Endotoxin detection is important in both clinical and research areas. In a clinical setting endotoxin detection can be a useful diagnostic device, and is vitally important to protect human health in the preparation of pharmaceuticals and vaccines.
In the laboratory, where endotoxins can damage cells and ruin experiments, endotoxin testing of cell culture reagents, from media to proteins, is vital.
Blue blood may have saved your assay. As well as your life. Though the aristocracy is not to thank for this.
As the use of injectable drugs began to increase in the late 19th and 20th centuries, doctors began to notice that even sterile drugs were causing some people to develop ‘injection fever’, which at its worst caused sepsis.
The reason was eventually tracked down to bacteria. But how? The solutions were sterile. There were no viable, living bugs left in the solutions to cause a fever. However, there were remnants of them still present following sterilisation: bits of bacterial cell outer membrane floating about.
What the doctors and scientists of the time didn’t know was that these portions of cell wall were adorned with large molecules called lipopolysaccharides, also called ‘endotoxin’. Endotoxins are heat stable integral parts of bacterial cells and differ from exotoxins, which are typically proteins produced and released by bacterial cells which can be destroyed by heating.
For gram negative bacteria, such as Neisseria meningitides, endotoxin is a key component of their outer membranes, helping maintain structure and fend off attack from different chemicals.
However, it serves one useful purpose for mammals. It’s the signature of infection by gram-negative bacteria. A receptor called toll-like receptor 4 (TLR4), found on many cells, including macrophages, B-cells and monocytes, detects this signature and, when activated, it kicks off the production of pro-inflammatory cytokines.
It’s a kind of knee-jerk reaction to the signature of gram-negative bacteria, which, if enough endotoxin is present, leads to fever and even sepsis. And, weirdly, it turns out human are especially sensitive to endotoxins compared to other mammals.
As such, pharmaceutical and vaccine manufacturers need to be able to be sure their products don’t contain endotoxin and are as safe as possible, and labs need to know their cell culture reagents aren’t contaminated by endotoxins, which may mess up their results.
Well, testing for endotoxin began in earnest. But it wasn’t great, relying on injecting rabbits with batches of drugs and seeing if they got sick. Not a great solution, especially for the rabbit.
The alternative? Crabs.
Horseshoe crabs in particular. Horseshoe crabs spend much of their time grubbing about in the mud and shallows around coasts, constantly surrounded by bacteria. As such, any cut or injury is immediately smothered in bacteria. So it needs a way to seal up any injuries fast. And it does.
The horseshoe crabs blue blood – which uses the copper containing heamocyanin rather than haemoglobin to transport oxygen – contains an immune cell called an ‘amoebocyte’. These cells detect endotoxin and form clots, fast and in response to very small quantities of endotoxin.
This same property can be used in the lab to test for endotoxin, giving almost instant, highly specific, result. This has come be known as the LAL or TAL method for detecting endotoxin, depending on the species of horseshoe crab used.
The blood is sustainably harvested from the crabs, draining around a third of their blood before re-releasing them back in to the ocean, before being processed in to a usable, consistent, product.
Crab blood harvest: Kayla Westerlund