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991000

Product FAQ

What is the purpose of an isotype control?

Answer

To estimate the contribution of the non-specific interaction and Fc receptor binding, staining protocols using an antibody directed to an irrelevant antigen (for example, DNP) having the same isotype as the antibody of interest may be analyzed in parallel with the antibody of interest. The antibody directed to the irrelevant antigen is known as the isotype control.

Antibodies are useful for identifying and localizing proteins and other antigens both on the surface of cells as wells as inside cells. The utility of antibodies lies in their specificity and avidity for their antigens, an interaction which is mediated by the antigen binding sites. Antibodies can also react with components other than their antigens in several ways. They can associate with cellular components by non-specific protein-protein interactions. They can associate with fatty components by hydrophobic interaction. Antibodies can also bind to antibody receptors expressed on the surface of some cell types. For example, antibodies of the IgG class can bind to the cell surface Fc receptors known as CD16, CD32, and CD64. Under ideal conditions, various types of non-specific binding will be prevented by including blocking proteins (BSA, milk, or animal serum products) in antibody incubation reaction mixtures. Antibody binding to Fc receptors can be prevented by preincubation of cells with serum preparations containing immunoglobulins.

Answer Id: E5165

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Product FAQ

I have an elevated optical density (OD) reading for my ELISA standard. What went wrong?

Answer

Here are possible causes and solutions:

Incorrect dilution of the anti-rabbit IgG HRP or streptavidin-HRP working solution.
Warm the solution of anti-rabbit IgG HRP or streptavidin-HRP (100X) to room temperature, draw it up slowly, and wipe the tip with a laboratory tissue (e.g., Kimwipe™ tissue) to remove the excess. Dilute only in the HRP diluent provided.
Incubation times extended. Follow incubation times outlined in the protocol.
Incubations performed at 37 degrees C. Perform incubations at room temperature (approximately 25 plus or minus 2 degrees C) when instructed in the protocol.

Answer Id: E12631

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Product FAQ

Does the IgG Subclass Human ELISA Kit (Cat. No. 99-1000) work with serum from cynomolgus monkeys? What about other nonhuman primates?

Answer

No. A customer notified us early in 2014 that Cat. No. 99-1000 did not detect IgG subclasses from cynomolgus monkeys. As far as other nonhuman primates are concerned, we do not know if this kit will work with samples from these animals.

Answer Id: E12620

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Product FAQ

How do I process plasma samples for use on the Luminex™ assay platform?

Answer

Separate the cells from the plasma samples by centrifugation at 2,000 x g for 10 min in a refrigerated centrifuge. Centrifugation at this force is necessary to deplete the sample of platelets. Transfer the supernatant to a chilled clean polypropylene tube with a sterile Pasteur pipette. Maintain the samples at 2-8 degrees C while handling.

If the plasma is to be analyzed at a later date, apportion it into aliquots in polypropylene microcentrifuge tubes and store at -80 degrees C. Avoid multiple freeze-thaw cycles. When you are ready to analyze them, allow the samples to thaw on ice. All plasma samples should be clarified by centrifugation (14,000 rpm for 10 min at 4 degrees C) in a refrigerated microcentrifuge immediately prior to analysis. Follow the assay procedure provided with the kit for appropriate dilutions.

Answer Id: E12641

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Product FAQ

I see very weak to no color development after my ELISA. What happened?

Answer

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.

Answer Id: E12632

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Product FAQ

My filter plate has clogged wells. Do you have any suggestions?

Answer

If wells are clogged after the first incubation with the standards:

When washing a partial plate, the vacuum may not be complete because air is being drawn through the empty wells. We recommend covering the plate with Parafilm™ film to create a seal, which will in turn raise the pressure high enough to empty the wells. If there is a small clog, it may be difficult to see it even though it is enough to prevent aspiration. For this, we suggest using the tip of a 15 mL conical tube to gently rub against the tip of the well bottom opening, going from left to right. This will dislodge small clogs only. Other ways to unclog a well are to apply pressure from above the well using a gloved finger or thumb with an absorbent paper towel under the plate, or unplugging the drain hole using a large syringe needle.

Answer Id: E12656

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Product FAQ

What is the difference between your total and phosphospecific ELISA kits?

Answer

Both types of ELISA kits capture total protein, regardless of its phosphorylation state, within the wells of a plastic 96-well plate. This is done by coating the wells with a “pan-antibody” that does not distinguish between the phosphorylated and non-phosphorylated forms of a protein and does not block the phosphorylation site to be studied. In addition, a phosphospecific ELISA kit quantifies the amount of that same protein that is phosphorylated on one or more specific amino acids. Instead of a second pan-antibody for detection, this assay uses an antibody that specifically recognizes an epitope that is only present on a protein when it is phosphorylated specifically (i.e., it is phosphospecific).

We recommend running the total and phosphospecific ELISAs simultaneously with the same samples. If this is not possible, make sure to test the same samples with both kits as soon as possible.

Answer Id: E12621

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Product FAQ

How do your phosphospecific ELISA kits compare to immunoprecipitation and western blotting?

Answer

Our phosphospecific ELISA kits have several advantages, including ease of use and increased sensitivity. Phosphospecific ELISA kits are typically 2-10 times more sensitive than western blots, so they are particularly useful for the detection of “low-expressing” proteins or for small sample sizes. In addition, with the use of the recombinant standards provided in the kit, phosphospecific ELISAs provide quantitative results without having to perform densitometry.

Answer Id: E12624

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Product FAQ

How do I process cell culture supernatants for use on the Luminex™ assay platform?

Answer

Cells should be in log-phase growth. Stimulate cells as desired in appropriate cell culture flasks. Using sterile technique, remove the desired volume of conditioned cell culture medium with a pipette and transfer the medium to clean polypropylene microcentrifuge tubes. Centrifuge the medium at 14,000 rpm for 10 min at 4 degrees C in a refrigerated microcentrifuge to remove any cells or cellular debris. Aliquot the clarified medium into clean polypropylene microcentrifuge tubes. These samples are ready for the assay. Alternatively, clarified medium samples can be aliquoted and stored at -80 degrees C for future analysis. Avoid multiple freeze-thaw cycles. Frozen samples should be allowed to thaw on ice just prior to running the assay. Thawed samples should be clarified by centrifuging at 14,000 rpm for 10 min at 4 degrees C in a refrigerated microcentrifuge prior to analysis to prevent clogging of the Luminex™ probe and/or filter plate. Follow the assay procedure provided with the kit for appropriate dilutions.

Answer Id: E12642

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Product FAQ

I got a poor standard curve after my ELISA. Why is this?

Answer

Here are possible causes and solutions:

Improper preparation of standard stock solution.Dilute the lyophilized standard as directed on the vial label, only with the standard diluent buffer or a diluent that most closely matches the matrix of your sample.
Reagents (lyophilized standard, standard diluent buffer, etc.) from different kits, with either different analytes or different lot numbers, were substituted. Never substitute any components from another kit.
Errors in pipetting the standard or in subsequent steps. Always dispense into wells quickly and in the same order. Do not touch the pipette tips on the individual microwells when dispensing. Use calibrated pipettes and the appropriate tips for that device.

Answer Id: E12633

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Product FAQ

The readout for my samples says that they are above the detectable limit. What should I do?

Answer

Here are some suggestions: Qualify your standard curve using the data analysis tools found here (https://www.thermofisher.com/content/dam/LifeTech/migration/files/cell-analysis/pdfs.par.20874.file.tmp/co23717-072511-magpix-data-analysis-tips-ga-flr.pdf). Make sure that your dilution factors are set correctly. Sample optimization may be needed: Dilute the sample with an appropriate diluent and re-read.

Answer Id: E12657

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Product FAQ

After I added the chromogen reagent to the plate, I incubated the ELISA as suggested in the manual, but the A450 of the highest standard was higher than what my plate reader can read. What should I do?

Answer

Our customers use a wide variety of plate readers. Some of these can’t read absorbances higher than 2 AUFS (Absorbance Units Full Scale), while others can’t go beyond 3 AUFS, for example. If you read your ELISA plates after 30 minutes of incubation at room temperature and 1 or 2 A450 values are off-scale, you can shorten the incubation time. For example, some customers find that 20 minutes is the ideal incubation time because the ambient temperature in their lab is higher than approximately 2 degrees F (22 degrees C). In this case, higher temperatures increase the rate of the HRP-driven ELISA. Conversely, if the A450 values you get are not high enough, you can increase the incubation time accordingly.

Answer Id: E12629

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Product FAQ

What can you tell me about the Human IgG Subclass Standard included in the IgG Subclass Human ELISA Kit (Cat. No. 99-1000)?

Answer

In the IgG Subclass Human ELISA Kit (Cat. No. 99-1000), the human IgG Subclass Standard is Part Number 50287HK. Each batch of 50287HK is calibrated against a WHO reference standard designated 67/97. More details about this standard are in the abstract from Klein F et al. (1985) Clin Chem Acta 150 (2):119-127.

Answer Id: E12619

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Product FAQ

What units of measurement do you use for the results of total and phosphospecific ELISAs?

Answer

The results of our total ELISAs are given in pg/mL of sample, or sometimes ng/mL. This measurement is always given in mass units because standards of known mass are used to prepare the standard curve. The results of the phosphospecific ELISAs are given in “units”, which we do not relate to a particular mass of protein. We use units because it is difficult to precisely know the efficiency of a particular phosphorylation reaction, and therefore the ratio of phosphorylated to unphosphorylated protein, in a particular preparation of phosphoprotein standard. Phosphorylation units will be unique to each phosphospecific ELISA and are described within the product manual that accompanies each kit.

For example, a typical unit description would be “1 unit = the amount of FAK [pY397] derived from 300 pg of auto-phosphorylated FAK protein”. Since there is no guarantee that the FAK in our standard preparation is 100% phosphorylated, we refrain from making the statement that this corresponds to 300 pg of phosphorylated FAK. Instead, we validate a large batch of phosphorylated protein and use this to develop our unit definition and standard curve for our original assay. Subsequent preparations of our protein standards are normalized to the original batch of protein to ensure that our unit definitions remain constant from lot to lot.

Answer Id: E12622

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Product FAQ

How does the IgG Subclass Human ELISA Kit (Cat. No. 99-1000) work?

Answer

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.

Answer Id: E12617

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