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Biomatik offers an extensive list of over 38,000 high quality ELISA Kits. Most kits can be shipped within 3-11 business days with door-to-door delivery worldwide. Overnight shipping within the US and Canada, 3-4 business days to reach European and most other countries.
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Highlights of ELISA Kits from Biomatik
1. High sensitivity and low detection range: Our lowest detection ranges are in the picogram range (1-100pg/mL, 3-200pg/mL).
2. A wide range of sample types: Serum, plasma, tissue homogenates, cell lysates, cell culture supernatants, and other biological fluids.
3. Enhanced specifications: Some of our kits feature a shortened experimental time, pre-diluted detection reagents, and enhanced stability.
4. Customer support: Thorough technical support - we stand behind our products and assist our customers promptly whenever the need arises.
An enzyme-linked immunosorbent assay also referred to as ELISA, is a highly sensitive, quantitative method used in the laboratory to assist in the detection of a wide range of analytes. This test measures the concentration of antibodies or antigens and detects their interaction with one another. Antigens are molecules with distinct features, which allow them to stimulate specific responses from the immune system. Once stimulated, the immune system releases an antibody specific to the antigen its responding to, acting like a lock and key combination. The ELISA Kit utilized these analytes for fast detection and amazing specificity.
This test can be performed on a number of different sample types, including:
Human, Mouse, Monkey, Porcine, Rat, Bovine, Sheep, Goat, Rabbit, Chicken, Guinea Pig, and Canine
Introduction to ELISA
The enzyme-linked immunosorbent assay (ELISA) is a sensitive immunochemical test involving an enzyme, plus an antibody (or antigen). ELISA kits are used for the detection of proteins, viruses or low molecular weight compounds (for example: hormones, pesticides and toxins) and their concentration.
In an ELISA kit, a polystyrene multiwell plate is coated with a specific antibody (capture antibody) against the sample (analyte or antigens). This immobilized antibody binds a specific amino acid sequence (epitope) on the analyte. This is followed by incubation with a second antibody (detection antibody) which can be both monoclonal origin (from hybridoma cells) as well as polyclonal (from animal serum). If these antibodies are already conjugated to a fluorophore or an enzyme, the detection can be done directly. In the event that the second antibody is uncoupled, a further conjugated antibody is necessary for the detection (indirect ELISA). Thus, a characteristic color change after the addition of a substrate or a monochromatic excitation (in the case of a fluorophore-conjugated antibody) can be achieved.
Understanding The ELISA Assay Types
The ELISA assay formats can also be divided into direct and indirect ELISA test, sandwich ELISA test, and competitive or inhibitory ELISA test.
The ELISA plate is coated with capture antibodies and the remaining surface is blocked with BSA or detergent. The plate is then loaded with a sample in which only the protein of interest binds to the capture antibodies and everything else gets washed away. Next, detection antibodies which are linked to enzymes are added to the plate to bind to existing proteins. Once a colorless substrate (TMB) is added, the enzymes that have found proteins will cause a color change in the substrate. The color intensity is dependent on the concentration of the protein of interest.
The ELISA plate is coated with capture antibodies and the remaining surface is blocked with BSA or detergent. The sample is then mixed with an enzyme conjugated version of the protein of interest. When this mixture is added to the plate, the proteins in the sample will compete with the enzyme conjugates to bind with the limited number of antibodies. After washing away the excess mixture, a colorless substrate is added to the plate (TMB). If the sample had a high amount of the protein of interest, there would be less enzyme conjugate bound to the plate, which would result in a lower color intensity.
The ELISA plate is coated with seed cells and allowed to adhere for fixation. A primary antibody is added and allowed to bind before any excess is washed away. Next, a secondary antibody is added to the plate and allowed to bind before any excess is washed away. The target protein or post-translational modification of the target protein are then quantified with an IR scanner or a spectrophotometer (if using an HRP labeled microplate). Cell staining is an optional step for additional data collection to measure relative cell density.
What are the ELISA Test Types?
ELISA assay is used for determining the concentration of target protein in biological samples. It can yield three types of ELISA data output.
Quantitative: A standard curve can be generated by ODs of serial diluted standard proteins and corresponding known concentrations. ELISA data of samples can be interpolated from standard curve to calculate the concentrations of target proteins in samples.
Qualitative: Simply get a negative or positive result from the assay to determine whether there is any target protein existing in a certain sample by comparing to the negative control.
Semi-Quantitative: With this type of ELISA kit, we are able to get a negative or positive result as well as to compare the target protein levels in assay samples, since the level of samples' absorbance will directly correspond to the level of target protein concentration. However, we can't calculate the exact concentration since there isn't any standard protein in the kit.
Generally speaking, with quantitative ELISA kit, you can quantify target protein in samples since you test a serial of standard proteins of known concentration. When you analyze ELISA data with quantitative ELISA kit, you have to plot the mean absorbance against the protein concentration and draw a curve fitting your standard result best, and then interpolate absorbance of samples to the curve to calculate the concentration. This ELISA standard curve protocol will give you a comprehensive instruction on how to calculate ELISA results with quantitative ELISA kit step by step.
What are The Basic ELISA procedures?
In general, an ELISA is an immunochemical test involving an enzyme, plus an antibody (or antigen). Enzyme-linked immunosorbent assays are used to detect substances that have antigenic properties (e.g., proteins, hormones, bacterial antigens and antibodies). Basically, an ELISA consists of a solid surface, such as a plate containing depressions or “wells,” onto which an antibody having an affinity for a “target” substance is coated. A mixture consisting of: a) purified target substance coupled to an enzyme, and b) the actual test specimen is added to the test system. If no target substance is present in the test specimen, then only the enzyme-linked purified target substance will bind to the antibody coated on the surface of the wells. Therefore, the more “target” present in the test specimen, the less enzyme-linked purified target substance will be bound. A substance (e.g., TMB- Tetramethylbenzidine dihydrochloride) the enzyme acts on is then added, and the amount of the product produced by the enzyme-catalyzed reaction is measured, such as by the change produced in the color of the solution. The resulting color change is indicative of the level of the target substance found in the test specimen.
For example, the test procedure of the mouse Glutathione S-Transferases ELISA Kit would be based on the sequential addition of specimen sample, antibody-enzyme conjugate and substrate to the microassay wells coated with rabbit polyclonal Immunoglobulin (Ig)G antibody directed against rat α945 GST.
Frequent Questions for ELISA Kits
1. How should I store my ELISA Kit?
Components in the kits either need to be stored cold (2-8°C) or frozen (-20°C). Different kits may have different storage conditions, please check the indicated storage conditions upon receiving.
2. How do I prepare my reagents?
Finish preparing the reagents 10 minutes before use. Concentrate all reagents to the bottom of the tube through centrifugation when using for the first time. Use precise measuring tools to ensure the most accurate preparation (pipette, graduated cylinder etc.). NEVER assume the quantity by adding directly to the supplied vials. Often, the amount in the tube is larger than specified. Ensure all labwear is clean before beginning.
3. How do I separate my ELISA plate?
The strips provided are movable and it is recommended that any strips (wells) you do not intend to use immediately stay in the dark between 2-8°C. Avoid exposing the whole plate unless you intended to use the whole plate immediately. As a side note, leakage from the aluminum foil bag is alright. This will not affect the quality of the plate.
4. How do I add my samples or reagents?
You should add all your samples within a 5 minute span. The time between each sample addition should be uniform to ensure reaction consistency.
5. How do I incubation my plates?
Ensure the proper use of new, clean plate sealers in order to prevent sample evaporation and contamination. Maintain a steady hand when moving the plate in order not to spill the liquid. Avoid excessive opening of the incubator door to keep the temperature a consistent 37℃.
6. How do I wash the wells?
Use the same volume of buffer per well, a multi-channel pipette is ideal. If you choose to use an ELISA washer, ensure it is clean and contamination free. Tap plates upside down on a clean paper towel to dry. For a detailed procedure, please refer to your manual.
7. How do I read my ELISA plate?
Begin reading the plate 5 minutes after the stop solution has been added. This helps to avoid a miss reading due to a buildup of precipitate. Ensure the microplate reader has been set correctly and the filter is properly calibrated. Avoid mixing reagents from different lot numbers, using a cap for any other tube than the one it originated, or replacing any components of a kit with those of another.
8. Can detection of reagent A, B and the reconstituted standard only be used once or multiple times?
Dilution for reagent A and B is not necessary to be done all at once. It is recommended they be kept in their stock solution to maintain stability. Only prepare as much reagent as you intend to use for that immediate experiment. The reconstitution standard however cannot be kept as it degrades after storage at low concentrations. It is recommended to only use the reconstitution standard once.
9. Can the standard curve be extended?
Results for outside of the standard curve are not supported. Only results within the standard curve are reproducible and therefore accurate and in accordance with the kit.
10. Why must I dilute my samples?
If your values fall above the standard curve, you must dilute your samples to bring them within the kit’s detection range.
11. Why is there a low sensitivity or low absorbance values?
Is the target protein not expressed in sample used, or is there a low level of target protein expression in sample used? Ensure the expression profile of the target protein will express in your samples. If there is a low level of expression, try increasing the amount of sample used. You may also need to switch to a more sensitive assay. Confirm you are using a positive control within the detection range for this assay.
12. How can I check if my kit is working before I begin my experiment?
The pre-coated plate, standards, detection reagent A, and detection reagent B are core reagents in the kit. If you are concerned about the kit quality after transit, you can run one or both of the following tests. If you are looking to conserve reagents and only validate TMB and detection buffer B, try Test #1. If you wish to validate the whole kit, run Test #2 or both Test #1 and #2.
Test #1: This test is used to validate the TMB substrate and detection B after transportation. Draw 100ul of TMB substrate and place it in a clean EP tube, this should be colorless. Using a new pipette, add in 1ul of the detection reagent B. Mix well and observe for color. If there is color present, it means that the TMB substrate and detection B are good - this is a good indicator that the whole kit is good after transit.
Test #2: This test is used to validate the whole kit (validate all reagents). Run a pilot experiment with different dilutions of the standard with one or two samples to test. The standard curve can be used to determine whether the whole kit is still good or not, and it can also be served as the positive control and negative control both.
Troubleshooting Tips and Common Problems for ELISA Failure
Many factors contribute to ELISA failure. Most of these results can be avoided by carefully reading and understanding the manual in its entirety before experiment. Please see the suggestions below for troubleshooting failed ELISA test:
1. Check the kit’s expiration date to ensure the test was carried out prior to expiry. Also ensure that all the reagents were stored properly, in accordance with the manual.
2. Look for signs of deterioration in reagent solutions or instability. Some indications are precipitation or discoloration. Also, ensure the substrate reagent is colorless.
3. It is recommended that you use disposable plastic pipettes, tips, and containers to prepare and store the reagent. This helps to minimize cross contamination. It is also recommended to change pipette tips after every use.
4. Ensure the manual indicated incubation time and temperature was achieved.
5. Ensure the correct kit reagents were used and not substituted from a different kit.
6. Finally, the samples and standards should be run in duplicates to improve accuracy.
1. Incomplete washing: This can contribute to a poor standard curve or poor precision. Ensure the apparatus was soaked for an adequate amount of time.
2. Inadequate aspiration: This can contribute to poor standard curve, poor precision, or too much signal. Ensure wells are completely dry after aspiration.
3. Pipetting error: This can contribute to a poor standard curve, poor precision, or too much signal.
Recalibrate the pipette if needed. Furthermore, consider setting duplicates for each assay.
4. Incomplete washing: This can contribute to poor precision, a poor standard curve, a high background, too much signal, or poor duplication. If the apparatus was not washed properly this can occur. Ensure the apparatus was soaked for an adequate amount of time and the wash solution is not contaminated.
5. Improper dilution of Standard: This can contribute to a poor standard curve. Ensure the diluent provided in the kit is used as a blank value, ensure the highest standard was diluted accurately, and the 2-fold dilution series was completed accurately.
6. Unequal equal mixing of reagents: This can contribute to poor precision, or too much signal.
7. Unequal volumes: This can contribute to a poor standard curve. Ensure equal volumes are added to each well. If automatic pipetting, check pipette calibration.
8. Lack of HRP-Conjugate: this can contribute to poor discrimination against the standard curve. Ensure the correct dilution for the experiment.
9. Plate incubation time: Short incubation period can contribute to poor discrimination against the standard curve, or a low reading across the entire plate. Ensure the plate is incubated according to the kit or increase the incubation time.
10. Re-used plate seal: This can contribute to poor duplication. Ensure a new plate seal is used for each step of the procedure.
11. Lack of plate seal: This can contribute to poor duplication. Ensure a plate seal is used for each step of the procedure.
12. Contamination of the buffer: This can contribute to poor duplication. If you suspect the buffer is contaminated, prepare a fresh buffer.
13. Sample is too concentrated: This can contribute to a normal standard curve but the sample OD can be too high. Dilute samples and run the test again.
14. Incorrect wavelength: This can contribute to a low reading across the entire plate. Ensure to check both the filter and the reader to determine the cause.
15. Neglecting to add stop solution: This can contribute to a low reading across the entire plate. Ensure to add stop solution to each well.
16. Variation in incubation temperature: This can contribute to a low reading across the entire plate. Check the incubator to ensure even temperature.
17. Lost activity: This kit is not functioning properly. Check expiration date. Try a new kit.