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General

Here are some possible causes and solutions for this problem:

Cause

Solution

Placing the plate on an absorbent material both at the bench and on the shaker (which results in wicking when there is contact between the absorbent material and the opening on the bottom of the well funnel).

 

Rest the plate currently in use on top of a second filter plate so that the filter plate currently in use only comes into contact with a nonabsorbent surface. This will also help locate any potential leaks. 

Prior to incubations, use a Kimwipe™ tissue and lightly press up on each well to dry off the bottom of the plate.

Putting any vertical pressure on the plate during the incubations (using either tape or clamps).

Use clamps that fit around the sides of the filter plate to secure the plate to the shaker during incubations.

Subjecting the plate to vacuum pressure greater than 5 mm Hg, even for a moment, which either tears the filter membrane or creates an opening that exceeds the design limits for volume retention. 

Prevent any vacuum surge by opening and adjusting the vacuum on the manifold before placing the plate on the manifold surface and checking the vacuum with a test plate (i.e., not the plate used for the assay). For our assays we recommend the setting not exceed 5 mm Hg.

Puncturing the filter membrane with pipet tips, by inserting the tips all the way into the wells during reagent loading.

Pipet solutions along the sides of the wells, rather than deep into the wells.

Separating the soft (opaque) plastic bottom from the hard (clear) plastic top of the filter plate before starting, which compromises the integrity of the seal even if the pieces appear to fit back together properly. 

If you accidentally separate these two layers of the plate, the plate should be discarded and a fresh plate used instead.

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. 

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.

Here are some suggestions:

  • Qualify your standard curve using the data analysis tools found here.
  • Make sure that your dilution factors are set correctly.
  • Sample optimization may be needed: Dilute the sample with an appropriate diluent and re-read.

Here are some suggestions:

  • Qualify your standard curve using the data analysis tools found here
  • 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.

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. 

Here are some suggestions:

  • Review the warning messages.
  • Review the instrument settings and make sure they are appropriate for the assay being run.
  • Review the workflow and reagents used in the assay: volumes, order added, incubation time and temperatures, etc.
  • View the bead maps and histograms generated by your run to look for irregularities. Compare the patterns with those in the “Troubleshooting” section of your instrument operation manual. This will guide further troubleshooting steps needed. For example: bubble in the system, probe height corrections, sheath fluid incompatibility. 

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.

Here are possible causes and solutions for this issue:

  • This usually indicates that the beads have been photobleached. This problem can also be caused by exposing the beads to organic solvents. Unfortunately, the assay will have to be repeated because the beads cannot be restored. The beads must be protected from light and organic solvents.
  • Alternatively, the instrument may be off in its measurements or you may have a calibration issue. Call the manufacturer for a service appointment.

Here are possible causes and solutions for this issue:

  • Insufficient bead mixing by sonication and vortexing: Vortex the beads for at least 30 seconds, and then sonicate the beads for at least 30 seconds before adding them to the assay. Be sure your plate shaker is set appropriately for optimal bead movement in the wells during the incubation steps.
  • Entry of air into the line of the Luminex™ instrument, due to insufficient bead suspension volume in the well for the height of the instrument probe: Verify that the beads are suspended in an appropriate amount of solution for analysis (see assay specific instructions). Adjust the sample probe height. 

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