Limulus amoebocyte lysate (LAL) test
1.     Purpose:-                         

  To lay down a procedure is to provide guidelines for Bacterial Endotoxin Test. 
      2. Scope:-

       This procedure is applicable for Bacterial Endotoxin Test.

3. HSE Statement:-

§     Gloves
§     Face Mask

4. Responsibilities:-

                               i)      Manager Quality Control is responsible to ensure that procedure & formats are followed entirely as approved.
                             ii)        Microbiologist is responsible to perform the test.
5. Materials Required:-
      5.1 Dry Heat Oven having the capacity for depyrogenation of glassware at 2000C
5.2 Incubator (37+10C)
5.3 Refrigerator and Freezer (2-80C and -200C)
5.4 Test Tube Rack
5.5 Micropipette
5.6 Pyrogen or Endotoxin free micro tips (100ml)
5.7 Reaction tubes (10 X 75mm, flint (soda lime) glass tube)
5.8 Vortex Mixer
5.9 Depyrogenated Glass Pipette (1.0ml, 2.0ml and 5.0ml)
5.10 Reference Standard Endotoxin (RSE of E. coli 055:B5) having strength of 10,000EU/ml Limulus amoebocyte Lysate (LAL) reagent having sensitivity of 0.25EU/ml
5.11 Endotoxin Free Water (LAL Reagent Water or LRW)
5.12 Aluminum foil    
6. Definitions:-

6.1 Bacterial Endotoxin Test:-
                                            This is a pharmacopeial test for the detection of Gram-negative endotoxins using the LAL methodology.
6.2 Gel Clot Limit Test:-
                                    The gel clot limit test is a qualitative test for the detection of gram-negative bacterial
      endotoxin.The assay is conducted by mixing LAL with test specimen followed by incubating the
      mixture undisturbed for 60 minutes at 370C .If a gel has formed and remains intact after inversion of
      1800, test is positive, indicating that the endotoxin concentration of test specimen is greater or equal to
       LAL’s labeled sensitivity. If intact gel has not formed test is negative.
6.3 PFW:-
               Pyrogen Free Water
6.4 CSE:-
               Control Standard Endotoxin
6.5 MVD:-
                 Maximum Valid Dilution
7.Flow Chart:-

 8. Description:-
 8.1 Preparation of Apparatus
 8.1.1 Thoroughly wash reaction tubes and glass pipettes with chromic acid solution.
 8.1.2 Rinse with tap water for least 3 minutes and then with purified water for at least 3 minutes.
 8.1.3 Allow it to dry, wrap with aluminum foil and depyrogenate at 2000C for 2 hours. 
8.2 Reconstitution of Reference Standard Endotoxin
8.2.1 Keep the vial at room temperature for at least 05 minutes.
8.2.2 Tap the base and top of vial to minimize the powder adhering with rubber stopper of vial.
8.2.3 Disinfect the seal and rubber stopper of the vial with 70% filtered IPA and aseptically open the vial.
         Do not insert LRW through the rubber stopper.
8.2.4  Reconstitute RSE vial with LRW by using depyrogenated glass pipette as per manufacturer’s instructions to obtain 10,000EU/ml i.e. E10,000.
8.2.5  Vortex vigorously for 30 minutes and if not in use, preserve the RSE reconstituted vial at 2-80C for not more than 14 days. And before use mix vigorously not less than 3 minutes.
8.2.6  Prepare serial dilutions of RSE with LRW in depyrogenated assay tubes to obtain Control Standard Endotoxin (CSE) as follows:
              i.      Aseptically add 0.1ml of E10, 000 in 0.9ml of LRW to obtain 1,000EU/ml i.e. E1, 000.
            ii.      Aseptically add 0.1ml of E1, 000 in 0.9ml of LRW to obtain 100EU/ml i.e. E100.
          iii.      Aseptically add 0.1ml of E100 in 0.9ml of LRW to obtain 10EU/ml i.e. E10. 
          iv.      Aseptically add 0.1ml of E10 in 0.9ml of LRW to obtain 1EU/ml i.e. E1.
            v.      Aseptically add 0.5ml of E1 in 0.5ml of LRW to obtain 0.5EU/ml i.e. E0.5.
          vi.       Aseptically add 0.5ml of E0.5 in 0.5ml of LRW to obtain 0.25EU/ml i.e. E0.25 or 2λ.
        vii.      Aseptically add 0.6ml of E0.25 in 0.4ml of LRW to obtain 0.125EU/ml i.e. E0.15 or λ.
      viii.      Aseptically add 0.5ml of E0.15 in 0.5ml of LRW to obtain 0.075EU/ml i.e. E0.075 or λ/2.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       
          ix.      Aseptically add 0.5ml of E0.075 in 0.5ml of LRW to obtain 0.0375 EU/ml i.e. E0.0375 or λ/4.                                           
               8.2.7 Vortex each dilution for at least 30 seconds before proceeding or making next dilution.
8.2.8 Do not store the CSE dilutions due to loss of activity by adsorption. CSE dilutions must be made immediately before use.
8.3     Reconstitution of Limulus amoebocyte Lysate (LAL) 
8.3.1        Keep the ampoule at room temperature for at least 05 minutes.
8.3.2        Tap the base and top of ampoule to minimize the powder adhering with ampoule.
8.3.3        Disinfect the seal of the ampoule with 70% filtered IPA and aseptically open the ampoule.
8.3.4        Reconstitute LAL reagent with LRW by using depryrogenated glass pipette as per manufacturer’s instructions. Mix the contents gently. Don’t vortex to avoid foaming.
8.3.5        Confirm the sensitivity of lysate as per SOP.
8.4       Performing the test
8.4.1        Prepare the assay solutions as follows:
Prepare Maximum Valid Dilution (MVD) of sample in LRW by using following formula.
         MVD (Sample): Endotoxin Limit in EU/ml   OR   Endotoxin Limit in EU/mg x Product Concentration
                                                 l                                                                         l
·               To prepare the MVD, dilute sample with LAL Reagent Water.  This will be the solution “A” or Assay sample.
·               To prepare solution B, aseptically add 0.1ml of 2λ and 0.1ml MVD diluted sample “A”
solution in depyrogenated assay or reaction tube. This will be Positive Product Control.
·               Solution C contains only 2λ potency (twice the sensitivity of lysate) of endotoxin. This will be Positive control.
·               Solution D contains only LRW. This will be Negative control.
8.4.2        Aseptically add 100ml of solution A, B, C and D into 100ml dispensed lysate separately and label appropriately. Mix gently up to 30 seconds to uniformly mix up the contents of the tube.
8.4.3        Run all the reactions in duplicates.
8.4.4        Incubate all assay tubes at 37+10C for 60+2 minutes. Do not disturb the reaction tubes during incubation.
8.4.5        Note the formation of gel in all reaction tubes after incubation.

8.5      Reading and recording the results
8.5.1        After incubation, if the gel forms and remains intact when the tube is inverted through 1800 in one smooth motion indicates a positive reaction.
8.5.2        A negative reaction is characterized by the formation of such gel that does not maintain its integrity when the tube is inverted through 1800 in one smooth motion.
8.5.3        The test is not valid unless
·               Both replicates of Solution B are positive.
·               Both replicates of Solution C are positive.
·               Both replicates of Solution D are negative.
8.5.4        The preparation under test complies with the test when a negative result is found for both tubes containing solution A.
8.5.5        The preparation under test does not comply with the test when a positive result is found for both tubes containing solution A.
8.5.6        Repeat the test when a positive result is found for one tube containing solution A and a negative result for other one.
8.6. Specifications:-
8.6.1 Both replicates of Solution A should be negative.
8.6.2 Both replicates of Solution B should be positive.
8.6.3 Both replicates of Solution C should be positive.
8.6.4 Both replicates of Solution D should be negative.
9. Records:-

9.1 Sterility and LAL test Log               
9.2 LAL Test (Gel Clot Limit Method)   

10. References:-
       Unites States Pharmacopeia 35

11. Distribution:-

This SOP has to be distributed in below mentioned Departments:-

Sr. NO
Distributed to
Quality Control Department

Quality Management Department

12. Revision History:-

Date                                        Changes


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