TITLE: BACTERIAL ENDOTOXINS
AUTHORS: Jiaxin Li

[STEPS]
?. [Confirmation of the labeled lysate sensitivity]
Confirm in 4 replicates the labeled sensitivity λ, expressed in IU/mL, of the lysate solution prior to use in the test. Confirmation of the lysate sensitivity is carried out when a new lot of lysate is used or when there is any change in the test conditions which may affect the outcome of the test.
?. [Confirmation of the labeled lysate sensitivity]
Prepare standard solutions of at least 4 concentrations equivalent to 2λ, λ, 0.5λ and 0.25λ by diluting the standard endotoxin stock solution with water for BET.
?. [Confirmation of the labeled lysate sensitivity]
Mix a volume of the lysate solution with an equal volume of 1 of the standard solutions (such as 0.1 mL aliquots) in each tube. When single test vials or ampoules containing lyophilized lysate are employed, add solutions of standards directly to the vial or ampoule. Incubate the reaction mixture for a constant period according to the recommendations of the lysate manufacturer (usually at 37±1℃ for 60±2 min), avoiding vibration.
?. [Confirmation of the labeled lysate sensitivity]
Test the integrity of the gel: for tubes, take each tube in turn directly from the incubator and invert it through approximately 180° in one smooth motion. If a firm gel has formed that remains in place upon inversion, record the result as positive. A result is negative if an intact gel is not formed.
?. [Confirmation of the labeled lysate sensitivity]
The test is considered valid when the lowest concentration of the standard solutions shows a negative result in all replicate tests. The end-point is the lowest concentration in the series of decreasing concentrations of standard endotoxin that clots the lystae. Determine the geometric mean end-point concentration by calculating the mean of the logarithms of the end-point concentrations of the 4 dilution series, take the antilogarithm of this value, as indicated by the following expression: Geometric mean end-point concentration ＝log-1(Σe/f)Σe = sum of the log end-point concentrations of the dilution series used,f = number of replicates.
?. [Confirmation of the labeled lysate sensitivity]
The geometric mean end-point concentration is the measured sensitivity of the lysate solution (IU/mL). If this is not less than 0.5λ and not more than 2λ, the labeled sensitivity is confirmed and is used in the test performed with this lysate.
?. [Test for interfering factors]
Prepare solutions A, B, C and D as shown in Table 1, and use the test solutions at a dilution less than the MVD, not containing any detectable endotoxins, operating as described under (i) Confirmation of the labeled lysate sensitivity.  ABCDEF1Solution Endotoxin concentration/solution to which endotoxin is addedDiluent Dilution factorEndotoxin concentrationNumber of replicates2ANone/Test solution---43B2λ/Test solutionTest solution12λ44   21λ45   40.5λ46   80.25λ47C2λ/Water for BETWater for BET12λ28   21λ29   40.5λ210   80.25λ 211DNone/Water for BET---2
ABCDEF1Solution Endotoxin concentration/solution to which endotoxin is addedDiluent Dilution factorEndotoxin concentrationNumber of replicates2ANone/Test solution---43B2λ/Test solutionTest solution12λ44   21λ45   40.5λ46   80.25λ47C2λ/Water for BETWater for BET12λ28   21λ29   40.5λ210   80.25λ 211DNone/Water for BET---2
?. [Test for interfering factors]
The test is considered valid when all replicates of solutions A and D show no reaction and the result of solution C confirms the labeled lysate sensitivity.If the sensitivity of the lysate determined with solution B is not less than 0.5λ and not greater than 2λ, the test solution does not contain interfering factors under the experimental conditions used. Otherwise, the test solution interferes with the test.
?. [LIMIT TEST]
Prepare solutions A, B, C and D as shown in Table 2.  ABC1Solution Endotoxin concentration/Solution to which endotoxin is addedNumber of replicates2ANone/Diluted test solution23B2λ/Diluted test solution24C2λ/Water for BET25DNone/Water for BET2
ABC1Solution Endotoxin concentration/Solution to which endotoxin is addedNumber of replicates2ANone/Diluted test solution23B2λ/Diluted test solution24C2λ/Water for BET25DNone/Water for BET2
?. [Interpretation]
The test is considered valid when both replicates of solution B and C are positive and those of solution D are negative.
?. [Interpretation]
When a negative result is found for both replicates of solution A, the preparation being examined complies with the test.
?. [Interpretation]
When a positive result is found for both replicates of solution A, the preparation being examined does not comply with the test.