LIMULUS AMEBOCYTE LYSATE TEST

LIMULUS AMEBOCYTE LYSATE TEST LAL TEST

1. LIMULUS AMEBOCYTE LYSATE TEST

2. ENDOTOXINS The toxic activity of LPS was first discovered and termed “endotoxin” by Richard Friedrich Johannes Pfeiffer. Endotoxins are part of the outer membrane of the cell wall of Gram- negative bacteria. These are invariably associated with Gram- negative bacteria whether the organisms are pathogenic or not. These are important because it is a pyrogen and survive sterilization. Injectable drugs and medical devices which will contact blood or spinal fluid are tested for endotoxins. They are thermostable, water- soluble, unaffected by the common bactericides, non- volatile and liberated when bacteria die and cell wall breaks apart. Consequences of endotoxin contamination:- Fever, Headache, Chills, Nausea/ Vomiting, Hypotension, Acute lung injury, Miscarriage, Death.

3. LAL TEST (BACTERIAL ENDOTOXIN TEST) LIMULUS:- Genera of horseshoe crab ( Limulus polyphemus ) AMEBOCYTE:- Crab blood cell from which active component is derived. LYSATE:- Component is obtained by separating amebocytes from the plasma and then lysing them. Developed in 1960’s by Drs. Bang and Levin. To measure the concentration of endotoxins of Gram- negative bacterial origin. Based on clotting reaction of horseshoe crab blood to endotoxin.

4. Principle The test is based on the primitive blood- clotting mechanism of the horseshoe crab. The addition of a solution containing endotoxins to a solution of the lysate produces turbidity, precipitation or gelation of the mixture. The rate of reaction depends on the concentration of endotoxin, the pH and the temperature. The reaction requires the presence of certain bivalent cations, a pro-clotting enzyme system and clottable proteins all of which are provided by the lysate. Gram-negative bacterial endotoxin catalyzes the activation of proenzyme in the LAL. The initial rate of activation is determined by the concentration of endotoxin present. The activated enzyme (coagulase) hydrolyzes specific bonds within a clotting protein (coagulogen) also present in LAL. Once hydrolyzed, the resultant coagulin self- associates and forms a gelatinous clot.

5. LYSATE REAGENT Bleeding adult crabs blood into an anticlotting solution. W ashing and centrifuging to collect the amoebocytes. L ysing in 3% NaCl L ysate is washed and lyophilized for storage.

6. 3 DIFFERENT TECHNIQUES GEL- CLOT TECHNIQUE Gel formation CHROMOGENIC TECHNIQUE The development of color after cleavage of a synthetic peptide- chromogen complex. 01 02 03 T URBIDIMETRIC TECHNIQUE T he development of turbidity after cleavage of an endogenous substrate

7. GEL CLOT METHOD A solid gel is formed in the presence of endotoxins. This technique requires positive and negative controls. Positive controls- a known concentration of endotoxin added to the lysate solution. Negative controls- water, free from endotoxins, added to the lysate solution.

8. TURBIDIMETRIC METHOD The test is based on the measurement of opacity change due to the formation of insoluble coagulin. Opacity is directly proportional to the endotoxin concentration This technique is used for water systems and simple pharmaceutical products. The specimen is incubated with LAL and either the rate of increase in turbidity or the time taken to reach a particular turbidity is measured spectrophotometrically and compared to standard curve.

9. CHROMOGENIC METHOD This is based on the measurement of color change which is caused by the release of the chromogenic chemical p-nitroanilide. The quantity of the p-nitroanilide produced is directly proportional to the endotoxin concentration. Endotoxin catalyzes the activation of a proenzyme in LAL which will cleave a colorless substrate to produce a colored end product which can be measured spectrophotometrically and compared to a standard curve

10. PROCEDURE F ormation of solid gel clot that withstands inversion of the tube constitutes a positive test Equal volume of test solution and LAL reagent are mixed in glass test tubes After incubation at 37° C for 1 h, the tubes are observed for clot formation after inverting them. 01 03 02

11. RESULT

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