Definition of Horseshoe Crab-Based Lysate Reagents
Horseshoe Crab-Based Lysate Reagents is a sterile, freeze-dried product derived from the lysate of amebocyte cells found in the blood of the marine arthropod Limulus polyphemus (horseshoe crab). It contains Coagulation enzyme and coagulogen, which can be activated by trace amounts of bacterial endotoxins and fungal (1,3)-β-glucans. The reagent is prepared by extracting the amebocyte lysate from the blue blood of horseshoe crabs, followed by low-temperature freeze-drying. This biological reagent enables accurate and rapid qualitative or quantitative detection of bacterial endotoxins and (1,3)-β-glucans in samples.
It is widely utilized in pharmaceutical, clinical, and research fields for the detection of bacterial endotoxins and fungal glucans.
Origin of the Horseshoe Crab
The horseshoe crab is a unique marine arthropod with a remarkable evolutionary history. Its ancestors first appeared during the Paleozoic era, a period in geological history dating back hundreds of millions of years. Over time, while many other species have either evolved significantly or gone extinct, the horseshoe crab has remained virtually unchanged for over 400 million years. This extraordinary evolutionary stability has earned it the title of a "living fossil."
Classification of Horseshoe Crab-Based Lysate Reagents
Horseshoe crab-based lysate reagents are classified into two main types based on their source species:
Limulus Amebocyte Lysate (LAL): Derived from the blood of the Atlantic horseshoe crab (Limulus polyphemus), this type is abbreviated as LAL and is primarily produced in the United States.
Tachypleus Amebocyte Lysate (TAL): Extracted from the blood of the Asian horseshoe crab (Tachypleus tridentatus), this type is abbreviated as TAL. TAL shares the same functional properties as LAL and is used interchangeably for endotoxin detection.
Applications of Horseshoe Crab-Based Lysate Reagents
Due to the specific reaction of orseshoe crab-based lysate reagents to bacterial endotoxins, it is not only used for pyrogen testing of drugs, but also for clinical and laboratory disease diagnosis and hygiene.
Preparation Process of Horseshoe Crab-Based Lysate Reagents
1.Fresh live horseshoe crabs are cleaned and disinfected with 75% ethanol. Blood is drawn from the cardiac chamber, mixed with an anticoagulant, and processed at 10–15°C to obtain horseshoe crab blood.
2.The blood is centrifuged at 750–800 rpm to separate the components. The blue serum is discarded, and the remaining white cells are mixed with a suspending wash solution at a volume ratio of 1:4. After treatment, a cell lysing agent is added, and the mixture is vigorously shaken until the blood cells are completely lysed. The intracellular lysate is then stored at 0°C.
3.Chloroform (CHCl₃) is added at a volume ratio of 1:0.8–0.6, and the mixture is vigorously shaken at 2–5°C. It is then centrifuged at 2500–3000 rpm to collect the intracellular lysate, which serves as the raw reagent solution. A sensitizing agent is added, followed by pre-testing and the addition of a sensitivity-protecting agent. The solution is then aliquoted and freeze-dried at -45°C to -55°C to obtain the final lysate reagent product.
Experimental Methods for Horseshoe Crab-Based Lysate Reagents
Horseshoe crab lysate testing methods can be categorized into quantitative and qualitative assays. Based on the testing methodology, they are further divided into the following types:
1.Gel-Clot Method (Qualitative/Semi-Quantitative):
Available in five sensitivity levels: 0.03 EU/mL, 0.06 EU/mL, 0.125 EU/mL, 0.25 EU/mL, and 0.5 EU/mL. Can be selected based on the number of tests, with options for 10 tests or 100 tests per kit.
2.Kinetic Chromogenic Method (Quantitative):
Supports up to 96 tests per kit.
3.Turbidimetric Method (Quantitative):
Provides quantitative results for endotoxin detection.
Market Prospects of Horseshoe Crab-Based Lysate Reagents
Endotoxemia and sepsis caused by Gram-negative bacterial infections remain one of the leading causes of clinical mortality. According to literature reports, approximately 500,000 people in the United States suffer from sepsis annually, with about 175,000 deaths, resulting in an average mortality rate of 35%. Among surgical patients, the mortality rate can be as high as 40%–70%.
Traditional diagnostic methods for Gram-negative bacterial infections, such as bacterial culture, are not only time-consuming but also exhibit low positivity rates due to the widespread use of antibiotics. Since the critical window for treating septic shock is within the first few hours, rapid and accurate detection of endotoxins in bodily fluids, along with timely treatment, is crucial.
Horseshoe crab-based lysate reagents enable precise quantification of endotoxin levels in clinical biological materials (e.g., blood, urine, ascitic fluid, pleural fluid, and cerebrospinal fluid) and offer the following advantages:
1. Rapid detection (results in less than 1 hour).
2. High sensitivity (detection limits as low as 0.01 EU/mL).
3. Strong anti-interference capability.
4. Low experimental costs.
These reagents are used to detect endotoxin levels in patients with unexplained fever, endotoxemia, sepsis, and meningitis. They can be used as an auxiliary reference indicator for clinicians to diagnose diseases and judge prognosis, and can also be used as an important auxiliary means to guide clinical treatment, judge efficacy, and screen appropriate drugs. This approach overcomes the limitations of traditional bacterial culture methods, such as low detection rates and prolonged processing times.
