LAL test

The LAL test or Limulus amebocyte lysate test is a test that is used for the detection and quantification of bacterial endotoxins.[1] There are three basic LAL test methodologies: gel-clot, turbidimetric, and chromogenic. The primary application for LAL is the testing of parenteral pharmaceuticals and medical devices that contact blood or cerebrospinal fluid. In the United States, the FDA has published a guideline for validation of the LAL test as an endotoxin test for such products.[2] The LAL cascade is also triggered by (1,3)-β-D-glucan. Both bacterial endotoxins and (1,3)-β-D-glucan are considered "Pathogen-Associated Molecular Patterns", or PAMPS, substances which elicit inflammatory responses in mammals [3]

Overcoming inhibition and enhancement

One of the most time consuming aspects of endotoxin testing using LAL is pretreating samples to overcome assay inhibition and enhancement.[4] Agents such as EDTA and heparin are known to affect the assay if they are present in sufficient concentrations. All assays, independent of methodology are standardized using endotoxin in water. Therefore, unless the sample is water, some components of the solution may interfere with the LAL test such that the recovery of endotoxin is affected. If the product being tested causes the endotoxin recovery to be less than expected, the product is inhibitory to the LAL test. Products which cause higher than expected values are enhancing. Overcoming the inhibition and enhancement properties of a product is required by the FDA as part of the validation of the LAL test for use in the final release testing of injectables and medical devices. Proper endotoxin recovery must be proven before LAL can be used to release product.[5]

Methodologies

Gel-Clot method

This method is based on the presence or absence of a clot of gel in the sample tube. When proteins are coagulated due to the presence of endotoxins, the gelation occurs. The limit of detection of the tests depends on the manufacturer of the kit containing the Limulus amebocyte lysate reagent. Using this method, the limit of detection is normally between 0.01-0.03 endotoxin units per 0.001 litre of the solution used in the test. This means that a solid gel does not come to be formed below this concentration of endotoxins when moving the test tube. one of the criteria which are used in this method of gelation is to turn the test tube 180 degrees and ascertain that the gel remains intact. This method can be used in a qualitative manner, yielding positive results or negative ones if the gel is not formed. The method can also be used in a semi-quantitative fashion.[6][7]

Turbidimetric method

In this method of LAL test, the turbidity of the solution when the coagulant begins to be formed is measured. The insoluble protein is responsible for the appearance of a gel in the tube of LAL test. Having the patterns of the endotoxin, the turbidity through a spectrophotometrical method can be measured and this measurement allows to quantify the amount of endotoxins that are present.[8] The measurement of the turbidity at a determined period of time can lead to manipulation errors. It has a disadvantage in that the reading cannot be taken correctly in a single sample and therefore the entire process will need to be repeated. Also there is only a single moment to make the measurement and the measurement needs to be precise. If the reading is not taken at the moment indicated, the gelation continues. For these reasons, this method is not used much.[9]

Chromogenic method

The detection of bacterial endotoxins with the chromogenic method in the LAL test can be carried out in two ways: the chromogenic endpoint method and the kinetic chromogenic method. The kinetic chromogenic method is based on the measurement of color at different intervals of time after the addition of the LAL reagent, which contains the chromogenic reagent, to the solution which could contain endotoxins. The advantages of this method are varied: it could be totally automated, it allows for the measurement of many samples in a short period of time and the results are processed easily. The main disadvantage is that many substances could cause interference: those that absorb around 400 nm, which is the zone where you find the absorption band of the p-nitroaniline, those that form chelates or denaturalise the proteins, etc. Moreover, it is needed to avoid using “cloudy” solutions, in order to make the measurement without errors that could be caused by turbidity in the absorbency data that is gathered.[10][11]

References

  1. ↑ Prior, editor, Richard B. (1990). Clinical applications of the Limulus amoebocyte lysate test. Boca Raton, Fla.: CRC Press. p. 112. ISBN 0849362091.
  2. ↑ "The necessity of LAL endotoxin testing". Wako Pyrostar. Retrieved 7 March 2015.
  3. ↑ Tim Sandle (31 August 2013). "Pharmaceutical Product Impurities: Considering Beta Glucans". 16 (5). pp. 16–19. Retrieved 12 March 2015.
  4. ↑ Interference with the LAL Test and How to Address It, LAL Update, October 2005
  5. ↑ Williams, edited by Kevin L. (2007). Endotoxins pyrogens, LAL testing and depyrogenation (3rd ed. ed.). New York: Informa Healthcare. p. 342. ISBN 1420020595. Retrieved 7 March 2015.
  6. ↑ Liu, Shigui; Tobias, Rowel; McClure, Shannon; Styba, Garth; Shi, Qinwei; Jackowski, George (August 1997). "Removal of Endotoxin from Recombinant Protein Preparations". Clinical Biochemistry 30 (6): 455–463. doi:10.1016/S0009-9120(97)00049-0.
  7. ↑ 8 October 2014. "Gel-Clot Method to quantify endotoxins via the LAL test". Retrieved 14 March 2015.
  8. ↑ 27 November 2014. "Limulus Amebocyte Lysate test using the Turbidimetric method". Retrieved 14 March 2015.
  9. ↑ Sakai, Hiromi; Hisamoto, Shuji; Fukutomi, Ippei; Sou, Keitaro; Takeoka, Shinji; Tsuchida, Eishun (February 2004). "Detection of lipopolysaccharide in hemoglobin-vesicles byLimulus amebocyte lysate test with kinetic-turbidimetric gel clotting analysis and pretreatment of surfactant". Journal of Pharmaceutical Sciences 93 (2): 310–321. doi:10.1002/jps.10525.
  10. ↑ Harthug, S.; Bjorvatn, B.; Østerud, B. (July 1983). "Quantitation of endotoxin in blood from patients with meningococcal disease using a Limulus lysate test in combination with chromogenic substrate". Infection 11 (4): 192–195. doi:10.1007/BF01641194. Retrieved 27 March 2015.
  11. ↑ "The Detection of Endotoxins Via the LAL Test, the Chromogenic Method". Pyrostar. Retrieved 27 March 2015.