I am observing liquid on the XY platform tray in the instrument after the run is over. What could have caused this?

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Answer

This may originate from the probe height being set too low, and the pressure from the fluid return is forcing liquid through the membrane. However, remember that as long as the required 100 beads are being read in a reasonable time frame, the sample is being read correctly. If there is difficulty reaching 100 beads, liquid may have leaked out prior to reading the sample. In this case, stop the run and check the plate for leakage. If leakage is found, remove the Wash Solution on the vacuum manifold and completely dry the bottom of the plate. Add fresh Wash Solution and shake. Continue the run from where it was stopped.

Note: Refer to the appropriate instrument hardware manual for instructions to check and to reset the sample probe (sample needle) height.

Answer Id: E12655

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

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

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

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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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The readout for my samples says that they are below the detectable limit. What are your suggestions?

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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.
It is possible that the functional sensitivity of the standard curve can be extended to better distinguish the data at the lower end of the curve. This can be done by adding one or two further dilutions to the curve and re-running the assay with the samples of lower concentrations.
It is possible that the levels of your protein of interest fall below the detection limits of the assay.

Answer Id: E12658

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

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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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How do I process bronchoalveolar lavage (BAL) for use on the Luminex™ assay platform?

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Answer

The bronchoalveolar lavage (BAL) should be collected in a sterile syringe and kept on ice until you are ready to analyze it. Alternatively, BAL can be aliquoted and frozen in usable sample sizes (such that exposure to freeze-thaw is limited to one time). All samples need to be clarified by centrifugation (14,000 rpm for 10 min) and/or filtered prior to analysis to prevent clogging of the filter plates. Dilute 2-fold with Assay Diluent before applying to the plate.

Answer Id: E12647

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

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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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During data analysis, beads do not appear in the region gated. What happened?

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Answer

This indicates that an incorrect buffer was used for the final step. The wash solution provided in the kit must be used for washing the beads and for resuspending the beads before loading them into the Luminex™ instrument. The osmolarity of the solution will impact the size of the bead, and any change in the bead size will alter detection by the instrument.

Answer Id: E12661

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I ran out of the streptavidin-HRP that came in your ELISA (or Antibody Pair) kit. Can I get more?

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Answer

No. The exact same streptavidin-HRP conjugate supplied in these kits is not available as a stand-alone product. However, it is derived from our ELISA-grade streptavidin-HRP (Cat. No. SNN2004), which we do sell. Remember that the streptavidin-HRP provided in each lot of ELISA or Antibody Pair kits is also lot-specific. So, if you use Cat. No. SNN2004 or another source of streptavidin-HRP, you will have to determine which dilution of SNN2004 works best for you.

Answer Id: E12626

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Can I store Luminex™ assay plates overnight and read them the next day?

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Answer

If the plate cannot be read on the day of the assay, cover and store the plate in the dark overnight at 2-8 degrees C for reading the following day without significant loss of fluorescence intensity. When you are ready to read the plate, bring the plate to room temperature on an orbital plate shaker (still protected from light). Aspirate the Working Wash Solution from the stored plates and add 100 μL of fresh Working Wash Solution. Place the plate on an orbital shaker for 2-3 min at 500-600 rpm prior to analysis.

Answer Id: E12653

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What is the purpose of an isotype control?

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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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I am getting an elevated background in my ELISA. What should I do?

Product FAQ

Answer

Here are possible causes and solutions:

Insufficient washing and/or draining of wells after washing. Residual solution containing anti-rabbit IgG HRP or streptavidin-HRP can elevate the background if left in the well.
Wash according to the protocol. Verify the function of the automated plate washer. At the end of each washing step, invert the plate on absorbent tissue on the countertop and allow it to completely drain, tapping forcefully if necessary to remove residual fluid.
Chromogen exposed to light prior to use, resulting in a blue color.
Keep chromogen in its vial until you are ready to dispense it into the plate, and then pour it into a reservoir to prevent contamination of the vial with equipment. Do not cover the plate with foil.
Incubation time is too long or incubation temperature is too high.
Reduce incubation time and/or temperature.
Contamination of pipette, dispensing reservoir, or substrate solution with anti-rabbit IgG HRP or streptavidin-HRP.
Do not use chromogen that appears blue prior to dispensing onto the plate. Obtain a new vial of chromogen.
Blanks that have been set up improperly.
Follow the protocol when designating blank wells. Blank wells contain only chromogen and stop solution. Subtract blank well results from all other wells.
Incorrect dilution of standard stock solution or standards diluted in serum, culture supernatant, or other.
Follow the protocol instructions regarding dilution of the standard. Dilute standards only in the Standard Diluent Buffer provided in the kit.

Answer Id: E12630

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How do I process synovial fluid on the Luminex™ assay platform?

Product FAQ

Answer

Collect synovial fluid into non-heparinized tubes and spin at 1,000 x g for 10 min within 30 min of sample collection. The acellular portion of synovial fluid should be stored at -80 degrees C before subsequent analysis. All samples need to be clarified by centrifugation (14,000 rpm for 10 min) and/or filtered prior to analysis to prevent clogging of the filter plates. Dilute samples 1:1 with Assay Diluent prior to addition to the assay. Reference: Raza K et al. (2005) Arthritis Research &Therapy 7(4): R784-R795.

Answer Id: E12645

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The bead count for my standard curve is correct and regular, but for my samples the bead count is erratic and I get a warning message saying “The acquisition had at least one region that did not reach the maximum count”. What should I do?

Product FAQ

Answer

This pattern is indicative of a sample matrix effect. Here are some suggestions:

Confirm that the sample has been clarified and is free of debris.
Confirm that the reagents appropriate for your sample type have been used.
Confirm that there is at least a 1:1 ratio of sample to assay diluent for serum, plasma, CSF, and culture supernatant samples. For cell lysates or tissue homogenates, confirm that the sample has been diluted appropriately to reduce the concentration of detergent from the lysis buffer to ?0.01%. For other sample types, further sample optimization may be required.

Answer Id: E12659

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