E.L.I.S.A PHM142 Fall 2019 Instructor: Chesa Dojo Soeandy Coordinator: Jeffrey Henderson Good morning, today we will be presenting on the lab technique ELISA that stands for enzyme-linked immunosorbent assay. My name is Christine and this is….. PRESENTERS: MINRONG (ERIN) LI, SEUNG BEOM (JASON) BONG, CARMEN LIN, CHRISTINE TAN

CONTENTS What is ELISA? How ELISA Works Applications of ELISA Summary Direct and Indirect Capture Competitive and Sandwich Capture Applications of ELISA Summary Before we jump in, here is a brief overview of what we will cover today. We will first talk about what ELISA is and its different forms, then end with some real-life applications and a summary for happy studying.

[1] DISCOVERY The lab technique of ELISA was discovered in 1966 by two separate groups in Sweden and the Netherlands simultaneously.

What is ELISA? Diagnostic immunoassay [1] Diagnostic immunoassay Commercially successful starting 1970s Fully automatic systems starting 1980s Laboratory technique staple And what ELISA is is a diagnostic immunoassay, commercially starting to be available in the 1970s and 80s. Now ELISIA is a staple in the laboratory Promotional photo of first EIA

Demonstration of general ELISA concept Before we jump into the nitty gritty, we want to introduce the concept of ELISA with a little demonstration. We have Carmen who is a human and everyone in this room are aliens. We heard a lot about humans having hands but we don’t know how many any one human has. So to investigate, we designed Erin and Jason to detect hands. When they bind to a hand they will wave. THat is how ELISA works, it is a lab technique to detect something specific then give off a signal like a linked enzyme or fluorophore to let us know.

How ELISA Works Specific binding of antibody to specific ligand General Method Immobilization of Antigen Detection Antibody Label How ELISA Works [2] ELISA solely relies on the specific binding of an antibody to a specific antigen, also known as a ligand. There’s multiple methods of ELISA as shown in the diagram on the slide; however, I’ll explain a general step by step process that all methods follow. Firstly, the antigen must be immobilized by either adsorption or a capture antibody. Next, a detection antibody is added which binds to the adsorbed ligand. Finally, a label must be shown to show quantity/quality data for the antigen. This could be from the detection antibody being conjugated with an enzyme where a substrate will then bind to produce a label, or it will provide a binding site for a labeled secondary antibody.

Direct and Indirect Capture Antigen directly attaches to wall by passive adsorption Detection antibody attaches to antigen directly Indirect Antigen adsorbed to wall and antigen-specific antibody attaches to antigen directly Separate Detection antibody attaches to antigen-specific antibody [2] In the direct capture method, the antigen is immobilized to the inner wall by adsorption where a detection antibody attaches directly. The detection antibody will be specific to the antigen and will also be conjugated to an enzyme or labelled. In the indirect capture method, the antigen is first adsorbed to the wall where a primary antigen-specific antibody attaches to the ligand. The secondary antibody will then attach to the primary antibody, acting as the detection antibody. This means that only the first antibody is specific to the antigen and the secondary antibody is not.

Competitive Capture Measures the concentration of an antigen in a sample by detecting interference in an expected signal output Used when the antigen is small and has only one antibody binding site [3]

Sandwich Capture Most specific form of ELISA The antigen is bound between a layer of capture antibodies and a layer of detection antibodies Each antibody is specific for a different antibody binding site on the antigen The capture antibody may be conjugated or unconjugated Conjugated - Direct ELISA Unconjugated - Indirect ELISA 1 2 3 4 5 6 7

Advantages vs. Disadvantages ELISA Direct Indirect Competitive Sandwich Fast Less error Inspecific No signal amplification Efficient Sensitive Extra step Cross-reactive The direct capture method uses the fewest step as it involves only the antigen and a single antibody. This is also the reason that it has less room for error since it uses so few reagents. The results can still be unclear as many different proteins will adsorb to the wall as well as the antigen. Since it also only relies on a single antibody, there’s no room for signal amplification which is done by using separate detection antibodies. The indirect method is more flexible with which antigens to look for while using the same label antibody. There is also a risk of cross-reactivity as the secondary antibody can bind to something that isn’t the primary antibody, causing error. Impure samples may be used High reproducibility Lower sensitivity and specificity Fast Highly specific Antigen does not require purification prior to use Antigen must contain at least 2 antibody binding sites

Applications of ELISA Food Allergens Cancer Screening Detecting Viruses Others Applications of ELISA

Application of ELISA: 1. Food Allergens ELISA is commonly used in the food industry to ensure safety Detects the presence of allergens Advantageous over other methods when testing for: Oils, egg whites, and milk Can detect at concentrations on the low parts per million (ppm) scale Photo: http://blog.denticle.com/2018/04/university-of-michigan-discovers.html Flucke, J. (2018). University of Michigan Discovers Vaccine Suppresses Peanut Allergy in Mice . Retrieved from http://blog.denticle.com/2018/04/university-of-michigan-discovers.html ELISA is commonly used in the food industry to ensure product safety before it is released for sale to consumers. ELISA kits can be used to detect the presence of food allergens such as peanuts, sesame, gluten, soy, and many more. ELISA is advantageous over other methods, such as polymerase chain reaction (PCR), when testing for allergens such as oils, egg whites, and milk because ELISA can detect at concentrations on the low parts per million (ppm) scale. [4] Common food allergens.

Application of ELISA: 2. Cancer Screening ELISA can detect tumor markers are synthesized by tumor cells Tumor markers may be used to: Detect early stages of cancer Monitor response to treatment Look at the initial level of tumour marker and compare it with the current level Determine whether the cancer has recurred after treatment Photo: https://www.creative-diagnostics.com/tumor-marker.htm Creative Diagnostics (CD). (n.d.). Microscopic tumor. Retrieved from https://www.creative-diagnostics.com/tumor-marker.htm ELISA can detect tumor markers, which consists of proteins and peptides, synthesized by tumor cells or by surrounding tissue. Tumor markers may be used to detect early stages of cancer, to monitor response to treatment, and to determine whether the cancer has recurred after treatment. For example, if ELISA is being used to monitor a response to treatment, then you would look at the initial level of tumour marker and compare it with the current level of tumour marker. If the levels went down, then that indicates that the treatment is working and vice versa. [5] Microscopic tumor cell.

Application of ELISA: 3. Detecting Viruses Viruses invade and colonize host tissues and induce the host to generate antibodies ELISA can detect and measure these antibodies ELISA has been used to detect: HIV West Nile virus Newcastle disease virus (NDV) Viral infection rates are high in developing countries Instant results at testing locations Photo: https://www.mirror.co.uk/news/technology-science/science/hiv-vaccine-trials-to-begin-6752566 Radowitz, J. von. (2015). Hiv under the microscope. Retrieved from https://www.mirror.co.uk/news/technology-science/science/hiv-vaccine-trials-to-begin-6752566 Infectious diseases caused by viruses invade and colonize host tissues and induce the immune system of the host to make antibodies against them. These antibodies can be detected and measured by ELISA. ELISA has been used to detect HIV, West Nile virus, and Newcastle disease virus (NDV). This is advantageous because these viral infection rates are high in developing countries and ELISA provides instant results at testing locations. [6] Microscopic HIV.

4. Other Applications of ELISA Veterinary Plant pathogen Pesticides Autoimmune disease Gastrointestinal disease Many more!

Enzyme-linked immunosorbent assay (ELISA) is a staple laboratory technique today Relies on specific binding of antibody to antigen General Method Immobilization Detection Label There are 4 different types of ELISA techniques: Direct Indirect Competitive - Highly reproducible Sandwich - Highly specific Some applications of ELISA are: Food allergens Cancer screening Detecting viruses SUMMARY

Barnett, C. (2019, June 20). ELISA Applications. Retrieved from https://www.news-medical.net/life-sciences/ELISA-Applications.aspx Bio-Rad. (n.d.). Retrieved from https://www.bio-rad-antibodies.com/blog/deciding-which-elisa-technique-is-right-for-you.html Creative Diagnostics. (2009). Products. Retrieved from https://www.creative-diagnostics.com/ELISA-Kits.htm ThermoFisher. (n.d.). Overview of ELISA. Retrieved from https://www.thermofisher.com/ca/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-elisa.html Images: Lequin, R. M. (2005). Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clinical chemistry, 51(12), 2415-2418. ELISA. Retrieved from https://www.sbhsciences.com/ELISA.asp. Konstantinou G.N. (2017) Enzyme-Linked Immunosorbent Assay (ELISA). In: Lin J., Alcocer M. (eds) Food Allergens. Methods in Molecular Biology, vol 1592. Humana Press, New York, NY. ELISA- Principle, Types and Applications. (2015, July 28). Retrieved from http://microbiologynotes.com/elisa-principle-types-and-applications/ Flucke, J. (2018). University of Michigan Discovers Vaccine Suppresses Peanut Allergy in Mice . Retrieved from http://blog.denticle.com/2018/04/university-of-michigan-discovers.html Creative Diagnostics (CD). (n.d.). Microscopic tumor. Retrieved from https://www.creative-diagnostics.com/tumor-marker.htm Radowitz, J. von. (2015). Hiv under the microscope. Retrieved from https://www.mirror.co.uk/news/technology-science/science/hiv-vaccine-trials-to-begin-6752566 Works Cited Remember this should be in alphabetical order Numbers should be the same as the images

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