What is the role of LAL, LRW and CSE in Bactiral Endotoxin Testing?

LAL :

• Limulus Amoebocyte Lysate (LAL) is an aqueous extract of blood cells (amoebocytes) from the Atlantic horseshoe crab, Limulus Polyphemus.
• Amoebocyte acts coagulation mechanism.
• Anti aggregation agent must be added to inhibit the aggregation N-Ethylmaleimade.
• LAL mainly used for detection and quantification of bacterial endotoxins.
• LAL reacts with bacterial endotoxin lipopolysaccharide(LPS), which is a membrane component of Gram negative bacteria.
• Frederick Bang reported in 1956 that Gram negative bacteria, even if killed will cause the blood of the horseshoe crab to turn in to a semi-solid mass.
• Amoebocytes contains granules with a clotting factor known as coagulogen.
• In 1970 the U.S. Food and Drug Administration (FDA) approved LAL for testing drugs, product and devices that come in contact with the blood. prior to that date, much slower and more expensive tests on rabbit test  had been used for this purpose.
• LAL test useful of identification and detection of endotoxins.

How to get the LAL?

• Collect big size of healthy horseshoe crabs.
• Remove  the blood by using of Syringe.
• Separate the white blood cells (serum) consist of Amoebocyte by using centrifugation.
• Amoebocytes is then freeze- dried for further process.
• Horseshoe crab (only blood removed) blood doner.
• 500 mL blood (serum) = 10 ng/vial 
• LAL made of Amoebocyte/Gametocyte of L-polyphemus 1.5 % v/v of 25 % human serum albumin (stabilizer), 3 % Nacl and other appropriate ions.
• LAL have ( sodium ions, magnesium ions) buffer for automatic pH adjustment purpose.

LAL Reagent:

• different sensitivity of the  Limulus Amoebocyte Lysate (LAL) available in market.
• Choose one of the following LAL reagent (0.25, 0.125, 0.0625, 0.03125, 0.015625 EU/mL).
• Always LAL reagent detect on endotoxins above on  LAL sensitivity. (Example 0.25 EU/mL Lysate TS (Limulus Amoebocyte Lysate Test Solution) sensitivity detect on above 0.25 EU/mL only, not detected on 0.03 EU/mL) 
• Re hydrate immediately prior to testing ( 5 minutes to allow to come to room temperature and dissolve).
  
• Do not vortex Limulus Amoebocyte Lysate, because Lysate have sensitivity protein that protein is break down.
• Cover with an endotoxin -free surface if not in use.
• Stored the LAL recommended as per manufactures.

LAL Reagent Water (LRW):

• LAL Reagent Water (LRW) is sterile purified water prepared by distillation or reverse osmosis.
• LAL Reagent Water (LRW) is equivalent to water for bacterial endotoxin test.
• Its contains <0.005 EU/ml Endotoxin(Charles river),<0.001 EU/ml Endotoxin(cape cod).

Controlled Standard Endotoxin (CSE) Reagent:

• Re-hydrate  following lot specific EU/ng ratio.
• Vortex vial for 5 minutes( As for manufactures recommendation)the first use and 1 minute for subsequent. usage.
• Vortex each dilution for 30 seconds and use dilutions as soon as possible.
• Storage of dilutions is only permitted based upon stability studies.
• Store Re-hydrate vial in refrigerator not the freezer.
• US Reference Standard Endotoxin (RSE) E.Coli -  O113 : H10 : K0

Combination of LAL and CSE:

• While LAL/ CSE Lots Remained the same Reaction times for each standard changed (verify the Co-A).
• CSE is freeze dried powder is available 10 EU/ng to 1000 EU/ng ampoules.
• CSE is intended for use to construct standard curves.
• This test use in-vitro laboratory purpose only, not for use in human and animals.
• The potency of CSE is referenced against the USP Referenced standard Endotoxin (RSE).

EU/ng Ratio:

• Referenced standard Endotoxin (RSE)------- convert------Controlled Standard Endo toxin (CSE).
• EU (Endotoxin Units)  = IU ( International Units).

EU/ng    X      ng/Vial   = EU/vial

EU/vial / mL vial  = EU/mL

Example : 1 

RSE = 10 EU/ng       Potency  = 10ng/Vial

=   10 EU/ng   X  10 ng/Vial   = 100 EU/Vial

=100 EU/Vial/ 5 mL of Vial  = 20 EU/mL

Example : 2

CSE = 0.5 ug/vial   (0.5 ug/vial x 1000 =500 ng/vial)      Potency : 14 EU/ng

 = 14 EU/ng x 500 ng/vial = 7000 EU/vial 

=    7000 EU/ml/ 5 ml LRW= 1400 EU/ml 

USP Reference to Glucans:

• Amoebocyte Lysate reacts to some beta -Glucans in addition to endotoxins. Amoebocyte Lysate preparation that do not react to glucans are available. They are prepared by removing the G-Factor reacting to glucans from Amoebocyte Lysate or by inhibiting the G-Factor reacting system of Amoebocyte Lysate and may used for endotoxin testing in the presence of glucans.
• Only (1-3) β-D Glucans have high reactivity with LAL.
• Because most  β-D Glucans are not soluble in water, some are soluble in alkaline solution (NaOH), DMSO.
• Most glucans interference can be eliminated using ES- Buffer to re-hydrate the LAL reagent.
• Use ES-Buffer containing β-D Glucans blocker.

Dilutions:

• Dilutions can be calculated as follows.
  
  
  Dilutions =   What we have  / What we want
  
  
  Example :  Need 2 EU/mL from 20 EU/mL tube make an 10 fold dilution.
  
  
  = 20 EU/mL / 2 EU/mL  
  
  
  = 10 Dilution
  
  
  1mL of sample  add 9 mL of diluent (LRW) this dilution is called 10 fold dilutions.
  
  

  

  
  

  Dilutions Verification:

   = Total Volume of the Solution ( Sample + Diluent) / Volume of the sample
  
  
  = 1 ml + 9mL  / 1     = 10
  
  
• Related: Gel- Clot Test