Bacterial Endotoxin Tests
- Endotoxin definition
- Endotoxin tests in pharmaceutical industries
It is a toxin which is stable ,resist destruction by steam sterilization and removed by filtration , present in outer membrane of Gram negative bacteria . It is actually not secreted by bacteria but released when bacterial cells get damaged. Bacteria both Gram positive and Gram negative consists peptidoglyacane layer which is made up of sugar and peptide chain but as we all know that in Gram negative bacteria this layer is thin and having outer membrane of LPS (lipopolysaccharide). In this LPS layer lipid A is present which is actually act as a toxin. When bacterial cell get distrupted by antibiotics or host immune system this LPS layer breaks and lipid A get release and act as a toxin , which is dangerous. It causes fever , haemorrhagic shock, sever diarrhea , comma and may cause death.
- Endotoxin tests in pharmaceutical industries –
As we know endotoxins are how much harmfull for us. In pharmaceutical industries, product should not contain any contamination. So it should be gone through various tests , among these tests endotoxin test also is there to check our final product should not contain endotoxin. For this assurance 3 tests are there.
2)Turbidometric LAL tests
3)chromogenic LAL tests
Limulous amebocyte lysate test based on biology of horseshoe crab. These animals produce LAL enzyme in blood cell which bind to endotoxin and inactivate them and inactivation of endotoxin also forms clot. Horseshoe crabs are the species present on earth from the ancient time (200 millions ago). It is world’s oldest and most fascinating creatures. Horseshoe crab blood is blue coloured blood because it is copper based instead of iron based. It has high medical value because it contains a protein called LAL which is used by pharmaceuticals and medical devices manufacturer to test their product for presence of endotoxin. Actually LAL binds to the endotoxin and form gel clot. Amebocyte (WBC of crab) contains a several serine protease zymogens which involves in a coagulation cascade trigger by endotoxin. Cascade also trigger by 1,3 β-D –glucan which is present on cell wall of yeast and fungi. Result of this coagulation pathway is formation of gel which ultimately shows presence of endotoxin. In cascade factor C, factor B, proclotting enzyme , clotting enzyme these factors are envolved. Clotting enzyme cleaves two bonds of coagulogen, which is fibrinogen like molecule to yield an insoluble coagulin gel. This tests is very sensitive because cascade can triggers by pico –nano grams of endotoxin. So it is very helpful and perfect test for detection of even small proportion of endotoxin present in product.
Small amount of sample of LAL solution, Incubate at 37°C for 1 hour. After incubation invert tube
Note- after inverting the tube gel formation is observed means endotoxin is present in sample. If gel is not form means endotoxin is not present in sample.
Advantage of LAL test-
- Simple and easy
- Short time required
- Rabbits are not used in this test.
2) Turbidimetric LAL assay-
This assay is used to detect the presence of endotoxin. It is quantative kinetic assay.
Sample + Reconstituted LAL reagent
Then it should be placed in incubating absorbance plate reader.
And it get monitored over a time of turbidity and absorbance is checked at 340nm.
- Time required for the change is inversely proportional to the amount of endotoxin present.
If endotoxin is present lysate will become a gel and makes solution turbid or give cloudy appearance.
3) Chromogenic LAL assay-
This assay is used to detect the presence of endotoxin. It is quantitative kinetic assay.
Sample + reconstituted LAL reagent
Placed in an incubating plate reader that measures absorbance at 405nm
Reaction is automatically monitored over time for the appearance of yellow colour.in this test lysate will begin to cleave the chromogenic substrate in the presence of endotoxin and solution become yellow. Time required for the change is inversely proportional to the amount of endotoxin present.
- This assay is usefull in testing of small volume parenterals, vaccines , antibiotics.
Factor C factor B proclotting enzyme clotting enzyme chromogenic substrate
405nm yellow colour
These tests are used in pharmaceutical industries for endotoxin detection.