BIOMEDICAL POTENTIAL OF AMOEBOCYTE LYSATE FROM CARCINOSCORPIUS ROTUNDICAUDA AND TACHYPLEUS GIGAS

Amoebocyte lysate were prepared from 2 Singaporean species of horseshoe crab, Carcinoscorpius rotundicauda and Tachypleus gigas. Whilst chloroform extraction adversely affected the potency of the lysate, no adverse effect was observed using bovine serum albumin as a protein stabilizer. The quality of the prepared lysate was enhanced by the addition of calcium, magnesium and sodium ions. The lysates were comparable to commercial lysates of Limulus Polyphemus and Tachypleus tridentatus in terms of the total protein content and sensitivity of endotoxin detection. Protein concentration, however, did not appear to be an absolute indicator of lysate potency. Our preparations were proven effective in the gelation, chromogenic and fluorimetric assays for endotoxin. Large-scale culture of amoebocytes was attempted with a view to reduce biological variations found in prepared lysates. The amoebocytes were cultured in Grace Insect Medium + 20% Foetal Bovine Serum. These cells survived for up to 2 weeks but did not appear to multiply, implying that the amoebocytes might have reached the end of their hemopoeitic pathway. In an attempt to reverse this arrest in the cell cycle, the amoebocytes ere stimulated by 4 known lymphocyte mitogens: pokeweed mitogen, phytohaemagglutinin, concanavalin A and endotoxin. Transformation was carried out using a range of 5 to 50 ug/ml of mitogen, over a time course. Based on the uptake of tritiated-thymidine as an indicator of DNA synthesis, pokeweed mitogen was found to be most effective. An increase of up to 55 fold of tritiated-thymidine uptake was observed in cultures stimulated by 2.5 ug/ml pokeweed mitogen for 48 h. The addition of insulin to promote glucose uptake did not have any apparent effect on the amoebocytes whereas the stimulation was completely reversed by the addition of 4-hydroxy urea. By gel filtration on Sephadex G-75, whole amoebocyte lysate was fractionated into 5 peaks. The first two peaks contained pro-clotting enzyme and Factor C, respectively. Coagulogen was found in the third peak. Reconstitution studies revealed that the fourth and fifth peaks did not have any significant role to play in the gelation cascade reactions. Biochemical similarities between the two types of lysates revealed that a major protein of 21 kDa was involved in the gelation reaction. This protein, later determined to be coagulogen, constitutes approximately 40% of the total lysate protein. By using reducing and non-reducing SDS-PAGE, the cleavage of coagulagen to coagulin, a 17 kDa protein, was monitored against time and varying concentrations of either lysate protein or endotoxin. Results indicated that the gelation reaction was completed in 15 minutes. It also shows that by gel densitometry, the sum of the coagulogen and coagulin were almost equal at all the time intervals tested. The presence of the 17 kDa coagulin proved to be a more definitive way to ascertain the integrity of fresh lysate preparations.

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• KIM JOW CHANG