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Background

What is Lipopolysaccharide?

Endotoxin is a Lipopolysaccharide (LPS) it consists of a core oligosaccharide, O-antigen a glycan polymer and the lipid A. The lipid A is a phosphorylated glucosamine disaccharide with multiple fatty acids and is the cause for endotoxin toxicity. LPS are bacterial poisons and can impact numerous biological activities. When gram-negative bacteria enter the body a complement immune response is initiated. Once the cell wall and/or bacteria are destroyed a significant of endotoxins are released which can lead to endotoxemia the symptoms of which are vomiting, nausea, diarrhea, fever, disseminated intravascular coagulation, vascular collapse, organ failure and possibly death. Antibiotics will not inactivate the endotoxins, therefore detection of the endotoxin before they enter the body is prudent.

What do horseshoe crabs have to do with endotoxin?

Limulus polyphemus (Atlantic horseshoe crab) is protected against infection by their immune system and blood coagulation system. They use hemocyanin to carry oxygen instead of hemoglobin. The blue color of their blood is due to the presence of copper in hemocyanin. The amebocytes (blood cells) are similar to white blood cells. Inside the amebocytes are proteins that are released in response to unwanted organisms like gram-negative bacteria. These proteins bind to and inactivate endotoxin. Assistance in wound control is moderated by components of their blood which prevent bleeding and form a physical barrier against additional infection.
In the presence of endotoxin, a clotting cascade is invoked to activate the proclotting enzyme which is used to transform coagulogen into coagulin. The zymogen Factor C is a glycoprotein that is 123kD, and the only enzyme that is endotoxin sensitive. It consists of an H chain (80kD) and L chain (43kD). Factor C activates in the presence of LPS and undergoes autocatalysis, the phenylalanine- isoleucine bond on the L chain is cleaved resulting in a B chain (34kD) and an A chain (8.5 kD). Factor C then activates Factor B which activates the proclotting enzyme. An activated proclotting enzyme is called the clotting enzyme which converts coagulogen into coagulin.

What is LAL assay?

Limulus Amebocyte Lysate test is an extract of the Limulus polyphemus (Atlantic horseshoe crab) blood cells (amoebocytes) it detects small concentrations of endotoxin. Horseshoe crabs are bleed through the pericardium. A third of their blood is taken and they are released back into the water. Through centrifugation, their blood cells are separated from the serum. In order to release the chemicals from inside the blood cells, they are placed in distilled water where they burst and form the lysate. Once the lysate is made the test becomes simple. A sample is mixed with lysate and water. If endotoxin is present if coagulation transpires.
An injectable healthcare product like a vaccine and any other healthcare product like implantables that come in contact with a patients blood or cerebrospinal fluid must be sterile. However, the process to kill bacteria results in the release of endotoxin into the product because the cell wall can withstand steam sterilization. The products must be tested for endotoxin before use.

So where does GSU come in to play?

Using horseshoe crab blood is an unsustainable practice due to the impact on the crabs and the expense of the LAL assay. A third of the blood is taken from the horseshoe crab before they are realsed back into the water. The theory is that the crabs eventually heal however up to 30% of bled crabs die. The total population is decreasing rapidly while producers are forced to increase harvests to keep up with global demand. The LAL test is expensive to make; a quart of blood is sold for $15,000. Part of affordable healthcare means that the production of the products used also need to be affordable.
Creating an alternative form of testing is unavoidable. Instead of creating a recombinant version of the entire clotting cascade it is more efficient to create a recombinant version of factor c and include a detection mechanism to detect the autocatalysis in the presence of LPS. Human chorionic gonadotropin (hCG) beta subunit is detected through a human pregnancy test. Using the subunit in tandem with the pregnancy test allows for an efficient and inexpensive autocatalysis detection method. The goal of our project is to create a fusion protein of Factor c with hCG and immobilize it in a solution. When the LPS is exposed to the factor c it will undergo autocatalysis and release hCG into the solution where it is then detected by the pregnancy test.

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References
- - -B. Akbar John, K.C.A. Jalal, Y.B. Kamaruzzaman and K. Zaleha, 2010.Mechanism in the Clot Formation of Horseshoe Crab Blood during Bacterial Endotoxin Invasion (July 10, 2010). Journal of Applied Sciences, 10: 1930-1936. Retrieved June 11, 2017, fromhttp://scialert.net/fulltext/?doi=jas.2010.1930.1936
- - - Elizabeth Cox. (2017,September, 21). Why do we harvest horseshoe crab blood? [Video file]. https://www.youtube.com/watch?v=VgEbcQxFUu8/
- - - Ecological Research & Development Group. (2013).
Horseshoe Crabs and Endotoxin Testing . . Retrieved October 31, 2017, from http://www.horseshoecrab.org/med/sustainable.html
- - - Ecological Research & Development Group. (2013). [Copper Based "Blue blood" and Cascade of Enzymes and Proteins ]. JRetrieved October 31, 2017, from
http://www.horseshoecrab.org/med/testing.html
- - - PBS. (2014, February 26). A still for the PBS Nature documentary Crash [Digital image]. Retrieved October 31, 2017, from
https://www.theatlantic.com/technology/archive/2014/02/the-blood-harvest/284078/