Assembly of fibrin. A light scattering study.

Using stopped flow light scattering, we show that assembly of fibrin following activation with non-rate-limiting amounts of thrombin or reptilase occurs in two steps, of which the first is end-to-end polymerization of fibrin monomers to protofibrils and the second is lateral association of protofibrils to fibers, in agreement with Ferry's original proposal. Polymerization is found to proceed as a bimolecular association of bifunctional monomers; the overall rate varies as the inverse first power of the concentration; end-to-end association of two monomers, of a monomer and an oligomer, and of two oligomers occurs with the same rate constant. The value of the rate constant is 8.2 C 10(5) M-1 s-1 in 0.5 M NaCl, is three times larger in 0.1 M NaCl (0.05 M Tris, pH 7.4), and is the same following activation by reptilase and by thrombin. The onset of growth of fibers from protofibrils takes 12 times longer in 0.5 than in 0.1 M salt, i.e. thick fibers ("coarse" gels) form from short protofibrils, and thin fibers ("fine" gels) form from longer protofibrils. Jumps of salt concentration at times when protofibrils, but not fibers, have formed result in immediate growth of thick fibers at low salt from long protofibrils formed at high salt. The rate of fiber growth in these experiments varies as the inverse first power of the concentration. 3the instant of gelation (formation of a network of fibers) falls in the later half of the time during which the scattering rises due to fiber growth; the rise of gel rigidity after gelation is found to continue beyond the end of this period. Jumps from low to high salt result in retention of whatever fibers have formed at low salt and a very small additional increase of the scattering due to further fiber growth at high salt. From a variety of evidence, we conclude that the properties of fibrin are determined by kinetics and not equilibria of assembly steps. Results obtained here agree with the following scheme of fibrin assembly: monomers polymerize to protofibrils; long protofibrils associate laterally to fibers; occasionally a long protofibril associates with two different fibers to form an interfiber connection; fiber growth does not reverse to yield stabler, more compact, structures and terminates in formation of a network of fibers. The typical delay of fiber growth is the time during which protofibrils form from monomers. Measurements at rate-limiting concentrations of thrombin have allowed estimation of turnover rates of fibrinopeptides that agree with kinetic parameters obtained with direct assay of fibrinopeptide. Release of fibrinopeptide B causes more rapid fiber formation. Addition of thrombin after activation by reptilase, at a time when protofibrils, but not fibers, have formed, is followed rapidly by fiber formation; this proves that thrombin readily removes fibrinopeptide B from protofibrils. On the basis of these new results and earlier work (in particular, Blomb?ck, B., Hessel, B., Hogg, D., and Therkildsen, L...     

Monoclonal antibodies as an instrument to study fibrin polymerization

Examples of using monoclonal antibodies (MAb) for studying the fibrin polymerization mechanism are considered. MAb with epitopes situated in the fibrin polymerization sites or in the recognition sites of enzymes thrombin, plasminogen, and factor XIII, which are the functional partners of fibrin, are primarily discussed. The MAb to epitopes in various regions of A alpha, B beta, and gamma polypeptide chains of the functionally important E, D, and alpha C domains of fibrin are successively described.     

The goodpasture autoantigen. Identification of multiple cryptic epitopes on the NC1 domain of the alpha3(IV) collagen chain

Goodpasture (GP) disease is an autoimmune disorder in which autoantibodies against the alpha3(IV) chain of type IV collagen bind to the glomerular and alveolar basement membranes, causing progressive glomerulonephritis and pulmonary hemorrhage. Two major conformational epitope regions have been identified on the noncollagenous domain of type IV collagen (NC1 domain) of the alpha3(IV) chain as residues 17-31 (E(A)) and 127-141 (E(B)) (Netzer, K.-O. et al. (1999) J. Biol. Chem. 274, 11267-11274). To determine whether these regions are two distinct epitopes or form a single epitope, three GP sera were fractionated by affinity chromatography on immobilized NC1 chimeras containing the E(A) and/or the E(B) region. Four subpopulations of GP antibodies with distinct epitope specificity for the alpha3(IV)NC1 domain were thus separated and characterized. They were designated GP(A), GP(B), GP(AB), and GP(X), to reflect their reactivity with E(A) only, E(B) only, both regions, and neither, respectively. Hence, regions E(A) and E(B) encompass critical amino acids that constitute three distinct epitopes for GP(A), GP(B), and GP(AB) antibodies, respectively, whereas the epitope for GP(X) antibodies is located in a different unknown region. The GP(A) antibodies were consistently immunodominant, accounting for 60-65% of the total immunoreactivity to alpha3(IV)NC1; thus, they probably play a major role in pathogenesis. Regions E(A) and E(B) are held in close proximity because they jointly form the epitope for Mab3, a monoclonal antibody that competes for binding with GP autoantibodies. All GP epitopes are sequestered in the hexamer configuration of the NC1 domain found in tissues and are inaccessible for antibody binding unless dissociation of the hexamer occurs, suggesting a possible mechanism for etiology of GP disease. GP antibodies have the capacity to extract alpha3(IV)NC1 monomers, but not dimers, from native human glomerular basement membrane hexamers, a property that may be of fundamental importance for the pathogenesis of the disease.     

Characterization of the kinetic pathway for liberation of fibrinopeptides during assembly of fibrin

The time dependence of the release of fibrinopeptides from fibrinogen was studied as a function of the concentration of fibrinogen, thrombin, and Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization. The release of fibrinopeptides during fibrin assembly was shown to be a highly ordered process. Rate constants for individual steps in the formation of fibrin were evaluated at pH 7.4, 37 degrees C, gamma/2 = 0.15. The initial event, thrombin-catalyzed proteolysis at Arg-A alpha 16 to release fibrinopeptide A (kcat/Km = 1.09 X 10(7) M-1s-1) was followed by association of the resulting fibrin I monomers. Association of fibrin I was found to be a reversible process with rate constants of 1 X 10(6) M-1s-1 and 0.064 s-1 for association and dissociation, respectively. Assuming random polymerization of fibrin I monomer, the equilibrium constant for fibrin I association (1.56 X 10(7) M-1) indicates that greater than 80% of the fibrin I protofibrils should contain more than 10 monomeric units at 37 degrees C, pH 7.4, when the fibrin I concentration is 1.0 mg/ml. Association of fibrin I monomers was shown to result in a 6.5-fold increase in the susceptibility of Arg-B beta 14 to thrombin-mediated proteolysis. The 6.5-fold increase in the observed specificity constant from 6.5 X 10(5) M-1s-1 to 4.2 X 10(6) M-1s-1 upon association of fibrin I monomers and the rate constant for fibrin association indicates that most of the fibrinopeptide B is released after association of fibrin I monomers. The interaction between a pair of polymerization sites in fibrin I dimer was found to be weaker than the interaction of fibrin I with Gly-Pro-Arg-Pro and weaker than the interaction of fibrin I with fibrinogen.     

Integrin alpha3beta1 expressed by human colon cancer cells is a major carrier of oncodevelopmental carbohydrate epitopes

Oncodevelopmental carbohydrate epitopes are a common feature of human colorectal carcinoma, yet their biological significance remains unclear. We have shown previously that monoclonal antibody (MAb) 3A7, which recognizes a determinant on type 2 chain blood group A and B oligosaccharides, detects oncodevelopmental changes in azoxymethane-induced rat colon tumors and some human colon cancer cell lines. (Laferté S et al. [19951 J. Cell. Biochem. 57:101-119). In this study, we set out to purify gp140, the major glycoprotein carrier of the 3A7 epitope expressed by human colon cancer cells, as a first step towards elucidating the contribution of the 3A7 epitope and its major glycoprotein carrier to colon cancer progression. To this end, gp140 was purified from HT29 cells and used for the preparation of polypeptide-specific monoclonal antibodies. Five monoclonal antibodies (7A8, 7B11, 8C7, 8H7, and 11D4) immunoprecipitated a 3A7-immunoreactive glycoprotein complex of 140 kDa which was subsequently identified by partial protein sequencing as alpha3beta1 integrin. Flow cytometric analysis of Chinese hamster ovary (CHO) cells expressing different human integrin chains revealed that MAbs 7A8 and 7B11 detect the alpha3 integrin subunit whereas MAbs 8C7 and 8H7 detect the beta1 integrin subunit. MAb 11D4, which did not bind to any of the CHO transfectants, detected type 2 chain blood group A determinant. Flow cytometric analysis of a panel of human colon carcinoma cell lines obtained from blood group A, AB, or B individuals revealed a direct correlation between cell-surface expression of the 3A7 epitope and alpha3 integrin subunit, suggesting that alpha3beta1 integrin is a preferred target of the 3A7 epitope in colon cancer cells. Using lectins and glycosidases to examine further the carbohydrate structure of alpha3beta1 integrin, we demonstrated that it is a sialoglycoprotein containing both N- and O-linked oligosaccharides. In addition, both alpha3 and beta1 integrin subunits express beta1-6 branched Asn-linked oligosaccharides and short poly-N-acetyllactosamine units (Galbeta1-4GlcNAc-R; n < or = 3), glycans previously implicated in cancer metastasis.Thus, alpha3beta1 integrin expressed by human colon carcinoma cells is a major carrier of oncodevelopmental carbohydrate epitopes whose presence may modulate tumor cell adhesion, migration, and/or invasion.     

Dynamics of the exposure of epitopes on OmpF, an outer membrane protein of Escherichia coli.

The OmpF protein is the major outer membrane trimeric porin of Escherichia coli B. The exposure of several cell-surface-exposed epitopes, that are recognized by various monoclonal antibodies directed against the protein, is investigated. Kinetic analyses show that two epitopes (E18 and E19) appear early during the in-vivo assembly on the folded monomer, just after the removal of the signal peptide, and are conserved in the native trimer. The trimerization that immediately follows or occurs in conjunction with the folding of monomers exposes another antigenic site (E21) at the surface of metastable forms. The binding of nascent lipopolysaccharide promotes the conversion of the heat-modifiable intermediate to a stable trimer and ensures the exposure of E20, E1, E3, E4 and E7. Late epitopes, E1, E3, E4 and E7 are only detected in the outer membrane fraction. These results suggest that different steps induce the sequential exposure of native antigenic sites. The detection of these epitopes depends on conformational changes occurring during the OmpF insertion into the outer membrane.     

Monoclonal antibodies to human low density lipoprotein identify distinct areas on apolipoprotein B-100 relevant to the low density lipoprotein-receptor interaction.

We have characterized the epitopes for ten murine monoclonal antibodies (Mabs) to human low density lipoprotein (LDL) and studied their ability to interfere with the LDL-receptor interaction. The epitopes for the antibodies were defined by using the following approaches: 1) interaction with apoB-48; 2) interaction with apoB-100 thrombolytic fragments; and 3) interaction with beta-galactosidase-apoB fusion proteins spanning different areas of the apoB-100 sequence. The results obtained are consistent with the following map of epitopes: Mab 6E, amino acids (aa) 1-1297, Mabs 5A and 6B, aa 1480-1693, Mabs 2A, 7A, 3B, and 4B, aa 2152-2377, Mabs 8A and 9A, aa 2657-3248 and 3H, aa 4082-4306. Four Mabs (2A, 5A, 7A, and 9A) whose epitopes are located in three different areas of apoB, dramatically reduced (up to 95%) the LDL-receptor interaction on cultured human fibroblasts; Fab fragments were as effective as the whole antibodies. Mab 3H, on the other hand, increased LDL binding up to threefold. These findings are consistent with the hypothesis that several areas of apoB-100 are involved independently or in concert in modulating the apoprotein B conformation required for interaction with the LDL receptor.     

Identification of an epitope in the C terminus of normal prion protein whose expression is modulated by binding events in the N terminus

We have characterized the epitopes of a panel of 12 monoclonal antibodies (Mabs) directed to normal human cellular prion protein (PrP(C)) using ELISA and Western blotting of recombinant PrP or synthetic peptide fragments of PrP. The first group of antibodies, which is represented by Mabs 5B2 and 8B4, reacts with PrP(23-145), indicating that the epitopes for these Mabs are located in the 23 to 145 N-terminal region of human PrP. The second group includes Mabs 1A1, 6H3, 7A9, 8C6, 8H4, 9H7 and 2G8. These antibodies bind to epitopes localized within N-terminally truncated recombinant PrP(90-231). Finally, Mabs 5C3, 2C9 and 7A12 recognize both PrP(23-145) and PrP(90-231), suggesting that the epitopes for this group are located in the region encompassing residues 90 to 145. By Western blotting with PepSpot(TM), only three of Mabs studied (5B2, 8B4 and 2G8) bind to linear epitopes that are present in 13-residue long synthetic peptides corresponding to human PrP fragments. The remaining nine Mabs appear to recognize conformational epitopes. Two N terminus-specific Mabs were found to prevent the binding of the C terminus-specific Mab 6H3. This observation suggests that the unstructured N-terminal region may influence the local conformation within the folded C-terminal domain of prion protein.     

Mapping the binding of monoclonal antibodies to histone H5

The binding sites of nine monoclonal antibodies along the polypeptide chain of histone H5 were mapped. Immunoblotting experiments with peptides generated from H5 by trypsin digestion, N-bromosuccinimide cleavage, and cyanogen bromide cleavage revealed that all of the monoclonal antibodies reacted with the globular region of H5 which is encompassed by amino acid residues 22-98. Within this globular segment, the epitopes could be subdivided into three regions. Monoclonals 1G11, 2E5, and 2H5 bind to residues 28-31. The close proximity of the epitopes was verified by a competitive enzyme-linked immunosorbent assay and by their binding pattern to a tryptic digest of H5. Monoclonals 4C6, 6E12, and 2E12 bind to a region encompassed by amino acids 28-53 while monoclonals 4H7, 1C3, and 3H9 bind to a region encompassed by residues 53-98. Precise localization of the epitopes in the primary sequence of H5 will allow detailed studies on the mode of binding of H5 to core particles in chromatin.     

Preparation of Monoclonal Antibodies against Human Ventricular Myosin Light Chain 1 （HVMLC1） for Functional Studies

Using purified recombinant human ventricular myosin light chain 1 (HVMLC 1) as the antigen,three monoclonal antibodies,designated C8,C9 and B 12,were prepared.Immunoblot experiments demonstratedthat all monoclonal antibodies could react with the ventricular myosin light chain 1 isolated from differentsources,such as human,rat or pig.It was also demonstrated that C8 was directed against the NN part of theN-fragment (amino acid 1-40) of HVMLC1,and both C9 and B12 against the C-fragment (amino acid 99-195).The affinity constants of C8,C9 and B12 were 3.20×10~8,8.60×10~7 and 1.77×10~8 M~(-1),respectively,determined by non-competitive ELISA.The isotype of B12 was determined as lgG2a,whereas the isotype ofboth C8 and C9 were IgG1.In the presence of C9 or B12,the actin-activated Mg~(2 )ATPase activity of myosinwas greatly inhibited,but there was almost no effect on the Mg~(2 )ATPase activity for C8.B12 and C9 alsoinhibited the superprecipitation of porcine cardiac native actomyosin (myosin B) and reconstituted actomyosin,but C8 did not.The results indicate that all three monoclonal antibodies could bind the intact myosin molecule,but B12 and C9 might more easily react with epitopes located in the C-fragment of HVMLC1.The inhibitoryeffects of B 12 and C9 on ATPase activity and superprecipitation assays show that light chain 1,particularlythe C-fragment domain,is involved in the modulation of the actin-activated Mg~(2 )ATPase activity of myosinand,as a consequence,plays an essential role in the interaction of actin and myosin.     

Immunochemistry of human Lp[a]: characterization of monoclonal antibodies that cross-react strongly with plasminogen.

Forty different monoclonal antibodies were produced from hybridomas that were raised against human Lp[a]. Of these, 14 strongly cross-reacted with plasminogen on ELISA screening assays while 16 clearly did not and 10 were only marginally cross-reactive. We took advantage of the homology between plasminogen and apo[a] to define the epitopes of 8 strongly cross-reacting monoclonal antibodies. We were able to subdivide these into four general categories based upon site competition assays (using both plasminogen and Lp[a]), and their reactivity with elastolytically derived plasminogen fragments. Group A monoclonal antibodies (F1 1E3, F2 3A3) recognized epitopes within the kringle 5 and protease domains (miniplasminogen) of plasminogen. The group B monoclonal antibody (F6 1A3) reacted solely with plasminogen kringle 4-like domains and appeared to recognize a limited number of sites on Lp[a]. Group C monoclonal antibodies (F6 1B5, F6 1G9) recognized a second, more frequently distributed site within these kringle 4-like domains. The final group, D, monoclonal antibodies (F6 2C3, F6 2G2, F6 3F4) reacted with a cluster of sites found associated with kringle 4-like domains but also reacted with the miniplasminogen domain. Interestingly, only the members of this group were able to interfere with the proteolytic activity of plasmin. Neither periodate treatment of Lp[a] nor incubation of Lp[a] with epsilon-aminocaproic acid affected the binding of any of our monoclonal antibodies.     

Fibrin protofibril and fibrinogen binding to ADP-stimulated platelets: evidence for a common mechanism

The molecular basis of platelet-fibrin binding has been elucidated by studying interactions between platelets and protofibrils, soluble two-stranded polymers of fibrin which are intermediates on the fibrin assembly pathway. The fibrinogen degradation product, fragment D, has been used to block fibrin assembly, thus enabling the preparation of stable solutions of short protofibrils, composed of fewer than twenty fibrin monomer molecules per polymer. Fibrin protofibrils bound to ADP-activated platelets in a time- and concentration-dependent process which was effectively blocked by excess unlabelled fibrinogen, i.e., the binding was specific and appeared to involve a common receptor. ADP-stimulated cells bound approx. 3 micrograms of fibrin protofibrils/10(8) platelets, compared to 4 micrograms of fibrinogen/10(8) cells, following a 30-min incubation period at room temperature. Binding of both ligands was inhibited by high concentrations of fragment D, further indicating a similar mechanism. The kinetic data obtained were well described by an apparent first-order mechanism in which the rate constant for fibrin protofibril binding was found to be 5-fold slower than that measured for fibrinogen. Two monoclonal antibodies, each directed against the platelet glycoprotein IIb-IIIa complex, inhibited the binding of fibrin protofibrils and fibrinogen in a similar, concentration-dependent manner, providing strong evidence for a common receptor. Binding of GPRP-fibrin (soluble fibrin oligomers formed in the presence of 1 mM Gly-Pro-Arg-Pro) to ADP-stimulated platelets was also inhibited by a monoclonal antibody directed against the GPIIb-IIIa complex. Neither fibrin protofibrils nor fibrinogen bound to Glanzmann's thrombasthenic platelets, which lack normal quantities of functional glycoprotein IIb-IIIa complex, further supporting the hypothesis that fibrinogen and fibrin bind to a common platelet receptor present on the glycoprotein IIb-IIIa complex.     

Substitution of the human αC region with the analogous chicken domain generates a fibrinogen with severely impaired lateral aggregation: fibrin monomers assemble into protofibrils but protofibrils do not assemble into fibers

Fibrin polymerization occurs in two steps: the assembly of fibrin monomers into protofibrils and the lateral aggregation of protofibrils into fibers. Here we describe a novel fibrinogen that apparently impairs only lateral aggregation. This variant is a hybrid, where the human αC region has been replaced with the homologous chicken region. Several experiments indicate this hybrid human-chicken (HC) fibrinogen has an overall structure similar to normal. Thrombin-catalyzed fibrinopeptide release from HC fibrinogen was normal. Plasmin digests of HC fibrinogen produced fragments that were similar to normal D and E; further, as with normal fibrinogen, the knob 'A' peptide, GPRP, reversed the plasmin cleavage associated with addition of EDTA. Dynamic light scattering and turbidity studies with HC fibrinogen showed polymerization was not normal. Whereas early small increases in hydrodynamic radius and absorbance paralleled the increases seen during the assembly of normal protofibrils, HC fibrinogen showed no dramatic increase in scattering as observed with normal lateral aggregation. To determine whether HC and normal fibrinogen could form a copolymer, we examined mixtures of these. Polymerization of normal fibrinogen was markedly changed by HC fibrinogen, as expected for mixed polymers. When the mixture contained 0.45 μM normal and 0.15 μM HC fibrinogen, the initiation of lateral aggregation was delayed and the final fiber size was reduced relative to normal fibrinogen at 0.45 μM. Considered altogether, our data suggest that HC fibrin monomers can assemble into protofibrils or protofibril-like structures, but these either cannot assemble into fibers or assemble into very thin fibers.     

Neutralization escape mutants define a dominant immunogenic neutralization site on hepatitis A virus.

Hepatitis A virus is an hepatotrophic human picornavirus which demonstrates little antigenic variability. To topologically map immunogenic sites on hepatitis A virus which elicit neutralizing antibodies, eight neutralizing monoclonal antibodies were evaluated in competition immunoassays employing radiolabeled monoclonal antibodies and HM-175 virus. Whereas two antibodies (K3-4C8 and K3-2F2) bound to intimately overlapping epitopes, the epitope bound by a third antibody (B5-B3) was distinctly different as evidenced by a lack of competition between antibodies for binding to the virus. The other five antibodies variably blocked the binding of both K3-4C8-K3-2F2 and B5-B3, suggesting that these epitopes are closely spaced and perhaps part of a single neutralization immunogenic site. Several combinations of monoclonal antibodies blocked the binding of polyclonal human convalescent antibody by greater than 96%, indicating that the neutralization epitopes bound by these antibodies are immunodominant in humans. Spontaneously arising HM-175 mutants were selected for resistance to monoclonal antibody-mediated neutralization. Fourteen clonally isolated mutants demonstrated substantial resistance to multiple monoclonal antibodies, including K3-4C8-K3-2F2 and B5-B3. In addition, 13 mutants demonstrated a 10-fold or greater reduction in neutraliztion mediated by polyclonal human antibody. Neutralization resistance was associated with reduced antibody binding. These results suggest that hepatitis A virus may differ from poliovirus in possessing a single, dominant neutralization immunogenic site and therefore may be a better candidate for synthetic peptide or antiidiotype vaccine development.     

Characterization of monoclonal antibodies reactive to several biochemically distinct human cytomegalovirus glycoprotein complexes.

Three monoclonal antibodies were characterized by examining their reactivity to human cytomegalovirus (HCMV) glycoproteins under reducing and nonreducing conditions and their reactivity to glycoproteins and disulfide-linked glycoprotein complexes isolated by ion-exchange high-performance liquid chromatography. One monoclonal antibody, 9E10, reacted with glycoprotein complexes which had molecular weights of 93,000 and 450,000 and eluted from the ion-exchange column at 0.3 and 0.9 M NaCl, respectively. All glycoproteins associated in these complexes could be immunoprecipitated under reducing conditions by 9E10, suggesting that they were related to one another. The most abundant glycoproteins immunoprecipitated by 9E10 had molecular weights of 50,000 to 52,000. In contrast to this antibody, two other monoclonal antibodies, 9B7 and 41C2, reacted with glycoprotein complexes which had molecular weights of 130,000 and greater than 200,000 and eluted from the ion-exchange column at 0.6 M NaCl. All glycoproteins associated in these complexes could be immunoprecipitated by 9B7 or 41C2 under reducing conditions, suggesting that they were also related to one another. The most abundant glycoprotein immunoprecipitated by 41C2 or 9B7 had a molecular weight of 93,000. In addition, it was also determined that a 93,000-molecular-weight glycoprotein which was not associated with other glycoproteins by disulfide bonds could not be precipitated by any of the three antibodies, suggesting that it was different from the other glycoproteins. The monoclonal antibodies were also examined for specificity and neutralizing activity. Monoclonal antibodies 41C2 and 9B7 were specific to HCMV as determined by immunofluorescent staining of skin fibroblast cells infected with several different viruses. However, 41C2 did not neutralize Towne strain HCMV, while 9B7 did. The neutralizing activity of 9B7 did require complement. These results suggested that 41C2 and 9B7 reacted with different antigenic sites on the same glycoproteins. Unlike 41C2 and 9B7, monoclonal antibody 9E10 was found to cross-react with adenovirus and herpes simplex virus as determined by immunofluorescent staining of infected skin fibroblast cells. Furthermore, 9E10 neutralized the Towne and Toledo strains of HCMV in the absence of complement.     

Conversion of fibrinogen to fibrin: mechanism of exposure of tPA- and plasminogen-binding sites

Conversion of fibrinogen into fibrin results in the exposure of cryptic interaction sites and modulation of various activities. To elucidate the mechanism of this exposure, we tested the accessibility of the Aalpha148-160 and gamma312-324 fibrin-specific epitopes that are involved in binding of plasminogen and its activator tPA, in several fragments derived from fibrinogen (fragment D and its subfragments) and fibrin (cross-linked D-D fragment and its noncovalent complex with the E(1) fragment, D-D. E(1)). Neither D nor D-D bound tPA, plasminogen, or anti-Aalpha148-160 and anti-gamma312-324 monoclonal antibodies, indicating that their fibrin-specific epitopes were inaccessible. The Aalpha148-160 epitope became exposed only upon proteolytic removal of the beta- and gamma-modules from D. At the same time, both epitopes were accessible in the D-D.E(1) complex, indicating that the DD.E interaction resulted in their exposure. This exposure was reversible since the dissociation of the D-D.E(1) complex made the sites unavailable, while reconstitution of the complex made them exposed. The results indicate that upon fibrin assembly, driven primarily by the interaction between complementary sites of the D and E regions, the D regions undergo conformational changes that cause the exposure of their plasminogen- and tPA-binding sites. These changes may be involved in the regulation of fibrin assembly and fibrinolysis.     

Epitope mapping of monoclonal antibodies to human plasma membrane Ca2-ATPase

Overlapping fragments of the fourth isoform of human plasma membrane Ca(2+)-ATPase (hPMCA4) and several fragments of hPMCA1 were expressed in bacterial cells and purified by metal affinity chromatography. Enzyme immunoassays of the fragments helped map epitopes for 4 monoclonal antibodies (2D8, 8B8, 7C8 and 5E6). The epitope for 2D8 was localized within the 222-249 site (i.e., in the putative transduction domain), the epitopes for 8B8 and 7C8 were localized within the 330-353 site, in which phospholipids are presumably bound, and the 5E6 epitope was found within the 791-843 site, where the putative hinge region is situated. 2D8 recognizes hPMCA1 and hPMCA4 isoforms, while 8B8 and 7C8 are specific for hPMCA4. The amino acid sequences of these epitopes and phage-displayed mimotopes were compared.     

Distribution of Epitopes of Trypanosoma cruzi Amastigotes During the Intracellular Life Cycle within Mammalian Cells

ABSTRACT. In this study we have examined the distribution of epitopes defined by monoclonal antibodies raised against Trypanosoma cruzi amastigotes during the intraceullar life cycle of the parasite. We have raised monoclonal antibodies towards amastigote forms and performed preliminary immunochemical characterization of their reactivities. MAB 1D9, 3G8, 2B7, 3B9, and 4B9, and 4B9 react with carbohydrate epitopes of the parasite major surface glycoprotein—Ssp-4 defined by MAB 2C2 [5]: MAB 4B5 reacts with a noncarbohydrate epitope in all developmental stages of the parasite, and MAB 3B2 also detects a noncarbohydrate epitope preferentially in T. cruzi flagellared forms. Vero cells infected with tissue culture-derived trypomastigotes of clone D11 (G strain) were fixed at different times during the intraceullular proliferation of parasites, and processed for immjno-electron microscopy and confocal immunoflurescence with the different monoclonal antibodies. We observed that while the surface distribution of MAB 2C2 and 4B9 epitopes was uniform throughout the cycle, MAB 1D9, 3G8, and 2B7 reacted with cytoplasmic membrance-bound compartments of the amastigotes. MAB 3B9 displayed a unique surface dentate pattern in some amastigotes. MAB 4B5 recognized a curved-shaped structure at the flagellar pocket region in some intracellular amastigotes and localized to the membrane in dividing forms. In intracellular trypomastigotes, MAB 4B5 also displayed a punctate pattern near the flagellar pocket.     

Flow cytometric analysis of erythrocyte populations in Tn syndrome blood using monoclonal antibodies to glycophorin A and the Tn antigen.

Flow cytometric analysis employing monoclonal antibodies to the Tn antigen and glycophorin A was used to characterize the erythrocyte populations present in blood samples from individuals with Tn syndrome. Four monoclonal antibodies specific for the Tn antigen, Gal-NAc monosaccharide, on human erythrocytes were obtained from a fusion of splenocytes from a Biozzi mouse immunized with red cells from a Tn individual. These monoclonal antibodies specifically recognize GalNAc monosaccharide sites located on the erythrocyte cell surface sialoglycoproteins, glycophorin A and glycophorin B, and do not bind to fixed normal red cells presenting the Neu-NAc alpha 2-3Gal beta 1-3(NeuNAc alpha 2-6)GalNAc alpha 1-O-Ser(Thr) tetrasaccharide or to fixed neuraminidase-digested cells presenting the Gal-GalNAc disaccharide. The percentages of Tn-positive red cells in samples from six unrelated Tn donors ranged from 28 to 99%. Binding of the glycophorin A-specific monoclonal antibodies showed that the erythrocytes composing the Tn-negative fraction presented normal amounts of the M and N epitopes on glycophorin A. The presumed somatic mutational origin of Tn-positive cells was tested in blood samples from five normal donors; three possible Tn cells were observed after analysis of a total of 1.1 x 10(7) erythrocytes, suggesting that the frequency of such cells in normal individuals is less than 1 x 10(-6).     

Critical epitopes in transmissible gastroenteritis virus neutralization.

Purified transmissible gastroenteritis (TGE) virus was found to be composed of three major structural proteins having relative molecular weights of 200,000, 48,000, and 28,000. The peplomer glycoprotein was purified by affinity chromatography with the monoclonal antibody (MAb) 1D.G3. A collection of 48 MAbs against TGE virus was developed from which 26, 10, and 3 were specific for proteins E2, N, and E1, respectively. A total of 14 neutralizing MAbs of known reactivity were E2 protein specific. In addition, MAb 1B.C11, of unknown specificity, was also neutralizing. These MAbs reduced the virus titer 10(2)- to 10(9)-fold. Six different epitopes critical in TGE virus neutralization were found, all of which were conformational based on their immunogenicity and antigenicity. Only the epitope defined by MAb 1G.A7 was resistant to sodium dodecyl sulfate treatment, although it was destroyed by incubation in the presence of both the detergent and beta-mercaptoethanol. The frequency of MAb-resistant (mar) mutants selected with four MAbs (1G.A7, 1B.C11, 1G.A6, and 1E.F9) ranged from 10(-6) to 10(-7), whereas the frequency of the putative mar mutant defined by MAb 1B.B11 was lower than 10(-9). Furthermore, the epitopes defined by these MAbs and by MAbs 1H.C2 and 1A.F10, were present in 11 viral isolated with different geographical locations, years of isolation, and passage numbers (with the exception of two epitopes absent or modified in the TOY 56 viral isolate), suggesting that the critical epitopes in TGE virus neutralization were highly conserved.