title: THE ROLE OF α₂-MACROGLOBULIN IN THE LIMULUS POLYPHEMUS CLOTTING SYSTEM
Creators: name: Herbstritt, Christopher
Publisher: name: Maryland Shared Open Access Repository
Year: 1998

description: The circulating amebocyte of the North American Horseshoe Crab, Limulus polyphemus, contains a rudimentary immune system that reacts to Gram-negative bacterial endotoxin. Lipopolysaccharide is a purified component of endotoxin and induces an enzymatic cascade in the Limulus blood system, by activating a series of serine protease zymogens, eventually forming a protein clot (Levin, J. and F. Bang 1964; 1968). A variety of commercial endotoxin detection assays have been developed using the Limulus amebocyte lysate, LAL. The pharmaceutical industry uses these products to detect endotoxin in their final products, since endotoxin is a potent fever-inducing pyrogen in humans. β (1-3) glucans, of fungal and yeast origin, also activate the LAL cascade, using an alternate pathway. Current LAL assays detect endotoxin and β (1-3) glucans, but do not allow differentiation between the two. Glucans are not toxic to humans, while endotoxins are toxic and their presence in medical products leads to severe immune responses and septic shock. An assay that could differentiate between the endotoxin and glucan would be useful. In addition to the proteins involved in the LAL cascade, the amebocyte lysate contains a variety of other proteins of unknown function. The third most abundant protein is a serine protease inhibitor named α₂-Macroglobulin, α₂-M. This complex protein is known to bind active serine proteases and remove them from circulation. The role of α₂-M in the LAL cascade is not known. Since α₂-M is a serine protease inhibitor, and the LAL cascade enzymes are serine proteases, it was suspected that removal or inactivation of α₂-M from the LAL system may increase the sensitivity or the rate of the LAL reaction. To address this question, a number of monoclonal antibodies were generated to the Limulus polyphemus α₂-M protein, as a means to study its role in the LAL enzymatic cascade, with minimal alteration of other components of the system. The antibody was characterized by studying cross-reactivity with human α₂-M and a number of mammalian clotting factors. The antibodies were generated by, injecting unreduced Limulus α₂-M into BALB/c mice, collecting the spleens and fusing the spleen cells with mouse myeloma cells. An ELISA screening assay was developed to identify clones producing the antibodies specific for α₂-M. Supernatant material from clones was also tested for specificity using western blot techniques. Clones producing α₂-M antibodies were expanded in mice and ascites was collected. The purification of the antibody was performed using Protein G columns. Large quantities of purified α₂-M antibodies were made from mouse ascites for two of the clones, 7A10 and 2D5. The clones 6E5, 8H12 and 5D6 produced antibodies to α₂-M, but did not produce sufficient quantities of ascites in mice. Antibody isotype determined using ELISA, indicated that all five of the antibodies were the IgG1 isotype. The 7A10 and 2D5 antibodies bound to the α₂-M protein, in its dimer and tetramer forms, on native LAL western blots. ELISA tests with a number of mammalian complement factors showed no cross-reactivity. An ELISA test with human α₂-M showed that the 2D5 antibody did cross-react. When added to the LAL assay, the 7A10 antibody showed almost complete inhibition of the endotoxin mediated pathway, but did not affect the ability of the assay to detect glucan. The 2D5 antibody did not affect either the glucan or endotoxin pathway of the LAL cascade. The antibody 7A10 may be useful in making a glucan specific assay.