I’m beginning this article exactly where the last one finished, with a cartoon showing how LAL is made.
Cartoon by Shruti Muralidhar – How LAL is made. Adapted from multiple references cited at the end of the article
Lets focus on what is done to the blood after collection. It is spun at very high speeds to separate out the cells in it, which are called amoebocytes. When amoebocytes are collected and put into distilled water, they burst and release their molecular contents. This phenomenon of cells bursting is called lysis and the stuff inside them that flows out is called lysate. So, blood from the horseshoe crab (Limulus) contains cells (Amoebocytes) which are burst to release their contents (Lysate). Hence, LAL.
Let us summarise what we know up to this point. LAL is a horseshoe crab blood extract, responsible from blood coagulation or clotting, that is switched on by very tiny amounts of bacterial endotoxin. This is all very convenient for humans to use in our quest for safer medicines. But, why does horseshoe crab blood have such properties?
The components of LAL, that we are interested in, are molecules that are a part of the horseshoe crab’s immune system. And, the immune system of the horseshoe crab, uses endotoxin as an early warning signal to detect and immobilise the pathogens (disease-causing microbes) that have produced it.
Detection of small numbers of pathogens at early stages of infection is necessary because it gives the immune system a good chance of destroying them. Using endotoxins to detect microbes is like using smoke to detect fire. So, if molecules of the immune system can detect minute amounts of endotoxin inside a horseshoe crab, these same molecules can be extracted to do the same in a test tube. This is exactly what the LAL test is.
Besides testing for their contamination in medicines, endotoxins are also used as a signal to quickly detect microbes, the source of endotoxins. Traditional methods for detecting microbes require culturing, which is growing them in controlled laboratory conditions. The time period required for growing them is, at least, a few hours. In contrast, scientists have developed novel methods that work much faster. Endotoxin detection is just one of these fast tests. The fastest version of the endotoxin test only requires fifteen minutes to show results.
Detecting the presence of microbes is important in many situations. Medically, it helps to diagnose whether an illness is caused by a pathogen. The quicker the diagnosis, the better the chances are of successful treatment and patient recovery. In terms of public health, quick detection of possible pathogens in food and water can prevent large-scale epidemics. Besides, there are other interesting situations where quickly detecting microbes is very useful. We will discuss these, just after we take a look at the following flowchart. It shows three sections of the horseshoe crab immune system which have been used to make tests to detect microbes. Of these tests, the LAL test is the most well-known.
Image: Selected portions of the horseshoe crab immune system. Adapted from multiple references cited at the end of the article
The first two sections in the flowchart (outlined in red and blue) are triggered by toxins released by different types of bacteria. The red box in the flowchart shows the section of the immune system that we use as the LAL test. LPS (LipoPolySaccharide) is the name for the endotoxin molecule that is detected by the test. There are many versions of the LAL test, each of them tweaked to provide different types of information. Some versions of the test just give ‘yes’ or ‘no’ results (endotoxin detected or not). Other versions provide quantitative information (how much endotoxin is present).
The blue box in the flowchart shows a section of the immune system that detects endotoxin-like molecules called LTA (LipoTeichoic Acid) that are secreted by other bacteria. The third section in the flowchart (outlined in green) is triggered by fungal molecules, called Beta glucans.
Finally, let’s take a look at an interesting situation in which detecting microbes can be useful: space travel. The components of all three sections of the horseshoe crab immune system have been used in a microbe detector on the International Space Station (ISS). Its called the LOCAD-PTS (Lab On a Chip Applications Development-Portable Test System). It has been jointly developed by NASA and Charles River Laboratories International.
Image: LOCAD-PTS, microbe detection device. Source: https://www.nasa.gov/mission_pages/station/research/experiments/LOCAD-PTS1.jpg
The LOCAD-PTS has already been used to detect bacteria and fungi at various locations on the ISS. It is expected that the device will eventually have expanded functionality. Then, it can be used to monitor the microbial load on spacecrafts, the health of astronauts, as well as the possible spread of Earth-based microbes to spacecraft and other planets that they visit.
Image: LOCAD-PTS, being used by Astronaut Sunita L. Williams, in the Destiny laboratory of the International Space Station. Source:http://spaceflight.nasa.gov/gallery/images/station/crew-14/html/iss014e18822.html
That’s it for this time, folks! Stay tuned for the final article in the sage of the horseshoe crab, where we will travel ‘back to the future’. We will start by looking at the past, at what we did before we knew about horseshoe crab blood and its amazing abilities. Following that, we will look at the problems that horseshoe crabs face in the present. And, for the finale, we will take a peek at what the future might look like!
Novitsky, T. J. 1984. Discovery to commercialization: the blood of the horseshoe crab
Kreamer, G. 2012 Biomedical use of horseshoe crabs slides for classroom use (including teacher notes and references). Delaware Division of Fish and Wildlife, Smyrna, DE.
Ding, Jeak L., and Bow Ho. “A new era in pyrogen testing.” Trends in biotechnology 19.8 (2001): 277-281.
Morris, Heather C., et al. “Setting a standard: the limulus amebocyte lysate assay and the assessment of microbial contamination on spacecraft surfaces.” Astrobiology 10.8 (2010): 845-852.
Heather C. Morris, Michael Damon, Jake Maule, Lisa A. Monaco, and Norm Wainwright. Astrobiology. September 2012, 12(9): 830-840. doi:10.1089/ast.2012.0863.