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

2. 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

3. 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.

4. 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.

5. Direct ELISA Methodology
Substrate

6. 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.

7. Indirect ELISA Methodology
Substrate

8. 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

9. Sandwich ELISA Methodology
Substrate

10. 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

11. 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

12. 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

13. 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

14. Thanks for listening!

15. 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–
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.
ELISA for Home Pregnancy Test. (n.d.). Retrieved September 18, 2017, from 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.
Types of ELISA | Bio-Rad. (n.d.). Retrieved September 18, 2017, from antibodies.com/elisa-types-direct-indirect-sandwich-competition-elisa-formats.html

16. 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