Endotoxin Testing – LAL Method

Endotoxins are components of bacterial cell walls, the most common of which is lipopolysaccharide (LPS).  Endotoxins cause toxicity even after bacteria die because they are release when the cells lyse.  Products used in humans must be endotoxin-free, or they will cause illness (via an inflammatory response).  For our purposes, endotoxin-free cell media is desirable so that cells are not reacting to low levels os inflammatory agents during routine cell culture (thus preventing the formation of tight monolayers).

Endotoxins can be detected via the limulus amebocyte lysate assay (LAL) assay.  The principle behind this assay is neat – the blood cells of horseshoe crabs are extremely sensitive to endotoxins due to a clotting mechanism.  In this assay, lysates from these cells are used and then modified with a chromogenic substance to induce a color change.

Genscript ToxinSensor Chromogenic LAL Endotoxin Assay Kit

Detects 0.005-1 EU/mL endotoxin; linear from 0.005 to 0.1 and 0.1 to 1 EU/mL

NOTE: Since LAL and chromogen are reconstituted in only 1.5mL water/buffer, only 15 total tests (including standards, controls, water, samples, etc.) can be performed per vial of these substances.

Protocol:

Solutions:

1. Reconstitute LAL in 1.5mL LAL reagent water.  Do not vortex.  Store < 1 week at -20C.
2. Reconstitute Chromogenic Substrate in 1.5mL LAL reagent water.  Store < 4 weeks @ 4C.
3. Reconstitute color stabilizer #1 with 10mL buffer S (stop solution).
4. Reconstitute color-stabilizers #2 and #3 with 10mL each LAL reagent water.  Store < 1 week @ 4C.
5. Reconstitute 10EU lyophilized endotoxin vial in 1mL LAL reagent water by vortexing for at least 1 minute.  This makes a 10EU/mL stock solution.  Store < 2 weeks @ -20C.

Assay:

1. Make EU/mL dilutions in LAL reagent water in endotoxin-free vials
   1. Concentrations should be between 0.005 and 1 EU/mL – 2 linear regimes
2. Add 100uL standards and samples to endotoxin-free vials.  Use 100uL LAL reagent water as negative control.
3. Add 100uL LAL to each vial and mix well
4. Place vials in rack and incubate at 37C, 45 minutes.  Or only 10 minutes if using EU standard curve for 0.1-1 EU/mL
5. Add 100uL chromogenic substrate to each vial; Mix well
6. Incubate @ 37C, 6 minutes
7. Add 500uL stop solution to each vial; Mix well
8. Add 500uL color-stabilizer #2 to each vial; Mix well
9. Add 500uL color-stabilizer #3 to each vial; Mix well
10. Measure ABS @ 545nm; Use DIH20 as blank (zero)