Limulus Amebocyte Lysate - AbsoluteAstronomy.com

Limulus amebocyte lysate (LAL) is an aqueous extract of blood cells (amoebocytes) from the horseshoe crab

Horseshoe crab

The Atlantic horseshoe crab, Limulus polyphemus, is a marine chelicerate arthropod. Despite its name, it is more closely related to spiders, ticks, and scorpions than to crabs. Horseshoe crabs are most commonly found in the Gulf of Mexico and along the northern Atlantic coast of North America...

, Limulus polyphemus. LAL reacts with bacterial endotoxin

Endotoxin

Endotoxins are toxins associated with some Gram-negative bacteria. An "endotoxin" is a toxin that is a structural molecule of the bacteria that is recognized by the immune system.-Gram negative:...

or lipopolysaccharide

Lipopolysaccharide

Lipopolysaccharides , also known as lipoglycans, are large molecules consisting of a lipid and a polysaccharide joined by a covalent bond; they are found in the outer membrane of Gram-negative bacteria, act as endotoxins and elicit strong immune responses in animals.-Functions:LPS is the major...

(LPS), which is a membrane component of Gram negative bacteria. This reaction is the basis of the LAL test, which is used for the detection and quantification of bacterial endotoxins.

Background

Fred Bang reported in 1956 that gram-negative bacteria, even if killed, will cause the blood of the horseshoe crab to turn into a semi-solid mass. It was later recognized that the animal's blood cells, mobile cells called amoebocytes, contain granules with a clotting factor known as coagulogen; this is released outside the cell when bacterial endotoxin is encountered. The resulting coagulation

Coagulation

Coagulation is a complex process by which blood forms clots. It is an important part of hemostasis, the cessation of blood loss from a damaged vessel, wherein a damaged blood vessel wall is covered by a platelet and fibrin-containing clot to stop bleeding and begin repair of the damaged vessel...

is thought to contain bacterial infections in the animal's semi-closed circulatory system

Circulatory system

The circulatory system is an organ system that passes nutrients , gases, hormones, blood cells, etc...

.

In 1970 the U.S. Food and Drug Administration (FDA) approved LAL for testing drugs, products and devices that come in contact with the blood. Prior to that date, much slower and more expensive tests on rabbit

Rabbit

Rabbits are small mammals in the family Leporidae of the order Lagomorpha, found in several parts of the world...

s had been used for this purpose.

Blood is removed from the horseshoe crab's pericardium

Pericardium

The pericardium is a double-walled sac that contains the heart and the roots of the great vessels.-Layers:...

; the crabs are returned to the water. LAL manufacturers have measured mortality rates of 3% in bled crabs, however recent studies indicate that this number may be closer to 15%. The blood cells are separated from the serum using centrifugation

Centrifugation

Centrifugation is a process that involves the use of the centrifugal force for the sedimentation of mixtures with a centrifuge, used in industry and in laboratory settings. More-dense components of the mixture migrate away from the axis of the centrifuge, while less-dense components of the mixture...

and are then placed in distilled water, which causes them to swell up and burst ("lyse"). This releases the chemicals from the inside of the cell (the "lysate"), which is then purified and freeze-dried

Freeze drying

Freeze-drying is a dehydration process typically used to preserve a perishable material or make the material more convenient for transport...

. To test a sample for endotoxins, it is mixed with lysate and water; endotoxins are present if coagulation occurs.

The LAL test

There are three basic LAL test methodologies: gel-clot, turbidimetric, and chromogenic. The primary application for LAL is the testing of parenteral pharmaceuticals and medical devices that contact blood

Blood

Blood is a specialized bodily fluid in animals that delivers necessary substances such as nutrients and oxygen to the cells and transports metabolic waste products away from those same cells....

or cerebrospinal fluid

Cerebrospinal fluid

Cerebrospinal fluid , Liquor cerebrospinalis, is a clear, colorless, bodily fluid, that occupies the subarachnoid space and the ventricular system around and inside the brain and spinal cord...

. In the United States, the FDA has published a guideline for validation of the LAL test as an endotoxin test for such products http://www.fda.gov/CBER/gdlns/lal.pdf.

The LAL cascade

Biochemical cascade

A biochemical cascade is a series of chemical reactions in which the products of one reaction are consumed in the next reaction. There are several important biochemical cascade reactions in biochemistry, including the enzymatic cascades, such as the coagulation cascade and the complement system,...

is also triggered by (1,3)-β-D-glucan

Glucan

A glucan molecule is a polysaccharide of D-glucose monomers linked by glycosidic bonds.Many beta-glucans are medically important.-Types:The following are glucans:-Alpha:...

. Both bacterial endotoxins and (1,3)-β-D-glucan are considered "Pathogen-Associated Molecular Patterns

Pathogen-associated molecular pattern

Pathogen-associated molecular patterns, or PAMPs, are molecules associated with groups of pathogens, that are recognized by cells of the innate immune system. These molecules can be referred to as small molecular motifs conserved within a class of microbes...

", or PAMPS, substances which elicit inflammatory responses in mammal

Mammal

Mammals are members of a class of air-breathing vertebrate animals characterised by the possession of endothermy, hair, three middle ear bones, and mammary glands functional in mothers with young...

Overcoming inhibition and enhancement

One of the most time consuming aspects of endotoxin testing using LAL is pretreating samples to overcome assay inhibition and enhancement. Agents such as EDTA

EDTA

Ethylenediaminetetraacetic acid, widely abbreviated as EDTA , is a polyamino carboxylic acid and a colourless, water-soluble solid. Its conjugate base is named ethylenediaminetetraacetate. It is widely used to dissolve limescale. Its usefulness arises because of its role as a hexadentate ligand...

and heparin

Heparin

Heparin , also known as unfractionated heparin, a highly sulfated glycosaminoglycan, is widely used as an injectable anticoagulant, and has the highest negative charge density of any known biological molecule...

are known to affect the assay if they are present in sufficient concentrations. All assays, independent of methodology are standardized using endotoxin in water. Therefore, unless the sample is water, some components of the solution may interfere with the LAL test such that the recovery of endotoxin is affected. If the product being tested causes the endotoxin recovery to be less than expected, the product is inhibitory to the LAL test. Products which cause higher than expected values are enhancing. Overcoming the inhibition and enhancement properties of a product is required by the FDA as part of the validation of the LAL test for use in the final release testing of injectables and medical devices. Proper endotoxin recovery must be proven before LAL can be used to release product.

External links

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