Enzyme Linked Immunosorbent Assay PHM142 Fall 2017 Instructor: Dr. Jeffrey Henderson Enzyme Linked Immunosorbent Assay Andrew De Jong Ira Gabriel Sulit Garcia Pei Lun Liu Sam Sateifard

ELISA: Enzyme Linked Immunosorbent Assay General concept Enzyme linked to antibody for detection Application Diagnostic tool in Medicine Food industry safety Types of ELISA Methodologies Direct Indirect Sandwich Competitive

ELISA Plate Setup and Data Analysis Decreasing Standard Concentrations Create Standard Curve and Determine Unknown Concentrations Load Standard and Samples onto 96-Well Plate Measure absorbance on spectrophotometer ELISAs utilize conjugated antibodies to catalyze chromogenic reaction so we can quantify unknown protein concentrations. Before we explain the biochemistry behind these reactions, it is important to known how ELISAs are often setup. ELISAs reactions are typically conducted on a 96-well polystyrene plate. CLICK. This plate map identifies which wells specific reactions are occurring in. CLICK. The wells are loaded in duplicate or triplicate fashion, to increase the accuracy of the experiment. The green wells will be loaded with known concentrations of the protein of interest. CLICK. Known protein concentrations decrease as you move down the column. The red wells will serve as a negative control, and the yellow wells will be loaded with the unknown samples. CLICK. Once the samples have been loaded onto the plate, it will incubate for a period of time, CLICK, before the absorbance reading for each well is measured using a spectrophotometer. CLICK. Using the absorbance readings of the known protein concentration standards, or the wells in green, a standard curve can be constructed. The concentrations of the unknown samples, in yellow, can be determined by plotting their absorbance values on the standard curve.

Direct ELISA Methodology Antigens from sample are immobilized on the bottom of each well Primary conjugated antibodies will specifically bind immobilized antigens Enzyme conjugated to primary antibodies catalyze a chromogenic reaction once substrate is added Named due to DIRECT binding of antigen to conjugated antibody catalyzing chromogenic reaction There are different types of ELISAs, depending on the needs of your specific assay. The direct ELISA is the most simple in terms of methodology. First, antigens from the sample must be bound to the bottom of each well. Wells are incubated with specially conjugated antibodies, which exhibit specific binding to the antigen of interest. Once substrate is added, the enzymes conjugated to the bound primary antibodies catalyze a chromogenic reaction. This reaction changes the color in the wells that is detected using the spectrophotometer. The direct ELISA assay is named due to the DIRECT binding of the antigen to the conjugated antibody.

Direct ELISA Methodology Substrate

Indirect ELISA Methodology Antigens from sample are immobilized on the bottom of each well Primary antibodies specifically bind immobilized antigens Secondary conjugated antibodies bind primary antibodies and catalyze the chromogenic reaction when substrate is added Antigen binds INDIRECT to conjugated antibody catalyzing chromogenic reaction Indirect ELISAs also require antigens to be immobilized onto the bottom of each well. Primary antibodies are then incubated and bind the immobilized antigens. Next,the wells are incubated with secondary conjugated antibodies that are able to bind the primary antibodies. It is important to note that multiple secondary antibodies can bind one primary antibody, which allows for signal amplification. Once the substrate is added, the conjugated secondary antibodies catalyze the chromogenic reaction. The indirect ELISA is named due to the INDIRECT contact between the antigen and the conjugated antibody.

Indirect ELISA Methodology Substrate

Capture Assay “Sandwich” ELISA The surface is prepared with an identified amount of capture antibodies The nonspecific binding sites are blocked and the antigen sample is administered to the plate Plate is washed Primary antibodies are then added to the plate to “sandwich” the antigens Enzyme-linked secondary antibodies are then added and they bind with the primary antibodies A chromogenic substrate is added that enzymatically creates a color signal

Sandwich ELISA Methodology Substrate

Type of ELISA Advantages Disadvantages Direct Fastest assay, fewest reagents Most simple and less error prone No signal amplification Highest background noise Indirect Signal amplification is possible Greater specificity than direct Longer assay, lower throughput Sandwich Highest sensitivity Antigens do not required purification Heterophilic antibodies may generate a false-positive signal

Competitive ELISA Looks for an amount of sample antigen Sample antigen, and primary antibody incubated together Labelled antigen coated on plate competes with sample antigen for primary antibody binding Secondary antibody that is conjugated to an enzyme is added onto plate The primary antibodies bound to labelled antigen on the plate bind to secondary antibodies

Competitive ELISA Cont. After washing plate, substrate is added and the amount of labelled antigen is detected through chromogenic or fluorescent signal Signal output is inversely correlated to the amount of sample antigen Competitive binding is concentration dependent

Real-Life Application: Pregnancy Tests Application of Sandwich ELISA Testing for hormone called human chorionic gonadotropin (hCG) hCG found in blood or urine samples hCG can be detected after implantation (6-12 days after fertilization) Looks for beta subunit of hCG https://mom.me/pregnancy/38614-3-things-i-never-knew-about-early-pregnancy-tests/

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References An ELISA avoiding interference by heterophilic antibodies in the measurement of components of the plasminogen activation system in blood. (2002). Journal of Immunological Methods, 268(2), 219–231. https://doi.org/10.1016/S0022-1759(02)00213-2 Aydin, S. (2015). A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides, 72, 4–15. https://doi.org/10.1016/j.peptides.2015.04.012 ELISA for Home Pregnancy Test. (n.d.). Retrieved September 18, 2017, from http://www.elisa- antibody.com/ELISA-applications/home-pregnancy-test Gan, S. D., & Patel, K. R. (2013). Enzyme Immunoassay and Enzyme-Linked Immunosorbent Assay. Journal of Investigative Dermatology, 133, 1–3. https://doi.org/10.1038/jid.2013.287 Types of ELISA | Bio-Rad. (n.d.). Retrieved September 18, 2017, from https://www.bio-rad- antibodies.com/elisa-types-direct-indirect-sandwich-competition-elisa-formats.html

Summary Slide ELISA: Enzyme Linked Immunosorbent Assay Used to detect proteins, antigens, and antibodies of interest Enzyme linked to antibodies catalyse chromogenic reaction that changes reaction color Direct ELISA Uses primary conjugated antibody that binds directly to antigen of interest Advantages: fastest assay, fewest reagents Disadvantages: no signal amplification, highest background noise Indirect ELISA Secondary conjugated antibodies binds primary antibody allow for signal amplification Advantages: greater specificity than direct ELISA Disadvantages: Assay takes longer to perform assay Sandwich ELISA Antigen is sandwiched between a capture and detection antibody Advantages: highest sensitivity and specificity Disadvantages: heterophilic antibodies may generate a false-positive signal Competitive ELISA Concentration-dependent competitive binding between sample antigen and labelled antigen Signal output is inversely correlated to amount of sample antigen Real World Applications Pregnancy test uses sandwich ELISA which tests for the presence of hCG