04 - Put the M into STEM: Quantitative Techniques for Biotechnology
Step 2: ELISA Have you ever heard of a pregnancy test, HIV test, or a rapid COVID test? If yes, then you have already heard about the ELISA, or the Enzyme-Linked ImmunoSorbent Assay. The ELISA is a highly sensitive technique that uses antibodies to detect the presence of specific molecules (i.e. peptides, proteins, and hormones) in complex samples. Because of its low cost and ease of use, researchers use ELISAs in many fields, including medical diagnostics, forensic science, and in quality control of foods. THE HISTORY OF THE ELISA In the 1890s, researchers identified that serum from animals that had been immunized against diphtheria or tetanus could confer resistance to the disease when transferred into a second animal. The hypothesis was that there was a molecule within the serum that could neutralize a foreign antigen. They called this molecule an antibody (Ab) or an immunoglobulin (Ig). Through careful experimentation, researchers learned that antibodies are specialized protein complexes that allow the immune system to distinguish between molecules that are either “self” and “non-self.” Each antibody is highly specific and only recognizes one epitope (a particular location within a foreign substance). These foreign molecules were named antibody generators, or antigens. Once bound, antibodies mark antigens for attack by other parts of the immune system. Each antibody is a y-shaped molecule composed of four polypeptide chains: two “heavy chains” and two “light chains”. The polypeptides are linked together by disulfide bonds. The vast majority of the amino acid sequence is the same if we compare antibodies that recognize different antigens. The differences lie in the amino acid sequence of the antigen-binding site (the little pocket at the end of the Y), allowing each antibody to recognize a unique antigen. Since the sequence can be so variable, antibodies can recognize a lot of different molecules. Due to their specificity, scientists imagined using antibodies as powerful tools to detect specific molecules in biological samples. In the early 1960s, Rosalyn Yalow and Solomon Berson developed an assay that used radioactivity to detect the interactions between antibodies and their target molecules. This assay, called the radioimmunoassay, or RIA, lead to the ability for researchers to calculate the concentrations of antigens in solutions (like insulin in blood). While this test revolutionized medical research, high levels of radioactivity can be hazardous to human health. In 1971, Peter Perlmann and Eva Engvall in Sweden, and Anton Schuurs and Bauke van Weemen in the Netherlands, independently linked antibodies to enzymes so that they could use colors (chromo- genic reporter) or light (fluorescent reporter) to detect antigens. This innovation allowed researchers to quickly detect the smallest amount of antigen present in a sample without using radioactivity. Since we can generate antibodies to lots of different molecules, the ELISA has been adapted for many uses. The ELISA is commonly used for medical diagnostics, as it is can be used to identify antigens in blood, saliva, urine and other biological samples. Figure 1: General Structure of an antibody. Antigen binding sites Light chain Heavy chain Fab region Fc region
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