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Limulidae, also known as horseshoe crab, appeared in the Devonian period 300 million years ago.
This period has experienced many major events such as the glaciers, the extinction of species, the drift of plates, and the collision of asteroids with the earth.
But so far, it still retains its original and ancient appearance. It is one of the creatures that has spent a long time in the history of the earth and is called a modern living fossil.

But in the past few decades, they have become human life-saving drugs.
The blood of sputum can be made into a reagent LAL dedicated to bacterial endotoxin detection.
This has now grown into a huge industry. Each drug that is authorized by the US Food and Drug Administration (FDA) must undergo LAL testing.

 

Catch it and get blue blood

In fact, capturing limulus is not that difficult. After being authorized by the Department of Natural Resources of the United States, the fishermen picked up the shoal in the shallows of Carolina, USA, and put it into a fishing boat.

The fishermen will not kill them immediately after catching them, but will wipe them clean, remove the barnacles and sand from their bodies, and then peel the shells apart.

Finally, it was fixed on a work bench and a stainless steel needle was used to pierce the sacral pericardium (a pericardium is a conical double-fiber sac sac) to extract the blood of the sputum.

Unlike other animals, the baboon blood is blue because it contains copper ions in their blood.
This blue blood is not clear blue, but like blueberry juice mixed with milk.

Why do humans need this blue blood?

In fact, the life-saving drugs mentioned above are mainly in this blue blood.
In circulatory amoebic cells of blood stasis, there is a lysate called Limulus Amebocyte Lysate (hereinafter abbreviated as LAL), which can be used to detect the cell wall of gram-negative bacteria. Ingredient lipopolysaccharide (endotoxin).

Endotoxin detection using LAL came from the discovery of Frederik Bang.

Before Bang discovered it, the standard method for detecting bacterial toxins was to inject the sample into rabbits.
This requires someone to check the rabbit’s body temperature once every 30 minutes, to see if there is any signs of fever within 3 hours, and if there are any bacterial toxins. Under the microscope, the rabbit’s blood cells also tend to accumulate around the toxins.

In the fifteen and a half years after the publication of the study in 1956, Bang observed that the Gram-negative infection of Limulus polyphemus resulted in fatal intravascular coagulation.
Later, Bang and a young pathologist named Jack Levin found that this condensation was caused by endotoxin interacting with a clottable protein in circulating amoebic cells in blood stasis. result.
Therefore, Levin and Bang prepared a lysate from the washed amoebic cells, which is a very sensitive indicator of endotoxin.

However, it was not until 1977 that the U.S. Food and Drug Administration (FDA) allowed pharmaceutical companies to replace the inefficient test for rabbit toxins with LAL kits.

 

Nowadays, you just need to add LAL to the test material, and then turn the vial over to see if it turns solid to know if it contains toxins.

 

What are the functions of LAL made from the blood of endangered Limulus?

LAL can accurately and quickly detect whether the internal tissues of the human body are caused by bacterial infections.
In the pharmaceutical and food industries, it can be used to monitor the deformed cells in the blood (a small amount, centrifuged to precipitate white matter) or even taken into outer space to detect organisms and protect astronauts from disease.
Hemorrhoidal blood also plays a decisive role in the modern biological industry, and its functional value is irreplaceable for a long period of time.

Therefore, if it disappears, it will have a huge impact on modern biomedicine.
Unfortunately, the survival of the existing limulus is not good.
Especially in the Asian region, the construction of seawalls has led to a gradual reduction in the area of ​​beaches where spawning takes place.
Marine pollution and overfishing have all contributed to the continuous decrease in the number of plutonium.

In the United States, although the earthworms used for biomedical purposes will eventually be shipped back to the sea, they will still face death after they are sent back. It is estimated that 50,000 beggars die every year after being transported back to the beach.

 

LAL has now become a multi-million dollar industry.
According to reports, a quart (a quart of approximately 0.946 liters) has a blood value of up to $15,000 for a horseshoe crab, and a set of reagents costs up to $1,000.
The price of expensive reagents and the number of endangered individuals have become the driving force for scientists to find alternatives to LAL.

Scientists came up with this method to replace LAL

We now know that factor C causes clotting of blood in sputum, and it is only necessary to find the relevant gene fragment. Scientists want to cut the genes that can bind to toxins through transgene technology and transfer them to yeast cells that are easy to grow in the lab.

This idea sounds simple, but the test is very difficult. Since earthworms are extremely sensitive to bacterial toxins, blood clotting in the laboratory environment will condense in the test tube because toxins are ubiquitous in normal environments, so it is extremely necessary to make a thorough sterile laboratory.

 

 

One scientist overcomes these difficulties. Ding Ling is a molecular biologist at the National University of Singapore. Since the 1980s, after more than ten years of hard work, he has finally developed an alternative to LAL based on genetically modified technology. The first non-hemoglobin test kit was in 2003. Year is coming.

But even then, the initial pharmaceutical company did not have much interest in it.
The reason is that this technology is based on Ding Ling’s personal patent and only one Swiss chemical company, Lonza, is allowed to supply this synthetic reagent.
What will happen if the Lonza supplier falls down in the future? Didn’t the other pharmaceutical companies follow it?

At the Charles River Laboratory in Charleston, South Carolina, horseshoe crabs were transported for bleeding

 

However, some things are changing recently.
First of all, Lonza is no longer the only supplier. In 2013, Hyglos became the second company to produce synthetic factor C. Second, in terms of regulation, the European Pharmacopoeia added factor C in 2016 as a recognized bacterial-toxin test, which also paved the way for the reform of blue blood reagents. Nowadays, more and more pharmaceutical companies are adding synthetic toxin indicator manufacturing.

Maybe you would think that this ancient species that has survived for several hundred million years will finally be able to squat on the beach and live in peace and tranquility. But even if the pharmaceutical companies no longer need to be embarrassed, the fishermen will use them as bait to capture moths, and their tragic destiny does not seem to end.

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