Exploratory studies into the therapeutic and diagnostic capability of blood from the horseshoe crab, Limulus polyphemus

Antimicrobial resistance is a major health challenge that causes serious morbidity and mortality worldwide. The overuse of antibiotics has sped up the rise of resistant pathogens and rendered many antibiotics useless. There has been a worldwide push to use antibiotics with more caution, but in order to do so, physicians need quicker diagnostic methods than blood cultures for confirming bacterial infections. Sepsis, which is when the body’s response to an infection harms its own tissues and organs, is a fast-acting syndrome. Delaying administration of antibiotics rapidly increases the risk for mortality, forcing physicians to prescribe broad spectrum antibiotics until blood cultures can provide more information. To combat these antimicrobial resistant superbugs, not only do we need a faster way to diagnose them, but we need more diverse methods to fight them. The horseshoe crab, Limulus polyphemus, has an innate property in its blood to coagulate in the presence of LPS at the pico- to nanogram level. The limulus amoebocyte lysate (LAL) assay has been used for decades by the biomedical industry to verify sterility of medical devices. Scientists have attempted to use the LAL assay as a diagnostic test for bacterial infections, with much difficulty over the years. The overall objective of this research is to investigate the diagnostic and therapeutic potential of blood components from Limulus polyphemus. To achieve this goal, the first aim will determine a simple protocol to detect bacteria or bacterial endotoxin in human blood. Preliminary data shows that anticoagulants that are often found in blood collection tubes will inhibit the LAL assay in the presence of endotoxin. We have demonstrated the ability to overcome these inhibitors by isolation and washing of the red blood cells. The second part of this aim quantifies the detection limits for endotoxin. We performed serial dilutions of pathogen concentrations to determine sensitivity of the LAL assay to bacteria and bacterial endotoxin. We also performed the LAL assay using E. coli. Our results demonstrate the ability to detect bacteria and bacterial endotoxin in human blood samples. The second aim of this research focused on the therapeutic potential of the horseshoe crab blood and its components. To achieve this, bioassays on different fractions and preparations of hemolymph were tested against two strains of Staphylococcus aureus to see if any fraction of the blood has bioactivity. We have found that antimicrobial activity was not observed in the hemolymph, plasma, and amoebocytes of the horseshoe crab blood. Further studies are needed to investigate isolated antimicrobial peptides and hemocyanin from amoebocytes for testing