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We do not recommend diluting the working reagent. It is better to dilute or otherwise optimize primary and or secondary antibody concentrations or use more washes between incubation steps.

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We do not recommend diluting the working reagent. It is better to dilute or otherwise optimize primary and or secondary antibody concentrations or use more washes between incubation steps.

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We provide the following components needed to run a successful ELISA. They include the following:

High-affinity binding NUNC Maxisorp (Cat. No. 44-2404) or Corning Costar 9018 ELISA Plates (Cat. No. 44-2504)
ELISA/ELISPOT Coating Buffer Powder (Cat. No. 00-0044-59)
5X ELISA/ELISPOT Diluent (Cat. No. 00-4202-56)
Avidin HRP (Cat. No. 18-4100-51)
Super AquaBlue ELISA substrate (Cat. No. 00-4203-56, 00-4203-58) or 1X TMB Solution (Cat. No. 00-4201-56)
For ELISA pairs, eBioscience recommends using the HRP compatible substrate, Super AquaBlue (read at 405nm). Our Ready-Set-Go! ELISA Sets include 1X TMB Solution (read at 450nm).

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QuantaBlu Fluorogenic Peroxidase Substrate is a fluorescent detection reagent for HRP and provides attamole sensitivity. SuperSignal ELISA Pico Chemiluminescent Substrate is strictly chemiluminescent.

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All ELISA kits are provided in the sandwich ELISA format with capture antibody already coated onto a 96 well plate. Typical detection uses a biotinylated detection antibody followed by Streptavidin-HRP and HRP substrate. Most kits are available as single 96-well plate kits, some are available as 2- and 5-plate kits. Kits typically contain:

96 Well Strip Plate coated with capture antibody
Standard protein
Wash buffer, 10x
ELISA buffer/Diluent, 10x
Detection antibody (in most kits, biotinylated)
HRP Reagent (either secondary antibody or streptavidin conjugated to HRP)(100x)
Substrate (usually TMB)
Stop solution
Adhesive plate covers

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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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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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Please review the following applications for TSP (Transferable Solid Phase) :
- Reactions such as the ELISA can be performed on the TSP. The pins are coated by submerging in the analyte solution contained in a 96-well plate. Washing and reaction with succeeding antibody or streptavidin conjugates can be performed by transferring the TSP into a washing tray or second 96-well plate filled with the appropriate solution. The TSP is placed into a substrate solution until color is observed and is then removed to ensure a simultaneous start and stop to the enzymatic reaction.
- TSP is available sterile for screening hybridoma cells for the production and secretion of monoclonal antibodies.
- TSP can be used for radioimmunoassays because it can be placed directly on X-ray film and exposed for several hours. Only the tips of the pins should be incubated with the radiolabeled reagent.
- TSP can be used with the OmniTray for performing dot blots and for replicating bacterial clones from a 96-well plate.

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Our standard procedure usually produces an assay with a background <0.2 OD. If a background higher than 0.2 OD is observed, we suggest the following steps:
1) Shorten incubation times, especially the substrate incubation. This will result in lowering the overall signal.
2) Decrease the concentration of the streptavidin-HRP conjugate.
3) Include a greater number of washing steps.
4) Increase soak time.
5) Try a lower binding capacity ELISA plate.
6) Increase blocking time or try an alternative blocking agent.
7) Optimize the standard diluent buffer formulation by adding additional animal protein.

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The formation of intact IgG molecules may be quantitated using a sandwich ELISA designed to capture and detect rabbit IgG. Besides the rabbit IgG positive control, reagents, and consumables that are included in the kit, you will also need purified rabbit IgG to be used as a standard, F(ab')2 goat anti-rabbit IgG HRP conjugate (Cat. No. A10547), Protein A-coated plates (Cat. No. 15130 for clear plates used in colorimetric detection), TMB colorimetric substrate (Cat. No. 34021), SuperBlock (TBS) Blocking Buffer (Cat. No. 37581), and PBS or TBS buffer for washes. There is an example procedure in our Protein A-coated plates manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011310_Pierce_ProteinA_G_AG_Coat_96Well_UG.pdf). Please note, our R&D scientists determine titer values from crude cell culture supernatants using a Pall Life Sciences FortéBio Octet instrument equipped with a protein A biosensor.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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The formation of intact IgG molecules may be quantified using a sandwich ELISA designed to capture and detect rabbit IgG. Besides the rabbit IgG positive control vector, reagents, and consumables that are included in the kit, you will also need purified rabbit IgG to be used as a standard, F(ab')2-Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP (Cat. No. A10547), Protein A Coated Plates, Clear, 96-Well (Cat. No. 15130), TMB Substrate Kit (Cat. No. 34021), SuperBlock (TBS) Blocking Buffer (Cat. No. 37535), and PBS or TBS buffer for washes. There is an example procedure in our Protein A Coated Plates manual (https://tools.thermofisher.com/content/sfs/manuals/MAN0011310_Thermo Scientific_ProteinA_G_AG_Coat_96Well_UG.pdf). Please note that our R&D scientists determine titer values from crude cell culture supernatants using a Pall Life Sciences FortéBio Octet instrument equipped with a Protein A biosensor.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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In general, high background can be a result of one or more of the following:

(1) Blocking was insufficient. Increase concentration of blocking agent. Increase incubation time with blocking agent. Try alternate blocking agent.

(2) Reagents were too concentrated. Dilute primary antibody, secondary antibody, and/or streptavidin or avidin conjugate. Use recommended substrate concentration.


ELISA-specific suggestions for reducing high background:
-Debris or other component interfered with plate reading. Centrifuge or filter turbid samples before use. Blood-derived samples exhibiting excessive hemolysis or lipemia may interfere with detection and increase background. Use care in pipetting into or out of the wells to avoid air bubbles in the wells (break any air bubbles remaining on plate). Increase wash volumes to more effectively remove residual reagents. Add one or more soaking steps to the plate washing protocol.
-Blocking was insufficient. Increase the concentration of blocking agent. Try alternate blocking agent.

Membrane-bound-specific suggestions for reducing high background:
-Blocking was insufficient. Try alternate blocking agents such as BSA, casein, or nonfat dried milk (nonfat dried milk may contain endogenous biotin or phosphatase activity that may interfere with detection). Increase the concentration of blocking agent.
-Bands or spots appeared in the absence of primary antibody. Dilute the secondary antibody or the streptavidin or avidin conjugate to avoid non-specific interactions. Try alternate reagents. Try alternate detection schemes.
Immunocytochemistry-specific suggestions for reducing high background:
-Blocking was insufficient. Use more blocking agent (such as BSA or control serum).
-Tissues were allowed to dry during incubation. Use humidified chambers for incubation steps.
-Cell or tissue contained large amounts of endogenous biotin (when using avidin/streptavidin system). Block with sequential applications of streptavidin and biotin before using a streptavidin-based system. If endogenous biotin is excessively high, switch to an antibody-based secondary detection system.
-If HRP-based detection is used, remember to inactivate endogenous peroxidase activity in the sample.
-Slide contained excessive adhesive. Reduce the amount of adhesive used.
-Paraffin residue remained on sample. Use more changes of xylene or longer time in xylene.
-Tissue was necrotic. Use alternate tissue, if possible.

Immunofluorescence microscopy-specific suggestions for reducing high background:
-Autofluorescence occurred. Use glass instead of plastic specimen support. Use different coverslip mounting medium. Use another fluorochrome that emits at a different wavelength.
-Filters in microscope were incorrect. Use combinations of excitation and emission filters to select only the wavelengths of interest.
-Dirt on objective. Clean objective.
-Non-specific binding caused by fluorescent dyes can be blocked with Image-iT FX signal enhancer (Cat. No. I36933).

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