USP-chapter85

GEL-CLOT TECHNIQUE

The Gel-Clot Technique is for detecting or quantifying endotoxins based on clotting of the lysate reagent in the presence of endotoxin. The minimum concentration of endotoxin required to cause the lysate to clot under standard conditions is the labeled sensitivity of the lysate reagent. To ensure both the precision and validity of the test, perform the tests for confirming the labeled lysate sensitivity and for interfering factors as described under Preparatory Testing.

Preparatory Testing

Test for Confirmation of Labeled Lysate Sensitivity— Confirm in four replicates the labeled sensitivity, , expressed in EU/mL of the lysate prior to use in the test. The test for confirmation of lysate sensitivity is to be carried out when a new batch of lysate is used or when there is any change in the test conditions that may affect the outcome of the test. Prepare standard solutions having at least four concentrations equivalent to 2, , 0.5, and 0.25 by diluting the USP Endotoxin RS with Water for BET.

Mix a volume of the Lysate TS with an equal volume (such as 0.1-mL aliquots) of one of the Standard Endotoxin Solutions in each test tube. When single test vials or ampuls containing lyophilized lysate are used, add solutions directly to the vial or ampul. Incubate the reaction mixture for a constant period according to the directions of the lysate manufacturer (usually at 37 ± 1 for 60 ± 2 minutes), avoiding vibration. To test the integrity of the gel, take each tube in turn directly from the incubator and invert it through about 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. The test is considered valid when the lowest concentration of the standard solutions shows a negative result in all replicate tests.

The endpoint is the smallest concentration in the series of decreasing concentrations of standard endotoxin that clots the lysate. Determine the geometric mean endpoint by calculating the mean of the logarithms of the endpoint concentrations of the four

replicate series and then taking the antilogarithm of the mean value, as indicated in the following formula:

Geometric Mean Endpoint Concentration = antilog (Σe/f)

where Σe is the sum of the log endpoint concentrations of the dilution series used, and f is the number of replicate test tubes. The geometric mean endpoint concentration is the measured sensitivity of the lysate (in EU/mL). If this is not less than 0.5 and not more than 2, the labeled sensitivity is confirmed and is used in tests performed with this lysate.

Test for Interfering Factors— Usually prepare solutions (A–D) as shown in Table 1, and perform the inhibition/enhancement test on the Sample Solutions at a dilution less than

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the MVD, not containing any detectable endotoxins, operating as described for Test for Confirmation of Labeled Lysate Sensitivity. The geometric mean endpoint concentrations of Solutions B and C are determined using the formula described in the Test for Confirmation of Labeled Lysate Sensitivity.

Table 1. Preparation of Solutions for the Inhibition/Enhancement Test for Gel-Clot

Techniques Endotoxin Concentration/ Solution to which Endotoxin Solutionis AddedNone/Sample SolutionAaBb2/Sample SolutionDiluent—Sample SolutionDilution Endotoxin Number of FactorConcentrationReplicates——41248Cc2/Water for BET248DdaNone/Water for BET——0.50.25—2220.254 Solution A: A Sample Solution of the preparation under test that is free of detectable endotoxins. b Solution B: Test for interference. c Solution C: Control for labeled lysate sensitivity. d Solution D: Negative control of Water for BETThe test is considered valid when all replicates of Solutions A and D show no reaction and the result of Solution C confirms the labeled sensitivity.

If the sensitivity of the lysate determined in the presence of Solution B is not less than 0.5 and not greater than 2, the Sample Solution does not contain factors that interfere under the experimental conditions used. Otherwise, the Sample Solution to be examined interferes with the test.

If the sample under test does not comply with the test at a dilution less than the MVD, repeat the test using a greater dilution, not exceeding the MVD. The use of a more sensitive lysate permits a greater dilution of the sample to be examined and this may contribute to the elimination of interference.

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Interference may be overcome by suitable treatment, such as filtration, neutralization, dialysis, or heating. To establish that the chosen treatment effectively eliminates

interference without loss of endotoxins, perform the assay described above using the preparation to be examined to which USP Endotoxin RS has been added and which has then been submitted to the chosen treatment.

Limit Test

Procedure— Prepare Solutions A, B, C, and D as shown in Table 2, and perform the test on these solutions following the procedure for Test for Confirmation of Labeled Lysate Sensitivity under Preparatory Testing.

Table 2. Preparation of Solutions for the Gel-Clot Limit Test

SolutionABCD*Endotoxin Concentration/Solution to which Endotoxin is AddedNone/Diluted Sample Solution2/Diluted Sample Solution2/Water for BETNone/Water for BETNumber of Replicates2222* Prepare Solution A and the positive product control Solution B using a dilution not greater than the MVD and treatments as for the Test for Interfering Factors under Preparatory Testing. The positive control Solutions B and C contain the Standard Endotoxin Solution at a concentration corresponding to twice the labeled lysate sensitivity. The negative control Solution D consists of Water for BET. Interpretation— The test is considered valid when both replicates of Solution B and C are positive and those of Solution D are negative. When a negative result is found for both replicates of Solution A, the preparation under test complies with the test. When a positive result is found for both replicates of Solution A, the preparation under test does not comply with the test.

When a positive result is found for one replicate of Solution A and a negative result is found for the other, repeat the test. In the repeat test, the preparation under test

complies with the test if a negative result is found for both replicates of Solution A. The preparation does not comply with the test if a positive result is found for one or both replicates of Solution A. However, if the preparation does not comply with the test at a dilution less than the MVD, the test may be repeated using a greater dilution, not exceeding the MVD.

Quantitative Test

Procedure— The test quantifies bacterial endotoxins in Sample Solutions by titration to an endpoint. Prepare Solutions A, B, C, and D as shown in Table 3, and test these solutions by following the procedure in the Test for Confirmation of Labeled Lysate Sensitivity under Preparatory Testing.

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Table 3. Preparation of Solutions for the Gel-Clot Assay Endotoxin Concentration/ Solution to which Endotoxin SolutionDiluentis AddedNone/Sample Water for AaSolutionBETDilution Factor1248Endotoxin Concentration————2Number of Replicates222222BbCc2/Sample Solution2/Water for BET—121280.25Calculation and Interpretation— The test is considered valid when the following three conditions are met: (1) Both replicates of negative control Solution D are negative; (2) Both replicates of positive product control Solution B are positive; and (3) The geometric mean endpoint concentration of Solution C is in the range of 0.5 to 2.

To determine the endotoxin concentration of Solution A, calculate the endpoint

concentration for each replicate by multiplying each endpoint dilution factor by . The endotoxin concentration in the Sample Solution is the endpoint concentration of the replicates. If the test is conducted with a diluted Sample Solution, calculate the

concentration of endotoxin in the original Sample Solution by multiplying by the dilution factor. If none of the dilutions of the Sample Solution is positive in a valid assay, report the endotoxin concentration as less than (if the diluted sample was tested, report as

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less than times the lowest dilution factor of the sample.) If all dilutions are positive, the endotoxin concentration is reported as equal to or greater than the greatest dilution factor multiplied by (e.g., initial dilution factor times 8 times in Table 3).

The preparation under test meets the requirements of the test if the concentration of endotoxin in both replicates is less than that specified in the individual monograph.

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