Common Reasons for Poor Assay Duplication and Poor Assay Quality

How well replicates match each other within an assay is an indication of how well the assay was conducted. Effectively running high quality assays with consistency is achievable, however there are a number of factors that can negatively affect sample duplication and therefore negatively affect the outcome of the assay. Being an assay service company, Eve Technologies is providing the following list as a resource to help you run high quality assays.

Assay Issues with Samples and Sample Handling.

  1. Insufficient mixing of samples.Failing to vortex your samples can cause variability. For samples that require centrifugation, vortex first to adequately mix the biomarkers the centrifuge to pull down unwanted debris.
  2. Samples with large amounts of particulate.In Multiplexing Bead Assays, particulates interfere with the accurate reading of the beads. In all assays, some particulates can cause significant sample-to-sample variability. To address this issue, centrifuging is highly recommended as well as filter purification (0.22 μm). If lipids are present they will separate on the top after centrifugation. If necessary, avoid lipids by not pipetting from the top and certainly avoid pipetting from the bottom where the particulates will be. If you have access to a good liquid handling robotic platform, you can program the exact location to pipette from in each sample (ie 1mm below liquid surface).
  3. Highly viscous samples.Viscous samples can also interfere with the accurate reading of the beads and can cause other problems in other assay platforms like ELISA. Viscous samples are also difficult to pipette accurately and consistently. Dilute viscous samples (ie 1:2 or 1:5) to ensure consistent assay dynamics. Some samples can be pretreated to help lower viscosity. For example, adding hyaluronidase to synovial fluid.
  4. Incompatible substances in samples.

Foreign substances in some samples can negatively affect assay performance and duplication. The following is a list of such substances that negatively affect Multiplexing Bead Assays:

  • Heparin should not exceed 10 IU per mL of sample. It can cause bead agglutination and increased signals.
  • DMSO and other similar organic solvents. May cause the leaching of dye from the beads making sample analyte levels undetectable.
  • SDS and other similar denaturing agents. Should not exceed 0.1% in sample. These reagents can cause improper capturing of analytes.

Assay Washing Step Errors

  1. Ensure all assay reagents are removed from the microwells in all wash steps.
  2. For magnetic beads, make sure magnetic separator is in close enough proximity to assay plate to ensure bead retention.

Filter Plate Leaking During the Assay

Many assays (such as BioPlex, and Milliplex) require the use of vacuum filter plates. Though these plates offer many advantages, they are prone to leaking. When this occurs during an assay incubation the results can be highly affected. The following are tips to avoid leaks:

  1. Avoid touching the filter with your pipette as this can puncture the filter or force liquid through the filter prematurely. Instead, pipette on the side of the wells.
  2. After vacuum washes, blot the bottom of the filter plate with lint-free absorbent towels. Do this until the bottom is dry to prevent capillary action which can wick out the well contents.
  3. Do not place the plate on an absorbent surface during incubations. Instead place the plate on a smooth uniform surface to prevent the spouts from touching the surface.
  4. If you are using a plate sealer, be careful not to apply pressure which can force the contents of the well through the filter and out of the well. Gently place the sealing tape on the plate and apply pressure around the perimeter of the sealing tape. Doing so will prevent evaporation, help prevent cross contamination, and will not cause leaks.

Plate Shaking During the Assay

  1. Unless directed otherwise, assay plates should be shaken for the duration of all assay steps to ensure even kinetics.
  2. Shake speed must be high enough to promote assay kinetics (ie keeping Multiplexing Beads in suspension) but low enough to ensure liquid is retained within the wells. 500- 800rpm is recommended on most microplate shakers for standard 300ul wells.

Reagent Issues

  1. Expired reagent lot. Check labels on bottles to confirm that the reagent is not passed its expiry date.
  2. Reagents exposed to extreme temperatures. Specifically for bead multiplexing assays, the beads and detection antibodies must never be frozen. Also, any reagent containing recombinant protein must not be brought to room temperature for extended periods of time. Follow recommended storage conditions for each reagent (usually 4-8° C).
  3. Reagents not brought to room temperature prior to use. This will change reaction rates within the assay.
  4. Incomplete mixing of reagents. If reconstituting a standard cocktail or other reagent from a freeze dried or lyophilized state, make sure that your solution is uniformly mixed by visually inspecting it. Mix all reagents prior to use.

Low Bead Counts

Sometimes, low bead counts can be a factor or a sign of poor duplication. Here are some reasons why low bead counts can occur:

  1. Sample incompatibility with assay. Samples may cause beads to agglutinate thus negatively affecting bead count and signals.
  2. Samples with large amount of particulate.
  3. Highly viscous samples. May need to dilute viscous samples.
  4. Inadequate vortexing and sonication of beads prior to use.

Assay Becomes Less Accurate at Extremes of the Standard Curve.

Some assay standard curves are characterized by ‘flat spots’ at the high and low end of the curve. For samples that have very high or low signals, the corresponding slope of the curve may be insufficient to give an accurate discrimination between them. Diluting the samples that have high signals will bring the samples down to the linear portion of the curve. However, a diluted sample that gets a low signal may become inflated due to the dilution factor multiplication and corresponding flat portion of the curve.

Inaccurate pipetting

a. Calibration needed

  • Calibrating pipettes every six months is standard laboratory practice.

b. Using single channel pipettes exclusively

  • Whenever possible, using multiple channel pipettes is preferred over using single channels. Multiple channel pipettes significantly reduce the number of pipetting steps required in immunoassays which in turn leads to an increase in accuracy reduces the chances of repetitive stress injuries.

c. Using electronic pipette.

  • With many electronic pipettes, when their motors switch from aspirate to dispense the percentage of error is greater in the first dispense.
  • Aspirating a ‘primer’ volume as well as your intended transfer volume will alleviate this issue. First, dispense the primer volume and then continue with your desired transfer.

d. Multi-dispensing

  • When using a pipette with a multi-dispense function, the cumulative percentage error from previous increments is passed on to the last dispense step. For that reason, discard the last pipette volume when multi-dispensing.

e. Aspirate slowly dispense quickly

  • When handling viscous liquids, aspirate and dispense slowly to ensure accuracy. For aqueous liquids aspirate slowly and dispense quickly to ensure proper evacuation from the pipette tip.

f. Poor connection between tip and pipette, faulty tips, dirty tip

• Visually inspect the tips before use to check for proper connection with the pipette, integrity and cleanliness.

g. Poor technician technique

  • Visually inspect tips after aspiration to make sure that the correct amount of liquid it present.
  • Drawing up liquid should be done at a 90 degree angle to the solution. Dispensing should be done at a 45 degree angle with the tip touching the side of the receiving vessel.