Endotoxin Detection by Gel-Clot Methods

Gel-Clot Methods

Methods A and B depend on the formation of a firm gel when a solution containing bacterial endotoxins is incubated after mixing with the lysate. Method A is conducted as a limit test wherein both the replicate solutions of the preparation under examination must contain endotoxin in the concentration less than the endotoxin limit concentration specified in the individual monograph. Method B determines the endotoxin concentration semi-quantitatively in the preparation under examination.

Sensitivity of the lysate

Confirm the labeled sensitivity of each new batch of lysate prior to use in the test using at least one vial of each batch of lysate. Prepare a series of dilutions of CSE to give concentrations of 2λ, λ, 0.5λ and 0.25 λ, where λ is the labeled sensitivity of the lysate in EU per ml. Perform the test as given under Method on these four standard concentrations in duplicate and include a negative control consisting of water BET. At least the final dilution in each series must give a negative result.

Calculate the average of the logarithms of the lowest concentration of endotoxin in each series of dilutions for which a positive result is found. The geometric mean end-point concentration is the measured sensitivity of the lysate in ED/ml, which is calculated using the following expression:

Geometric mean end-point concentration = antilog (∑e/f)
Where, ∑e = sum of the log end-point concentrations of the series of dilutions used
f = number of replicate test-tubes.
This average gives the estimated lysate sensitivity which must lie between 0.5λ and 2λ.
Related: Bacterial Endotoxin Test (BET or LAL Test) Validation

Test for interfering factors

The possibility of interference with the bacterial endotoxins test by certain factors should be borne in mind. For validation of the test results, it must be demonstrated that the test preparation does not inhibit or enhance the reaction or otherwise interfere with the test. The validation must be repeated if the lysate vendor or the method of manufacture or the formulation of the sample is changed.
Dilution of the test preparation with water BET is the easiest method for overcoming inhibition.

The allowable dilution level or Maximum Valid Dilution (MVD) is dependent on the concentration of the product, the endotoxin limit for the product and the lysate sensitivity. It is calculated by the following expression:

MVD = Endotoxin limit x Concentration of the test solution
                                                        λ
Preparation of test solutions. Prepare replicates of solutions A to D as indicated in the table.

Solution A = Solution of the product at a dilution at or below MVD (test solution).
Solution B = Test solution spiked with indicated CSE concentrations (Positive Product Control; PPC).
Solution C = Standard solution with indicated CSE concentrations in water BET.
Solution D = Water BET (Negative Control; NC).

Method 

Carry out the following procedure in receptacles such as tubes, vials or wells of micro-titre plates. Into each of the chosen receptacle, add an appropriate volume of negative control (NC), standard CSE solutions in water BET, test solution and positive product control (PPC). At intervals that will permit the reading of each result, add to each receptacle an equal volume of the appropriately constituted lysate unless single test vials are used. Mix the sample-lysate mixture gently and place in an incubating device such as a water-bath or a heating block, accurately recording the time at which the receptacles are so placed. Incubate each receptacle at 37°± 10 undisturbed for 60 ± 2 minutes. Remove the receptacles and examine the contents carefully. A positive reaction is characterized by the formation of a firm gel that retains its integrity when inverted through 180° in one smooth motion.

Record this result as positive (+). A negative result is characterized by the absence of such a gel or by the formation of a viscous gel that does not maintain its integrity. Record such a result as negative (-). Handle the receptacles with care to avoid subjecting them to unwanted vibrations as false negative observations may result.

Calculate the geometric mean end-point concentration of solutions of series Band C by using the formula described under sensitivity of the lysate.

Calculation and interpretation of results

The test for interfering factors is valid if
(a) solutions of series A and D give negative results;
(b) the results obtained with solutions of series C confirm the labeled sensitivity of the lysate;
(c) the geometric mean of the end-point concentration of solutions of series B is not more than 21 or not less than 0.51.

If the result obtained is outside the specified limit, the test preparation under examination is acting as an inhibitor or activator. The interfering factors may be eliminated by further dilution (not greater than MVD), filtration, neutralization, inactivation or by removal of the interfering substances. The use of a more sensitive lysate permits the use of greater dilution of the preparation under examination. Ultrafiltration may be used, if necessary, when the interfering factor passes through a filter with a nominal separation limit corresponding to a molecular weight of 10,000 to 20,000, such as asymmetrical membrane filters of cellulose triacetate. 

Such filters should be checked for the presence of any factors causing false positive results. The material retained on the filter, which contains the endotoxins, is rinsed with water BET or tris-chloride buffer pH 7.4 BET. The endotoxins are recovered in the water BET or the buffer. The endotoxin concentration in the test volume and the final volume are determined for each preparation under examination.

Establish that the chosen treatment effectively eliminates interference without removing endotoxins by repeating the test for interfering factors using the preparation under examination to which the CSE has been added and which has been submitted to the chosen treatment.
Also see:
Method A. Gel-Clot Limit Test Method
Method B. Semi-quantitative Gel-Clot Method

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