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Reagent Sets contain Capture Antibody, Detection Antibody, Recombinant Standard, HRP Conjugate, TMB Substrate and Stop Solution. Each contains enough reagents to process five 96-well plates. Reagent Sets are included in the main list of ELISA (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/elisa/antibody-pair-kits.html) kits (search by “Reagent Set”).

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Antibody pair kits contain capture antibody, detection antibody, recombinant standard and HRP conjugate. Each contains enough reagents to process forty 96-well plates. A list of Antibody Pair Kits (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/elisa/antibody-pair-kits.html) is available by target.

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Here are possible causes and solutions:

Reagents not at room temperature (approximately 25 plus or minus 2 degrees C) at start of assay. Allow all reagents to warm to room temperature prior to commencing the assay.
Incorrect storage of components, e.g., not stored at 2-8 degrees C. Store all components exactly as directed in the protocol and on labels.
Anti-rabbit IgG HRP or streptavidin-HRP working solution made more than 15 minutes before use in assay. Use the diluted anti-rabbit IgG HRP or streptavidin-HRP within 15 minutes of dilution.
Expired reagents.Check expiration dates upon receipt of kit and use the kit prior to expiration.
Plate read at incorrect wavelength. The correct wavelength to read ELISAs using the TMB substrate is 450 nm.
TMB solution lost activity. Ensure that the TMB solution is clear before it is dispensed into the plate wells. A blue color and/or the presence of particulate matter indicate that the product is contaminated. Please contact Technical Support if this problem is noted. To avoid contamination, we recommend that the quantity required for an assay be dispensed into a previously unused disposable trough for pipetting. Discard any TMB solution left in the trough and do not put it back in the bottle. Avoid contact between the TMB solution and items containing metal ions. Do not cover your plates with aluminum foil or aluminum-coated Mylar sheets because this can cause color development in the absence of HRP.
Attempt to measure analyte in a matrix for which the ELISA assay is not optimized. Contact Technical Support when using alternative sample types.
Wells have been scratched with pipette tip or washing tips. Use caution when dispensing into and aspirating out of microwells.
Incorrect chromogen or stop solution used. Use only the chromogen and stop solution supplied with the kit.
Standard diluent buffer added to all wells rather than the designated wells. Follow the protocol and only add the standard diluent to the designated wells and to the samples where it is required, or to samples producing signals greater than that of the highest standard.
Use of buffer containing azide, which is not compatible with HRP. Avoid the use of azide in the assay.

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Each Antibody Pair kit contains capture (coating) antibody, biotinylated detection antibody, recombinant standard, and streptavidin-HRP. Other reagents required are listed in the Antibody Pair manual included with the kit, and can also be purchased separately (Antibody Pair Buffer Kit, Cat. No. CNB0011; 5X Assay Buffer, Cat. No. DS98200; etc.). The manual also provides a specific procedure and illustrates an example of a standard curve that can be obtained when the specific procedure is followed.

A general procedure is summarized here:
1) Coat the microplate with diluted capture (coating) antibody overnight at 2-8 degrees C; wash the plate.
2) Incubate the standards or samples in the coated microplate; wash the plate.
3) Incubate diluted biotinlyated detection antibody in the plate; wash the plate.
4) Incubate streptavidin-HRP in the plate for 15-45 min; wash the plate.
5) Incubate the plate with TMB substrate for 10-60 min, and then stop the reaction with Stop solution.
6) Read the microplate at 450 nm.
We recommend determining optimal buffer formulations, concentrations, and incubation times for individual applications.

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Here are possible causes and solutions:

Errors in pipetting the standards or samples or in subsequent steps. Always dispense into wells quickly and in the same order. Do not touch the pipette tip on the individual microwells when dispensing. Use calibrated pipettes and the appropriate tips for that device. Check for any leaks in the pipette tip.
Repetitive use of tips for several samples or different reagents. Use fresh tips for each sample or reagent transfer.
Wells have been scratched with the pipette tip or washing tips. Use caution when dispensing into and aspirating out of microwells.
Liquid transferred from well to well during incubations. Adjust the orbital shaker or check for correct rotator rpm. Peel the adhesive plate cover off carefully.
Incorrect volumes of materials dispensed into the microwells. Follow the protocol for dispensing volumes of reagents. Check calibration of the pipettes.
Standard diluted with the serum, culture medium, or other buffer. Dilute the standard with the standard diluent buffer provided in the kit.
Particulates or precipitates present in the samples. Remove any particulates/precipitates by centrifugation prior to dispensing into the assay.
Dirty microwells: visible debris within or on bottom of microwells. Inspect the microwells and invert the plate to remove debris. Wipe the bottom of the plate with an absorbent tissue after each wash step. Never insert tissue into the microwells.
“Edge effect” due to uneven temperature between the outer-edge wells and the wells in the center of the plate. Seal the plate completely with a cover during incubations, and place the plate in the center of the incubator when 37 degrees C incubation is indicated.

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Each CytoSets contains capture (coating) antibody, biotinlyated detection antibody, standard and Streptavidin-HRP. Other reagents required are listed in the CytoSets information sheet included with the kit and can be purshased from us separately (Antibody Pair Buffer kit CNB0011, 5x Assay Buffer DS98200, etc.). The information sheet also provides a specific procedure and illustrates an example standard curve which can be obtained when the specific procedure is followed. A general procedure is summarized here:

1) Coat the microplate with diluted capture (coating) antibody overnight at 2-8 degrees C; Wash the plate
2) Incubate standards or samples with the coated microplate; Wash the plate
3) Incubate diluted biotinlyated detection antibody with the plate; Wash the plate
4) Incubate Streptavidin-HRP with the plate for 15-45 minutes; Wash the plate
5) Incubate the plate with TMB substrate for 10-60 minutes and stop the reaction with Stop solution
6) Read microplate at 450 nm.

Investigators are advised to determine optimal buffer formulations, concentrations and incubation times for individual applications.

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In the IgG Subclass Human ELISA Kit (Cat. No. 99-1000), all of the wells of the 8-well strips provided are coated with a goat polyclonal anti-FITC antibody, which serves as a general capture reagent. When you add the individual FITC-labeled, subclass-specific monoclonals to the wells (step 2 in the assay procedure on page 2 of the manual - https://tools.thermofisher.com/content/sfs/manuals/PI99-1000_Human%20IgG%20ELISA%20Kit%20Rev%201208.pdf), they bind to the goat capture antibody. This second layer of the ELISA sandwich now performs the subclass-specific capture function. When you add samples (i.e., serum, standards, and controls) to the wells, any human IgG present in the samples will bind to the subclass-specific antibodies that are captured on the plate via their FITC labels. The subclass-specific detection is enabled by each of these subclass-specific mouse monoclonal anti-human IgG antibodies provided in the kit. The actual subclass detection occurs after you add the HRP-labeled anti-human IgG antibody and the TMB chromogen solution to the wells. The amount of each IgG subclass is determined separately in its own set of wells.

An example of one of these subclass-specific sets is shown under step 1 in the assay procedure on page 2 of the manual - https://tools.thermofisher.com/content/sfs/manuals/PI99-1000_Human%20IgG%20ELISA%20Kit%20Rev%201208.pdf. In this case, the setup shown is for IgG1, but if you wanted to measure only IgG4, for example, you would follow the same setup. However, instead of loading these wells with mouse anti-human IgG1, you would use the mouse anti-human IgG4 instead. If all you want to detect is IgG4, the rest of the wells in the plate can be used for other samples instead of additional standard curves for the other subclasses and their respective samples. However, we suggest running a standard curve for the IgG subclass of interest on each plate that you prepare, and each time you run the assay.

The control in the kit consists of lyophilized human serum, and the human IgG standard provided contains all four subclasses at the concentrations indicated on the lot-specific manual. Even though you may want to measure only 1 or 2 subclasses, you'll be using a standard that contains all of them. The other 3 subclasses don't interfere with detection of IgG3, for example, because the capture antibody is IgG3-specific. The detection antibody in the kit is an HRP conjugate of an anti-human IgG that detects all of the subclasses equally effectively and detects all of the human IgG captured in the wells.

Note that the antibody-coated plates in the kit come as 8-well strips that you snap into the frame provided. You do not have to run an entire plate or both plates at one time. Store any unused 8-well strips at 2-8 degrees C and keep them dry. Unused wells in individual strips should be sealed securely to prevent the entry of moisture while running the assay.

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Sorry, we have not tested the compatibility of Tissue Extraction Reagent I with our protein assays.

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ProcartaPlex multiplex assays, which are based on Luminex xMAP technology, provide a versatile platform that gives users more flexibility and a greater array of options for analyte detection. Whether you are testing for single or multiple analytes, ProcartaPlex multiplex assays deliver accurate analytical performance using efficient, easy-to-follow protocols. Each of these assays has undergone the same development, validation, manufacturing, and quality control standardization we conduct for our ELISAs. Each lot of ProcartaPlex multiplex assays as well as ELISA assays is fully qualified with the appropriate sample type (i.e., species-specific serum, plasma, and cell culture supernatants), and each lot is evaluated based on the following performance characteristics:

Specificity-each analyte is screened to make sure there is no significant cross-reactivity with other analytes in the multiplex test
Sensitivity-each analyte is evaluated for both functional sensitivity (differentiation from background) and lower limit of detection (LLOD)
Precision/accuracy-multiplex assays have good intra-assay precision (<10% CV), inter-assay precision (<10% CV), and lot-to-lot consistency (<20% CV); these values are comparable to or better than most ELISA tests
ProcartaPlex multiplex assays are regularly tested against the matching ELISAs. Therefore, you can switch easily from ProcartaPlex assays to ELISA and vice versa with reliable results. Most of our ProcartaPlex assays use the same antibody pairs as our traditional plate-based ELISAs, resulting in high correlation (R2 > 0.9) between the two assays.

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The chromogen blank and zero standard provide you with different indicators of assay performance. The chromogen blank-comprising only chromogen and stop solution-should have an A450 of less than 0.03. The A450 of the chromogen blank can be used to blank your plate reader and, in any case, it should be subtracted from all other A450 values you obtain from the plate.

If the chromogen blank is higher than approximately 0.03, you should check the color of the chromogen reagent straight from the bottle. It can range from clear and bluish to clear and slightly yellow. If it is a stronger blue color, then it is most likely contaminated and should not be used. The most frequent cause of contamination is transfer of unused chromogen solution back into the bottle. The chromogen solution should always be transferred to a clean reservoir in the first place.

The other blank wells that you run are the zero standard wells, which are sometimes referred to as the “zero wells”. The A450 of the zero standard wells in your assay should be less than 0.35, and preferably lower than this. Although the zero standard A450 value should be low, it is usually higher than the chromogen blank. It serves as the critical first point on the standard curve, so if the zero standard value is too high, it reduces the dynamic range covered by the standard curve. Expected values for the zero standard are provided as part of typical data in the kit manual. If the zero standard value is higher than expected, this usually indicates that there is some other problem with the ELISA.

We strongly recommend that you include both the chromogen blank and the zero standard when you run an ELISA. Since we recommend that all blanks, standards, controls, and samples be tested in duplicate, these blanks take up only 4 wells. If you are short of wells for samples, you can omit the highest standard in the standard curve as well as the next-highest one, if absolutely necessary. You should still make all of the standard dilutions as instructed in the manual, but don't add the one(s) that you're omitting to the plate. This recommendation is based on the assumption that the A450 values for your samples will still fall on a standard curve drawn with the remaining points. If they do not, then you will need to run the assay again with all of the standards or dilute the samples so that they fall on the abbreviated standard curve.

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All reagents necessary to perform 1 or 10 plate ELISA assays, including optimized capture-antibody pre-coated plates.

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Yes, our Universal Assay Buffer (Cat. No. EPX-11110-000) can be bought separately. We offer most of our ProcartaPlex buffers and reagents as stand-alone items. A complete list of what is currently available can be found on our website here, under "Accessories": https://www.thermofisher.com/us/en/home/life-science/antibodies/immunoassays/procartaplex-assays-luminex/procartaplex-immunoassays/procartaplex-preconfigured-panels.html.

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