Determination of Exposure to A Contagious Disease Using ELISA

Determination of Exposure to A Contagious Disease Using ELISA
Name and Names of Lab Partners
Date of Submission
Determination of Exposure to A Contagious Disease Using ELISA
1.0 Introduction
1.1 Background information
Antibodies are known to be specific animal and human proteins, which are generated by the white blood cells following the introduction of foreign materials. Such foreign materials, which are referred to as antigens, include pathogens and a number of environmental foreign materials (Brimberg et al., 2015). Biological antigens often present as high-molecular weight molecules such as carbohydrates, proteins and nucleic acids that freely circulate or form part of complexes of the bacterial cell surface or virus coats. Antibodies are generated as a response to antigen introduction, and they bind to the antigens, playing a critical role in the eventual removal of antigens from circulation (Alberts, Johnson & Lewis, 2002). For instance, exposure to viral pathogens will cause the body to elicit an antibody response that will eventually result in the production of plasma antibodies that bind to the virus proteins and/or to different parts of the same viral polypeptide.
As the antibody binds to a specific antigen, it can precisely recognize specific sequences, chemical charges or structural conformational molecules. Such structural binding properties form the specific fingerprint for antigens. All antibody molecules bind two specific antigen molecules, and the recognition and binding are highly specific, which makes it possible to differentiate between two circulating viruses that may have a close relationship. Antibody molecules are able to differentiate between two different strains of the same virus, such as in the case of HIV-1 and HIV-2. The antigens and antibodies form complexes through immunoprecipitation, which is a highly specific binding process (Lal, Haynes & Gorospe, 2005). Complex precipitation results from the binding of different polyclonal antibodies to antigens forming a meshwork. Traditionally, in immunoprecipitation assays, antibodies are harvested from animal sera that have been exposed to certain antigens. This serum, also referred to as plasma, is often prepared through the removal of red blood cells, and it contains specific proteins for that individual and the antibodies that are against the antigen introduced in the individual by infection or design (Acker, Marks & Sheffield, 2016). These antibodies are then purified from the samples of plasma and can be utilized in detecting specific antigens, such as infectious agents.
Originally, enzyme-linked immunosorbent assay (ELISA) tests were developed to measure antibodies. With the advancement in immunological techniques, these tests can today detect samples containing antigens (Americano do Brasil, Castro & de Castro, 2016). ELISA tests are performed in microtiter plates that are made of either polyvinyl chloride or polystyrene. These plates are transparent and often contain numerous small wells where liquid samples can be deposited. The antigens are first added to these wells where part of them remain adsorbed by hydrophobically associating with the wells upon washing away the surplus. Antigens are usually the whole infectious agent such as bacteria or specific proteins, lysates or a combination of the two (Magnarelli, Norris, & Fikrig, 2012). While there is no specificity in the adsorption process, some substances can exhibit low binding to the walls. In some cases, antigens may covalently be cross-linked to the plastic through UV light. Upon washing the unadsorbed materials, the unbound sites on the walls of the wells are then blocked using milk proteins, gelatin or bovine serum albumin. In real world, ELISA tests have been exploited in medicine, agriculture and environmental studies. For instance, ELISA is used in detecting pregnancy, diseases in humans, plants and animals, and in the testing of air quality (ELISA Lab, n.d).
1.2 Hypothesis
1. Samples of simulated ‘body fluids’ shared with classmates could be exposed to a ‘contagious disease’.
2. Antigen-treated wells could show positive results
1.3 Objectives and Purpose of the Lab
The objective of this experiment was to perform ELISA test and master the concepts as well as the procedures involved in ELISA. This experiment was designed to detect the circulating antibodies directed towards the introduced antigens in the wells. Using the simulated “body fluids” this lab activity aimed to employ an indirect antibody capture ELISA to determine if class members had been exposed to a contagious disease by determining antibody specificity and the development of color.
2.0 Materials and Methods
2.1 Materials
4 yellow tubes for student samples (1 per student in the group)
1 violet tube for positive control (+)
1 blue tube for negative control (-)
1 green tube for primary antibody (PA)
1 orange tube for secondary antibody (SA)
1 brown tube for enzyme substrate (SUB)
12-well microplate strips
10-20 50 µl fixed-volume micropipet or 20-200 µl adjustable micropipet
5 yellow tips
1 disposable plastic transfer pipets
One 70-80 ml wash buffer in beaker
2 large stack of paper towels
1 black marking pen
2.2 Methods
Each well of the 12-well strip was labelled on the outside, and on each strip, the first three wells were labelled with a “+” sign for positive controls and the other three wells labelled with a “-” sign to denote negative controls. The 6 remaining wells were marked with the initials of the names of students in the group as shown bel...