Horseshoe crabs are marine and brackish water arthropods of the family Limulidae, suborder Xiphosurida, and order Xiphosura. Since its inception more than 400 million years ago, it still retains its original and ancient appearance, so it is known as the "living fossil". There are only 3 genera and 4 species of existing horseshoe crabs：Limulus polyphemus, Tachypleus tridentatus, Tachypleus gigas, Carcinosvorpius rotundicauda.
Fig. 1 Horseshoe crabs
The blood circulation system of horseshoe crab is a relatively open pattern: There is a large blood chamber in the body, which is well-connected and allows direct contact between blood and tissue. Therefore, bacteria can easily enter the body of a horseshoe crab by drilling through a slit in the shell, and there is no white blood cell in the horseshoe crab that can fight infection. In order not to be swallowed by bacteria, the blood of horseshoe crabs has evolved a protection mechanism different from vertebrates. The horseshoe crab has a magical amebocyte cell in the blood, and the amebocyte cells are fluid. When contacted to bacteria, amebocyte cells do not engulf the bacteria like white blood cells, but instead immediately shrink and rupture, and the chemical substances released after rupture will cause local coagulation of the blood. This can enclose harmful bacteria and block the spread of disease. Amebocyte cells are extremely sensitive to Gram-negative bacteria or LPS. When horseshoe crab blood is exposed to Gram-negative bacteria or LPS, amebocyte cells mediate blood clotting. This response not only prevents blood from leaking out, but also engulfs invading microorganisms. LAL/TAL is a biological reagent obtained by lyophilizing and drying the amebocyte cell lysate extracted from the blue blood of horseshoe crab.
The molecular mechanism of horseshoe crab coagulation is a complex enzymatic cascade. The molecular basis of this reaction is three serine zymogens: factor C, factor B, factor G, and coagulogen and coagulogen. In the presence of LPS, the first enzyme, Factor C is activated which activates Factor B. Factor B activates the proclotting enzyme to clotting enzyme. And each step of activation is restricted proteolysis. Finally, the clotting enzyme cleaves coagulogen into coagulin. Coagulin molecules stick together to form a clot to protect the horseshoe crab from an exposure to Gram-negative bacteria or endotoxin. This enzymatic cascade can also be activated by (1,3)-β-D glucan, which activates another serine zymogens (named G factor) mediated coagulation system.
Fig. 2 Enzymatic Cascade of LAL/TAL
The existing LAL/TAL is divided into two types according to different species of horseshoe crab. One is the Limulus Amebocyte Lysate (LAL) extracted from the blood of the Limulus Polyphemus. The other is the Tachypleus Amebocyte Lysate (TAL) extracted from the blood of the Tachypleus tridentatus.
The recombinant factor C (rFC) is an evolution of the LAL, which is the activation of a recombinant form of factor C by endotoxin to complete the enzymatic cascade of horseshoe crabs. The rFC assay acts through a single enzymatic step as compared to the multi-step enzymatic process required for LAL assay.
