Induction of arthritis with monoclonal antibodies to collagen.

mAb were developed from DBA/1 mice immunized with chick type II collagen. A total of 69 IgG antibodies was isolated and characterized. The majority (36%) reacted with a CNBr-derived peptide CB11 previously identified as containing a major immunogenic and arthritogenic epitope(s). Seven of the antibodies reactive with CB11 crossreacted strongly with mouse type II collagen. These were administered to DBA/1 mice in an attempt to induce arthritis. Individual antibodies were able to induce mild lesions consisting of minimal synovial proliferation but not overt arthritis. However, a combination of antibodies induced severe arthritis with marked destruction of articular cartilage. The minimal effective combination consisted of three antibodies. Arthritis developed within 48 to 72 h after injection of the antibodies and persisted for the duration of the observation period of 3 wk. Antibody levels were measured at intervals and persisted for the 3 wk observation period although at diminishing levels. Competitive binding assays demonstrated that each of the effective antibodies bound independently suggesting that some spatial or quantitative relationship was important possibly related to their ability to activate complement.     

Immunochemistry, genuine size and tissue localization of collagen VI

Collagen VI was solubilized with pepsin from human placenta and used for preparing rabbit antisera. Major antigenic determinants were located in the central region of the antigen including triple-helical and globular structures. Antisera prepared against a constituent-chain showed preferential reactions with unfolded structures. Antibodies were purified by affinity chromatography and failed to cross-react with other collagen types I-V and with fibronectin. These antibodies demonstrated intracellular and extracellular collagen VI in fibroblast and smooth muscle cell cultures. Immunoblotting identified a disulfide-bonded constituent chain about twice as large as those of the pepsin fragments in both cell cultures and tissue extracts. Rotary shadowing electron microscopy indicated that the increase in mass is due to larger globular domains present at both ends of collagen VI monomers. Indirect immunofluorescence demonstrated a wide occurrence of collagen VI in connective tissue particularly of large vessels, kidney, skin, liver and muscle. Collagen VI is apparently not a typical constituent of cartilage or of basement membranes. Ultrastructural studies using the immunoferritin technique showed collagen VI along thin filaments or in amorphous regions of aortic media or placenta but not in association with thick, cross-striated collagen fibrils or elastin. This supports previous suggestions that collagen VI is a constituent of microfibrillar structures of the body.     

Poliovirus neutralization epitopes: analysis and localization with neutralizing monoclonal antibodies.

Two hybridomas (H3 and D3) secreting monoclonal neutralizing antibody to intact poliovirus type 1 (Mahoney strain) were established. Each antibody bound to a site qualitatively different from that to which the other antibody bound. The H3 site was located on intact virions and, to a lesser extent, on 80S naturally occurring empty capsids and 14S precursor subunits. The D3 site was found only on virions and empty capsids. Neither site was expressed on 80S heat-treated virions. The antibodies did not react with free denatured or undenatured viral structural proteins. Viral variants which were no longer capable of being neutralized by either one or the other antibody were obtained. Such variants arose during normal cell culture passage of wild-type virus and were present in the progeny viral population on the order of 10(-4) variant per wild-type virus PFU. Toluene-2,4-diisocyanate, a heterobifunctional covalent cross-linking reagent, was used to irreversibly bind the F(ab) fragments of the two antibodies to their respective binding sites. In this way, VP1 was identified as the structural protein containing both sites.     

Radioimmunocytochemical localization of retinal S-antigen with monoclonal antibodies

Two monoclonal antibodies (RSA1/83 and RSA2/83) were developed against a homogeneous preparation of bovine retinal S-antigen. The two hybridomas produced by mouse X mouse hybrid myeloma cells secrete immunoglobulin G. Indirect autoradiography on glutaraldehyde-fixed preparations of bovine explants was used to locate the antigenic site. Antibody RSA1/83 recognizes the antigen primarily in the apical region of the rod outer segment, while antibody RSA2/83 located the antigen both in the outer and inner segments of the rod photoreceptor cells. A distinct band of silver grains also appeared along the inner limiting membrane with both antibodies. Control explants showed no specific labeling pattern over the various retinal compartments.     

Subcellular localization of glycogen synthase with monoclonal antibodies

Two monoclonal antibodies, designated 7H5 and 8E11, were produced against glycogen synthase purified from rabbit skeletal muscle. Both antibodies were of the IgG1 (k) isotype. Western blot analysis of extracts of rat and rabbit tissues showed that antibody 7H5 recognized glycogen synthase from skeletal and cardiac muscles, but not from liver. Antibody 8E11 gave similar results but the responses were weaker. Antibody 7H5 also recognized a 69,000 dalton tryptic fragment of glycogen synthase whereas antibody 8E11 did not bind this fragment. Immunocytochemical staining of rabbit skeletal muscle with antibody 7H5 indicated two major sites of glycogen synthase localization. A granular localization present in the cytoplasm and a band-like staining associated with the Z-disk region of the myofibrils. Rabbit cardiac muscle presented a similar pattern though less cytoplasmic staining was apparent. An assay of subcellular fractions for glycogen synthase indicated that the enzyme in cardiac and skeletal muscles is distributed between the soluble (80-90%) and myofibrilar (10-20%) fractions of the tissues. These results provide direct evidence for the presence of glycogen synthase in subcellular fractions other than the soluble fraction of skeletal and cardiac muscles.     

Isolation from bovine elastic tissues of collagen type VI and characterization of its form in vivo.

Foetal-bovine nuchal ligament and aorta, together with adult-bovine aorta and pregnant uterus, were extracted under dissociative conditions in the absence and in the presence of a reducing agent. A collagenous glycoprotein of Mr 140000 [designated component 140K(VI)], identified in these extracts as the major periodate/Schiff-positive component, was shown to be related to collagen type VI. Digestion of non-reduced extracts with pepsin yielded periodate/Schiff-positive peptides that, on the basis of their electrophoretic mobilities, amino acid analyses and peptide 'maps', were identical with type VI collagen fragments prepared by standard procedures. It is concluded that collagen type VI occurs in vivo as molecule comprising three chains of Mr 140000 in which the helical domains account for about one-third of each polypeptide. Biosynthetic experiments with nuchal-ligament fibroblasts in culture demonstrated that a bacterial-collagenase-sensitive [3H]fucose-labelled glycoprotein, Mr 140000, was immunoprecipitated from culture medium by a specific antibody to the pepsin-derived form of collagen type VI. This result suggests that the collagenous polypeptides [140K(VI) components] represent the biosynthetic precursors of type VI collagen that do not undergo processing to smaller species before deposition in the extracellular matrix. Analyses of 5M-guanidinium chloride extracts of tissues with markedly different elastin contents and at different stages of development suggested that there was no relationship between collagen type VI and elastic-fibre microfibrils, a conclusion supported by the observation that the immunoprecipitated glycoprotein, Mr 140000, was distinct from the glycoprotein MFPI, Mr 150000, believed to be a constituent of these microfibrils [Sear, Grant & Jackson (1981) Biochem. J. 194, 587-598].     

Studies on the structure and conformation of brush border myosin using monoclonal antibodies

We have produced and characterised five monoclonal antibodies against myosin isolated from chicken intestinal epithelial brush border cells. The binding sites of the antibodies on the rod portion of brush border myosin were localised using rotary shadowing/electron microscopy of myosin-antibody complexes. Two antibodies were shown to bind to the tip of the myosin tail, two antibodies to sites about two thirds down the length of the rod, and one antibody about one third down the length of the rod. Brush border myosin was digested with papain, trypsin and alpha-chymotrypsin, and they myosin fragments obtained were analysed by western blots with the monoclonal antibodies and polyclonal antiserum, and by gel overlay with 125I-labelled light chains. Using this approach we were able to identify and map the protease cleavage sites and thus characterise the proteolytic substructure of brush border myosin. Solid-phase assays, western blots and immunofluorescence were used to study the cross-reactivity of these monoclonal antibodies against a variety of myosins from different species and cell types, to assess the immunological relatedness between brush border myosin and homologous molecules present in different tissues and species. Finally, we used a competitive solid-phase assay to measure the 'relative affinities' of the antibodies towards the three possible conformational states of brush border myosin, i.e. filament, extended monomer and folded monomer.     

Studies on the antigenic structure of Japanese encephalitis virus using monoclonal antibodies

The antigenic relationships among 11 strains of Japanese encephalitis (JE) virus were analyzed by using monoclonal antibodies (NARMA) against the Nakayama-RFVL strain in hemagglutination-inhibition (HI) and neutralization (Nt) tests. Of the 14 JE virus-specific HI antibodies, all except NARMA 5 showed Nt reactivity with the homologous strain. The HI and Nt titers of these antibodies were not parallel. The 14 antibodies included the following characteristic antibodies: NARMA 3 is a species-specific antibody with HI and Nt reactivities against JE virus, NARMA 13 is a species-specific HI antibody, NARMA 6 is a Nakayama strain-specific antibody with HI and Nt reactivities, and NARMA 5 is a Nakayama strain-specific HI antibody. The 11 strains of JE virus were divided into four major antigenic groups. However, slight antigenic differences were found among some strains of the same group. Furthermore, competitive binding assays were performed to determine the distribution of antigenic determinants by enzyme-linked immunosorbent assay. The results suggest the existence of at least five HI sites on the JE virus virion, and indicate that the JE species-specific HI site and the flavivirus genus-specific HI site are topologically distinct.     

胶原蛋白与Ⅵ型胶原蛋白的结构概述

描述了胶原蛋白的结构和Ⅵ型胶原蛋白的结构以及胶原蛋白的应用 ,并对人类胶原蛋白的生产进行了展望。     

Probing myosin head structure with monoclonal antibodies

Monoclonal antibodies that react with defined regions of the heavy and light chains of chicken skeletal muscle myosin have been used to provide a correlation between the primary and the tertiary structures of the head. Electron microscopy of rotary shadowed antibody-myosin complexes shows that the sites for three epitopes in the 25,000 Mr tryptic fragment (25k) of subfragment-1, including one within 4000 Mr of the amino terminus of the myosin heavy chain, are clustered 145(+/- 20) A from the head-rod junction. An epitope in the 50,000 Mr fragment maps even further out on the head. These antibodies bind to the head in several orientations, suggesting that each of the heads can rotate can rotate 180 degrees about the head-rod junction. The epitopes are accessible on subfragment-1 bound to actin when they were probed with Fab fragments; therefore, none of these heavy chain sites is is on the contact surface between the head and actin. Two of the anti-25k antibodies affect the K+-EDTA-and Ca2+-ATPase activities of myosin in a manner that mimics the effect on activity of the modification of the reactive thiol, SH-1. These two antibodies also inhibit the actin-activated ATPase non-competitively with respect to actin. None of the other eight antibodies tested had any marked effect on activity. A monoclonal antibody that reacts with an epitope in the amino-terminal third of myosin light chain 2 maps close to the head-rod junction. A polyclonal antibody specific for the amino terminus of light chain 3 binds further up in the "neck region" of the head, indicating that these portions of the two classes of light chains are located at different sites.     

Studies of murine malaria antigens using monoclonal antibodies. Production, selection, and characterization of antibodies

A panel of ten monoclonal antibodies made against Plasmodium chabaudi and Plasmodium yoelii infected mouse erythrocytes were used for characterization of antigens present in murine malaria. Screening of the antibodies in ELISA with different fractions of infected erythrocytes revealed both species-specific and fraction-specific monoclonal antibodies (MAbs), but also MAbs cross-reacting between the species. Two MAbs bound normal erythrocyte components. Subcellular localization of the target antigens was studied by immunofluorescence and their molecular identity by immunoblotting after SDS-PAGE. Of the MAbs to P. yoelii, one reacted with a cytoplasmic granule component of 137 k and two others reacted with vacuole-associated antigens of 26 k and 25/70/73 k, respectively. The latter antibodies cross-reacted with P. chabaudi antigens. Of the MAbs to P. chabaudi, all were species specific, one reacting with parasite surface antigens of 79 and 250 k and two with a vacuole-associated antigen of 70 k.     

Analysis of the interaction of platelet collagen receptor glycoprotein VI (GPVI) with collagen. A dimeric form of GPVI,but not the monomeric form,shows affinity to fibrous collagen

Glycoprotein VI (GPVI) is a platelet-specific glycoprotein that has been indicated to react with collagen and activate platelets. Its structure was recently identified by cDNA cloning (Clemetson, J. M., Polgar, J., Magnenat, E., Wells, T. N., and Clemetson, K. J. (1999) J. Biol. Chem. 274, 29019-29024). However, the mechanism of the interaction between collagen and GPVI has not been analyzed in detail because both collagen and GPVI are insoluble molecules. In this study, we expressed the extracellular domain of GPVI as soluble forms as follows: the monomeric form (GPVIex) and the dimeric form of GPVI fused with the human immunoglobulin Fc domain (GPVI-Fc(2)). Purified GPVIex strongly inhibited convulxin (Cvx)-induced platelet aggregation but only weakly inhibited that induced by collagen-related peptide. However, only GPVI-Fc(2), and not GPVIex, inhibited collagen-induced platelet aggregation. The dimeric form of GPVI exhibits high affinity for collagen, as concluded from measurements of GPVI binding to immobilized collagen by both the enzyme-linked immunosorbent assay and surface plasmon resonance methods. GPVI-Fc(2) bound to the surface of immobilized collagen with a dissociation constant (K(D)) of 5.76 x 10(-7) m, but the binding of GPVIex was too weak to allow estimation of this parameter. Cvx did not inhibit the binding of dimeric GPVI to collagen, indicating that the binding site of GPVI to collagen was different from that to Cvx. Taken together, our data indicate that the high affinity binding site for collagen is composed from two chains of GPVI. Furthermore, they suggest that the binding sites for Cvx are different from the collagen-binding sites and do not need to be formed by two GPVI molecules. Because dimeric GPVI is the only form that shows high affinity to fibrous collagen, our results indicate that GPVI would be present as a dimeric form on the platelet. Moreover, surface plasmon resonance indicated that there is no detectable interaction between soluble collagen and GPVI, supporting our previous observation that GPVI only reacts with fibrous collagen.     

Ultrastructural localization of human kidney antigens using monoclonal antibodies.

A highly sensitive method of ultrastructural-immunoperoxidase staining was developed for use with monoclonal antibodies which have been raised in this laboratory to a variety of antigens of the human kidney. Because of the susceptibility of the antigens to fixation and processing, a four layer, pre-embedding method of staining was used. Results confirmed and clarified previously reported light microscopy results, indicating that an antigen recognized by the PHM5 antibody was found on the podocyte cell membrane within the glomerulus and was not present within the glomerular basement membrane. The antigen was also present on the extraglomerular endothelial cell membrane. The study also demonstrated the presence of an antigen specific to endothelial cells throughout the renal cortex, and gave further insight into the precise localization of glomerular basement membrane components including fibronectin. The method of staining is now being used together with detailed ultrastructural studies to identify the cells produced from isolated glomeruli in tissue culture.     

The use of monoclonal antibodies to fragments of chicken type IV collagen in structural and localization studies

In previous experiments, three pepsin-resistant fragments of type IV collagen were isolated from chicken gizzards and designated 7S, F3, and (F1)2F2 (Mayne, R., and Zettergren, J. G. (1980) Biochemistry 19, 4065-4072). In the present experiments, a series of monoclonal antibodies to type IV collagen were prepared, each one of which recognized an epitope present in only one of the three fragments. A high molecular weight fraction of type IV collagen (designated 7S + arms (215 nm)) was isolated after agarose gel filtration and characterized by electron microscopy after rotary shadowing and by gel electrophoresis. Analysis of 7S + arms (215 nm) by inhibition enzyme-linked immunosorbent assay demonstrated the presence of the epitopes for 7S and F3 but not for (F1)2F2. This result, therefore, provides additional evidence that the order of the pepsin-resistant fragments of chicken type IV collagen is 7S-F3-(F1)2F2.     

Human specific anti-type IV collagen monoclonal antibodies,characterization and immunohistochemical application

Summary This paper describes two new monoclonal antibodies reactive with human specific type IV collagen epitopes in frozen as well as routinely fixed and processed tissue sections. The antibodies (1042 and 1043) were raised against human placental type IV collagen and were shown by immunoblotting and ELISA tests to react exclusively with type IV collagen determinants. Extensive immunohistochemical survey studies on panels of tissues from various species, using unfixed cryosat sections, demonstrated that antibody 1042 reacted only with human type IV collagen whereas antibody 1043 in addition reacted with rabbit type IV collagen. All tissues showed homogeneous staining of the basement membrane, indicating that the detected epitopes did not show organ-specific distribution.Tissue processing protocols for using these monoclonal antibodies on routinely processed paraffin embedded tissues were developed. It was found that whereas polyclonal antitype IV collagen antisera required pepsin digestion, our monoclonal antibodies required pronase or papain digestion to restore type IV collagen immunoreactivity in paraffin sections.It is concluded that these monoclonal anti-type IV collagen antibodies detect species specific epitopes which can be detected in routinely processed paraffin embedded tissues after appropriate enzyme pretreatment.     

Tumor localization of Lewis lung carcinoma with radiolabeled monoclonal antibodies

Summary Mouse 6D6 IgG_2a and 5B5 IgM monoclonal antibodies which specifically bind murine lung carcinoma cells (3LL cells) were injected to healthy and tumor-bearing mice. In vivo localization was analyzed by counting the tissue radioactivity and by external gamma ray scintigraphy at various times after IV injection of ¹²⁵I- or ¹³¹I-labeled antibodies. The clearance of the two monoclonal antibodies was not modified by the presence of the tumor, and the 6D6 IgG_2a was cleared at a rate slower than the 5B5 IgM. Both antibodies gave a high specific uptake at the tumor level; the tumor-to-healthy tissue ratios were higher with the 6D6 IgG_2a than the 5B5 IgM; unspecific mouse immunoglobulins (IgG₂) did not localize in the tumor. The amount of 6D6 IgG_2a antibody still associated with the tumor after 2 days following IV injection was 10 times higher than that of 5B5 IgM, and was still high enough to localize the tumor after 5 days.Imaging experiments confirmed the ability of 6D6 IgG_2a to detect the presence of tumor cells. The targeting kinetics determined by computer analysis of camera images indicated a rapid targeting of antibodies in tumor with a maximal concentration after 4–6 h; after 48 h the background was quite low and the 6D6 IgG_2a appeared to be specifically localized in the tumor.