ELISA Assays: Indirect, Sandwich, and Competitive
1. Indirect ELISA
An indirect ELISA is one where the primary antigen-specific antibody is recognized by a secondary conjugated antibody. The following protocol is an example of an indirect ELISA method, where the serum samples of of influenza A virus (IAV)-infected mice are tested for the presence of IAV-specific IgG antibody. One strength of this example is that different secondary antibodies can be used that recognize all antibody isotypes or specific isotypes (e.g., IgG).
Coating antigen to the microplate
- Coat the wells of a 96-well ELISA plate with purified antigen by pipetting 50 µL of purified antigen (2 mg/mL of purified A/PR/8 Influenza A virus in 0.05M Tris-HCl buffer (pH 9.5)) into each well of the plate.
- Cover the plate with an adhesive cover and incubate it overnight at 4°C to allow the antigen to bind to the plate.
- Upon incubation completion, remove the coating solution by flicking the plate over a sink.
Blocking
- Block the remaining protein-binding sites in the coated wells by adding 200 µL blocking buffer, 5% donkey serum in 1X PBS is used here, per well. Alternative blocking reagents include 5% non-fat dry milk or BSA in PBS or normal serum from an animal in which the secondary antibody was generated.
- Incubate for at least 2 hours at room temperature or overnight at 4°C.
- Following the incubation, remove the blocking buffer by flicking the plate and then wash plate with PBS containing 1% Tween-20.
Incubation with the primary antibody
- Prepare a serial dilution of the serum sample, which contains the primary antibody, to obtain a dilution range of 1 to 204,800, using 1X PBS. To do this, first dilute the serum 1:12.5 and then perform a 4X dilution (dilution range - 1:12.5 to 1:204,800).
- Add 100 µL of the serially-diluted serum samples to the wells.
- Cover plate with adhesive cover and incubate at room temperature for 1-2 h.
- Following the incubation, flick the plate over a sink and wash plate with PBS containing 1% Tween-20.
Incubation with the secondary antibody
- Add 100 µL of an enzyme-conjugated secondary antibody, horseradish peroxidase, HRP-conjugated donkey anti-mouse secondary in this experiment, to each well.
- Incubate the plate for 1 hour at room temperature.
- Following the incubation, flick the plate over a sink and then wash plate with PBS containing 1% Tween-20.
Detection
- Add 100 µL of the indicator substrate (3,3',5,5'-tetramethylbenzidine (TMB)) at a concentration of 1 mg/mL to each well.
- Incubate the plate with the substrate for 5-10 min at room temperature.
- After 10 min, stop the enzymatic reaction by adding 100 µL 2N Sulfuric acid (H2SO4).
Within 30 min of adding the stop solution, read the plate using a microplate reader at 405 nm to determine the absorbance of the wells.
2. Sandwich ELISA
In this ELISA version, the experimental sample is "sandwiched" between an unconjugated capture antibody and a conjugated detection antibody, both of which are specific to the same protein but at different epitopes. In the following sandwich ELISA example, concentration of human TNFα was determined in unknown sample using a standard curve generated from 2.5X serial dilution of a known standard, recombinant human TNFα (stating at concentration of 75 pg/mL).
Coating capture antibody to the microplate
- Coat the wells of a 96-well ELISA plate with purified capture antibody by adding 100 µL of capture antibody (1-10 µg/mL range) to each well of the plate.
- Cover plate with an adhesive plate cover and incubate it overnight at 4°C.
- After incubation, remove the coating solution from plate by flicking the plate over a sink.
Blocking
- Block the remaining protein-binding sites in the antibody coated wells by adding 200 µL blocking solution, 5% nonfat dry milk containing PBS, to the wells.
- Incubate for at least 2 h at room temperature or overnight at 4°C.
- Following the incubation, remove the blocking buffer by flicking the plate and then wash plate with PBS containing 1% Tween-20.
Add antigen containing test samples
- Add 100 µL of the test sample to the wells. Seal the plate with an adhesive cover.
- Incubate for 1-2 h at room temperature or overnight at 4°C.
- After incubation, remove the samples by flicking the plate over the sink and then wash the wells with 200 µL 1X PBS containing 1% Tween-20.
Add enzyme-conjugated detection antibody
- Add 100 µL of enzyme-conjugated detection antibody to the wells at a preoptimized concentration.
- Seal the plate with an adhesive cover and incubate at room temperature for 2 h.
- Remove the unbound detection antibody by flicking the plate over a sink and wash the wells with 200 µL 1X PBS containing 1% Tween-20.
Detection
- Add 100 µL of the indicator substrate at a concentration of 1 mg/mL. Any bound enzyme-conjugated detection antibody will convert the substrate to a detectable signal.
- Incubate the plate for 5-10 min at room temperature.
- After 5-10 min, stop the enzymatic reaction by adding 100 µL 2N H2SO4 to the wells. Within 30 min of adding the stop solution, read the plate using a microplate reader to determine the absorbance of the wells.
3. Competitive ELISA
The steps of a competitive ELISA are different from those used in indirect and sandwich ELISA, with the main difference being the competitive binding step between the sample antigen and the "add-in" antigen. The sample antigen is incubated with the unlabeled primary antibody. These antibody-antigen complexes are then added to the ELISA plate, which has been pre-coated with the same antigen. After an incubation period, any unbound antibody is washed away. There is an inverse correlation between the amount of free antibody available to bind the antigen in the well and the amount of antigen in the original sample. For example, a sample with abundant antigen would have more antigen-primary antibody complexes, leaving little unbound antibody to bind to the ELISA plate. An enzyme-conjugated secondary antibody specific to the primary antibody is then added to the wells, followed by the substrate.
Coating antigen to the microplate
- Coat the wells of a 96-well ELISA plate with 100 μL of purified antigen at a concentration of 1-10 μg/mL.
- Cover plate with an adhesive plate cover and incubate the plate overnight at 4°C.
- Following incubation, remove the unbound antigen solution from the wells by flicking the plate over a sink.
Blocking
- Block the remaining protein-binding sites in the coated wells by adding 200 μL of blocking buffer to each well, which can be either 5% non-fat dry milk or BSA in PBS.
- Incubate the plate for at least 2 h at room temperature or overnight at 4°C.
Incubation sample (antigen) with the primary antibody
- While blocking the wells, prepare the antigen-antibody mixture by mixing 150 μL sample antigen and 150 μL of primary antibody for each well in the assay.
- Incubate this mixture for 1 h at 37°C.
Add antigen-antibody mixture to the well
- Now, remove the blocking buffer from the wells by flicking the plate over a sink.
- Then, wash the wells with 1X PBS containing Tween-20.
- Add 100 μL of the sample antigen-primary antibody mixture.
- Incubate the plate at 37°C for 1 h.
- Remove the sample mixture by flicking the plate over a sink.
- Then, wash the wells with 1X PBS containing 1% Tween-20 to remove any unbound antibody.
Add the secondary antibody
- Add 100 μL of an enzyme conjugated secondary antibody, which in this case is AP-conjugated antibody, to each well.
- Incubate the plate for 1 h at 37°C.
- Following incubation, wash the plate with 1X PBS containing 1% Tween-20.
Detection
- Add 100 μL of the substrate solution to each well.
- Wait for 5-10 min.
- After 10 min, stop the enzymatic reaction by adding 100 μL 2N sulfuric acid to the wells. Then, measure the absorbance in a microplate reader within 30 min of adding the stop solution
Source: Whitney Swanson1,2, Frances V. Sjaastad2,3, and Thomas S. Griffith1,2,3,4
1 Department of Urology, University of Minnesota, Minneapolis, MN 55…
1. Indirect ELISA
An indirect ELISA is one where the primary antigen-specific antibody is recognized by a secondary conjugated antibody. The following protocol is an example of an indirect ELISA method, where the serum samples of of influenza A virus (IAV)-infected mice are tested for the presence of IAV-specific IgG antibody. One strength of this example is that different secondary antibodies can be used that recognize all antibody isotypes or specific isotypes (e.g., IgG).
Coating antigen to the microplate
- Coat the wells of a 96-well ELISA plate with purified antigen by pipetting 50 µL of purified antigen (2 mg/mL of purified A/PR/8 Influenza A virus in 0.05M Tris-HCl buffer (pH 9.5)) into each well of the plate.
- Cover the plate with an adhesive cover and incubate it overnight at 4°C to allow the antigen to bind to the plate.
- Upon incubation completion, remove the coating solution by flicking the plate over a sink.
Blocking
- Block the remaining protein-binding sites in the coated wells by adding 200 µL blocking buffer, 5% donkey serum in 1X PBS is used here, per well. Alternative blocking reagents include 5% non-fat dry milk or BSA in PBS or normal serum from an animal in which the secondary antibody was generated.
- Incubate for at least 2 hours at room temperature or overnight at 4°C.
- Following the incubation, remove the blocking buffer by flicking the plate and then wash plate with PBS containing 1% Tween-20.
Incubation with the primary antibody
- Prepare a serial dilution of the serum sample, which contains the primary antibody, to obtain a dilution range of 1 to 204,800, using 1X PBS. To do this, first dilute the serum 1:12.5 and then perform a 4X dilution (dilution range - 1:12.5 to 1:204,800).
- Add 100 µL of the serially-diluted serum samples to the wells.
- Cover plate with adhesive cover and incubate at room temperature for 1-2 h.
- Following the incubation, flick the plate over a sink and wash plate with PBS containing 1% Tween-20.
Incubation with the secondary antibody
- Add 100 µL of an enzyme-conjugated secondary antibody, horseradish peroxidase, HRP-conjugated donkey anti-mouse secondary in this experiment, to each well.
- Incubate the plate for 1 hour at room temperature.
- Following the incubation, flick the plate over a sink and then wash plate with PBS containing 1% Tween-20.
Detection
- Add 100 µL of the indicator substrate (3,3',5,5'-tetramethylbenzidine (TMB)) at a concentration of 1 mg/mL to each well.
- Incubate the plate with the substrate for 5-10 min at room temperature.
- After 10 min, stop the enzymatic reaction by adding 100 µL 2N Sulfuric acid (H2SO4).
Within 30 min of adding the stop solution, read the plate using a microplate reader at 405 nm to determine the absorbance of the wells.
2. Sandwich ELISA
In this ELISA version, the experimental sample is "sandwiched" between an unconjugated capture antibody and a conjugated detection antibody, both of which are specific to the same protein but at different epitopes. In the following sandwich ELISA example, concentration of human TNFα was determined in unknown sample using a standard curve generated from 2.5X serial dilution of a known standard, recombinant human TNFα (stating at concentration of 75 pg/mL).
Coating capture antibody to the microplate
- Coat the wells of a 96-well ELISA plate with purified capture antibody by adding 100 µL of capture antibody (1-10 µg/mL range) to each well of the plate.
- Cover plate with an adhesive plate cover and incubate it overnight at 4°C.
- After incubation, remove the coating solution from plate by flicking the plate over a sink.
Blocking
- Block the remaining protein-binding sites in the antibody coated wells by adding 200 µL blocking solution, 5% nonfat dry milk containing PBS, to the wells.
- Incubate for at least 2 h at room temperature or overnight at 4°C.
- Following the incubation, remove the blocking buffer by flicking the plate and then wash plate with PBS containing 1% Tween-20.
Add antigen containing test samples
- Add 100 µL of the test sample to the wells. Seal the plate with an adhesive cover.
- Incubate for 1-2 h at room temperature or overnight at 4°C.
- After incubation, remove the samples by flicking the plate over the sink and then wash the wells with 200 µL 1X PBS containing 1% Tween-20.
Add enzyme-conjugated detection antibody
- Add 100 µL of enzyme-conjugated detection antibody to the wells at a preoptimized concentration.
- Seal the plate with an adhesive cover and incubate at room temperature for 2 h.
- Remove the unbound detection antibody by flicking the plate over a sink and wash the wells with 200 µL 1X PBS containing 1% Tween-20.
Detection
- Add 100 µL of the indicator substrate at a concentration of 1 mg/mL. Any bound enzyme-conjugated detection antibody will convert the substrate to a detectable signal.
- Incubate the plate for 5-10 min at room temperature.
- After 5-10 min, stop the enzymatic reaction by adding 100 µL 2N H2SO4 to the wells. Within 30 min of adding the stop solution, read the plate using a microplate reader to determine the absorbance of the wells.
3. Competitive ELISA
The steps of a competitive ELISA are different from those used in indirect and sandwich ELISA, with the main difference being the competitive binding step between the sample antigen and the "add-in" antigen. The sample antigen is incubated with the unlabeled primary antibody. These antibody-antigen complexes are then added to the ELISA plate, which has been pre-coated with the same antigen. After an incubation period, any unbound antibody is washed away. There is an inverse correlation between the amount of free antibody available to bind the antigen in the well and the amount of antigen in the original sample. For example, a sample with abundant antigen would have more antigen-primary antibody complexes, leaving little unbound antibody to bind to the ELISA plate. An enzyme-conjugated secondary antibody specific to the primary antibody is then added to the wells, followed by the substrate.
Coating antigen to the microplate
- Coat the wells of a 96-well ELISA plate with 100 μL of purified antigen at a concentration of 1-10 μg/mL.
- Cover plate with an adhesive plate cover and incubate the plate overnight at 4°C.
- Following incubation, remove the unbound antigen solution from the wells by flicking the plate over a sink.
Blocking
- Block the remaining protein-binding sites in the coated wells by adding 200 μL of blocking buffer to each well, which can be either 5% non-fat dry milk or BSA in PBS.
- Incubate the plate for at least 2 h at room temperature or overnight at 4°C.
Incubation sample (antigen) with the primary antibody
- While blocking the wells, prepare the antigen-antibody mixture by mixing 150 μL sample antigen and 150 μL of primary antibody for each well in the assay.
- Incubate this mixture for 1 h at 37°C.
Add antigen-antibody mixture to the well
- Now, remove the blocking buffer from the wells by flicking the plate over a sink.
- Then, wash the wells with 1X PBS containing Tween-20.
- Add 100 μL of the sample antigen-primary antibody mixture.
- Incubate the plate at 37°C for 1 h.
- Remove the sample mixture by flicking the plate over a sink.
- Then, wash the wells with 1X PBS containing 1% Tween-20 to remove any unbound antibody.
Add the secondary antibody
- Add 100 μL of an enzyme conjugated secondary antibody, which in this case is AP-conjugated antibody, to each well.
- Incubate the plate for 1 h at 37°C.
- Following incubation, wash the plate with 1X PBS containing 1% Tween-20.
Detection
- Add 100 μL of the substrate solution to each well.
- Wait for 5-10 min.
- After 10 min, stop the enzymatic reaction by adding 100 μL 2N sulfuric acid to the wells. Then, measure the absorbance in a microplate reader within 30 min of adding the stop solution
Enzyme-linked Immunosorbent Assay, or ELISA is a highly sensitive quantitative assay commonly used to measure the concentration of an analyte like cytokines and antibodies in a biological sample. The general principle of this assay involves three steps: starting with capture, or immobilization, of the target analyte on a micro plate, followed by the detection of the analyte by target-specific detection proteins, and lastly, enzyme reaction, where a conjugated enzyme converts its substrate to a colored product. Based on different methods of capture and detection, ELISA can be of four types: direct, indirect, sandwich, and competitive.
For direct ELISA, the target antigen is first bound to the plate, and is then detected by a specific detection antibody. This method is commonly used for screening antibodies for a specific antigen. Indirect ELISA is used for detecting antibodies in a sample in order to quantify immune responses. The plate is first coated with a specific capture antigen, which immobilizes the target antibody, and this antigen-antibody complex is then detected using a second antibody.
In the case of sandwich ELISA, the target analyte is an antigen, which is captured on the plate using a capture antibody and then detected by the detection antibody, hence forming an antibody-antigen-antibody sandwich. This method is useful for measuring the concentration of an antigen in a mixed sample.
Competitive ELISA is used when only one antibody is available for a target antigen of interest. The plate is first coated with the purified antigen. Meanwhile, the sample containing the antigen is pre-incubated with the antibody and then added to the plate, to allow any free antibody molecules to bind to the immobilized antigen. The higher the signal from the plate, the lower the antigen concentration in the sample. In all of the four types of ELISA, direct, indirect, sandwich, and competitive, the detection antibody is either directly conjugated to the enzyme or can be indirectly linked to it through another antibody or protein.
The enzymes commonly used for the reaction are horseradish peroxidase or alkaline phosphatase with their respective substrates, both producing a soluble, colored product that can be measured and quantified using a plate reader. In this video, you will observe how to perform indirect ELISA, sandwich ELISA, and competitive ELISA, followed by examples of quantification of the target analyte from the indirect and sandwich ELISA methods.
The first experiment will demonstrate how to use indirect ELISA to determine the presence of anti-influenza virus antibodies in serum obtained from influenza-infected mice.
To begin, add 50 microliters of purified antigen - in this case, 2 milligrams per milliliter of purified A/PR/8 Influenza A virus- to each well of a 96-well ELISA plate. Next, cover the plate with an adhesive cover and incubate it overnight at 4 degrees celsius to allow the antigen to bind to the plate. The following day, remove the coating solution by flicking the plate over a sink. Next, block the remaining protein-binding sites in the coated wells by adding 200 microliters of a blocking buffer- here, 5% donkey serum in 1X PBS- to each well. Leave the plate to incubate for at least 2 hours at room temperature. Following the incubation, remove the blocking buffer and then wash the plate by adding 200 microliters of 1X PBS containing 1% Tween-20. Flick the plate over the sink once more to remove the wash.
Then, prepare the test samples by adding 460 microliters of PBS to a fresh tube, and then adding 40 microliters of serum to make a 1 to 12.5 dilution. Then, add 300 microliters of PBS to a second tube, and then add 100 microliters of the first dilution. Continue this serial dilution range until obtaining a final sample with a dilution of 1 to 204,800. Add the serially diluted serum samples in triplicate to the wells. Cover the plate with an adhesive cover and incubate at room temperature for an hour. Next, remove the samples by flicking the plate into the sink and then wash the plate by adding 200 microliters of 1X PBS containing 1% Tween-20. Once again, flick the plate to remove the wash.
Now, add 100 microliters of an enzyme-conjugated secondary antibody, which in this experiment is a horseradish peroxidase, or HRP, conjugated donkey anti-mouse secondary, to each well. Incubate the plate for one hour at room temperature, and flick the plate to remove any excess liquid. Wash the plate with 1X PBS containing 1% Tween-20 and then apply 100 microliters of the indicator substrate at a concentration of one milligram per milliliter to each well. Incubate the plate with the substrate for 5 to 10 minutes at room temperature. In this example, the colorless 3,3', 5,5' - tetramethylbenzidine, or TMB, substrate turns a blue color when HRP is present. After 10 minutes, stop the enzymatic reaction by adding 100 microliters of 2N sulfuric acid. The samples will turn a yellow color.
Within 30 minutes of adding the stop solution, insert the plate into a microplate reader and read the plate at the appropriate wavelength for the substrate to determine the absorbance of the wells.
To begin the sandwich ELISA, the plate must be coated with purified capture antibody. To do this, add 100 microliters of the capture antibody at a concentration within the 1-10 microgram per milliliter range, to each well of a 96-well ELISA plate. Next, cover the plate with an adhesive plate cover and then incubate the plate overnight at 4 degrees celsius. After the incubation, remove the coating solution by flicking the plate over a sink.
Now, block the remaining protein- binding sites in the coated wells by adding 200 microliters of 5% nonfat dry milk to the wells. Incubate the plate at room temperature for at least 2 hours. Next, remove the blocking buffer, and then wash the wells with 1X PBS containing 1% Tween-20. Remove the wash by flicking the plate over the sink. Now, add 100 microliters of the test sample to the wells, seal the plate with an adhesive cover, and then incubate it at room temperature for 2 hours. After incubation, remove the samples by flicking the plate over the sink and then wash the wells with 200 microliters of 1X PBS containing 1% Tween-20. Flick the plate over the sink to remove the wash and then add 100 microliters of enzyme-conjugated detection antibody to the wells.
Seal the plate with an adhesive cover. Leave the plate to incubate at room temperature for 2 hours. After the incubation, remove the unbound detection antibody by flicking the plate over a sink and wash the wells with 200 microliters of 1X PBS containing 1% Tween-20. Next, add 100 microliters of the indicator substrate at a concentration of 1 milligram per milliliter, and incubate the plate for 5 to 10 minutes at room temperature. After 10 minutes, stop the enzymatic reaction by adding 100 microliters of 2N sulfuric acid to the wells and then read the plate within 30 minutes of adding the stop solution in a microplate reader.
To perform a competitive ELISA, first coat the wells of a 96-well ELISA plate with 100 microliters of purified antigen at a concentration of 1-10 micrograms per milliliter. Cover the plate with an adhesive plate cover and then incubate overnight at 4 degrees celsius. Following this, remove the unbound antigen solution from the wells by flicking the plate over a sink.
Next, block the remaining protein-binding sites in the coated wells by adding 200 microliters of blocking buffer to each well- here, 5% nonfat dry milk in PBS. Incubate the plate for at least 2 hours at room temperature. While blocking the wells, prepare the antigen-antibody mixture in a 1. 5 milliliter tube by adding 150 microliters of sample antigen to 150 microliters of primary antibody for each well in the assay. Incubate this mixture for 1 hour at 37 degrees celsius. Now, remove the blocking buffer from the wells by flicking the plate over a sink. Then, wash the wells with 1X PBS containing Tween 20 and then add 100 microliters of the sample antigen- primary antibody mixture.
Leave the plate to incubate at 37 degrees celsius for one hour. Next, remove the sample mixture by flicking the plate over a sink and then wash the wells with 1X PBS containing 1% Tween-20 to remove any unbound antibody. Add 100 microliters of an enzyme-conjugated secondary antibody to each well and incubate the plate for one hour at 37 degrees celsius. Following this, wash the plate with 1X PBS containing 1% Tween-20 and then add 100 microliters of the substrate solution to each well. Wait for 5-10 minutes. After 10 minutes, stop the enzymatic reaction by adding 100 microliters of 2N sulfuric acid and then measure the absorbance in a microplate reader within 30 minutes of adding the stop solution.
For the semi-quantitative indirect ELISA assay, the presence of influenza A virus antibodies in serially diluted samples of serum from influenza A- infected mice was determined by reading the absorbance of each well at 405 nanometers in a plate reader. This raw data is exported to a spread sheet for calculation purposes. In this experiment, the serially diluted serum samples, which range from 1 - 12.5, to 1 - 204,800, were repeated in triplicate.
To analyze the data, the mean absorbance value is therefore calculated for each set of triplicates by adding all the values for each dilution and dividing the sum by 3. Once the mean for each set of triplicates is determined, the mean OD450 readings are plotted against the serial dilutions. The OD readings decrease as the serum is diluted, indicating that less antibodies are found in the more diluted samples. In the quantitative sandwich ELISA, dilutions of known standard, in this case recombinate Human TNFalpha, were added to a 96-well plate and read along with the unknown samples.
To create the standard curve, the mean absorbance value for each set of readings of the known concentrations was calculated. Then, the mean absorbance value was plotted on the y-axis, against the known protein concentrations on the x-axis. A best fit curve is added through the points in the graph.
Once the standard curve is generated, the amount of TNFalpha protein in the test sample can be determined by first calculating the mean absorbance value for the test sample. In this example, the test samples gave OD450 readings of 0.636 and 0. 681. Adding these values and dividing the sum by 2 gives an average of 0.659. From the y-axis on the standard curve graph, extend a horizontal line from this absorbance value to the standard curve. At the point of intersection, extend a vertical line to the x-axis and read the corresponding concentration which, in this test sample, corresponds to a TNFalpha concentration of 38.72 picograms per milliliter.
Q1: What are the three main steps in an ELISA assay?
ELISA involves three sequential steps: capture or immobilization of the target analyte on a microplate, detection of the analyte using target-specific detection proteins, and enzyme reaction where a conjugated enzyme converts its substrate to a colored product. This three-step process forms the foundation for all ELISA variants, enabling sensitive quantification of biomolecules in biological samples.
Q2: How does indirect ELISA differ from sandwich ELISA?
Indirect ELISA coats the plate with a specific capture antigen to detect antibodies in a sample, measuring immune responses. Sandwich ELISA uses a capture antibody to coat the plate and detects antigens sandwiched between capture and detection antibodies. Sandwich ELISA is better suited for analyzing complex samples like tissue culture supernatants where the antigen is part of a mixed sample.
Q3: When should competitive ELISA be used instead of other ELISA types?
Competitive ELISA is used when only one antibody is available for a target antigen or when detecting small antigens with a single epitope that cannot accommodate two different antibodies due to steric hindrance. The plate is coated with purified antigen, and higher signal indicates lower antigen concentration in the sample, creating an inverse relationship.
Q4: What enzymes are commonly used in ELISA and what do they do?
Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the most common enzymes conjugated to antibodies in ELISA. These enzymes convert colorless substrates into soluble, colored products that can be measured and quantified using a plate reader, enabling detection and quantification of antigen-antibody complexes.
Q5: How is a standard curve created and used in sandwich ELISA?
A standard curve is generated by adding known concentrations of purified recombinant protein to designated wells and processing them alongside test samples. Mean absorbance values for known concentrations are plotted on the y-axis against protein concentrations on the x-axis. Test sample absorbance values are then compared to this curve to determine the unknown antigen concentration in the sample.
Q6: Why is blocking an important step in ELISA protocols?
Blocking prevents non-specific binding of antibodies and proteins to empty sites on the microplate. After coating the plate with antigen or antibody, blocking buffer fills remaining protein-binding sites, reducing background signal and improving assay specificity. This step ensures that only target-specific interactions are detected.
Q7: What information does serial dilution provide in indirect ELISA analysis?
Serial dilution of serum samples reveals antibody concentration patterns by showing how absorbance decreases as samples become more diluted. When mean absorbance values from serially diluted samples are plotted against dilution factors, the resulting curve demonstrates that less antibody is present in more diluted samples, enabling semi-quantitative assessment of immune response.
Chapters in this video
0:01
Concepts
3:09
Indirect ELISA
6:49
Sandwich ELISA
9:17
Competitive ELISA
11:44
Results
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