1. Enzyme-Linked Immunosorbent Assay ELISA
Bahiya Osrah

2. Introduction
Enzyme-Linked Immunosorbent Assay is the most commonly used immunological assay to detect the presence of antibody or an antigen in a sample
Qualitative results: positive or negative results
Quantitative results: the amount of colored product is direct proportional to the amount of Enzyme-Linked antibody or antigen by using a standard curve.
The unknown antigen or antibody can be determined
Example: Therapeutic drug monitoring

3. Introduction
Readings: can be detected by spectrophotometer (microreader plate) + ELISA reader

4. Advantage Safest (no radioactive material) High level of accuracy
Easy visualization of results
Sensitive and specific
Large number of tests can be done
Require minimal reagents
Qualitative and quantitative
Well can be coated with antigen or antibody

5. Requirements Sample Purified antigen to detect and quantify antibody
or purified antibody to detect and antify antigen
Standard solutions: +ve and –ve controls
Microtiter plates
Washing fluids (buffers)
enzyme-labeled antibody and enzyme substrate
ELISA reader: spectrophotometer or spectrofluremeter for quantitative measurments

6. Types of ELISA Labeling Signals detection methodology
The basic approaches by fixing either antigen or antibody and detecting antibody-antigen complex
ELISA
In-direct
sandwish
competitive
direct

7. Direct Attached of antigen to the solid surface
Enzyme labeled antibody is added to react with the fixed antigen
Addition of enzyme substrate
Signal is produced as color
Color is directly proportional to antibody or antigen in sample

8. In-direct Attached of antigen to the solid surface
Primary antibody not labeled added to the fixed antigen
Labeled secondary antibody is added to recognizes the primary antibody
Addition of enzyme substrate
Signal is produced as color
Color is directly proportional to antibody or antigen is the sample

9. Sandwish
Purpose:
Measures the amount of antigen between two layers of antibodies
The capture antipody
The detection antibody
The antigen must contain at least two antigenic sites
Capture antibody is fixed on solid surface
Antigen is added
Primary Enzyme labeled antibody is added and sometime also secondary
Enzyme substrate is added
The color or signal after addition of substrate is proportional to antigen
Ex: HIV antibody

10. Competitive Purpose: Measures the amount of antigen in a sample
Antigen is labeled instead of antibody
Unlabeled antigen
Competition is between labeled and unlabeled antigen to bind with the capture antibody
The color is inversibly prportional to antigens in the sample
The absence of antigen  dark color
The presence of antigen  light color

11. Competitive Steps: Coat the plate with Capture antibody
Block the plate with BSA or detergent
Mix the sample+ enzyme conjugated (HRP)
Add the Mix to the ELISA plate
Wash the plate
Add colorless TMB substrate
Add stop solution (would change the blue to yellow)
The amount of yellow color is inversely proportional to antigen in the sample

12. Competitive Antigen in sample >> conjugated antigen  less color
Antigen in sample << conjugated antigen  more color

13. KIT: QUANTA Lite ASCA (S.cerevisiae) IgG
Used for semi quantitative detection of anti-S.cerevisiae antibodies in the human serum
It is used for the diagnosis of Crohn’s disease
Crohn’s disease is inflamatory bowel disease occurs in the small intestine and causes ulcer and inflammation in the top layers of colon and rectum.
ASACA antibodies have been found to be significantly more prevalent in the patients with crohn’s disease compared to control healthy people.
These antibodies are directed against mannose sequence in the cell wall of S.cerevisiae

14. procedure All reagent should be at room temperature
Add 100 ml of ASCA IgG low positive control to two wells
Add 100 ml of ASCA high positive control to two wells
Add 100 ml negative control to 2 wells
Add 100 ml of sample (serum) to 2 wells
Cover the plate and incubate for 30 min
Wash step: add ml of HRP washing buffer to the wells aspirate 3 times
Add 100ml of the HRP IgG conjugated
Incubate 30 min
Wash
Add 100ml of TMB chromogen  incubate in the dark 30 min at RT
Add 100ml HRP stop solution  tap to mix
Read OD at 450nm within an hour of stopping the reaction