Indirect-ELISA in immunology

Practical Immunology And Serology(Indirect-ELISA)

1. Practical Immunology And Serology (Indirect-ELISA) By Ahmed Riyadh Abdul Rahman Al-Noor University College 1

2. The Indirect Enzyme-linked immunosorbent assay (Indirect-ELISA) Principle The Indirect-ELISA utilizes an unlabeled primary antibody which is specific for the antigen attached to a polystyrene plate, followed by an enzyme-conjugated secondary antibody which can react with both the primary antibody and substrate and produces a colored product when a substrate is added. 

3. 3 Objective Screening antisera or  hybridoma for specific antibodies. Detect the presence of an antibody in a sample and to use it as a diagnostic tool in medicine. detect potential food allergens, such as milk , peanuts , walnuts , almonds , and eggs and as serological blood test for coeliac disease Used in toxicology as a rapid screen for certain classes of drugs.

4. 4 A-Coating antigen to microplate 1. Coat microtiter plate with dilute antigen: Dispense 50 μl antigen solution (coating reagent) into the wells of a microtiter plate. 2. Cover the plate with an adhesive plastic and incubate for 2 hr at room temperature, or 4°C overnight in an incubator. General procedure

5. 5 3. Remove the coating solution and wash the plate three times with washing solution (ex: 200 µl PBS per well). 4. Excess wash solution must be removed in the final wash step to prevent the dilution of the reagents added in the subsequent stage. This is done by tapping the plate on a paper towel. Note: wash steps are important to remove unbound materials .

6. 6 B - Blocking step Blocking is necessary to prevent the non-specific binding of detection antibodies to the plate surface itself. ​1. Block the remaining protein-binding sites in the coated wells by adding blocking buffer per well. 2. Cover the plate with an adhesive plastic and incubate for at least 2 hr at room temperature, Wash the plate twice with PBS. Note: This is a general protocol in which antigen coating and blocking may not be required if the wells have been pre-adsorbed with the antigen.

7. 7 C- Incubation with primary antibody 1. Add 100 µl of diluted primary antibody to each well. 2. Cover the plate with an adhesive plastic and incubate for 2 hr at room temperature. 3. Wash the plate four times with PBS to remove unbound antibody. D- Incubation with secondary antibody 1. Add 100 µl of conjugated secondary antibody, diluted in blocking buffer immediately before use. 2. Cover the plate with an adhesive plastic and incubate for 1-2 hr at room temperature. 3. Wash the plate four times with PBS to remove unbound antibody.

8. 8 E- Detection A substrate is added, and enzymes on the antibody elicit a chromogenic or fluorescent signal. The two widely enzymatic markers used to labeling secondary antibody: Horse radish peroxidase (HRP) catalyze TMB (3,3',5,5'-tetramethylbenzidine) substrate turns blue when catalyzed and turns yellow after the addition of stop solution. Alkaline phosphatase (ALP) Catalyze p- nitrophenyl phosphate substrate (PNPP) turns yellow when detecting. 1. Dispense 100 µl of the substrate solution per well incubate for 1 hr at room temperature .  

9. 9 2. After sufficient color development this reaction can be stopped by adding stop solution to the wells. 3. The substrate hydrolysed by enzyme attached to secondary antibody and give color. The color in reaction can be read visually or the reaction is detected by reading the optical density (estimated colorimetrically ) using microassay plate reader i.e. ELISA Spectrophotometer reader. 4. Read the absorbance (optical density) of each well with a plate reader.

10. 10 F- ELISA Data Interpretation The ELISA assay yields three different types of data: 1. Qualitative: ELISAs determines the presence or absence of the target and is just giving a positive or negative results, By comparing the absorbance of each sample with the cutoff value which represents the cut-off point in distinguishing between positive and negative results. Positive: If the sample's optical density value is equal or higher than the cut-off value. Negative: If the optical density value of the sample is less than the cut-off. The amount of color produced is proportional to the amount of primary antibody bound to the antigen proteins on the bottom of the wells.

11. 11 2. Quantitative & Semi-Quantitative: ELISA data can be interpreted in comparison to a standard curve (a serial dilution of known, purified antigen dilutions with concentration on the x-axis vs. absorbance on the Y-axis, in order to calculate the concentrations of antigen in samples. The unknown concentration can be determined directly on the graph or with curve fitting software which is typically found on ELISA plate readers.

12. 12 Advantage High sensitivity & High specificity Flexible: Different primary detection antibodies can be used with a single labeled secondary antibody. Cost-saving: Fewer labeled antibodies are required Disadvantages Cross-reactivity might occur with the secondary antibody, resulting in nonspecific signal. Required many incubation steps.

13. 13 THAKS