratory Immunology 12(12), 1409. Icon Group International (2011) The 2011 report on enzyme-linked immunosorbent assay (Elisa) technologies: world market segmentation by city. ICON Group International, Inc. Perlman, E. P., and Engvall, E. (1971) Enzyme-Linked Immunosorbent Assay (ELISA): Quantitative Assay of Immunoglobulin G. Immunochemistry 8(9), 871. Read More