Carcinoscorpius and Tachypleus lysates assay for detecting endotoxin in milk and groundwater: Toward reducing reliance on Limulus amebocyte lysate

As one of the emerging pollutants, the presence of endotoxins became an increasingly urgent issue due to their impact on human health when found in drinking water and groundwater. A simple and rapid assay for assessing bacterial biomass in these water samples was essential, particularly in situations where access to laboratory facilities was limited. The bacterial endotoxin test (BET) using the Limulus amebocyte lysate (LAL) test was established as an effective method. However, the application of BET employing Carcinoscorpius or Tachypleus Amebocyte Lysate (CAL/TAL), derived from Carcinoscorpius rotundicauda and Tachypleus gigas, remained rarely performed. This study aimed to explore the CAL and TAL potential for detecting bacterial endotoxins in milk and groundwater. In this assay, 94 blood samples of horseshoe crabs collected from the Banyuasin Waters of South Sumatra (79 samples of C. rotundicauda and 15 samples of T. gigas) were used. The assay was carried out using the gel-clot method. The results revealed that the CAL and TAL were able to detect small concentrations of endotoxin (up to a concentration of 0.0156 EU/mL) in raw milk, pasteurized milk, well water, and L1 HPV 52 protein based on the gel-clot test. Both CAL and TAL could be a potential substitute for the LAL assay, especially for detecting bacterial biomass in milk and groundwater. Furthermore, these findings were essential as initial scientific information for developing the CAL/TAL tests toward the development of recombinant Factor C (rFC).