Principles, protocols, methods and kits
At Assay Genie, we've created a comprehensive guide that describes everything you need to know about ELISA assays!
Jump to a section:
- - The ELISA type
- - ELISA sample preparation
- - ELISA protocols
- - The ELISA test plate
- - Precoat vs. DIY ELISA
*Auteur: John McFerraridoctorate
What is the ELISA test?
ELISA (Enzyme-Linked Immunosorbent Assay) is an antibody-based technique for measuring the levels of proteins, peptides, hormones or chemicals in biological samples.
In a sandwich ELISA assay, a capture antibody is immobilized on the surface of a 96-well plate, followed by the addition of a sample containing the analyte of interest and a complex formed between the antibody and the sample.
After an incubation step, the well is washed using a wash buffer to remove unbound analyte. Detection occurs after addition of a detection antibody conjugated to an enzyme. After incubation, another washing step is performed to remove excess antibody and non-specifically bound proteins.
Substrate is then added and a colorimetric change occurs. The amount of analyte in the sample correlates with the increase in intensity of the color change. Finally stop solution is added. Once the protocol is complete, the samples are then analyzed in an ELISA plate reader and a software program is used to graph and calculate the results.
Types of ELISA Assays
There are 6 main types of ELISA assays that researchers use in their daily studies, the most common being the Sandwich & Competitive ELISA assays, followed by ELISpot assays and Indirect ELISA assays. Below you will find an overview of each type of ELISA.
| ELISA-type | Description |
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Sandwich ELISA-assay | Sandwich ELISA tests are the most common form of ELISA and are named for the formation of the capture and detection antibody that interposes the analyte for detection. As mentioned above, Sandwich ELISA tests have their capture antibody immobilized on a polystyrene ELISA plate. The sample is then incubated in the well of the ELISA plate, followed by a washing step. An enzyme-linked detection antibody is then added, followed by further incubation and finally the addition of a substrate and stop solution to measure analyte levels. |
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Competitive ELISA test | Competitive ELISA assays are mainly used for the detection of hormones. In a competitive ELISA, the analyte of interest is immobilized on the polystyrene ELISA plate. Competitive ELISA is named for the process in which the analyte in the sample competes with the immobilized analyte on the plate for the capture antibody. Therefore, higher amounts of analyte in a sample will result in a reduced signal and is the opposite of the graph you see in a Sandwich ELISA assay. |
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ELISpot ELISA-test | An ELISpot assay is commonly used for vaccine development, veterinary research, monocyte/macrophage/dendritic cell characterization. The principle of an ELISpot is similar to a sandwich ELISA, in which a plate is coated with capture antibodies. The cells are then incubated in the ELISA plate for up to 3 hours, which may depend on the application. The cytokines produced by the cells are then bound by the capture antibody immobilized on the ELISA plate. Cells are then washed off the ELISA plate and a detection antibody is added, followed by substrate and stop solution to allow detection of cytokines. |
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FluoroSpot ELISA-assay | A FluoroSpot ELISA assay is identical to an ELISpot ELISA, but instead of using an enzyme-linked detection antibody, the detection antibody is conjugated to a fluorophore to enable detection and analysis. |
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Indirect ELISA-test | In an indirect ELISA assay, the analyte of interest is bound by the primary capture antibody. A secondary antibody is then used to bind the primary antibody. This is similar to the method of a Western blot test. |
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Direct ELISA-test | The direct ELISA technique is a test that uses an enzyme-labeled antibody to bind to an analyte in a solution. Once bound, the enzyme-labeled antibody can react with a substrate to produce a color change, allowing the analyte to be quantified. This eliminates the need for a secondary detection antibody, as the capture antibody performs both functions. |
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Multiplex ELISA-test | A multiplex ELISA allows researchers to analyze multiple analytes in a single sample. This is very useful when researchers want to measure multiple cytokines in a sample at the same time. Multiplex ELISA assays can be performed through multiple formats, including flow cytometry, plate-based multiplex, or using PVDF or nitrocellulose membranes.Learn more! |
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CLIA testing | CLIA tests are similar in principle to sandwich tests, but instead of using a chromogenic substrate to detect samples, CLIA tests rely on chemiluminescence. In a CLIA assay, the detection antibody converts a substrate into light. The amount of photons produced is proportional to the amount of analyte in a sample. To measure the amount of sample in an assay, luminescence is measured in relative light units (RLU) with a photometer. CLIA assays are typically used to detect low concentration analytes (detection limit = 10-21 zeptomol). |
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ELISA in de cel | An In-Cell ELISA is an indirect ELISA technique and is performed using cells that have been plated and grown overnight on a polystyrene ELISA plate. The cells are then fixed, permeable and blocked. Target proteins are then detected using a primary antibody that is enzyme-conjugated or fluorescently labeled. Fluorescently labeled antibodies allow detection by a fluorescent plate reader or a microscope, while enzyme-conjugated secondary antibodies would allow detection by a plate reader. |
Sandwich Test & Competitive ELISA
While there are a number of different types of ELISA assays, the two most common are the Sandwich and the Competitive ELISA. The main differences between these two approaches are described below!
Key Differences of Sandwich and Competitive ELISA
What is an ELISA sandwich?
Sandwich ELISA (Enzyme-Linked ImmunoSorbent Assay) is an antibody-based technique that allows researchers to quantify the amount of protein, hormone or analyte of interest in a sample. Capture and detection antibodies bind to non-overlapping epitopes on the protein to sandwich the protein, hence the name Sandwich ELISA. After the detection antibody is added, a chemical substrate (such as TMB) is added to produce a colorimetric signal that can be read by an ELISA plate reader.
ELISA sandwich PDF
ELISA-sandwichvideo:An overview of a BDNF sandwich ELISA principle and method.
Capture and detect antibody
Detection of single antibodies and capture sandwich ELISA plot:A schematic illustration of a sandwich ELISA where the capture antibody and the detection antibody are bound to the protein of interest.
Sandwich ELISA is a commonly used method to measure the concentration of a specific protein in a sample. This test is based on the binding of two antibodies to the protein of interest, forming a "sandwich" around the protein. The first antibody binds to the protein and the second antibody (conjugated to an enzyme) binds to the first antibody. When an enzyme substrate is added, the Sandwich ELISA can measure the protein concentration in the sample by measuring the amount of enzyme activity.
Sandwich ELISA is a quick and easy way to measure the concentration of a protein in a sample, making it a valuable tool for research and diagnostics. In addition, this assay can be used to measure the concentration of proteins that are difficult to purify or otherwise quantify.
Collect antibodiesare the main components of a sandwich ELISA, a test used to detect and quantify proteins and other biomolecules in a sample. Capture antibodies bind to specific targets on the surface of the analyte molecule and capture it for detection by a detection antibody.
Detection antibodiesact as indicators of the presence or amount of target proteins in a sample. They are often linked to enzymes that can produce a detectable signal, such as a change in light or color, when they bind to their targets.
Antigen binding
Sandwich ELISA is a popular method for detecting antigens in samples. The principle behind this test is that two antibodies are used to capture and detect the antigen of interest. The first antibody is used to capture the antigen from the sample and the second antibody is used to detect the bound antigen. This technique is very sensitive and can be used to detect very low levels of antigens in a sample. Sandwich ELISAs are commonly used to detect proteins, viruses and other macromolecules in various samples.
Sandwich versus competitive ELISA
Sandwich ELISA and competitive ELISA are two popular methods for measuring the concentration of a specific analyte in a sample. Both methods use antibodies to bind to the analyte, but there are some key differences in how these procedures are performed.
| ELISA-type | Description |
| ELISA-sandwich | The sandwich ELISA uses two different antibodies, one of which is attached to a solid surface. The analyte is then added to the mixture and allowed to bind to the surface-bound antibody. Then a second antibody is added, which is conjugated to a detectable marker. This second antibody binds to the analyte and the signal from the indicator is used to determine the concentration of the analyte in the sample. |
| Competitive ELISA | Competitive ELISA, on the other hand, uses a single antibody conjugated to a detectable marker. The analyte and a known amount of "competing" analyte are added to the mixture. The competitor analyte competes with the sample analyte for binding to the antibody. The signal from the indicator is then used to determine the amount of analyte in the sample. |
So, what are the main differences between these two methods? The sandwich ELISA is more sensitive than the competitive ELISA, which means it can detect lower analyte concentrations. The sandwich ELISA is also less subject to interference from other substances in the sample, making it a more specific method. Competitive ELISA is faster and easier to perform than sandwich ELISA, making it a more convenient choice in some cases.
Antibody sandwich ELISA
The antibodies used to generate a sandwich ELISA can be polyclonal or monoclonal, depending on the specificity, sensitivity, and analyte being detected.
> Polyclonal antibodies
Polyclonal antibodies are often used to remove as much analyte from a sample as possible. Polyclonal antibodies can bind to multiple aspects of an epitope, providing greater capture capability for detecting proteins of interest.
> Monoclonal antibodies
Monoclonal antibodies allow researchers to pull down a single antigen. Therefore, let researchers distinguish subtle differences in proteins. Monoclonal antibodies also provide more data consistency than polyclonal antibodies.
Benefits of an ELISA sandwich
| Benefit | Description |
| No sample cleanup is required | Sandwich ELISA tests allow the measurement of proteins/analytes in complex samples without the need for purification. |
| High specialty | Since capture and detection antibodies are used, a sandwich ELISA has increased sensitivity over a direct or indirect ELISA assay. |
| Quantification | Compared to other EIA methods such as Western Blot, the Sandwich ELISA test allows researchers to quantify the amount of protein in a sample. |
What is Competitive ELISA?
Competitive ELISA assays are used by researchers to determine the amount of analyte in their sample and changes in the level of the analyte under different conditions.Competitive ELISA assays allow researchers to efficiently, quantitatively and inexpensively measure a range of metabolites, hormones and proteins in a sample. Similar to a sandwich ELISA, a competitive ELISA uses antibodies to determine the amount of analyte in a sample and uses an enzyme-linked secondary antibody to quantify the amount of analyte in a sample.
Figure 1:Schematic principle of a competitive ELISA, according to which the primary antibody has bound the analyte placed on the ELISA plate. The secondary enzyme-linked detection antibody has bound the capture antibody.
The competitive ELISA is an excellent tool for measuring key analytes including universal hormones and signaling molecules such as cortisol, T3, hydroxyproline, serotonin. Because many researchers study these types of molecules, competitive ELISA offers a cheap, efficient and fast way to measure these analytes without the high cost of expensive machines.
Competitive ELISA Advantages and Disadvantages
| Advantages | Cons |
| Measure your analyte in composite samples:An important advantage offered by a competitive ELISA is the ability to measure analytes of interest in complex biological samples. Since the most important step in a competitive ELISA is to mix the capture antibody with your sample, this ensures efficient formation of the capture analyte complex. | Negative controls can produce positive results:If the wells are not properly blocked, antigens or secondary antibodies may non-specifically bind to the well. |
| The signal is inversely proportional to the amount of antigen in a sample:This means that for samples with high antigen concentrations, a weaker signal is produced, affecting the range of the assay. Sample dilution may be required to measure samples over a significant range. |
Competitive ELISA standard curve
Figure 2:Competitive ELISA standard curve. The amount of analyte in a sample is inverse to the absorbance values due to the binding of the antigen to the captured antibody in the complexing step.
Competitive ELISAs are based on the principle that the capture antibody competes for an antigen in a sample. First, an unlabeled capture antibody is incubated with a sample containing the antigen of interest. This leads to the formation of an antibody-antigen complex. Excess antibody is added to the mixture during this step, leaving free antibody after the incubation step.
The next step is to add the antibody-antigen complex to an ELISA plate pre-coated with the antigen of interest (blocking antigen). The free antibody remaining from the initial complexing step is then able to bind the inhibitory antigen on the plate, while the pre-formed antibody-antibody complexes are washed away during the washing steps.
An enzyme-linked detection antibody is then added to the plate, which binds the primary antibody bound to the inhibitory antigen. Finally, a substrate is added and a colorimetric change occurs, where the intensity of the signal is inversely proportional to the amount of antigen in the sample.
ELISA sample preparation
When performing an ELISA assay, it is important to prepare your samples to achieve the best possible results. Below we have a list of sample preparation procedures for different types of samples.
| Kind of sample | Protocol |
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Serum | If using serum separator tubes, allow the samples to solidify at room temperature for 30 minutes. Centrifuge at 1000 x g for 10 minutes. Collect the serum aliquot and analyze immediately or store the samples at -80°C. Avoid multiple freeze and thaw cycles. If serum separator tubes are not used, allow samples to solidify overnight at 2-8°C. Centrifuge at 1000 x g for 10 minutes. Remove serum and analyze immediately or store samples at -80°C. Avoid multiple freeze and thaw cycles. |
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Blood plasm | Collect plasma with EDTA or heparin as anticoagulant. Centrifuge samples at 4°C for 15 min at 1000 × g within 30 min of collection. Collect the plasma fraction and analyze immediately or store the samples at -80°C. Avoid multiple freeze and thaw cycles.Remark:Ultraperfused specimens are not suitable for use with this kit. |
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Urine and cerebrospinal fluid | Collect urine (mid-stream) in a sterile container, centrifuge at 2000-3000 rpm for 20 minutes. Remove supernatant and taste immediately. If sediment is detected, repeat centrifugation step. A similar protocol can be used for CSF. |
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Cell culture supernatant | Collect the cell culture media by pipetting followed by centrifugation at 4°C for 20 min at 1500 rpm. Collect the clear supernatant and test immediately. |
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Cellysate | Solubilize cells in lysis buffer and let sit on ice for 30 min. Centrifuge the tubes at 14,000 x g for 5 minutes to remove insoluble material. Separate the supernatant into a new tube and discard the remaining whole cell extract. Quantify the total protein concentration using a total protein assay. Test immediately or aliquot and store at ≤ -20°C. |
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The tissue is homogenized | The preparation of tissue homogenates will vary depending on the type of tissue. Rinse the tissue with 1X PBS to remove excess blood and homogenize in 20 ml 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. It takes two cycles of freezing and thawing to break the cell membranes. To further disrupt cell membranes, samples can be sonicated. Centrifuge homogenize for 5 minutes at 5000xg. Discard supernatant and analyze immediately or aliquot and store at -20°C or -80°C. |
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Tissue waste | Rinse the tissue with PBS, cut into 1-2 mm pieces and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse the tissues for 30 minutes at room temperature with gentle agitation. Centrifuge to remove dirt. Quantify the total protein concentration using a total protein assay. Test immediately or aliquot and store at ≤ -20°C. |
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Breastmilk | Collect milk samples and centrifuge at 10,000 x g for 60 minutes at 4°C. Dispense the supernatant and taste. Store samples at -80°C for extended use. Minimize freeze/thaw cycles. |
ELISA protocols
It is important to follow the correct ELISA protocol when performing an ELISA to obtain precise and accurate results. In Assay Genie, we have grouped our protocols in one convenient place. Click on a protocol to expand the information!
Sandwich ELISA-protocol
| Step | Procedure |
| 1. | Place the standard, test sample, and control (zero) wells, respectively, on the pre-coated plate and note their positions. It is recommended to measure each standard and sample in duplicate. Wash the plate 2 times before adding standards, sample and control (zero) wells! |
| 2. | Dispense 0.1 ml of standard solutions into the standard wells. |
| 3. | Add 0.1 ml of sample buffer/standard dilution to the control well (zero). |
| 4. | Add 0.1 ml of an appropriately diluted sample (human serum, plasma, tissue homogenates, and other biological fluids) to the sample wells. |
| 5. | Seal the plate with a cover slip and incubate for 90 minutes at 37 °C. |
| 6. | Remove the lid and discard the contents of the plate, tapping the plate on absorbent filters or other absorbent material. Do not allow the pits to dry completely at any time. Washing machine plate X2. |
| 7. | Add 0.1 ml of Biotin Detection Antibody Working Solution to the above wells (standard, test sample, and blank wells). Add the solution to the bottom of each well without touching the side wall. |
| 8. | Seal the plate with a coverslip and incubate for 60 min at 37°C. |
| 9. | Remove the coverslip and wash the plate 3 times with wash buffer. Allow the wash buffer to rest in wells for 1 minute between each wash. |
| 10. | Add 0.1 ml SABC working solution to each well, cover the plate and incubate for 30 minutes at 37°C. |
| 11. | Remove the coverslip and wash the plate with wash buffer 5 times, allowing the wash buffer to remain in the wells for 1-2 min each time. |
| 12. | Add 90 µl of TMB substrate to each well, cover the plate and incubate at 37°C in the dark for 15-30 min. (Note: This incubation time is for reference only, the optimal time should be determined by the end user. ) And shades of blue can be seen in the first 3-4 wells (with the most concentrated standard solutions), the other wells show no apparent color. |
| 13. | Add 50 µl of Stop solution to each well and mix well. The color immediately changes to yellow. |
| 14. | Read the O.D. absorbance at 450 nm in a microplate reader immediately after addition of the termination solution. |
Figure 3:Sandwich ELISA protocol for precoated ELISA plate. Step-by-step diagram of the steps involved in a sandwich ELISA assay. Prepare standards first, followed by addition of samples to the ELISA plate and incubation. Once incubated, wash the plate followed by the addition of labeled antibody and incubate. After incubation, wash the plate and add SABC working solution. Wash the plate and add TMB substrate followed by incubation. Finally, add the stop solution and measure.
DIY SandwichELISA-protocol
Cover plate with capture antibody
| Step | Procedure |
| 1. | Add 100 µl of diluted Capture Antibody to each well. |
| 2. | Cover with a plastic plate lid and incubate overnight at 4°C. |
| 3. | Remove the lid and wash the bowl as follows: |
| 4. | Add 100 µl of blocking buffer to each well. |
| 5. | Cover with a plastic plate and incubate for 2 hours at room temperature (18 to 25°C). |
| 6. | Remove the lid and wash the bowl as follows: |
| 7. | For immediate use of the plate(s), proceed to the next section. To store the coated and blocked plates for future use, dry each plate on a bench at room temperature (18 to 25°C) for 24 hours. It is then stored for up to 12 months at 2-8°C in a sealed plastic bag with a desiccant. |
Protocol
| Step | Procedure |
| 1. | Prepare the standard curve. |
| 2. | Add 100 µl of each standard, sample, blank (standard dilution buffer) in duplicate to appropriate wells. |
| 3. | Add 50 µl of diluted detection antibody to all wells. |
| 4. | Cover with a plastic plate and incubate at room temperature (18 to 25°C) for 1 hour. |
| 5. | Remove the lid and wash the bowl as follows: |
| 6. | Add 100 µl of Streptavidin-HRP solution to all wells. |
| 7. | Cover with a plastic plate and incubate for 30 minutes at room temperature (18 to 25°C). |
| 8. | Repeat washing step 5. |
| 9. | Add 100 µl ready-to-use TMB substrate solution to all wells. |
| 10. | Incubate in the dark for 5-15 minutes* at room temperature. Avoid direct exposure to light by wrapping the plate in aluminum foil. |
| 11. | Add 100µl Stop Reagent to all wells. |
| 12. | Read the absorbance value of each well (immediately after step 11) in a spectrophotometer using 450 nm as the primary wavelength and optionally 630 nm as the reference wavelength (610 nm to 650 nm are acceptable). |
Schematic
Image 4:Sandwich ELISA protocol for ELISA kit development. Step-by-step diagram of the steps involved in a sandwich ELISA assay.
Competitive ELISA protocol
The following protocol is an example protocol for competitive ELISA kits. Competitive ELISA kits allow the detection and quantification of an analyte in a sample. This can be very useful when looking for increases in protein concentration, protein phosphorylation or decreases in protein concentrations.
| Step | Procedure |
| 1. | Place the standard, test sample, and control (zero) wells, respectively, on the pre-coated plate and note their positions. It is recommended to measure each standard and sample in duplicate. Wash the plate 2 times before adding standard, sample and control (zero) wells. |
| 2. | Addition of Sample and Biotin Detection Antibody: Add 50 µL of standard, blank (sample/standard dilution buffer), or sample per well. The blank well is spiked with sample/standard dilution buffer. Immediately add 50 µL of Biotin Detection Antibody Working Solution to each well. Cover with the dishwasher we provide. Gently tap the plate to ensure complete mixing. Incubate for 45 min at 37°C. (Solutions are added to the bottom of the micro-ELISA plate, avoiding contact with the plate walls and foaming). |
| 3. | Wash: Aspirate each well and wash, repeat process x3 as directed. |
| 4. | HRP Streptavidin Conjugate (SABC): Add 100 µL of SABC Working Solution to each well. Cover with new plate sealant. Incubate for 30 min at 37°C. |
| 5. | Washing: Repeat the suction/washing process x5 times as directed. |
| 6. | TMB substrate: Add 90 µL of TMB substrate to each well. Cover with a new dish sealer. Incubate at 37°C for approximately 15-20 minutes. Protect from light. The reaction time can be shortened or extended depending on the actual color change, but not more than 30 minutes. When an apparent gradient occurs in standard wells, you can terminate the reaction. |
| 7. | Stop: Add 50 µL of stop solution to each well. The pits immediately turn yellow. The order of addition of the stop solution should be the same as the substrate solution. |
| 8. | OD measurement: Simultaneously determine the optical density (OD value) of each well using a microplate reader set at 450 nm. You need to open the microplate reader from the front, preheat the instrument and set the test parameters. |
Schematic
Image 5:Step-by-step scheme for the competitive ELISA protocol.
Direct ELISA-protocol
The direct ELISA technique is a test that uses an enzyme-labeled antibody to bind to an analyte in a solution. Once bound, the enzyme-labeled antibody can react with a substrate to produce a color change, allowing the analyte to be quantified.
| Step | Procedure |
| 1. | Coat the microtiter plate with antigen/analyte. |
| 2. | Cover the plate and incubate overnight at 4°C. |
| 3. | Wash the plate three times with 300 µl of wash buffer. |
| 4. | Add blocking buffer and incubate for 1 hour at 37°C. |
| 5. | Wash four times with 300 µl wash buffer. |
| 6. | Add samples and standards to the selected wells at the appropriate concentrations. |
| 7. | Incubate for 90 min at 37°C or overnight at 4°C. |
| 8. | Wash three times with 300 l wash buffer. |
| 9. | Add biotin-conjugated streptavidin to the wash buffer. |
| 10. | Incubate for 1 hour at 37°C. |
| 11. | Wash three times with wash buffer. |
| 12. | Add the substrate solution to the selected wells. |
| 13. | Incubate at room temperature until the desired color change is observed. |
| 14. | Add stop solution. |
| 15. | Read the absorbance values. |
Indirect ELISA-protocol
An indirect ELISA is similar to a Western Blot, where a secondary antibody binds to a primary antibody.
| Step | Procedure |
| 1. | Coat the microtiter plate with antigen/analyte. |
| 2. | Cover the plate and incubate overnight at 4°C. |
| 3. | Wash three times with 300 µl wash buffer. |
| 4. | Add blocking buffer and incubate for 1 hour at 37°C. |
| 5. | Wash four times with 300 µl wash buffer. |
| 6. | Add samples and standards to the selected wells at the appropriate concentrations. |
| 7. | Incubate for 90 min at 37°C or overnight at 4°C. |
| 8. | Wash three times with 300 µl wash buffer. |
| 9. | Add the detection antibody in wash buffer to the selected wells. |
| 10. | Incubate for 1 hour at 37°C. |
| 11. | Wash three times with wash buffer. |
| 12. | Add the substrate solution to the selected wells. |
| 13. | Incubate at room temperature until the desired color change is observed. |
| 14. | Add stop solution. |
| 15. | Read the absorbance values. |
Popular ELISA-kits
- HIF-1 alpha ELISA-kit
- Aflatoxine M1 ELISA-kit
- Catalase ELISA-kit
- GDNF ELISA-kit
- Insulin ELISA-kit
- Human Leptin ELISA Kit
- Human TAC1/Dust P ELISA
- Human IGFBP3 ELISA Kit
- Human HMGB1 ELISA kit
- Mouse ALT ELISA kit
- Muis HGF ELISA-kit
- Muis IL-18 ELISA-kit
- Human P1NP ELISA Kit
- Estradiol ELISA kit for rats
- Rat TGF beta ELISA Kit
- Human BDNF ELISA Kit
- Human IL-10 ELISA Kit
- Human IL-6 ELISA Kit
Immunoassay versus EIA
An immunoassay is a test that uses antibodies to detect an antigen of interest. An EIA (Enzyme Immunoassay) is a test in which an enzyme is conjugated to a detected antibody to enable detection and quantification.
Two examples of EIAs are a Western Blot and an ELISA. Western Blot is an EIA that used nitrocellulose or PVDF to immobilize proteins. This is followed by the addition of a primary antibody to bind the protein of interest, followed by incubation with an enzyme-conjugated secondary antibody to detect the analyte of interest. In an ELISA assay, the capture antibody is immobilized on a polystyrene plate, followed by incubation of a sample containing the analyte of interest, followed by detection with a detection antibody and colorimetric change with TMB as substrate.
Image 6:An example of detection using an EIA format such as Western Blot vs Sandwich ELISA format for capturing and analyzing targets.
ELISA test plate
ELISA assays are performed in 96 or 384 well polystyrene plates. ELISA plate charts, plate standards, plate readers, and plate washing are all important considerations when performing an ELISA assay. Below we take a closer look at each of these considerations.
ELISA plates
Proteins and antibodies can be immobilized on the 96-well ELISA plate after incubation. In a sandwich ELISA assay, a capture antibody is immobilized on the ELISA plate. However, in a competitive ELISA, the analyte of interest is bound to the ELISA plate. The ELISA plate is then blocked with BSA blocking solution to prevent binding of non-specific proteins during the ELISA assay.
The binding of antibodies or analytes to the plates allows washing steps with a wash buffer and thus the removal of non-specifically bound analytes.
ELISA plates have a flat bottom to allow the plate reader to read the absorbance on an ELISA plate reader at 450 nm.
Image 7:Cartoon of a 96-well polystyrene ELISA plate
ELISA plate card
When performing an ELISA assay, it is important to plan where to place your standards, blanks, controls, and samples on the ELISA plate to analyze your results. Each 96-well ELISA plate allows researchers to analyze up to 40 samples simultaneously in an ELISA plate reader.
Since most Sandwich & Competitive ELISA kits have a 7-point dilution of their standard, 14 wells are absorbed according to the standards in the ELISA plate. In addition to the standards, it is also necessary to have a blank that is just a sample/standard dilution buffer. Researchers can also use additional wells for positive, negative, or recovery control pins on the plate.
Figure 8:ELISA plate chart for setting up an assay. Purple circles = Standards, Gray circles = Samples, White circles = Blanks.
ELISA Plate Standards
ELISA standards allow researchers to determine the amount of analyte in their sample by projecting absorbance measurements of the samples onto known concentrations of standards, allowing researchers to quantify the amounts of analyte. In an ELISA kit, researchers receive a stock standard solution that is diluted by the sample/standard dilution buffer.
ELISA standards allow researchers to determine the amount of analyte in their sample by projecting absorbance measurements of the samples onto known concentrations of standards, allowing researchers to quantify the amounts of analyte. In an ELISA kit, researchers receive a stock standard solution that is diluted by the sample/standard dilution buffer.
Image 9:7-point serial dilution of the stock standard using standard dilution buffer.
Standard dilution instructions
*Remark:Refer to the data sheet provided with the kit for detailed sample dilution instructions.
Dilute each standard vial supplied with 1 ml (volume depends on kit) of Standard Dilution Buffer to make the standard stock solution. Keep the tube at room temperature for 10 minutes and mix well.
To make the standard series, label 6 microcentrifuge tubes and dispense 300 µl of the Sample Buffer/Standard Dilution into each tube. Add 300 µl of the standard stock solution to the 1st tube and mix well. Transfer 300 µl from the 1st tube to the 2nd tube and mix well. Repeat this dilution process until the standard run is complete (see Figure 1 above for details).
ELISA plate readers
What is an ELISA microplate reader?
A microplate reader is an instrument used by researchers to detect and analyze a range of biochemical changes. An ELISA plate reader can read and analyze multiple wells simultaneously over a range of wavelengths. The ELISA plate reader allows researchers to improve throughput and save time and operating costs, while automating data analysis for a range of applications.
How does an ELISA plate reader work?
An ELISA plate reader works by detecting light at a specific wavelength. For the final steps of the ELISA assay, TMB (3,3',5,5'-tetramethylbenzidine) is converted to a blue colored solution by HRP and finally converted to a yellow solution upon addition of Termination Solution. The amount of color in the well is proportional to the amount of analyte present in a sample.
Once the plate is placed in the ELISA plate reader, the absorbance of each sample is read at 450 nm to determine the amount of analyte in each well. Researchers can then plot their sample value against their known standard curve values to estimate the amount of analyte they have in each sample.
96-well ELISA plate readers
When performing an ELISA assay, you run your assay in a 96-well plate to measure the amount of analyte in each sample. Your plate layout consists of a standard 8-point curve and double spaces. This will occupy 20 wells in the 96 well ELISA plate. The investigator will measure 38 samples in duplicate in the 96-well ELISA plate.
There are a number of ELISA plate readers on the market that will allow you to measure the ELISA assay, including the SPECTROstar Nano from BMG LABTECH, as well as any plate reader from Bio-Rad, Thermo, Tecan, Molecular Devices, or Biotech. Each platform has its own dedicated plate reading software that helps researchers interpret their data.
| Company | Plate reader |
|
BMG Labtech |
SPECTROSTAR nano |
|
Biotech |
PowerWave HT microplaat spectrofotometer |
|
BYONOY |
ABSORPTION 96 READER PLATE |
|
Bio-Rad |
xMark™ microplaatabsorptiespectrofotometer |
Plate Reader settings for ELISA measurements
Depending on the ELISA plate reader you have, the settings may differ. However, the following settings may apply when reading your sample.
| Type | Institutions |
|
Optical settings |
Absorption spectrum, endpoint test |
|
General setting |
Number of flashes: 45 |
Wash ELISA plates
| ELISA-type | Description |
|
TMB response | To measure the performance of an ELISA experiment, different substrates can be used to detect the final bound antibody-antigen complex. Colorimetric, chemiluminescence and fluorescence testing are the three main technologies. Typically, ELISA refers to the colorimetric measurement of samples, where TMB (3,3',5,5'-tetramethylbenzidine) acts as a hydrogen donor to reduce hydrogen peroxide in water in the presence of horseradish peroxidase. This reaction results in the production of a diimine that changes the color of the solution to blue. This color change to blue can be read in a spectrophotometer at 370 & 650 nm. To stop this reaction, sulfuric acids are added to the sample and this results in a yellow color change. The ELISA plate can then be analyzed using a spectrophotometer and read at 450 nm. |
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ELISA wasstappen | An important step in the ELISA protocol are the washing steps. The washing steps are critical to reduce background signal, which may be due to unbound, conjugated antibody resulting in an increase in signal-to-noise ratio. Therefore, washing steps ensure that only high-fidelity binding interactions between antigen and antibody appear. When inefficient washing occurs, it can result in high background levels, which can hinder data collection, result in large variability between samples, ultimately resulting in poor results. |
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ELISA volumes and wash parameters | The volume of wash buffer added to the ELISA plate is key to effectively washing your plate. Depending on whether you are using an ELISA plate washer or a multichannel pipettor, you need to consider the volume required when washing your plate. The volume of wash buffer should be greater than the volume of coating buffer added to each well. For example, if your plate is coated with 100 µL of coating buffer, you will need to add a larger volume of wash buffer to ensure complete washing of the well, for example 200 – 350 µL. |
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ELISA plate wash cycles | After the wash volume, the number of wash cycles is important to increase background removal, but also to avoid unnecessary washing of bound antigen-analyte. Washing the samples too much can reduce signal strength and make it difficult to measure and analyze your data. Depending on where you are in the protocol, 1-3-5 wash steps may be required. In general, pre-coated plates require fewer washing steps than do-it-yourself coated plates, but this may depend on the manufacturer and the technology used to read the ELISA plate. |
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Aspiration of buffers when washing ELISA plates - manual | Aspiration of buffers during an ELISA protocol is key to removing residual buffer, unwanted complexes or antibodies. If you are using a multichannel pipette, insert new pipette tips into the multichannel pipette between steps. While aspirating the buffer, place the multichannel into the well at a title angle, taking care not to touch the side or bottom of the wells. After removing the buffer, you can turn the plate upside down and tap the plate on a paper towel to remove any remaining buffer. Do not allow the plate to dry between washing and conditioning steps. |
ELISA sensitivity and range
An ELISA assay allows researchers to determine the amount of analyte they have in their sample (serum, plasma, supernatant, milk, urine) within a certain range using a set of known standards. When performing an ELISA assay, a known concentration of analyte is used as a reference point for the amount of analyte in a sample. These are called standards.
During an ELISA assay, a stock of the standard is provided, usually in ng/ml or pg/ml, this stock is then diluted 6-7 times to provide a range of known concentrations of an analyte in a volume. Plotting values creates a standard curve and calculates unknown sample concentrations against these values.
Detection strategies for ELISA
The detection step in an ELISA is the last step in measuring the amount of analyte you have in your sample. The signal produced during the detection step is proportional to the amount of one. There are three options for detecting analytes in an ELISA. radioactive (Radioimmunoassay) or fluorescent labels or using a chromogenic substrate.
Chromogen is the most popular and widely used technique for ELISA detection and includes the substrate TMB (3,3',5,5'-tetramethylbenzidine) which oxidizes and turns yellow upon addition of sulfuric acid. Allowing samples to be read at 450 nm in an ELISA plate reader.
Other detection methods such as chemiluminescence can be used based on HRP with Luminol as a substrate that emits light at 425 nm.
Pre-coated ELISA & DIY ELISA
At Assay Genie, we offer a range of ready-to-use pre-coated ELISA kits and ELISA development kits for the detection of cytokines, chemokines, hormones, signaling proteins and thousands of other biomarkers.
| Characteristics | DIY-ELISA | Precoated ELISA | Multiplex-ELISA |
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Read out | HRP-TMB | HRP-TMB | EP/APC |
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Incubation period | 2-3 hours | 2-3 hours | two o'clock |
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Sensitivity | pg/mL | pg/mL | pg/mL |
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Number of targets per pit | 1 | 1 | 24 |
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Instruments required | ELISA plate reader | ELISA plate reader | Flowcytometer |
PharmaGenie High Sensitivity ELISA Kit
PharmaGenie ELISA Kits from Assay Genie are high quality ELISA kits designed to meet the needs of pharmaceutical and biotechnology research. Focusing on high quality monoclonal antibody pairs and reagents validated under ISO 9001:2000 quality systems, PharmaGenie ELISA kits are exceptional tests that help discover our future.
Verken PharmaGenie
- Accuracy and reliability are guaranteed as all our reagents are validated under ISO 9001:2000 quality systems
- It recognizes the specificity of both native and recombinant antigen
- No cross-reactivity with other human cytokines has been tested
- Standard calibration at NIBSC
- Development ELISA kit (antibody pairs)
- Antibody pairs enable research
Development of ELISA kits (antibody pairs)
Development ELISA kits enable researchers to create their own ELISA plates. ELISA development kits are supplied with antibody pairs (matched capture and detection antibodies) and buffers. This allows researchers to coat and plate their own ELISA kits. ELISA Genie offers a range of high quality ELISA development kits called ELISA SuperSet kits.
Discover the DIY ELISA
- Highly optimized pair of monoclonal antibodies.
- Purified high quality recombinant protein to generate stable standard curves.
- The reagents have been validated according to ISO 9001:2000 quality systems.
- Native and recombinant antigen specificity.
- No cross-reactivity with other cytokines tested.
- Standard calibration at NIBSC.
- ELISA kits were developed with the pharmaceutical and biotechnology research sectors in mind.
- Different kit sizes for efficient research!
Types of antibodies for ELISA
ELISA kits are created using monoclonal or polyclonal antibodies to detect and capture antibodies. Monoclonal agents offer the advantage of recognizing a single epitope, providing an accurate analysis of a specific antigen. However, polyclonals have the advantage of binding larger amounts of antigen. Recently, recombinant monoclonal antibodies have been used to generate ELISA kits, providing increased specificity and consistency.
The PharmaGenie Assay Genie ELISA kit line is manufactured using high quality monoclonal antibodies against major cytokine targets including IL-1 beta, IFN gamma, IL-2, IL-4, IL-6, CXCL8/IL-8 and many other targets .
ELISA Buffers and Recipes
What is a block buffer?
To prevent non-specific binding of proteins to an ELISA test plate, blocking buffers are used to coat a plate. The binding capacity of an ELISA plate is greater than the amount of protein (Capture Antibody/Antigen) coated on the plate. Therefore, the remaining region must be blocked to prevent non-specific binding of antibodies or other proteins during subsequent incubation steps. Therefore, a blocking buffer is used using a protein that will not bind or form complexes with other proteins or detection antibodies in subsequent steps. Blocking buffer therefore increases ELISA sensitivity as it prevents binding of non-specific proteins, reduces background noise and thereby increases the signal-to-noise ratio.
During ELISA development, different blocking buffers should be tested to optimize the assay and improve the signal-to-noise ratio. Factors that can influence non-specific protein binding are the formation of protein:protein interactions. When optimizing a buffer block, the key is optimizing for an increased signal-to-noise ratio, which is determined by using a control well without addition of the target analyte sample.
When optimizing buffer blocking, it is also important not to use excessive concentrations of inhibitor, which can inhibit antibody-antigen interactions or potentially inhibit enzyme activity, reducing the signal-to-noise ratio. When optimizing for blocking, different buffers can be tried to optimize for the best signal.
| Recipe for blocking buffers |
| Phosphate Buffered Saline (PBS) |
| 1% BSA |
Coatingbuffer
An ELISA coating buffer is used to immobilize proteins/analytes or antibodies on microtiter plates. Key factors in the immobilization of analytes/antibodies on microplates may be the pH of the coating buffer. Choosing a coating buffer between pH 7.4 and pH 9.6 can affect the steric structure of the protein/antibody/analyte binding and thus affect their immobilization. Testing of coating buffers can help increase the mobility and yield of immobilized antibodies. In SuperSet Development ELISA Kits for one plate, add 100 µl Capture Antibody to 10 ml Coating Buffer.
| Coating Buffer Recept (1L) | amounts of ingredients |
| Na2CO2 | 1,5 gr |
| NaHC03 | 2,93 gr |
| Distilled water | 1L (pH 9,6) |
TMB
In the presence of conjugated HRP (horseradish peroxidase) enzymes, TMB and peroxide react to form a blue by-product with an absorption maximum at 605 nm. The intensity of the color produced by the HRP activity is proportional to the levels of the analyte in the ELISA assay. After incubation with TMB (3,3',5,5'-tetramethylbenzidine), 0.16 M sulfuric acid stop solution is added to stop the reaction. The amount of stop solution added should be equal to the amount of TMB substrate added to each well of the ELISA plate (typically 50 – 100 µL per well). After addition of sulfuric acid stop solution. The color changes from blue to yellow, which stabilizes the color development and allows accurate measurement of the intensity at 450 nm using a spectrophotometer.
PNPP (p-nitrofenylfosfaat)
pNPP is a chromogenic substrate for alkaline phosphatases. pNPP for use with alkaline phosphate-conjugated antibodies. Alkaline phosphatase catalyzes the hydrolysis of pNPP to pNP. This results in the production of a yellow phenolic salt with an absorption maximum at 405 nm. pNNP is sensitive to light and must therefore be protected.
Wasbuffer
The goal of an ELISA wash is to remove all signal altering residues and to preserve the ELISA components. ELISA plates are washed before adding standards and samples, after addition of detection antibody and after addition of HRP-conjugated antibody. Both Tris-based and PBS-based wash buffers can be used in ELISA protocols.
| PBS wash buffer recipe |
| Phosphate Buffered Saline (PBS) |
| 0,05% V/V TWEEN-20 |
| Recipe for Tris-Based Wash Buffer (1L) |
| 6,06 g Tris-basis |
| 8.2 g NaCl |
| 6,0 ml 6 Μ HCL |
| 1 liter of distilled water |
| pH should be 7.2 to 7.8, conductivity should be 14,000 to 16,000 |
| Washing solution recipe (1L) |
| 1 liter TBS |
| 5ml Tween 20 10%. |
ELISA data analysis
After completion of the ELISA protocol, the next step is to obtain and analyze the data using an ELISA plate reader.
When performing an ELISA, it is recommended that you run your samples in duplicate or triplicate to ensure statistical significance of your results. In addition, positive controls such as standards, known positive controls, and negative controls such as blanks or samples without the antigen of interest may be required.
- Negative Controls: Samples without the presence of your analyte
- Positive Controls: Samples with a known presence or amount of your analyte
Depending on the type of ELISA used (qualitative, semi-quantitative or quantitative) the data output will vary. Therefore, you choose the specific ELISA you want to use based on the data you want to analyze. Data is presented as a plot of optical density (OD) versus log sample concentration. Standards of known concentrations are used to generate a standard curve from which to determine the concentration of an unknown analyte.
coefficient of variation
The coefficient of variation helps identify any inconsistencies and inaccuracies in the results. It is expressed as a percentage variation from the mean. The greater the variation, the greater the inconsistency and errors. The coefficient variation (CV) is the ratio of the standard deviation σ to the mean μ:
Cv= s/µ
A high CV can be attributed to one or more of the following:
- Pipette inaccuracies
- Contamination of samples with bacteria/fungi or other reagents
- Temperature fluctuations - plates should be incubated at a constant temperature away from drafts.
- Drying wells - plates should be covered at all stages of incubation
Calculation of results
Calculate using the following equation:
Relative OD450 = (OD450 of each well) - (OD450 of zero well)
The standard curve can be plotted as the relative O.D.450 of each standard solution (Y) against the corresponding concentration of the standard solution (X). The concentration of the samples can be determined from the standard curve. It is recommended to use professional software such as curve expert 1.3.
Note: If the measured samples are diluted, multiply the dilution factor by the concentrations of
interference to obtain the concentration before dilution.
Horseradish peroxidase (HRP) and alkaline phosphatase are the most commonly used enzymes for the detection of sandwich ELISA analytes and provide different options for researchers depending on the application.
| Ingredient | Description |
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p-nitrofenylfosfaat (pNPP) | pNPP is the ALP substrate. After addition of pNPP, incubate the samples at room temperature for 10-30 minutes. Stop the reaction by adding 0.75 M NaOH and reading samples at 405 nm. |
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hydrogen peroxide | Hydrogen peroxide is the substrate for HRP, which allows the color to change during the reaction. |
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TMB (3,3',5,5'-tetramethylbenzidine) | TMB undergoes a color change upon hydrogen peroxide reduction in the presence of HRP. To quench the reaction, sulfuric acid is added and the reaction results in a color change that can be read at 450 nm by an ELISA plate reader. |
Hemolysis & ELISA
Why Does Hemolysis Affect ELISA?
Hemolysis may interfere with an enzyme-linked immunosorbent assay (ELISA) as it may affect the accuracy of the results. Hemolysis is the breakdown of red blood cells, releasing hemoglobin into the sample. Hemoglobin can bind to the detection reagent used in ELISA, causing false positive results.
ELISA works by detecting the presence of a specific target molecule in a sample using antibodies specific for the target molecule. Antibodies are immobilized on a solid surface, such as a microplate, and the sample is added. If the target molecule is present in the sample, it will bind to the antibodies. Then a detection reagent is added, which is also specific for the target molecule. The detection reagent usually consists of an enzyme linked to a second antibody that recognizes a different part of the target molecule. If the target molecule is present, the detection reagent will bind to it and the enzyme will catalyze a reaction that produces a detectable signal.
However, if hemoglobin or other substances released by hemolysis are present in the sample, they may bind to the detection reagent instead of the target molecule. This can lead to a false positive signal, indicating the presence of the target molecule when it is not really there. In addition, hemolysis can lead to the release of other substances, such as enzymes or cytokines, which can interfere with the ELISA reaction or cause non-specific binding.
Therefore, it is important to minimize hemolysis during sample collection and handling for ELISA to ensure accurate results. If hemolysis is present, it may be necessary to repeat the analysis with a new, non-hemolytic sample or to use a different detection method that is unaffected by the interference caused by hemolysis.
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FAQs
What is the principle of ELISA kit? ›
Principle of ELISA
ELISA works on the principle that specific antibodies bind the target antigen and detect the presence and quantity of antigens binding. In order to increase the sensitivity and precision of the assay, the plate must be coated with antibodies with high affinity.
Basic ELISA principles
ELISA (which stands for enzyme-linked immunosorbent assay) is a technique to detect the presence of antigens in biological samples. An ELISA, like other types of immunoassays, relies on antibodies to detect a target antigen using highly specific antibody-antigen interactions.
The Direct ELISA Procedure can be summarised into 4 steps: Plate Coating, Plate Blocking, Antibody Incubation, and Detection.
What are the components of the ELISA kit? ›The standard components in an ELISA kit include antibodies, antigens, dried protein stabilizers and blockers, wash buffers, in-solution protein stabilizers, substrates, stop solutions and sample/assay diluents if needed.
What is the principle of ELISA quizlet? ›The basic principle of an ELISA is to use an enzyme to detect the Ag-Ab binding (antigen- antibody binding). The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag: Ab binding.
What are 3 limitations of ELISA? ›- Inadequate inhibition of immunogenic antigen in ELISA results in false results.
- Refrigerated transport and storage are necessary because antibodies are proteins.
- ELISA is labor-intensive and costly to prepare antibodies.
The standard ELISA reagents used include a detection antigen or antibody, ELISA coating buffer, washing buffer, dried protein stabilizers/blockers, sample or assay diluents should the sample needed to be treated, in-solution stabilizers or conjugate stabilizers, a detection substrate and a stop solution.
How long is ELISA protocol? ›Incubate for 1.5 minutes to 90 minutes at room temperature or 37°C.
What are the 4 types methods of ELISA tests? ›- Direct ELISA. In direct ELISA, only an enzyme-labeled primary antibody is used, meaning that secondary antibodies are not needed. ...
- Indirect ELISA. In indirect ELISA, both a primary antibody and a secondary antibody are used. ...
- Sandwich ELISA. ...
- Competitive ELISA.
ELISAs can be quite complex, including various intervening steps and the ability to measure protein concentrations in heterogeneous samples such as blood. The most complex and varying step in the overall process is detection, where multiple layers of antibodies can be used to amplify signal.
What are examples of ELISA assays? ›
The enzyme-linked immunosorbent assay (ELISA) is an immunological assay commonly used to measure antibodies, antigens, proteins and glycoproteins in biological samples. Some examples include: diagnosis of HIV infection, pregnancy tests, and measurement of cytokines or soluble receptors in cell supernatant or serum.
What are the ordered steps of an ELISA protocol quizlet? ›- Add antigen (sample)
- Add primary antibody.
- Add secondary antibody with enzyme.
- Add substrate.
- Look for color change and amount of color change.
every ELISA will have 2 components. 2) each ELISA has a reporter system. an enzyme reporter system produces a colored product when the appropriate substrate is added. - substrate is added to produce color in the enzymes that are attached to the antibodies that are attached to the antigen on the wells.
What are the basic principles of antibody mediated? ›What basic principles of antibody-mediated immunity are utilized in an ELISA assay? ELISA assays are based on the principles that antibodies are produced in response to infection and that these antibodies are designed to specifically target particular antigens and bind tightly to them.
What are the common errors in ELISA test? ›Some of the most common reasons assay developers might have this problem include insufficient plate washing, conjugate oversaturating the wells, standard curve range is too high, adding too much detection reagent, and not stopping the reaction at the appropriate time.
What is the most controversial aspect of ELISA? ›ELISA results are reported as a number; the most controversial aspect of this test is determining the "cut-off" point between a positive and a negative result. A cut-off point may be determined by comparing it with a known standard.
What is the major advantage of ELISA? ›ELISA exhibits the following advantages: (i) Simple procedure. (ii) High specificity and sensitivity, because of an antigen–antibody reaction. (iii) High efficiency, as simultaneous analyses can be performed without complicated sample pre-treatment.
What equipment is needed for ELISA test? ›Usual equipment includes pipettes, a device for plate washing, an ELISA reader (photometer) and a computer with an evaluation software. Some test kits require further devices, such as an incubator for constant temperature during test implementation. Pipettes are part of the standard equipment of laboratories.
How many samples can you run with an ELISA kit? ›The assay comes in a 96-well format; in addition to the standards, positive control, and negative control, up to 26 samples may be run in triplicate.
What type of ELISA is a Covid test? ›The COVID-19 ELISA IgG Antibody Test consists of two serial direct Enzyme-Linked Immunosorbent Assays (ELISA) for the qualitative detection of human IgG antibodies in serum and plasma specimens collected from individuals suspected of prior infection with the virus that causes COVID-19 by their healthcare provider.
What is the difference between PCR and ELISA? ›
Use ELISA to: • Confirm that herds are free from important pathogens. Assess the progression of a herd through pathogen elimination. Manage herds with endemic disease to reduce the impact of disease on health and production. Use PCR to: • Identify the DNA or RNA of a given disease agent.
Is ELISA a type of PCR? ›PCR-ELISA is an immunological method to quantify the PCR product directly after immobilization of biotinylated DNA on a microplate.
What is ELISA kit for gluten determination? ›Gluten ELISA kit is a procedure intended for analysing quantitative concentrations of gliadin or gluten in foods (such as baby food, bakery products, meat, beer and chocolate). This assay has a minimum analytical sensitivity limit of 0.3 ppm.
What is the principle of enzyme immunoassay? ›The basic principle of ELISA is to use an enzyme to detect the binding of antigen (Ag) or antibody (Ab). Enzymes such as alkaline phosphatase, horseradish peroxidase, lactoperoxidase, and β-galactosidase are used in ELISA.
What is the principle of ELISA test for Covid 19? ›The test kit is an Enzyme-Linked Immunosorbent Assay (ELISA) for the qualitative detection of anti-SARS-CoV-2 total antibodies in human serum specimens. The assay uses recombinant spike protein in a two-step sandwich format. In the first reaction step, patient samples are incubated in the antigen-coated microwells.
What is the principle of dot ELISA in reference to the reaction? ›Dot-enzyme linked immunosorbent assay (Dot - ELISA): The Dot-ELISA depends upon the principle that when test serum is layered on the nitrocellulose membrane bound with the cysticercus antigen, the specific cysticercus antibodies, if present in the serum, will bind to the cysticercus antigen dot.
How ELISA is used for detection of food allergens? ›In ELISA, allergens can be detected by the colorimetric reaction after binding of the antigen with the specific enzyme-labeled antibody. Earlier studies reported the allergenic proteins quantification in peanut-based products through ELISA and HPLC technique (29, 33–35), however, no information on peanut seeds.
How does ELISA test for food sensitivities? ›The Enzyme-Linked Immunosorbent Assay (ELISA) method is the most common and preferred technique in detecting food allergens. ELISA methods detect the allergen protein molecule by binding antibodies to the allergen. The samples are processed and change color to confirm the presence of the allergen protein.
What method is used to determine gluten quality? ›On the other hand, quantitative detection of gluten protein in food is currently performed by immunochemical methodologies. The only commercially available method approved by the FDA and the Codex Alimentarius for gluten detection in food products are immunological tests such as ELISA.
What are the basic principles of immunoassay? ›An immunoassay is based on the selective binding between an antibody and its antigen (analyte). Antibodies are produced as part of the immune response when a foreign agent or antigen is detected. Antibodies are composed of four polypeptide chains: two identical heavy chains and two identical light chains.
What is the difference between immunoassay and ELISA? ›
EIA (Enzyme Immunoassay) also known as ELISA (Enzyme-Linked ImmunoSorbent Assay), detects and measures antibodies in human samples as a reaction to specific antigens. ELISA-tests are also used to test the concentration of Vitamin D, insulin, COVID-19 or hepatitis antigens in samples.
What enzymes are used in ELISA? ›The most commonly used enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Other enzymes have been used as well; these include β-galactosidase, acetylcholinesterase, and catalase.
What is the difference between ELISA and PCR? ›Use ELISA to: • Confirm that herds are free from important pathogens. Assess the progression of a herd through pathogen elimination. Manage herds with endemic disease to reduce the impact of disease on health and production. Use PCR to: • Identify the DNA or RNA of a given disease agent.
Is ELISA an antigen test? ›There are four major types of ELISA: Direct ELISA (antigen-coated plate; screening antibody) Indirect ELISA (antigen-coated plate; screening antigen/antibody) Sandwich ELISA (antibody-coated plate; screening antigen)
Can ELISA test be false positive for Covid? ›Globally, the false-positive HIV ELISA rate was 1.3% [95% confidence interval (95% CI) 0.66–2.22; χ2 = 4.68, P = 0.03, when compared with the expected 0.4% false-positive rate].
What is the difference between ELISA and DOT ELISA? ›The main difference between ELISA and Dot ELISA is that ELISA helps in the detection and quantification of antibodies, hormones, peptides or other proteins in a biological sample whereas, in Dot-ELISA, the chromogenic substrate only precipitates into the area of enzyme activity.
What is the principle of T3 ELISA test? ›TEST PRINCIPLE
The T3 ELISA is a competitive immunoassay. Competition occurs between T3 present in calibrators, controls, specimen samples and an enzyme-labelled antigen (HRP conjugate) for a limited number of anti-T3 antibody binding sites on the microplate wells.