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Common Testing Problems

Inhibition

Inhibition is characterized by a Positive Product Control (PPC) being recovered at less than 50% in a chromogenic or turbidimetric assay or a failure of the 2λ PPC to clot. Inhibition can also cause a result to be lower than it should be. There are several possible causes for this:

  • The pH. The LAL assay works best between pH 6.0 to 8.0. The further the pH of the testing solution is from this range, the more inhibition will be seen. Check the pH of the sample to make sure it is within the proper range. If the sample is close, addition of Pyrosol, a buffer agent, will help bring the sample into the right range. Other samples will require neutralization.
  • Organic solvents. Organic solvents inhibit the assay by inhibiting the activity of enzymes. Dilute the sample with water.
  • Precipitates. Precipitates will adversely affect the chromogenic assay because the light path will be blocked. Dilute the sample.
  • Salt. If the concentration is too high, the enzymes of the assay will be unable to work efficiently. Dilute the sample.

 

Enhancement

Enhancement is characterized by a Positive Product Control (PPC) being recovered at more than 200% in a chromogenic or turbidimetric assay. Most gel-clot assays cannot detect enhancement. In the Inhibition-Enhancement assay, enhancement in a gel-clot assay is assumed when the 0.25λ Product Standard clots. Enhancement can also cause results to be reported as higher than they should be. There are several possible causes for this:

  • Proteolytic enzymes. The LAL assay is a series of serine protease enzymes. Serum, plasma, and other biological samples may have enzymes that can cause the reaction to proceed, even when no endotoxin is present. An example is Trypsin. Heat the sample to denature the enzymes is an easy way to remove this interference.
  • Glucans. (1→3)-ß-D-glucans may enhance the assay by activating an alternative pathway in the LAL assay. Glucans are biologically active compounds that can cause effects similar to endotoxin. There are two ways to determine if glucans are causing enhancement. One is to use an agent that blocks glucan. If the endotoxin result is lower, then glucan was causing enhancement. Two is to use Glucatell™ which is specific for (1→3)-ß-D-glucans and can quantify the amount of glucans present in the sample.
  • Surfactans.Surfactants can reduce the size of endotoxin aggregates, thereby making the endotoxin standard in the PPC tube appear as if there were a higher level of endotoxin present. Conversely, too much surfactant can inhibit the ability of enzymes to work efficiently, which will appear as inhibition. Dilute the sample.