The document discusses the bacterial endotoxin test (BET) which uses the limulus amoebocyte lysate (LAL) from horseshoe crabs to detect endotoxins from gram-negative bacteria. The BET involves using LAL to detect endotoxins via gel clot tests or kinetic chromogenic/turbidimetric assays. Standards and sample solutions are prepared according to USP guidelines to determine if samples meet specified endotoxin limits. The kinetic LAL assays provide quantitative results while gel clot tests provide binary positive/negative results. Interference testing is also conducted to identify non-interfering sample dilutions for accurate BET results.

1. Bacterial Endotoxin Test <85>
Definition : Pyrogens are fever-inducing agents in humans and
animals include endotoxin, gram + cell debris, fungi
Endotoxins are components from the outer membrane of gram-
negative bacteria
The Bacterial Endotoxins Test (BET) is a test to detect or quantify
endotoxins from Gram-negative bacteria using amoebocyte lysate
from the horseshoe crab (Limulus polyphemus or Tachypleus
tridentatus).

2. Endotoxin - Introduction
• Endotoxin - Component from outer membrane
• of gram-negative bacteria
• Environmental (unpurified) endotoxin: complex of protein,
• carbohydrate & lipid
• Purified endotoxin: lipopolysaccharide molecule of
• polysaccharide and lipid a, without protein

3. Why Test for Endotoxins?
• To protect against adverse reactions (sepsis)
• in humans and animals
• FDA and USP guidelines require final product
• testing on all parenterals and medical devices
• To safeguard against diminished
• effectiveness of a product due to endotoxin

4. BET
• LAL
• Limulus - genus of Horseshoe Crab
• Amoebocyte - blood cells
• Lysate - blood extract
LAL is a FDA regulated biological that reacts chemically in the
presence of gram-negative cell debris. This reaction can be
measured and quantified.

5. BET
• LAL Assay
• LAL is > 100 times more sensitive, specific and accurate
• Economy of LAL permits more testing
• LAL Testing Types
• Gel Clot(Limit Test)
• Gel Clot(Semi-Quantitative)
• Endpoint Chromogenic
• Endpoint Turbidimetric
• Kinetic Chromogenic
• Kinetic Turbidimetric

6. USP requirements
• LAL
• A lyophilized product obtained from the lysate of
amoebocytes (white blood cells) from the horseshoe crab
(Limulus polyphemus or Tachypleus tridentatus). This reagent
refers only to a product manufactured in accordance with the
regulations of the competent authority.
• [NOTE—AMOEBOCYTE LYSATE REACTS TO SOME Β-GLUCANS IN
ADDITION TO ENDOTOXINS. AMOEBOCYTE LYSATE PREPARATIONS
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 BE USED
FOR ENDOTOXIN TESTING IN THE PRESENCE OF GLUCANS.]

7. USP requirements
• PREPARATION OF SOLUTIONS
• Standard Endotoxin Stock Solution
• A Standard Endotoxin Stock Solution is prepared from a USP Endotoxin Reference Standard that
has been calibrated to the current WHO International Standard for Endotoxin. Follow the
specifications in the package leaflet and on the label for preparation and storage of the Standard
Endotoxin Stock Solution. Endotoxin is expressed in Endotoxin Units (EU). [NOTE—ONE USP
ENDOTOXIN UNIT (EU) IS EQUAL TO ONE INTERNATIONAL UNIT (IU) OF ENDOTOXIN.]
• Standard Endotoxin Solutions
• After mixing the Standard Endotoxin Stock Solution vigorously, prepare appropriate serial dilutions
of Standard Endotoxin Solution, using Water for BET. Use dilutions as soon as possible to avoid
loss of activity by adsorption.

8. USP requirements
• Sample Solutions
• Prepare the Sample Solutions by dissolving or diluting drugs using Water for
BET. Some substances or preparations may be more appropriately dissolved,
or diluted in other aqueous solutions. If necessary, adjust the pH of the solution
to be examined (or dilution thereof) so that the pH of the mixture of the lysate
and Sample Solution falls within the pH range specified by the lysate
manufacturer, usually 6.0–8.0. The pH may be adjusted by use of an acid,
base, or suitable buffer as recommended by the lysate manufacturer. Acids and
bases may be prepared from concentrates or solids with Water for BET in
containers free of detectable endotoxin. Buffers must be validated to be free of
detectable endotoxin and interfering factors.

9. Sample dilution
• DETERMINATION OF MAXIMUM VALID DILUTION (MVD)
• The maximum valid dilution is the maximum allowable dilution
of a specimen at which the endotoxin limit can be determined.
Determine the MVD from the following equation:MVD =
(endotoxin limit × concentration of Sample Solution)/(λ)

10. Gel Clot Test
• Gel Clot Test
• Endpoint sought by 180 inversion of
• sample tube
• Positive = Firm Gel
• Negative = Flow down side of tube

11. Gel Clot Test
• In general
• Lowest reagent cost
• Simple reporting (+-)
• Found in all Pharmacopoeia
• Good stability
• Simple technique
• Gel clot test problems
• Use of wrong EU/ng ratio
• use of improper reaction or dilution tubes
• Incubating temperature too hot or too cold
• Improper dilutions
• Improper handling of lysate

12. BET
• Kinetic LAL Advantages
• Quantitative results
• Higher throughput (96 wells)
• Lower sensitivity (to .001)
• Wider standard curve range
• Clear concise reporting
• Allows for data trending
• Measure changes in optical density (od)
• Relates changes in od to endotoxin concentration
• Automated calculations
• Reports results of analysis

13. Kinetic Test Assay
• Summary of Kinetic Assay
• The amount of endotoxin present is inversely proportional to the time of
reaction
• The more endotoxin present, the quicker the well or tube will react (change
color or become turbid)
• A standard curve is generated based on how long each of the standards
take to reach an onset OD
• Unknowns are then compared to the standard curve to generate an
endotoxin values

14. Kinetic Test assay
• LAL - Test Interference
• Inhibition:
• Recovery < Expected Amount
• Enhancement:
• Recovery > Expected Amount
• Non-interference:
• Recovery = Expected Amount

15. Calculations
• Calculate EL, MVD, MVC
• Make sample dilutions between undiluted and MVD
• Screen these dilutions to determine noninterfering test concentration
• Once a non-interfering sample dilution is determined perform validation on
three lots of this product

16. Thank You