Leukocyte complement: a possible role for C5 in lymphocyte stimulation

The results presented here show that Fab' antibody fragments directed to complement proteins C5, C6, and C7 inhibit lymphocyte stimulation in mixed lymphocyte culture (MLC) by up to 65%, as determined by decreased incorporation of 3H-thymidine. Lymphocyte stimulation induced by PHA-mitogen was also inhibited up to 100% by anti-C5 Fab'. Specificity of these reactions was established by the findings that goat anti-C5 or murine hybridoma anti-C5 both inhibited MLC; the inhibitory activity of anti-C5 Fab' was absorbed with highly purified C5 (but not with C3), and antibody directed to C3 did not inhibit lymphocyte stimulation by MLC or PHA. The effects of anti-C5 were exerted in a nontoxic manner. Cleavage of lymphocyte associated C5 with factor B (Bb) or with trypsin resulted in stimulation of lymphocyte thymidine incorporation. Purified C5a was found to induce lymphocyte stimulation in serum-free medium in pulse-chase types of experiments. Anti-C6 and C7 Fab' also inhibited lymphocyte stimulation induced in one-way MLC. These results suggest that C5, C5a, and/or C6 and C7 may play a role in triggering of lymphocyte blastogenesis.     

抗CD20嵌合抗体片段F(ab′)2突变体在大肠杆菌中的高效分泌表达

构建抗CD20嵌合抗体片段F(ab′)₂ 突变体 ,研究其在大肠杆菌中的高效表达及其表达产物的生物学活性。采用PCR法构建抗CD20嵌合抗体片段F(ab′)₂ 突变体 ,并用双脱氧终止法测定DNA序列 ;采用 19L发酵罐高密度发酵抗CD20嵌合抗体片段F(ab′)₂ 突变体 ,采用亲和色谱和分子筛色谱法纯化表达产物 ,并用SDS-PAGE和薄层激光扫描鉴定纯化产物 ;采用活细胞间接免疫荧光法测定纯化产物与靶细胞的结合活性 ;MTT法测定纯化产物对Raji细胞的生长抑制作用 ,并研究其作用机理。DNA序列测定结果表明 ,抗CD20嵌合抗体片段F(ab′)₂ 突变体已成功构建 ,表达可溶性产物的产量达 360mg L ,具有与Raji细胞 (CD20⁺)结合的活性 ,并抑制Raji细胞的生长 ,其作用机理为诱导Raji细胞凋亡。此突变体有望成为治疗非何杰金氏B细胞淋巴瘤的药物。     

Further evidence for the antibody nature of C3 nephritic factor (C3NeF).

C3 nephritic factor (C3NeF), found in the sera of some patients with membranoproliferative glomerulonephritis, has been shown to be composed of two heavy and two light chains, like IgG; in addition it shares antigenic determinants with IgG. Purified C3NeF binds to the amplification convertase of complement, C3b,Bb, and thereby prevents decay of its C3-cleaving potential. The capability of C3NeF to bind to C3b,Bb was used as a means for purifying C3NeF to homogeneity. The investigation described in this report suggests that binding of C3NeF to C3b,Bb occurs via the Fab portion of the molecule. Pepsin treatment of eight C3NeF preparations resulted in an average loss of 76% of C3NeF functional activity. Papain treatment induced a loss of approximately 90%. The decrease in functional activity could be attributed to the accelerated rate of dissociation of 125I-F(ab')2 and 125I-Fab fragments from stabilized cell-bound C3b,Bb. The dissociation rate of 125I-F(ab')2 from C3b,Bb was comparable with the decay of the functional activity of C3b,Bb stabilized by F(ab')2 or Fab fragments of C3NeF. Although these results suggest that the stabilizing activity of C3NeF is mediated by the Fab portion of the molecule, it was found that the Fc portion also contributes to its functional activity.     

Preparation and performance of bispecific F(ab' gamma)2 antibody containing thioether-linked Fab' gamma fragments

A simple and efficient method is described for the production of pure bispecific F(ab' gamma)2 heterodimers, in which the individual antibody Fab' gamma fragments are joined via a stable thioether linkage. Hybrid molecules were constructed from both mouse monoclonal and rabbit polyclonal antibodies with equal efficiency, in the combinations mouse-rabbit and mouse-mouse. Peptic F(ab' gamma)2 fragments from the two chosen antibodies were first reduced to provide Fab' gamma SH. The SH groups on one of the Fab' gamma SH partners were then fully alkylated with o-phenylenedi-maleimide to provide free maleimide groups. Finally the two preparations, Fab' gamma mal and Fab' gamma SH, combined under conditions which allowed cross-linking of the maleimide and SH groups and avoided reoxidation of SH groups. The major product isolated from the reaction mixture after chromatography was always the F(ab' gamma)2 heterodimer (50 to 70%), other products being unreacted Fab' gamma and trace amounts of putative F(ab' gamma)3. Immunochemical analysis revealed that the thioether-linked F(ab' gamma)2 molecules were essentially all heterodimers, most of which had been joined via their Fd chains. The dual specificity of F(ab' gamma)2 heterodimers was tested functionally in three systems: 1) the combination (anti-idiotype + anti-phycoerythrin) linked L2C cells to the fluorochrome phycoerythrin, allowing fluorescence analysis; 2) the combination (anti-idiotype + anti-saporin) linked L2C cells to the ribosome-inactivating protein saporin, and transformed a subtoxic dose of saporin into a highly toxic mixture which prevented further protein synthesis by L2C cells; and 3) the combination of anti-idiotype with 3G8 (antibody to the Fc gamma receptor CD16) subjected L2C cells to cytotoxic attack by human mononuclear effectors.     

Radiolocalisation of an anti-CEA monoclonal antibody (FO23C5) and its fragments in a colon carcinoma xenograft model

A new monoclonal antibody designated FO23C5 against a protein component of carcinoembryonic antigen (CEA) has been developed. A xenograft system of human colon cancer was used to compare the intact monoclonal IgG with its fragments (Fab')2 and Fab) and with an established anti-CEA antibody (MAb35) and the antibody AUA1 raised against the colon carcinoma cell line. We demonstrate that FO23C5 compares well with the existing anti-CEA antibody and with AUA1, and that F(ab')2 fragments perform best in achieving optimal tumour to normal tissue ratios compared with intact IgG and Fab fragment.     

Studies on the mechanism of bacterial resistance to complement-mediated killing. VI. IgG increases the bactericidal efficiency of C5b-9 for E. coli 0111B4 by acting at a step before C5 cleavage

Our previous experiments showed that immune IgG and F(ab')2, but not Fab', mediated serum killing of Escherichia coli 0111B4, strain 12015 (12015), without significantly increasing the extent of terminal complement (C) component attachment to the bacterial surface. We concluded that bactericidal antibody must change either the site or the nature of C5b-9 bacterial attachment. To pursue this possibility, conditions necessary for elution of C5b-9 from the bacterial surface were examined. Forty-two to 44% of 125I-C9 was released from the serum-resistant nonpresensitized 12015 by 1 M NaCl or 0.1% trypsin, compared with the 21 to 24% release from the serum-sensitive presensitized isolate under the same condition. When strain 12015 bearing 125I-C9 was lysed in a French pressure cell, 73.1% of 125I-C9 was released with the capsular fraction if the organisms had not been presensitized. In contrast, on presensitized 12015, 70.2% of 125I-C9 remained associated with the outer membrane after such lysis. These results suggested that C5b-9 was trapped within or underneath the capsule of 12015 in the absence of bactericidal antibody, but that addition of antibody led to C5b-9 insertion into the outer membrane with bacterial killing. The requirement of C components preceding C5 for bacterial killing was next examined. Minimal killing of presensitized 12015 occurred when a terminal C complex was formed by acid activation from purified C5, C6, C7, C8, and C9 in the absence of C3 or earlier components. In contrast, between 1.2 and 3 log killing of nonpresensitized rough Salmonella minnesota and rough E. coli was observed in the same system. Killing of 12015 was examined with bacteria incubated in C5-deficient serum (C5D), followed by washing and the addition of purified C5, C6, C7, C8, and C9 to permit C5b-9 formation. Antibody was added before or after incubation in C5D serum, or after the addition of purified C5-C9. Under conditions of equivalent C3 and C9 binding, significant killing occurred only when antibody was added before incubation in C5D serum. These results show that antibody must be present at or before the time of C5 convertase formation to mediate killing of 12015 by C5b-9. Therefore, antibody is unlikely to be functioning primarily to alter the bacterial surface to expose sites for C5b-9 insertion, nor is the effect of antibody simply to increase C3 and terminal component binding. We postulate that antibody mediates killing of 12015 by localizing C5b-9 around antibody-clustered sites of C3 and C5 convertase formation.     

Purification of recombinant chimeric B72.3 Fab' and F(ab')2 using streptococcal protein G.

Streptococcal protein G has been used extensively for the purification of antibodies using the interaction of the Fc region with protein G. Many antibodies also interact with protein G through a low-affinity binding site for the Fab region. The exploitation of this low-affinity interaction for the purification of Fab' fragments is described here. Chimeric mouse-human B72.3 Fab' and F(ab')2 fragments were expressed by CHO cells and purified from CHO cell supernatant using protein G-Sepharose. Since chimeric B72.3 Fab' bound weakly to the protein G-Sepharose it could be separated from F(ab')2 and eluted with a pH 7 wash whereas B72.3 F(ab')2 required elution at pH 2. Both Fab' and F(ab')2 were recovered with full immunoreactivity and could be further purified using gel-filtration chromatography to greater than 99% purity. This method allows the simple purification of directly expressed Fab' or F(ab')2 fragments from CHO cell supernatant.     

Triggering of histamine release from rat mast cells by divalent antibodies against IgE-receptors.

Antibodies against receptor molecules for IgE on rat basophilic leukemic (RBL) cells were prepared by immunization of a rabbit with immune precipitates composed of IgE-receptor complexes and anti-IgE. Antibodies against cell surface components were specifically purified by using RBL cells and rendered specific for mast cells by appropriate absorption. The major antibodies in the final preparation (anti-RBL) were directed against receptor molecules. It was found that the F(ab')2 fragments of anti-RBL induced histamine release from rat mast cells and caused immediate skin reactions in normal rats. These reactions by anti-RBL or its F(ab')2 fragments were inhibited if the receptors on mast cells had been saturated with IgE. The Fab' fragments of anti-RBL could bind with receptors on RBL cells and blocked passive sensitization of mast cells with IgE antibodies, but failed to induce skin reactions and histamine release from normal mast cells. Sensitization of normal rat skin with the Fab' fragment followed by an i.v. injection of anti-rabbit IgG induced skin reactions. The results indicated that bridging of receptor molecules by divalent anti-receptor antibody triggered mast cells for histamine release.     

Ligation of low-density lipoprotein receptor-related protein with antibodies elevates intracellular calcium and inositol 1,4, 5-trisphosphate in macrophages

We have probed the signaling characteristics of the macrophage low-density lipoprotein receptor-related protein (LRP) with monoclonal antibody 8G1, its Fab and F(ab')(2) fragments directed against the ligand binding heavy chain, and monoclonal antibody 5A6 directed against the membrane-spanning light chain of LRP. Ligation of LRP with 8G1, its Fab and F(ab')(2) fragments, or 5A6 increased intracellular Ca(2+) levels two- to threefold. Prior ligation of LRP with 8G1 did not affect the increase in [Ca(2+)](i) observed on subsequent ligation of LRP with lactoferrin, P. exotoxin A, or lipoprotein lipase. Binding to LRP by 8G1, its Fab and F(ab')(2) fragments, or 5A6 increased inositol 1,4,5-trisphosphate (IP(3)) levels by 50 to 100%. Incubation of macrophages with guanosine 5', 3'-O(thio)-triphosphate (GTP-gamma-S) before treatment with antibody potentiated and sustained the 8G1-induced increase in IP(3) levels. Treatment of macrophages with guanyl-5'-yl thiophosphate prior to GTP-gamma-S treatment abolished the GTP-gamma-S-potentiated increase in IP(3) levels in 8G1-treated macrophages. Antibody-induced increases in IP(3) and [Ca(2+)](i) in macrophages on ligation of LRP were pertussis toxin sensitive. Binding of 8G1 or its Fab or F(ab')(2) fragments to LRP stimulated macrophage protein kinase C (PKC) activity as evaluated by histone IIIs phosphorylation by about two- to sevenfold. Staurosporin inhibited the anti-LRP antibody-induced increase in PKC activity. Ligation of LRP with 8G1 increased cellular cAMP levels about twofold. Preincubation of macrophage with the LRP-binding protein receptor-associated protein suppressed the 8G1-induced increase in cAMP levels. Thus, binding of antibodies directed against either chain of LRP triggers complex signaling cascades.     

Mechanism of human monocyte activation via the 40-kDa Fc receptor for IgG

It is shown that a mAb specific for the human 40-kDa FcR (FcRII) leads to activation of human monocytic cells but that extensive cross-linking of the receptor is required. Calcium mobilization can be induced in immature monocytic cells (undifferentiated U937 cells) and peripheral blood monocytes with an intact IgG1 anti-FcRII antibody (CIKM5) but not by F(ab')2 fragments of this antibody. The intact antibody can bind in a tripartite manner by its two F(ab') sites and its Fc-binding site whereas the F(ab')2 fragments of this antibody can only bind in a divalent fashion. A rise in intracellular free calcium ion concentration occurs when F(ab')2 fragments are cross-linked with F(ab')2 anti-mouse Ig indicating that more extensive cross-linking of FcRII is required rather than an obligatory requirement for an Fc-FcRII interaction. Calcium mobilization in response to intact or cross-linked F(ab')2 fragments of CIKM5 is associated with superoxide production only in IFN-gamma-primed peripheral blood monocytes and IFN-gamma differentiated U937 cells indicating that the activation signal produced via FcRII is inadequate to fully stimulate non-"primed" cells. A second mAb reactive with FcRII (2E1) does not cause calcium mobilization in monocytes or U937 cells, and partially blocks the effects of CIKM5. 2E1 also blocks CIKM5 superoxide production in IFN-gamma-primed monocytes and differentiated U937 cells. This may be explained in part by the fact that 2E1 is an IgG2a antibody and can only participate in bipartite binding with FcRII. When 2E1 is cross-linked with F(ab')2 anti-mouse Ig there is a small calcium response. This does not cause superoxide generation in IFN-primed monocytes but does do so in IFN-gamma differentiated U937 cells. FcRII is also expressed on granulocytes and some B cells but the effects of cross-linking the receptor on these cells differ from those seen in monocytes.     

The role of A-cells in affecting the immunostimulating effect of F(ab')2-fragments

The immunoregulatory effect of F(ab')2 fragments on normal rabbit IgG and that preincubated with A-cells from spleen have been compared. Both products were tested for their ability to enhance primary immune response of rabbit spleen cells to SRBC. It was demonstrated that low molecular mass product appeared after F(ab')2 fragments incubation with A-cells at 37 degrees C and possessed immunostimulating activity similar to that of initial F(ab')2 fragments. In addition, it was shown that F(ab')2 reduction to monovalent Fab' fragment with the following alkylation of SH-group abolished the ability of Fab' fragment to enhance the immune response. It may signify that half cystein Fab' fragment residue is essential for processing of the fragment in A-cells and (or) for immune response enhancement.     

Formation and structure of the C5b-7 complex of the lytic pathway of complement.

The formation and structure of the complement cytolytic intermediary complex, C5b-7, were studied with the aim of determining the interactive regions of C5, C6, and C7. The structure of human complement component C5 was elucidated by the application of limited proteolysis which generated well characterized major polypeptide fragments of this molecule. Plasmin, thrombin, and kallikrein cleave C5b with greater facility than C5. The most useful cleavage of C5b was effected by plasmin because the fragmentation pattern was similar to the processing of C3b by factors H, I, and kallikrein. Plasmin hydrolyzes peptide bonds within the alpha'-chain of C5b, resulting in a four-chain fragment, C5c (M(r) = 142,000), and a single chain fragment, C5d (M(r) = 43,000). Circular dichroism spectroscopic analyses indicated that C5d is substantially richer in alpha-helical content than is C5c (27 versus 9%). Polyclonal antibodies directed against C5c blocked the interaction of C5b-6 with C7, whereas antibodies directed against C5d inhibited the binding of C5 with C3b. Chemical cross-linking using a cleavable radioiodinated photoreactive reagent revealed that both C6 and C7 associate preferentially with the alpha'-chain of C5b. The reversible interactions of C5 with C6, C7, and major polypeptide fragments derived from these were investigated with solid phase binding assays. The results indicate that the carboxyl-terminal domains of C6 and C7, which have cysteine-rich modules homologous to those found in factors H and I, have the capacity to link specifically with C5.     

Monoclonal antibodies against seven sites on the head and tail of Dictyostelium myosin

Ten monoclonal antibodies (My1-10) against Dictyostelium discoideum myosin were prepared and characterized. Nine bound to the 210-kD heavy chain and one (My8) bound to the 18-kD light chain. They defined six topographically distinct antigenic sites of the heavy chain. Five binding sites (the My1, My5, My10 site, and the My2, My3, My4, and My9 sites) are located on the rod portion of the myosin molecule. The position of the sixth site (the My6 and My7 site) is less certain, but it appears to be near the junction of the globular heads and the rod. Three of the antibodies (My2, My3, and My6) bound to myosin filaments in solution and could be sedimented in stoichiometric amounts with the filamentous myosin. In contrast, My4, which recognized a site on the rod, inhibited the polymerization of monomeric myosin into filaments. A single antibody (My6) affected the actin-activated ATPase of myosin. The nature of the effect depended on the valency of the antibody and the myosin. Bivalent IgG and F(ab')2 fragments of My6 inhibited the actin-activated ATPase of filamentous myosin by 50% whereas univalent Fab' fragments increased the activity by 50%. The actin-activated ATPase activity of the soluble chymotryptic fragment of myosin was increased 80-90% by both F(ab')2 and Fab' of My6.     

Defining the role of complement in experimental pemphigus vulgaris in mice

Parenteral passive transfer of human pemphigus vulgaris IgG (PV IgG) into neonatal mice reproduces the cutaneous disease. We used this model to study the role of complement in the development of acantholysis in three steps. Peptic F(ab')2 fragments were prepared from PV IgG and were injected into seven newborn mice, and all animals developed acantholytic skin blisters without local complement activation, as shown by direct immunofluorescence. These fragments were reduced and alkylated to produce Fab' fragments with equivalent in vitro binding activity. The monovalent fragments were given in an identical fashion to five littermates but failed to produce disease even though they were bound in the epidermis in vivo. Intact PV IgG was injected in 20 genetically C5-deficient neonates (B10-D2-OSN strain) and 20 control neonates (B10-D2-NSN, normal complementemic). Extensive blistering, with a positive Nikolsky sign, was produced in all 40 animals. PV IgG was given to 34 BALB/c neonates that were complement depleted by pretreatment with cobra venom factor (CoF) for 24 hr, and to 38 untreated neonates from the same litters. There was no difference in the disease produced after CoF treatment in animals that received high doses of PV IgG (5 to 15 mg/g/day). In animals receiving 2.5 mg PV IgG/g/day, blister formation was delayed and the final extent of the cutaneous lesions was less in CoF-treated mice (n = 12) than in normal complementemic controls (n = 12, p less than 0.02). These results show that complement activation is not an essential mechanism in PV IgG-induced acantholysis in vivo, but it does have an amplifying effect on the development of cutaneous lesions under certain conditions, and lesions can be induced in vivo by bivalent F(ab')2 fragments of PV IgG, but not by the monovalent Fab' fragments, suggesting that cross-linking of the cell surface antigen is an initiating signal in acantholysis.     

Neutralizing activities of early and late IgG fragments from rabbits immunized with herpes simplex virus.

Rabbits immunized with herpes virus were bled periodically and bivalent and univalent fragments of IgG from each serum sample were prepared by enzymatic digestion. The 2-week F(ab')2 showed a low neutralizing activity only after addition of anti-IgG. F(ab')2 of the 4-week serum retained almost all of the neutralizing activity of IgG, while its univalent fragments demonstrated none even when tested with anti-IgG. In contrast to these early IgG fragments, univalent fragments of the 9-week and 20-week IgG neutralized the virus to considerable extents in the absence of anti-IgG; after addition of anti-IgG the activity equaled that of intact IgG in the cases of Fab' and Fab-II, though the activity of Fab-I was relatively low. Three three univalent fragments were all sensitive to heating at 70 C and to ultraviolet irradiation, whereas intact IgG resisted these treatments. F(ab')2 was resistant to the heating and less sensitive to ultraviolet irradiation than univalent fragments. Neutralization kinetic curve experiments to test blocking effects of IgG fragments against the neutralization by intact IgG suggested that the early Fab' did combine with the virus and that the late Fab' exerted a higher blocking effect than the early Fab'.     

Synthesis of bifunctional antibodies for immunoassays

The synthesis of bifunctional antibodies using the principle of solid-phase synthesis is described. Two Fab' fragments were chemically linked together via a bismaleimide crosslinking reagent. The F(ab')(2) fragments from intact immunoglobulin G (IgG) were prepared using an immobilized pepsin column. Goat, mouse, and human antibodies were digested completely within 4 h. The F(ab')(2) fragments thus produced did not contain any IgG impurities. Fab' fragments were produced by reducing the heavy interchain disulfide bonds using 2-mercaptoethylamine. Use of the solid-phase reactor in the preparation of the bifunctional antibodies eliminated many of the time-consuming separation steps between the fragmentation and conjugation steps. This procedure facilitates the automation of bifunctional antibody preparation and the rapid optimization of reaction conditions.     

Inhibition of the formation of the complement membrane-attack complex by a monoclonal antibody to the complement component C8 alpha subunit.

The effect of nine monoclonal antibodies to complement component C8 on the interaction of C9 with preformed cell-surface C5b-8 complexes and on the functional insertion of C8 into the membrane-attack complex (MAC) was investigated. None of the antibodies prevented C9 insertion into a preformed C5b-8 complex. One antibody (F1) directed to the C8 alpha subunit clearly inhibited formation of a functional MAC. It is proposed that this antibody prevents the C8 alpha subunit unfolding and distorting the bilayer to allow C9 insertion.     

Modulation of FcR function by complement: subcomponent C1q enhances the phagocytosis of IgG-opsonized targets by human monocytes and culture-derived macrophages

We have investigated the interaction of C1q, a subunit of the first component of complement, with human monocytes and culture-derived macrophages. Adherence of these mononuclear phagocytes to surfaces coated with C1q induced a marked enhancement of the phagocytosis of sheep erythrocytes opsonized with IgG anti-Forssman antibody (EA-IgG). This C1q-mediated enhancement of phagocytosis was dose dependent, and was specifically blocked by pretreatment of the C1q-coated surfaces with F(ab')2 anti-C1q. The augmentation of FcR-mediated phagocytosis by C1q was determined to be a result of the interaction between the C1q and the phagocytic effector cell, and was not due to interaction between the surface-bound C1q and the EA-IgG. Neither resting nor N-formyl-methionyl-leucyl-phenylalanine-stimulated polymorphonuclear leukocytes were induced by C1q to increase FcR-mediated phagocytosis. Experiments conducted with purified fragments of C1q suggest that the C1q phagocytosis enhancement signal resides in the collagen-like tail domain of the molecule. This region is the same portion of the molecule previously shown to interact with the cell surface C1q receptor. Native type I collagen was unable to enhance FcR-mediated phagocytosis by mononuclear phagocytes. It has been demonstrated that C1q can be localized to areas of inflammation, and additionally C1q can be secreted by macrophages in culture. In view of these findings and the results of our present study, we hypothesize that C1q could provide local, direct, and non-opsonic enhancement of phagocytosis by mononuclear phagocytes in areas of infection and inflammation.     

Conjugation and evaluation of 7E3 x P4B6, a chemically cross-linked bispecific F(ab')2 antibody which inhibits platelet aggregation and localizes tissue plasminogen activator to the platelet surface.

A bispecific F(ab')2 monoclonal antibody which recognizes both the platelet GPIIb/IIIa receptor and human tissue plasminogen activator was produced to target tPA to platelets for enhancement of thrombolysis. A stable, thioether-cross-linked bispecific F(ab')2 (7E3 X P4B6) combining the GPIIb/IIIa-specific monoclonal antibody 7E3, which inhibits platelet aggregation, and a nonneutralizing anti-tPA monoclonal antibody (P4B6) was produced. This was performed by coupling each of the parental Fab' moieties with the homobifunctional cross-linker bis(maleimido methyl) ether (BMME). 7E3 X P4B6 was sequentially purified using gel-filtration chromatography and hydrophobic interaction (HIC) HPLC. HIC was shown to completely resolve each of the parental F(ab')2 species from the bispecific one. 7E3 X P4B6 was shown to retain completely each of the parental immunoreactivities in GPIIb/IIIa and tPA binding EIA's. The bispecific antibody inhibited platelet aggregation in vitro at levels comparable to those for 7E3 Fab. Recruitment of tPA activity to washed human platelets was demonstrated using the S-2251 chromogenic substrate assay. 7E3 X P4B6 recruited 12-fold more tPA to the washed platelets than a mixture of the parental F(ab')2 molecules used as controls.     

Preparation and properties of monoclonal antibodies against lipopolysaccharide-sensitive serine protease zymogen, factor C, from horseshoe crab (Tachypleus tridentatus) hemocytes.

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)