Applied Immunology: Enzyme-Linked Immunosorbent and Enzyme Linked Immunospot Assays - Research Paper Example

Applied Immunology Enzyme-Linked Immunosorbent Assay (ELISA) First introduced in the early 1970s, the enzyme linked immunosorbent assay, is a highly sensitive method employed to detect presence of antigens in a variety of samples. The technique, which uses an enzyme as a label, involves binding antigen to the solid phase, incubating then wash in the ELISA plate to clear unbound excess antigen. The diluted serum suspected to be containing specific immunoglobulin antibody for bound antigen is added to the ELISA plate/ well and allowed time to react. Enzyme labeled immunoglobulin is then added, allowed time for attachment then wells washed. The substrate of the enzyme labeled antibody is then added. The enzyme hydrolyses or breaks down the substrate causing color production. The optical density of the final color is directly proportional to the quantity or amount of unknown antibody or antigen present. (NOTE: Same procedure is repeated for the detection of antigen in a serum) (Bach, 1982, 58) ELISA is mainly applied in immunodiagnosis of infectious disease including measurement of viral antigens such as herpes, coxsackie and so on, measurement of bacterial and mycotic antigen such as brucella antigen, salmonella antigen and aflatoxin antigen, measurement of antigens to Trypanosoma cruzi and Schistosoma mansoni and the measurement of antibodies to viruses such as rubella, measles, herpes, rabies and so on. Various components in the blood sample of non-infected individuals can also be measured through ELISA, for example, hormone-insulin (Guttmann, 1981, 365). Some of the advantages of the Enzyme linked immunosorbent assay include the fact that it does not require a radioactive substance/ radioisotope or a costly radiation counter (radiation counting apparatus). It is also advantageous in the sense that it is a highly sensitive and specific method and hence relatively very accurate. However, there are a few disadvantages relating its application in immunology. For one, it can be difficult to develop an ELISA and the microtitre plates cannot be re-used as the antibodies bind to the surface and are difficult to remove. Also, specificity varies by target and the assay measures exposure and vaccine response but not acute infection. For example, once a bird is ELISA antibody positive, the infection is probably over. In addition, the process of labeling ELISA samples are costly and time consuming and the outcome of the experiment depends on the quality of ELISA kit in use and the competence of the user (Klein, 1990, 411). ELISPOT The Enzyme Linked Immunospot (ELISPOT) assay, developed by Cecil Czerkinskdy in 19832, measures the frequency of cytokine-secreting cells at a single cell level. Cells are cultured in a surface coated with a specific capture antibody in the presence or absence of stimuli. The specific antibodies on the surface capture the cell-secreted proteins such as cytokines. These cells are then removed after incubation for a prescribed period of time and secreted molecule detected using similar procedures as in ELISA. Spots are visualized on the surface with each spot corresponding to a cytokine secreting cell using a precipitating substrate rather than a soluble one (Rose & Oss, 1973, 312). ELISPOT assay is used in the prediction of infectious risk vaccine development, t-cell regulation analysis, monocyte and dendrite cell analysis, viral infection monitoring and treatment, organ transplants and the identification of auto-immune diseases and allergies. The ELISPOT technique is also useful in the measurement of antigen specific responses post-vaccination on peripheral blood cell preparations (Lydyard & Whelan, 2011, 525). The ELISPOT assay is beneficial as it captures cytokine as soon as secretion occurs with very minor impairments by receptor binding or protease activity. It is also fast and highly efficient with the limit of detection as low as one cell in 100, 000. Apart from being applicable to frozen/ thawed biological samples, the assay is highly throughput with high content analysis and wide range of detection, for example of cytokines and granyzyme B. another advantage of the ELISPOT is its relative affordability. However, the ELISPOT is slightly more expensive than the ELISA and as like the ELISA, there is the possibility of detecting non-biologically active cytokines (Hyde & Patnode, 1978, 377). Intracellular Staining (ICS) by Flow Cytometry Flow cytometry, routinely used for the diagnosis of health disorders such as blood cancers, is a laser-based biological technology which permits simultaneous multi-parametric analysis of the physical and chemical characteristics of up to thousands of particles per second. It is essential for cell counting, cell sorting, biomarker detections and protein engineering (Roitt & Male, 1998, 600). Other applications of the intracellular staining by flow cytometry include its uses in transplantation immunology, hematology, tumor immunology and chemotherapy, prenatal diagnosis, genetics and sperm sorting for sex pre-selection (Weir & Stewart, 1993, 413). It is also used in the metabolic studies of micro-organisms, offers possibility of studying gene expression using reporter genes in yeast, studying virus-cell interactions and discerning interaction between pathogen and phagocytic cells (Hood & Wood, 1978, 400). The assay is advantageous in that very large number of particles can be evaluated in a very short time while also ensuring very accurate and detailed results. The technique, which offers a wide area of application for analysis and diagnosis, takes of any debris and dead cells when providing final data. The process further allow for remote control and the measurement of single cells. As with the other assays, the technique also has its fair share of disadvantages. Flow cytometers are expensive to purchase and maintain (Kuby, 1997, 418). Additionally, operating a high speed sort is another recurring expense that typically costs hundreds of dollars for each turn. The assay requires highly skilled and trained operators and since the resulting data is at aggregate level, it is not easy to observe and measure individual cell behavior. With a low throughput rate, analysis is also restricted to only one cell suspension solution as technique requires passage of cells through a fluid stream. In addition, solid tissue cells have to be disintegrated by treating intact tissues with appropriate enzyme so as to release individual cells for further analysis (Cushing & Campbell, 1957, 345). References Bach, J., 1982, Immunology (2nd Ed.). New York: Wiley, 57-60. Cushing, J. E., & Campbell, D. H., 1957, Principles of immunology. New York: McGraw-Hill, 345. Guttmann, R. D., 1981, Immunology. Kalamazoo, Mich.: Upjohn, 365. Hood, L. E., Weissman, I. L., & Wood, W. B., 1978, Immunology. Menlo Park, Calif.: Benjamin/Cummings Pub. Co., 400. Hyde, R. M., & Patnode, R. A., 1978, Immunology. Reston, Va.: Reston Pub. Co., 377. Klein, J., 1990, Immunology. Boston: Blackwell Scientific Publications, 411. Kuby, J., 1997, Immunology (3rd Ed.). New York: W.H. Freeman, 418. Lydyard, P. M., & Whelan, A., 2011, Immunology (3rd Ed.). New York: Garland Science, 525. Roitt, I. M., Brostoff, J., & Male, D. K., 1998, Immunology (5th Ed.). London: Mosby, 600. Rose, N. R., Milgrom, F., & Oss, C. J., 1973, Principles of immunology. New York: Macmillan, 312. Weir, D. M., & Stewart, J., 1993, Immunology (7th Ed.). Edinburgh: Churchill Livingstone, 413. Read More