The document summarizes the indirect enzyme-linked immunosorbent assay (indirect-ELISA) method. It describes the principle of indirect-ELISA which uses an unlabeled primary antibody and an enzyme-conjugated secondary antibody. The general procedure involves coating a plate with antigen, blocking remaining sites, incubating with primary antibody, secondary antibody, adding a substrate to produce a detectable signal, and interpreting the data either qualitatively or quantitatively. The indirect-ELISA method has advantages of high sensitivity and specificity but requires multiple incubation steps and potential for cross-reactivity.

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

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

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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

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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THAKS