Preparation and Properties of Monoclonal Antibodies against Lipopolysaccharide-Sensitive Serine Protease Zymogen, Factor C, from Horseshoe Crab (Tachypleus tridentatus) Hemocytes1

Abstract

Seventeen murine monoclonal antibodies (mAbs) against horseshoe crab clotting factor, factor C, were prepared and characterized. When the binding sites of these mAbs were analyzed by immunoblotting, ten mAbs recognized nonreduced factor C, five mAbs were directed against the heavy chain, and two mAbs were directed against the B chain. Three mAbs, 1H4, 2C12, and 2A7, one selected from each group, were used for further study. The mAb 1H4, which recognized only nonreduced factor C molecule, inhibited the factor C activity in a dose-dependent manner. It also inhibited lipopolysaccharide (LPS)- and alpha-chymotrypsin-mediated activations of the zymogen factor C, suggesting that 1H4 binds close to the active site and/or the substrate-binding site located in the serine protease domain (B chain) of factor C. On the other hand, 2C12 and 2A7 recognized, respectively, an epitope located in the heavy and the B chains, and inhibited LPS-mediated activation of factor C, but not alpha-chymotrypsin-mediated activation of factor C or factor C activity. Both F(ab')2 and Fab' fragments derived from 2C12 inhibited LPS-mediated activation in the same manner. These three mAbs did not bind with LPS, although a factor C-mAb complex was able to bind LPS, suggesting that the LPS-mediated activation of the zymogen factor C was induced through intermolecular interaction between the LPS-bound factor C molecules. The dissociation constants (Kd) for 1H4, 2C12, and 2A7 binding to factor C were determined as 1.9 x 10(-9), 0.6 x 10(-10), and 1.8 x 10(-10) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

A serine protease zymogen functions as a pattern-recognition receptor for lipopolysaccharides

“…Preincubation of the anti-factor C antibody with an excess amount of purified factor C completely inhibited binding to hemocytes. In addition, six kinds of mAbs against factor C all reacted with hemocytes (data not shown): five mAbs (2C12, 1B6, 2F6, 3D2, and 4H1) are directed against the H-chain of the two-chain form of factor C, and one mAb (2A7) is directed against the L-chain containing the serine protease domain (33). In contrast, a polyclonal antibody against TL-2, a major component of the large granules of hemocytes, did not specifically react with hemocytes, indicating the specific expression of the factor C antigen on hemocytes (data not shown).…”

A Limulus Amoebocyte Lysate Activating Activity (LAL Activity) That Lacks Biological Activities of Endotoxin Found in Biological Products

“…The mechanism of the LAL activity of IHA vaccine was compared with the mechanism of the activity of endotoxin by using a monoclonal antibody, 2C12, which recognizes the LPS-binding site of factor C of LAL (14). Twelve batches of IHA vaccine with varied levels of LAL activity were supplemented with an appropriate amount of the monoclonal antibody and the mixtures were assayed for LAL activity.…”

“…2), perchloric acid (18) and enzymatic treatments using protease type XVI, proteinase K and trypsin (data not shown) and was inhibited by the anti-factor C monoclonal antibody (2C12), which does not affect α-chymotrypsin-mediated activation of factor C (14). Acidic phospholipids, such as phosphatidylinositol, phosphatidylglycerol and cardiolipin were also reported to activate factor C, but this activation was suggested to be inhibited at the ionic strength of 0.1 M NaCl, in contrast to the factor C activation mediated by endotoxin (16).…”

“…LAL-active IHA vaccines were shown to exhibit the LAL activity by the specific activation of LAL factor C, which was abolished by an anti-factor C monoclonal antibody, 2C12. The fact suggested that the LAL-active IHA vaccine activated LAL on account of specific binding to the LPS-binding site of factor C (14). However, the vaccine showed neither an inhibiting nor an enhancing effect on pyrogenicity and TNF-α inducing activity of endotoxin (Tables 1 and 2) to suggest that the LALactive substance in IHA vaccines lacks affinity with the endotoxin receptors of mammalian cells.…”

Purification, Characterization, and cDNA Cloning of a 27-kDa Lectin (L10) from Horseshoe Crab Hemocytes

“…Purification procedures were carried out at 4°C, except that Mono S chromatography was done at room temperature. The lysate prepared from 43 g (wet weight) of hemocytes was first fractionated on a dextran sulfate-Sepharose CL-6B column (5 ϫ 23 cm), as described elsewhere (30), and a typical elution pattern is shown in Fig. 1.…”