Sunday, 22 October 2017

Gel- Clot Test

  • 1977 FDA grants approval of first commercial LAL (Limulus  Amybiocyte Lysate) reagent.
  • Gel-clot technique based on the gel formation.

  • Kinetic turbimetric based on the turbidity.
  • Kinetic chromogenic based on the colour formation.
  • "Frederick Bag" Gel-clot method is developed.
  • Gel-clot method is pass or fail test.
  • LAL (Limulus  Amybiocyte Lysate) + Endotoxin  = Clotting
  • LAL test is in-vitro test for semi quantitative analysis of endotoxin ( it means given the results less than or more than of specified quantity).
Apparatus:
  • Depyrogenated dilution tubes(16 X 100 mm)
·         Depyroginated Assay tubes(10 X 75 mm), (polystyrene test tubes)

·         Calibrated Micropipettes.

·         Dipyrogenated Tips( Endotoxin free pipette tips)

·         pH Indicator

·         calibrated stop watch

Equipment:

Calibrated Heating Block.

Materials:

·         CSE (Controlled Standard Endotoxin)

·         LAL ( Limulus  Amybiocyte Lysate)

·         LRW ( LAL Reagent Water)

·         Test sample

Sample Preparation :

  • Dispense 100 µL of sample and 100 µL of LAL  in to 10 x 75 mm assay tubes ( Endotoxin free dilution tubes- Borosilicate glass or equivalent  polystyrene plastic).
  •  Mix the sample solution.
pH Checking:

·         The LAL reaction requires a neutral pH and is time and concentration dependent.

·         Sample and Lysate mixture take with the help of the depyrogenated tip  (Measure pH with one to one ratio of sample dilution and LAL to obtain an accurate pH reading).

·         Put the one drop on pH indicator

·         Read the pH

·         Lysate having some pH adjustment buffers .

·         Not maintain in the pH , please add the sterile 0.1N Tri-NaoH, 0.1 NTri-Hcl.(Tri sodiumhydroxide, tri hydoxy chloride)

·         The combination of LAL and prepared product will have a neutral pH 6-8.    
   

Observation:

·         Temperature 37°C ±1°C for 60 ± 2 min avoiding vibrations.
·         Check the result 180° smoothly & gently

·         Check the PPC results inverse position.

Select Lambda ( λ ) 

  • Labelled LAL sensitivity.
  • Choose one of the following LAL reagents (0.25 EU/mL,  0.125 EU/mL, 0.0625 EU/mL, 0.03125 EU/mL and 0.015625 EU/mL)
  • Work in 2 fold dilutions for gel-clot tests.
  • Run standard series from 2λ, λ, λ/2 and λ/4.
  • 2λ - must always clot.
  • λ/4 - must never clot.
  • Negative controls must not clot.

PPC preparation 2 methods:

1. Hot Spike method :
In reaction Test tube 0.01 mL of 20λ + 0.1 mL (100 µL)of test Solution  + 0.1 mL(100 µL) of LAL.
 

2. 50/50 method or Half strength method  or Double Strength method:
  • Use equal volumes of 4λ and sample.
  • Sample should be 2x desired dilution/concentration.
  • Accounts for 1: 2 dilution.
  • It means 50 µL of 4λ + 50 µL of test solution + 100 µL LAL.
Example : if want 500 dilution,
you can do 250 dilution, take 250 dilution of 50 µL sample and 50 µL LRW.
this dilution equal to 500 Dilution.

Routine Test Control:

  • Each test has four controls.
  1. Negative Water Control (NWC)
  2. Positive Water Control (PWC)
  3. Negative Product Control (NPC)
  4. Positive Product Control (PPC)

Results observation:

End point sought by 180° inversion
positive =  firm gel
Negative = anything other than firm gel.

What is the fumigation and fogging?

What is the fumigation and fogging?