bacterial endotoxin tests


Bacterial Endotoxin Tests

  • Endotoxin definition
  • Endotoxin tests in pharmaceutical industries
  • Endotoxins-

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.

1)LAL test

2)Turbidometric LAL tests

3)chromogenic LAL tests

1)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.

LAL test-

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 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.