Where metrics and morals align – Switching to a new endotoxin measurement kit
Green Team Blog 3
A new way to side-step the use of crabs in endotoxin detection.
In a previous blog, we discussed the amazing horseshoe crab and why its blue blood is so special. Specifically, how horseshoe crab blood is used as a component of the Limulus amoebocyte lysate (LAL) test to measure endotoxin levels. Endotoxins are lipopolysaccharide (LPS) components of the outer membrane of Gram-negative bacteria such as E. coli. Excessive exposure to these endotoxins can cause septic shock and organ failure in humans, thus making the measurement of endotoxin concentration crucial for anything that will enter the body. Endotoxin contamination of proteins can cause issues in cell-based and in-vivo assays and may even affect in vitro assays.
In horseshoe crabs such as Limulus polyphemus, a coagulation cascade has evolved to resist infections caused by bacteria. Unlike warm blooded creatures, they can’t raise temperature to kill infections. They have amoebocytes in their blood that detect bacterial infection and trigger clotting. Clotting is triggered by the Factor C pathway. This is a serine proteinase cascade that begins with LPS endotoxins binding to a protein called Factor C.
Figure 1: the Factor C pathway
Harm of Harvesting Horseshoe Crabs
For decades the LAL test (Limulus Amoebocyte Lysate) has made use of the Factor C pathway to detect endotoxin contamination using a qualitative gel clot. Although we’ve moved onto a quantitative method, LAL remains the key ingredient and is generated by bleeding live horseshoe crabs.
Crab bleeding is estimated to cause 70,000 horseshoe crab deaths per year in the USA [1], as many crabs do not survive the bleeding process (10-30%), and the long-term effects on those that do are poorly understood. Overharvesting has led to a drastic decline in horseshoe crab populations, which additionally threatens the survival of other species that rely on them. Horseshoe crab eggs are a crucial food source for many species, including the red knot shorebird, which undertakes an annual 19,000-mile migration [2].
A Recombinant Alternative
At Sygnature Discovery Protein Science, we have been trialling a new endotoxin detection test based on recombinant Factor C (rFC) and a small fluorescent peptide. By switching to this recombinant Factor C alternative, we are helping to reduce the number of live crabs that need to be bled, aligning with our commitment to more environmentally friendly practices. We have carried out internal experiments to ensure that it gives comparable results to previously used LAL assay.
In addition to the environmental benefits of the new test, the measurable range of endotoxin levels is much wider, from 0.05 EU/mL to 50 EU/mL, compared to just 0.1 EU/mL to 1 EU/mL in the traditional LAL method. Furthermore, the new assay is quicker and easier to run, requires less sample, and generates the standard curve more reliably.
Figure 2: Standard curve from original assay kit
Figure 3: Standard curve from new assay kit
The graphs above show the old (endogenous Factor C) and new (rFC alternative) standard curves. The new standard curve is plotted on a logarithmic scale, this illustrates the wider range of endotoxin concentrations that are detectable with this kit.
Conclusion – Better Data and Happier Crabs
Switching to the recombinant Factor C (rFC) endotoxin detection not only enhances the efficiency and accuracy of our lab tests, but it also aligns with our commitment to environmental sustainability.
References
[1] https://doi.org/10.3389/fmars.2018.00185
[2] https://earthjustice.org/experts/ben-levitan/a-pathway-to-end-the-medical-harvest-of-horseshoe-crabs
