1. Introduction
Enzyme Linked Immunosorbent Assay (ELISA) is a powerful technique for identifying and quantifying a particular protein in a complex mixture. Originally described by Engvall and Perlmann (1971), the method allows analysis of protein samples immobilized in a microplate well using specific antibodies. This technique has revolutionized immunology and is generally used in research laboratories. ELISA also has commercial uses, including the detection of disease markers and allergens in the diagnostic and food industries.
The development of ELISA was made possible because of scientific advances and improvements in several related fields. Technology developed by Kohler and Milstein (1975) enabled the production of antigen-specific monoclonal antibodies leading to their use as probes for detecting individual molecules in protein mixture. In the beginning, detection was attained by radioimmunoassay using antibodies labeled with radioisotopes, but due of health risks replacements were sought. Avramais (1966, 1969) and Pierce (1967) developed methods to chemically link antibodies to biological enzymes whose activities produce a measurable signal with solutions containing appropriate substrates. With the development of fluorescence technology, signal production using fluorophore-tagged antibodies has also become prevalent.
The elementary enzyme-linked immunosorbent assay (ELISA), or enzyme immunoassay (EIA), is distinguished from other antibody-based assays because separation of specific and non-specific interactions happen via serial binding to a solid surface, usually a polystyrene multiwell plate, and because quantitative results can be reached. The ELISA methodology results in a colored end product which correlates to the amount of analyte present in the original sample. ELISAs are fast and simple to carry out, and since they are planned to rapidly handle a large no. of samples in parallel, they are a very standard choice for the assessment of various research and diagnostic targets. They remain in widespread use in their original format and in expanded formats with variations that allow for several analytes per well, extremely sensitive readouts, and direct cell-based output.
Although many variants of ELISA have been developed and used in different situations, they all depend on the same basic elements:
1. Coating/Capture: where the first layer, either an antigen or an antibody, is adsorbed to a well in an ELISA plate
2. Plate Blocking: A buffer containing unrelated protein is used to block free sites in the wells
3. Probing/Detection: Enzyme conjugated detection antibody binds antigen
4. Signal Measurement: Substrate is catalyzed by enzyme to generate colored readout which is then detected and measured
The detection antibody may be directly labeled with signal-generating enzymes like HRP (Horse Radish peroxidase) or AP (Alkaline Phosphatase) or fluorophore or it may be secondarily probed with an enzyme- or fluor-labeled secondary antibody (avidin-biotin-Streptavidin chemistry). For enzymatic method, the appropriate enzyme substrate is used. The signal detected is proportionate to the amount of antigen in the sample. Washing in between the steps makes sure that only specific (high-affinity) binding events are retained to cause signal at the final step.
2. ELISA Formats
There are multiple formats in which an ELISA experiment can be performed.
2.1. Direct ELISA
In direct ELISA, the antigen itself is coated on the wells of micro testplates i.e. ELISA plate. The antigen is then detected by antibody directly conjugated with enzymes like HRP and AP. Direct ELISA is very fast as compared to other ELISA formats as very few steps are required. Chances of error is very low due to low reagent requirement and few steps. However there are some cons of using this method as the immobilization of antigens is not specific which may lead to background noise. This is because all the proteins in the applied sample will bind to the plate. Direct ELISA uses primary antibody which is specific and so new antibodies are needed for every protein. This makes it less flexible and costly. Assay sensitivity is reduced because signal amplification by secondary antibody is not occurring.
2.2. Indirect ELISA
In indirect ELISA antigen is adsorbed to a microplate well. A primary antibody is added which is specific to antigen and binds with it. Then a second antibody is added which then binds to the primary antibody. This secondary antibody is conjugated with an enzyme which converts the substrate applied to coloured product which is then measured. This method is highly sensitive since more than one secondary antibody can bind to a single primary antibody. This method is very economic and flexible as compared to “Direct ELISA” because a single secondary antibody can be used with multiple primary antibodies. The disadvantage of this method is that there may be a possibility that the secondary antibody may be cross reactive. Another disadvantage of this method is that the procedure is longer than the Direct ELISA method as an additional incubation step for the secondary antibody is needed.
2.3. Sandwich ELISA
Sandwich ELISA uses two antibodies which are called matched pair antibodies to capture and detect the antigen. Each antibody is specific to non-overlapping portions/ epitope of the antigen. It’s very essential that the antibody pair are matched and tested specifically in sandwich ELISA so as to ensure that they detect different epitopes and have no cross reactivity, so as to minimize error and achieve accurate results. The first antibody is the capture antibody which is used to capture/bind the antigen. This is done by coating the ELISA micro test plate with the primary antibody, and then adding the antigen to the coated wells. After the capturing step, detection antibody is added. If this antibody is conjugated then this ELISA is known as ‘Direct Sandwich ELISA’. If this detection antibody is unconjugated and another enzyme conjugated antibody is used to bind to detection antibody then it is known as ‘Indirect Sandwich ELISA’.
The major advantage of the sandwich ELISA is the high sensitivity which is more than 2-5 times than that of indirect ELISA and many times than that of direct ELISA. Another advantage is that the sandwich ELISA is highly specific as it requires 2 antibodies to detect antigen. It is highly flexible since both direct and indirect methods can be used. However it also has many drawbacks. The significant of them is if there is unavailability of a standardized ELISA kit or matched antibody pair, antibody optimization has to be carried out so as to determine the cross-reactivity.
2.4. Competitive/ Inhibition ELISA
Competition ELISA aka inhibition ELISA is most complex of all the ELISA formats. However it has its uses in finding out the concentration of antigen/antibodies in a sample by detecting interference in expected signal output. Sample competes with a reference to bind to the limited amount of labelled antibody. The higher the sample conc. the weaker will be the signal.
3. ELISA Results
The ELISA is done for of three type results.
3.1. Quantitative
ELISA data can be plotted against a standard curve, which is a serial dilution of known amount of purified antigen) in order to calculate the conc. of antigen.
3.2. Qualitative
ELISA can also be used for a yes or no answer indication whether a specific antigen is present of not as compared to a blank well containing a negative control.
3.3. Semi-Quantitative
Since varying concentration of antigen will give a variable signal, hence these signal can be compared to find their relative value.
4. Standard Curve
The standard curve is a graph which is plotted using OD (on Y axis) vs Log concentration (on X axis) to produce a sigmoidal curve. The linear portion of the graph is used to analyze the result output data.
Standards should be acquired so as to last for the expected service life of the assay not just for the developmental phase.
5. Controls
The controls are another important point which should be taken into account while developing ELISA. The most basic control is the blank sample control. Spiked sample control can also be used to as to determine percentage recovery of the ELISA readout.
6. ELISA Components and considerations
The following points are to be kept while developing ELISA.
6.1. ELISA Plates
6.1.1. Plate Format
Flat bottomed 96- well plate made of polystyrene is used in majority of ELISA assays. Strip well plated can also be used. 385 and 1536 well plates are also used, which can process more samples at a time. But these large well plates are automated and therefore are only used in high throughput applications. Chemiluminiscent or fluorescent signals may require opaque plates.
6.1.2. Plate readout
Generally flat bottomed plates specifically designed for ELISA should be used for the ELISA instead of round bottom plates. Polystyrene plates can bind from 100-500 ng of IgG/cm2. Pre-coated plates are also available with optimized ELISA kits.
6.2. Buffers
6.2.1. Coating Buffer
Coating buffer is first step in ELISA. It’s the process where a diluted antibody or antigen is adsorbed to the surface of well by incubation. Adsorption occur passively as the result of hydrophobic interactions between the plastic surface of the well and the side chains of the amino acids of the antigen or antibody used for coating. This interaction depends on time, temperature, concentration of coating agent and the pH of coating solution.
Usually coating condition involves adding of 50-100 µl of coating buffer per well. The concentration of the coating agent (antigen/antibody) can be between 1-10 µg/ml. The incubation condition varies from overnight at 4˚C to incubation for 1-2 hrs. at 37˚C.
6.2.2. Blocking Buffer
Generally the blocking buffer used is an unrelated, irrelevant protein or mixture of proteins. Casein is sort of an universal blocking agent used for blocking the wells. 2% casein solution is used for this purpose. You can also use ready made blocking buffers from reputed brands like Sigma Aldrich, Merck, Thermo Fisher. Although these buffers come at high cost so it's very cost effective to prepare your own buffer. The blocking buffer acts by blocking all passively to the binding sites.This prevents non-specific binding of sample proteins to portion of wells unbound by primary antibody, this reducing the noise of the experiment.
6.2.3 Washing Buffer
The washing buffer is the buffer used to wash the ELISA plate in between the different steps after the incubation.
Most common washing buffer used in ELISA, western blotting, and other immuno-assays is the PBST buffer with 0.05% or 0.1% Tween™ 20 detergent. The PBST does not harms the antigen-antibody coupling, and the Tween component is very excellent in cleaning the microtitre plate from solutions of previous steps.
6.2.4 Substrate buffer
The substrate buffer is the substrate of the enzyme which is conjugated with the identification antibody. The identification antibody, gets attached to the sample. When the substrate is added to the microtitre plate wells, the enzyme acts on the substrate. Typically this generates colour which is then used to find concentration by using the samples OD against the standard curve OD. However many other substrate-enzyme couple exist which release fluorescent light and are detected by fluorescent detector.
7. Buffer Preparation
7.1. Coating buffer (10mM Sodium carbonate pH 9.3 Buffer)-1000mL
Take 800 mL purified water/ Milli Q Water. Add 717 mg Sodium bicarbonate, 155 mg Sodium carbonate (anhydrous). MIx it properly. Check the pH of the solution. If it's not 9.3 ± 0.1. Adjust the pH by using 0.1M NaOH or 0.1M Carbonic Acid (H2CO3). When the pH is set, make up the volume to 1000mL and mix it. Filter it through 0.2μ filter and store it at RT.
7.2. Wash Buffer ( 10X PBST- 200mM PB + 150mM NaCl + 1%Tween20)- 1500mL
Prepare 10X PBST and dilute it every time in 1:10 ratio with purified water/ Milli Q water before use.
7.2.1 Solution A (200mM NaH2PO4)
Take 1000 mL purified water/ Milli Q Water. Add 36 gm of NaH2PO4 to it. After dissolving it, makeup the volume to 1500 mL.
7.2.2 Solution B (200mM Na2HPO4)
Take 1000 mL purified water/ Milli Q Water. Add 42.59 gm of NaH2PO4 to it. After dissolving it, makeup the volume to 1500 mL.
7.2.2 200mM Phosphate buffer with 150mM NaCl
Add solution B to solution A till the pH reaches 7.4. Take 1000 mL of the phosphate buffer pH 7.4. Now add 13.15 gm NaCl to it. Make up the total volume to 1500 mL with remaining Phosphate buffer pH 7.4. Filter it through 0.2μ filter. Remove 15 ml of the PBS, and add 15 mL of Tween20 to it. For diluting it to 1X, take 100 mL of the PBST and add it to 900 mL purified water/ Milli Q Water.
Enzyme Linked Immunosorbent Assay (ELISA) is a powerful technique for identifying and quantifying a particular protein in a complex mixture. Originally described by Engvall and Perlmann (1971), the method allows analysis of protein samples immobilized in a microplate well using specific antibodies. This technique has revolutionized immunology and is generally used in research laboratories. ELISA also has commercial uses, including the detection of disease markers and allergens in the diagnostic and food industries.
The development of ELISA was made possible because of scientific advances and improvements in several related fields. Technology developed by Kohler and Milstein (1975) enabled the production of antigen-specific monoclonal antibodies leading to their use as probes for detecting individual molecules in protein mixture. In the beginning, detection was attained by radioimmunoassay using antibodies labeled with radioisotopes, but due of health risks replacements were sought. Avramais (1966, 1969) and Pierce (1967) developed methods to chemically link antibodies to biological enzymes whose activities produce a measurable signal with solutions containing appropriate substrates. With the development of fluorescence technology, signal production using fluorophore-tagged antibodies has also become prevalent.
The elementary enzyme-linked immunosorbent assay (ELISA), or enzyme immunoassay (EIA), is distinguished from other antibody-based assays because separation of specific and non-specific interactions happen via serial binding to a solid surface, usually a polystyrene multiwell plate, and because quantitative results can be reached. The ELISA methodology results in a colored end product which correlates to the amount of analyte present in the original sample. ELISAs are fast and simple to carry out, and since they are planned to rapidly handle a large no. of samples in parallel, they are a very standard choice for the assessment of various research and diagnostic targets. They remain in widespread use in their original format and in expanded formats with variations that allow for several analytes per well, extremely sensitive readouts, and direct cell-based output.
Although many variants of ELISA have been developed and used in different situations, they all depend on the same basic elements:
1. Coating/Capture: where the first layer, either an antigen or an antibody, is adsorbed to a well in an ELISA plate
2. Plate Blocking: A buffer containing unrelated protein is used to block free sites in the wells
3. Probing/Detection: Enzyme conjugated detection antibody binds antigen
4. Signal Measurement: Substrate is catalyzed by enzyme to generate colored readout which is then detected and measured
The detection antibody may be directly labeled with signal-generating enzymes like HRP (Horse Radish peroxidase) or AP (Alkaline Phosphatase) or fluorophore or it may be secondarily probed with an enzyme- or fluor-labeled secondary antibody (avidin-biotin-Streptavidin chemistry). For enzymatic method, the appropriate enzyme substrate is used. The signal detected is proportionate to the amount of antigen in the sample. Washing in between the steps makes sure that only specific (high-affinity) binding events are retained to cause signal at the final step.
2. ELISA Formats
There are multiple formats in which an ELISA experiment can be performed.
2.1. Direct ELISA
In direct ELISA, the antigen itself is coated on the wells of micro testplates i.e. ELISA plate. The antigen is then detected by antibody directly conjugated with enzymes like HRP and AP. Direct ELISA is very fast as compared to other ELISA formats as very few steps are required. Chances of error is very low due to low reagent requirement and few steps. However there are some cons of using this method as the immobilization of antigens is not specific which may lead to background noise. This is because all the proteins in the applied sample will bind to the plate. Direct ELISA uses primary antibody which is specific and so new antibodies are needed for every protein. This makes it less flexible and costly. Assay sensitivity is reduced because signal amplification by secondary antibody is not occurring.
2.2. Indirect ELISA
In indirect ELISA antigen is adsorbed to a microplate well. A primary antibody is added which is specific to antigen and binds with it. Then a second antibody is added which then binds to the primary antibody. This secondary antibody is conjugated with an enzyme which converts the substrate applied to coloured product which is then measured. This method is highly sensitive since more than one secondary antibody can bind to a single primary antibody. This method is very economic and flexible as compared to “Direct ELISA” because a single secondary antibody can be used with multiple primary antibodies. The disadvantage of this method is that there may be a possibility that the secondary antibody may be cross reactive. Another disadvantage of this method is that the procedure is longer than the Direct ELISA method as an additional incubation step for the secondary antibody is needed.
2.3. Sandwich ELISA
Sandwich ELISA uses two antibodies which are called matched pair antibodies to capture and detect the antigen. Each antibody is specific to non-overlapping portions/ epitope of the antigen. It’s very essential that the antibody pair are matched and tested specifically in sandwich ELISA so as to ensure that they detect different epitopes and have no cross reactivity, so as to minimize error and achieve accurate results. The first antibody is the capture antibody which is used to capture/bind the antigen. This is done by coating the ELISA micro test plate with the primary antibody, and then adding the antigen to the coated wells. After the capturing step, detection antibody is added. If this antibody is conjugated then this ELISA is known as ‘Direct Sandwich ELISA’. If this detection antibody is unconjugated and another enzyme conjugated antibody is used to bind to detection antibody then it is known as ‘Indirect Sandwich ELISA’.
The major advantage of the sandwich ELISA is the high sensitivity which is more than 2-5 times than that of indirect ELISA and many times than that of direct ELISA. Another advantage is that the sandwich ELISA is highly specific as it requires 2 antibodies to detect antigen. It is highly flexible since both direct and indirect methods can be used. However it also has many drawbacks. The significant of them is if there is unavailability of a standardized ELISA kit or matched antibody pair, antibody optimization has to be carried out so as to determine the cross-reactivity.
2.4. Competitive/ Inhibition ELISA
Competition ELISA aka inhibition ELISA is most complex of all the ELISA formats. However it has its uses in finding out the concentration of antigen/antibodies in a sample by detecting interference in expected signal output. Sample competes with a reference to bind to the limited amount of labelled antibody. The higher the sample conc. the weaker will be the signal.
3. ELISA Results
The ELISA is done for of three type results.
3.1. Quantitative
ELISA data can be plotted against a standard curve, which is a serial dilution of known amount of purified antigen) in order to calculate the conc. of antigen.
3.2. Qualitative
ELISA can also be used for a yes or no answer indication whether a specific antigen is present of not as compared to a blank well containing a negative control.
3.3. Semi-Quantitative
Since varying concentration of antigen will give a variable signal, hence these signal can be compared to find their relative value.
4. Standard Curve
The standard curve is a graph which is plotted using OD (on Y axis) vs Log concentration (on X axis) to produce a sigmoidal curve. The linear portion of the graph is used to analyze the result output data.
Standards should be acquired so as to last for the expected service life of the assay not just for the developmental phase.
5. Controls
The controls are another important point which should be taken into account while developing ELISA. The most basic control is the blank sample control. Spiked sample control can also be used to as to determine percentage recovery of the ELISA readout.
6. ELISA Components and considerations
The following points are to be kept while developing ELISA.
6.1. ELISA Plates
6.1.1. Plate Format
Flat bottomed 96- well plate made of polystyrene is used in majority of ELISA assays. Strip well plated can also be used. 385 and 1536 well plates are also used, which can process more samples at a time. But these large well plates are automated and therefore are only used in high throughput applications. Chemiluminiscent or fluorescent signals may require opaque plates.
6.1.2. Plate readout
Generally flat bottomed plates specifically designed for ELISA should be used for the ELISA instead of round bottom plates. Polystyrene plates can bind from 100-500 ng of IgG/cm2. Pre-coated plates are also available with optimized ELISA kits.
6.2. Buffers
6.2.1. Coating Buffer
Coating buffer is first step in ELISA. It’s the process where a diluted antibody or antigen is adsorbed to the surface of well by incubation. Adsorption occur passively as the result of hydrophobic interactions between the plastic surface of the well and the side chains of the amino acids of the antigen or antibody used for coating. This interaction depends on time, temperature, concentration of coating agent and the pH of coating solution.
Usually coating condition involves adding of 50-100 µl of coating buffer per well. The concentration of the coating agent (antigen/antibody) can be between 1-10 µg/ml. The incubation condition varies from overnight at 4˚C to incubation for 1-2 hrs. at 37˚C.
6.2.2. Blocking Buffer
Generally the blocking buffer used is an unrelated, irrelevant protein or mixture of proteins. Casein is sort of an universal blocking agent used for blocking the wells. 2% casein solution is used for this purpose. You can also use ready made blocking buffers from reputed brands like Sigma Aldrich, Merck, Thermo Fisher. Although these buffers come at high cost so it's very cost effective to prepare your own buffer. The blocking buffer acts by blocking all passively to the binding sites.This prevents non-specific binding of sample proteins to portion of wells unbound by primary antibody, this reducing the noise of the experiment.
6.2.3 Washing Buffer
The washing buffer is the buffer used to wash the ELISA plate in between the different steps after the incubation.
Most common washing buffer used in ELISA, western blotting, and other immuno-assays is the PBST buffer with 0.05% or 0.1% Tween™ 20 detergent. The PBST does not harms the antigen-antibody coupling, and the Tween component is very excellent in cleaning the microtitre plate from solutions of previous steps.
6.2.4 Substrate buffer
The substrate buffer is the substrate of the enzyme which is conjugated with the identification antibody. The identification antibody, gets attached to the sample. When the substrate is added to the microtitre plate wells, the enzyme acts on the substrate. Typically this generates colour which is then used to find concentration by using the samples OD against the standard curve OD. However many other substrate-enzyme couple exist which release fluorescent light and are detected by fluorescent detector.
7. Buffer Preparation
7.1. Coating buffer (10mM Sodium carbonate pH 9.3 Buffer)-1000mL
Take 800 mL purified water/ Milli Q Water. Add 717 mg Sodium bicarbonate, 155 mg Sodium carbonate (anhydrous). MIx it properly. Check the pH of the solution. If it's not 9.3 ± 0.1. Adjust the pH by using 0.1M NaOH or 0.1M Carbonic Acid (H2CO3). When the pH is set, make up the volume to 1000mL and mix it. Filter it through 0.2μ filter and store it at RT.
7.2. Wash Buffer ( 10X PBST- 200mM PB + 150mM NaCl + 1%Tween20)- 1500mL
Prepare 10X PBST and dilute it every time in 1:10 ratio with purified water/ Milli Q water before use.
7.2.1 Solution A (200mM NaH2PO4)
Take 1000 mL purified water/ Milli Q Water. Add 36 gm of NaH2PO4 to it. After dissolving it, makeup the volume to 1500 mL.
7.2.2 Solution B (200mM Na2HPO4)
Take 1000 mL purified water/ Milli Q Water. Add 42.59 gm of NaH2PO4 to it. After dissolving it, makeup the volume to 1500 mL.
7.2.2 200mM Phosphate buffer with 150mM NaCl
Add solution B to solution A till the pH reaches 7.4. Take 1000 mL of the phosphate buffer pH 7.4. Now add 13.15 gm NaCl to it. Make up the total volume to 1500 mL with remaining Phosphate buffer pH 7.4. Filter it through 0.2μ filter. Remove 15 ml of the PBS, and add 15 mL of Tween20 to it. For diluting it to 1X, take 100 mL of the PBST and add it to 900 mL purified water/ Milli Q Water.