A Brief History of Enzyme Testing : ELISA



By David Frank


Over the past few decades, ELISA (enzyme-linked immunosorbent assay) has become a crucial component of research and testing applications across a wide range of fields. Prior to the development of ELISA in 1971, researchers were limited to radioimmunoassay tests for detecting the binding of antigens and antibodies. This method involved the use of radioactive, carcinogenic compounds for labeling, and the level of radioactivity indicated the presence and concentration of specific analytes. Handling these substances could be incredibly dangerous, even with proper procedures and protective gear. To avoid these health and safety risks, researchers discovered a way to biochemically mark analytes with enzyme labels to allow for safe, effective detection and quantification of desired antibodies.



A Brief History of ELISA Development


American immunologist Albert H. Coons and his colleagues become the first researchers to label antibodies with fluorescent dyes and use the signals created from dyes to detect antibodies, antigens, and other foreign proteins in cell and tissue samples. Referred to as immunofluorescence, this technique is now an indispensable tool in biomedical research and continues to drive major discoveries in the fields of cell biology and immunology.


Nuclear physicist Rosalyn Sussman Yalow and medical doctor Solomon Berson developed radioimmunoassay to quantify and label minute amounts of biological substances or chemical compounds in the human body. Yalow received a Nobel Prize in Physiology or Medicine in 1977 for her research, but Berson passed away before this point and was therefore not eligible.


Based on studies by Graham and Karnovsky on the ultrastructural detection of peroxidase, two independent research groups performed the first successful immunoenzyme labeling during the same year. One group at the Pasteur Institute in Paris, led by Stratis Avrameas, outlined the protocol for conjugating an enzyme (peroxidase) to an antibody (anti-immunoglobulin G) with glutaraldehyde as the bifunctional reagent. GB Pierce and Dr. Paul Nakane at the University of Michigan used the same conjugates for enzyme linking but used p,p’- difluoro-m,m’-dinitrodiphenyl sulfone (FNPS) as the reagent. 


The ELISA was conceptualized and developed simultaneously by two independent groups of researchers, Peter Perlmann and Eva Engvall at Stockholm University and Doctors Anton Schuurs and Bauke van Weemen at Organon Research Laboratories in the Netherlands. Both groups used enzyme labeling of antibodies to detect the presence of hormones or viruses in biological samples and published their techniques in research papers, with the team at Stockholm University coining the term ELISA. They shared the 1976 Nobel Prize in Biochemistry.


At Cambridge’s MRC Laboratory of Molecular Biology, German biologist George Kohler and Argentinian biochemist Cesar Milstein invented a method for stimulating cells to generate unlimited amounts of long-lasting monoclonal antibodies with precisely defined specificity. They accomplished this through hybridoma technology. Hybridomas can rapidly, indefinitely grow and divide, and produce and secrete a specific antibody. This allowed researchers to produce large numbers of identical antibody-producing cells that can be targeted against specific antigens. They received a Nobel Prize in Physiology or Medicine in 1984 for their work.


Competitive ELISA was developed by using a conjugated substrate to compete against an antigen of interest for binding. It was implemented in detecting the human choriogonadotropin hormone, which is responsible for the development of eggs in the ovary and stimulating the egg’s release during ovulation. This type of ELISA offers good reproducibility and flexibility, is less sensitive to experimental errors than other types, and requires minimal sample processing.


Sandwich ELISA was invented by coating the microplate surface with a detection antibody before adding the protein of interest. Because it involves two antibodies, this ELISA generates highly specific reactions and offers higher sensitivity than direct or indirect ELISAs, and it does not require purification of the antigen, but it is vulnerable to cross-reactivity.


Indirect ELISA was created by adding a secondary antibody to detect interactions between the primary antibody and the target antigen, and this method was used to detect human serum albumin, the predominant protein in vertebrate blood plasma. This enabled researchers to use one type of secondary antibody to label multiple different primary antibodies, making it more cost-effective than a sandwich ELISA, but creating cross-reactivity and background noise that requires an extra step to incubate a secondary antibody. 


After the identification of HIV in 1984, researchers began to adopt ELISA to recognize the antibodies generated in response to the virus. The first ELISA-based blood test for HIV was approved for use in 1985 to screen donated blood, and the focus of this testing soon expanded to include screening individuals. 


As the COVID-19 pandemic continues to spread across the world, researchers are now using standard, commercially available ELISA test kits to test blood samples, saliva, and upper respiratory specimens for the presence of IgG SARS-CoV-2 antibodies. The ELISA developed by the CDC offers greater than 99% specificity and 96% sensitivity to these antibodies.