LAL Test

LAL TEST(Limulus Amebocyte Lysate):

Introduction:

Limulus Amebocyte Lysate(LAL) is an aqueous extract of blood cells ( amebocytes) from th horseshoe crab, Limulus polyphemus  .In December 1987, the U.S Food and Drug Administration (FDA)          published the Guidelines on the validation of the Limulus Amebocyte Lysate test as an end-point endotoxin test for human & animal parenteral drugs., biological products & medical devices. The LAL test for pyrogen in parenterals was first applied by Cooper  et  al . The LAL test was found to be more accurate & time saving than pyrogen test.

Definition: 

LAL(Limulus Amebocyte Lysate) Test is the most sensitive and specific means to detect and measure endotoxin, a fever producing byproduct of  gram negative bacteria such as E.coli and Pseudononas commonly known as pyrogen. Endotoxin produces an opacity and gelation in LAL. The simplicity and economy of the LAL test encourages the testing of in- process solutions and raw materials, The LAL test is used as the replacement for the rabbit pyrogen test.

Principle:

Proenzyme  ----->  Endotoxin  -----> Coagulase

Coagulagen  -----> Coagulase  -----> Coagulin

Gram-negative bacterial endotoxin catalyzes the activation of proenzyme in the Limulus Amebocyte Lysate.  The concentration of endotoxin present  is determine the initial rate of activation . The activatied enzyme ( Coagulase ) hydrolyzes specific bonds within a clotting protein ( Coagulogen ) also present in Limulus Amebocyte Lysate . Once hydrolyzed, the  resultant coagulin self associates and forms a getatinous  clot .

 

Methods of Lat Test:

There are five methods of LAL test:

1)      Gelation Method : Limit Test

2)      Semi quantitative Getation Method

3)      Turbidimetric  Kinetic Method

4)      Chromogenic peptide Kinetic Method

5)      Chromogenic peptide endpoint Method .

Gelation  Method:

The Gelation or Gel–Clot LAL test Method  is a simple reproducible test that is conducted by mixing equal parts of Endosafe. LAL reagent and test specimen and promptly incubating the mixture in disturbed for 60 minutes at 37 °C . A positive response indicates that there is an amount of endotoxin in the sample which equals or exceeds the reagents labeled sensitivity.

Test Procedure :

1)   Add 0.1 ml /0.25 ml of each dilution to the assay tube ; test at least in duplicate.

2)   Add 0.1ml /0.25 ml of reconstituted standard to each tube starting with the – ve control & ending with the highest endotoxin concentration.

3)   Place the reaction tube in a 37°C water or dry bath for 60 ± 2minutes

4)   Observe the tubes for Gelatin . If gel is formed which retains after turning the tubes to 180°C, then the test is +ve for endotoxin . And if the gelation does not take  place, the test is – ve for endotoxin presence.

Conclusion:

As LAL is capable of detecting a very minute amount of endotoxin in product, hence it is now widely accepted throughout the world  in instead Pyrogen test.