Ph. Eur. 2.6.14. recommends three principal Limulus Amebocyte Lysate (LAL) techniques: gel-clot, turbidimetric and chromogenic.
Precisely, there are 6 methods:
Method A: Gel-clot method: limit test
Method B: Gel-clot method: quantitative test
Method C: Turbidimetric kinetic method
Method D: Chromogenic kinetic method
Method E: Chromogenic end-point method
Method F: Turbidimetric end-point method
Are the methods comparable when they are used for the analysis of biotechnological preparations?
A comparison study is attached. Based on Fig.3, it seems that the results depend on the method (in Fig 3, EC-6 is the current FDA Reference Endotoxins Standard).
I have been using Charles River System with LOL kinetic assay ( option D). It is working really good and strongly recommend this method for accurate quantification of endotoxin.
Thanks a lot.
However, it is also interesting to compare the performance of the six methods, otherwise it will be difficult to compare the results (e.g. doing a meta-analysis).
The regulatory agency EMA (European Medicines Agency) recommends the Ph. Eur. 2.6.14, without specifying the method, therefore, comparison of the studies it is the main issue.
Thanks a lot Ana.
I am still not sure about the performances of different methods, I have not found data. The final decision based on gel-clot limit test seems an historical reference, instead of a reference to a golden standard method (methods better performing).
Dear Sir. Concerning your issue about the performances of the bacterial endotoxins test . The Bacterial Endotoxins Test (BET) is a test to detect or quantify endotoxins from Gram-negative bacteria using amoebocyte lysate from the horseshoe crab (Limulus poly- phemus or Tachypleus tridentatus). There are three techniques for this test: the gel-clot technique, which is based on gel formation; the turbidimetric technique, based on the development of turbidity after cleavage of an endogenous substrate; and the chromogenic technique, based on the development of color after cleavage of a synthetic peptide-chromogen complex. Proceed by any of the three techniques for the test. In the event of doubt or dispute, the final decision is made based upon the gel-clot technique unless otherwise indicated in the monograph for the product being tested. The test is carried out in a manner that avoids endotoxin contamination.For more information, ,the following below attached file may help you in your analysis:
Thanks
Thanks a lot for the clarification and the official test provided.
It would help if there were information on comparative results, e,g, testing the same sample with the different techniques to assess analytical biases.
It would be good to write an article by gathering comparative data for selected samples. This can be achieved through collaboration. I am keen to test samples if someone else also open for collaboration to do a collective paper.
Please respond here to take it further.
Cheers
Jagan
Thanks Jagan, it is an interesting proposal. I can launch further this idea, other people can also be interested. From my side, I am involved in validation and evaluation of analytical methods (more statistical part than laboratory). Now, I am interested in this part of the proposal.
Thanks Roberto. We need 10 to 15 people from different countries to make this work successful. Please respond those who are keen on this proposal.
Regards
Jagan
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