Cross-reactive and specific monoclonal antibodies against cellobiohydrolases I and II and endoglucanases I and II of Trichoderma reesei

Splenocytes derived from mice inoculated with a commercial cellulase preparation or purified cellulases were fused with a stable myeloma cell line (SP2/0). Specific monoclonal antibodies to cellobiohydrolases I and II and endoglucanases I and II were established. In addition to specific monoclonal antibodies, we were also able to establish stable hybridoma cell lines which produced monoclonal antibodies that recognized similar epitopes possessed by two or more of the above cellulases. By obtaining monospecific antibodies for all four individual cellulases, the role and function of the individual cellulases can thus be studied in greater detail.     

Monoclonal antibodies against core and cellulose-binding domains of Trichoderma reesei cellobiohydrolases I and II and endoglucanase I.

Cellulases from Trichoderma reesei form an enzyme group with a common structural organization. Each cellulase enzyme is composed of two functional domains, the core region containing the active site and the cellulose-binding domain (CBD). To facilitate the specific detection of each domain, monoclonal antibodies (mAb) against cellobiohydrolase I (CBHI), cellobiohydrolase II (CBHII) and endoglucanase I (EGI) were produced. Five mAb were obtained against CBHI, ten against CBHII and eight against EGI. The location of the antigenic epitope for each antibody was mapped by allowing the antibodies to react with truncated cellulases, synthesized from deleted cDNA in Saccharomyces cerevisiae. Proteolytic fragments of Trichoderma cellulases, obtained by papain digestion, were used to confirm the results. Specific antibodies were detected against the core and the CBD epitopes for all three cellulases. Using the truncated enzymes, it was possible to locate the epitopes to a reasonably short region within the protein. To obtain a quantitative assay for each enzyme, a specific mAb against each antigen was chosen, based on the affinity to the corresponding antigen on Western-blot staining and on filter blots of the cellulolytic yeasts. The mAb were used to quantitative the corresponding enzymes in T. reesei culture medium. Specific quantitation of each cellulase enzyme has not been possible by biochemical assays or using polyclonal antibodies, due to their cross-reactions. Now, these mAb can be specifically used to recognize and quantitate different domains of these three important cellulolytic enzymes.     

Murine monoklonale Antikörper gegen Vibrio anguillarum vom Serotyp I und II

Murine monoclonal antibodies against Vibrio anguillarum serotype I und II Four stabil hybridoma cell lines against the Vibrio anguillarum strain 820/15/8 Kopenhagen—a typical serotype I strain—were raised. Three hybridoma cell lines producing monoclonal antibodies against the Vibrio anguillarum strain 134/82/1 Kiel (Serotype II) were established. The specifity of the antibodies was screened by ELISA. Monoclonal antibodies reacted with the characteristic determinant of the respective serotype only.     

Cross-reactions among carbonic anhydrases I,II, and III studied by binding tests and with monoclonal antibodies

Cross-reactions among carbonic anhydrases (CAs) I, II, and III were studied using a variety of antisera: (1) a rabbit antiserum to bovine CA III, (2) mouse antisera to human CA I, CA II, and CA III; and (3) five monoclonal antibodies prepared by the hybridoma technique using splenocytes from a mouse immunized with human CAs I and II and bovine CA III. Cross-reactions between CAs were readily found by binding assays using these antisera. Human CA I, but not human CA II, inhibited the reaction of the rabbit anti-CA III with its homologous antigen. Mouse antisera to CA I or CA II bound the homologous I or II with nearly as great efficiency as the autologous isozyme and sometimes weakly bound CA III. Mouse antisera to CA III frequently bound CA I or II. These cross-reactions were confirmed by the first use of hybridoma-prepared, monoclonal antibodies to CAs. The mouse monoclonal antibodies to CA isozymes varied in the amount of cross-reactivity among I, II, and III: at one extreme, one monoclonal was highly specific for the autologous CA III; at the other extreme, one monoclonal weakly reacted with some examples of CAs I, II, and III.This work was supported by NIH Grant GM-24681 and a grant from the National Foundation-March of Dimes.     

Role of class I and class II antigens in the allogenic stimulation: class I and class II recognition in allogenic stimulation; blocking of MLR by monoclonal antibodies and F(ab')2 fragments

Purified monomorphic monoclonal antibodies against Class I and Class II antigens in the inhibition of in vitro allogenic response were assayed. As expected, anti-Class II antibodies are highly inhibitory when used in concentrations greater than 5 micrograms/ml in MLRI and 50 micrograms/ml in MLRII. Surprisingly, anti-Class I monoclonal antibodies are as effective as anti-Class II in inhibiting primary MLR although they have no effect in MLRII. These results were confirmed by using F(ab')2 fragments. The inhibitory effect of anti-Class I has been shown to occur at the stimulator cell level. It is proposed that the allogenic stimulation is elicited after Class I and Class II recognition although only Class II differences are responsible for the proliferative response.     

Monoclonal antibodies specific to apical surfaces of rat alveolar type I cells bind to surfaces of cultured, but not freshly isolated, type II cells

The alveolar surface of the lung is lined by two classes of epithelial cells, type I and type II cells. Type I cells cover more than 97% of the alveolar surface. Although this cell type is felt to be essential for normal gas exchange, neither unique identifying characteristics nor functions have been described for the type I cell. We have produced monoclonal antibodies to (a) component(s) of molecular weight 40,000 and 42,000 of the apical surface of rat alveolar type I cells. The antibodies are specific to the lung in Western blots of organ homogenates. In immunocytochemical studies of frozen lung at the level of both light and electron microscopy, the monoclonal antibodies appear to react specifically with the apical plasma membrane of type I cells. Airway, vascular, interstitial cells, type II cells and macrophages are not immunoreactive. Western blots of isolated type I cells (approx. 70% pure) also demonstrate immunoreactivity at molecular weights of 40,000 and 42,000. When the lung is injured, type I cells may be damaged and sloughed from the alveolar surface. Alveolar repair occurs when the second type of alveolar cell, the type II cell, divides. Cell progeny may retain type II cell morphology or may differentiate into type I cells. Western blots of freshly isolated type II cells (approx. 85% pure) do not display immunoreactivity with our monoclonal antibodies. However, type II cells maintained in culture acquire immunoreactivity to monoclonal antibodies, demonstrating that type II cells in vitro have the capacity to develop a characteristic associated with type I cells in situ. The availability of markers for a specific membrane component of type I cells should facilitate the study of many questions on alveolar functions, development and response to injury.     

Role of HLA class I and class II antigens in activation and differentiation of B cells

The monoclonal antibodies (MoAb) CR10-214, CR11-115, and Q1/28 to distinct monomorphic determinants of HLA class I antigens, the MoAb CL413 and PTF29.12 recognizing monomorphic determinants of HLA-DR antigens, the anti-HLA-DQw1 MoAb KS11, the anti-HLA-DPw1 MoAb B7/21, and the anti-HLA-DR,DP MoAb CR11-462 were tested for their ability to modulate human B-lymphocyte proliferation and maturation to IgM-forming cells. Purified tonsillar B cells were stimulated with Staphylococcus aureus bacteria of the Cowan first strain (SAC) or anti-human mu-chain xenoantibodies, as well as in growth factor- or T-cell-dependent activation cultures. The B-cell proliferative responses induced by SAC or by mitogenic concentrations of anti-mu-chain xenoantibodies were inhibited by some of the anti-HLA class I and anti-HLA class II monoclonal antibodies tested. The same antibodies were effective inhibitors of the proliferation of B cells stimulated with interferon-gamma (IFN-gamma) or interleukin-2 (IL-2) and with submitogenic concentrations of anti-mu-chain xenoantibodies. The proliferation induced by IL-2 of SAC-preactivated B cells was inhibited by some of the anti-HLA class II monoclonal antibodies, but not by the anti-HLA class I monoclonal antibodies tested. This inhibition appeared to reflect at least in part a direct effect on later events of the B-cell activation cascade, since some anti-HLA class II monoclonal antibodies still exerted considerable inhibitory activity when added together with IL-2 to SAC-preactivated B cells after the third day of culture. Anti HLA-DR, DQ, and DP monoclonal antibodies consistently inhibited the IgM production induced in B cells by T cells alone, T cells plus pokeweed mitogen (PWM), SAC plus IL-2, or IL-2 alone. In contrast, two of the three anti-HLA class I monoclonal antibodies tested inhibited the IgM production in cultures stimulated with SAC plus IL-2 and one the IgM production induced by IL-2 alone, but none of them had inhibitory effects on T-cell dependent IgM production. The results reported herein indicate that HLA class II molecules directly participate in different phases of the B-cell activation cascade. In addition, our data also suggest that HLA class I molecules can be involved in the events leading to B-cell proliferation and differentiation into immunoglobulin-secreting cells.     

Purification of annexin I and annexin II from human placental membranes by high-performance liquid chromatography.

Annexin I and annexin II were extracted from human placental membranes with ethylene glycol bis(beta-amino-ethyl ether)-N,N'-tetraacetic acid (EGTA) and purified by high-performance liquid chromatography by measuring their ability to inhibit phospholipase A2 activity in vitro. Neither protein was capable of binding to a DEAE-5PW HPLC column at neutral pH; however, they were resolved through binding to a Mono S column and passage through size-exclusion HPLC columns. Annexin I and its covalently linked dimer (36 and 66 kDa, respectively, by sodium dodecyl sulfate (SDS)-gel electrophoresis) reacted in one-dimensional immunoblots with monoclonal antibodies to annexin I and calpactin II, and with monoclonal and polyclonal antibodies to lipocortin I, confirming that annexin I, calpactin II, and lipocortin I are the same or closely related proteins. Milligram amounts of monomeric annexin I containing negligible amounts of the cross-linked dimeric annexin I were selectively isolated from placental membranes by using buffers containing the sulfhydryl reagent iodoacetic acid. Milligram amounts of cross-linked annexin I were selectively isolated when placental membranes were initially treated with buffers that did not contain iodoacetic acid and then extracted with Triton X-100, suggesting that sulfhydryl-dependent transglutaminase activity contributes to the selective isolation of this protein. A third phospholipase A2-inhibitory protein (35 kDa by SDS-gel electrophoresis) that reacted in immunoblots with monoclonal antibodies to calpactin I and annexin II, indicating their similar identity, was isolated. The procedure employed allows the rapid purification of annexins I and II in milligram amounts from placental membranes within 2 days.     

Monoclonal antibodies directed against mammalian RNA polymerase I. Identification of the catalytic center

Mouse myeloma cells were fused with splenocytes from a mouse that had been immunized with RNA polymerase I purified from a rat hepatoma. Hybridoma cells were selected and colonies secreting antibodies directed against the enzyme were detected by analysis of cell culture supernatants in a solid phase radioimmunoassay. Two of these cell lines were grown on a larger scale and the interaction between the immunoglobulins obtained from them and RNA polymerase I was studied in detail. Antibodies from both of the hybridoma cell lines were able to inhibit DNA-dependent RNA synthesis catalyzed by RNA polymerases I and III, but not that catalyzed by polymerase II. The antibodies were also capable of reducing the RNA chain elongation reaction catalyzed either by RNA polymerase I associated with isolated nucleoli or by enzyme preinitiated in vitro on calf thymus DNA. Inhibition of RNA polymerase I activity by the monoclonal antibodies was inversely related to the nucleotide concentration. In contrast, the DNA concentration had relatively little effect on inhibition by the antibodies. Analysis of immune complex formation between the antibodies and isolated individual enzyme subunits demonstrated that the monoclonal antibodies were directed against the largest (Mr = 190,000) polypeptide of the polymerase I. These data indicate that the largest subunit of RNA polymerase I contains an immunological determinant in common with RNA polymerase III and suggest that the polymerase I polypeptide of Mr = 190,000 contains a catalytic center involved in RNA chain elongation.     

Lack of expression of HLA [corrected] class I and class II molecules on the human oocyte

The expression of histocompatibility leukocyte antigen (HLA) class I and class II antigens on human oocytes was investigated by the indirect immunofluorescence assay using well-defined monoclonal antibodies. Oocytes were obtained from an in vitro fertilization program or were studied on frozen sections from human ovaries. Neither HLA class I, beta 2-microglobulin, nor HLA class II molecules were detected on cultured oocytes or frozen sections. The zona pellucida also lacked these antigens, but granulosa cells expressed HLA class I molecules. Our results also indicate the presence of certain types of class II molecules on granulosa cells. The present experiments demonstrate that the human oocyte belongs to those few cell types in the human body which are devoid of both types of HLA molecules.     

Accessory cell function of human endothelial cells. I. A subpopulation of Ia positive cells is required for antigen presentation

The expression of class I and class II HLA antigens on preparations of human endothelial cells, isolated from umbilical cord veins, was investigated by immunofluorescence. While virtually all endothelial cells expressed class I antigens, less than 1% were positive for class II antigens, as detected with a panel of 10 different monoclonal antibodies. Antigen specific T cell lines proliferated in response to mumps antigen in the presence of endothelial cells or blood monocytes from HLA-DR matched donors. However, these T cell lines failed to respond in the absence of accessory cells or when accessory cells from HLA-D-region mismatched cord donors were used. The ability of both monocytes and endothelial cells to present antigen was abolished by treatment of the cells with monoclonal antibodies specific for either class I or class II HLA antigens plus complement. Similar treatment with monoclonal antibodies specific for monocytes greatly reduced antigen presentation by endothelial cells. These results indicate that preparations of endothelial cells contain a subpopulation of Ia positive cells, distinct from monocytes, which are required for antigen presentation.     

Monoclonal antibodies to human DNA topoisomerase I and the two isoforms of DNA topoisomerase II: 170- and 180-kDa isozymes

Several monoclonal antibodies of different isotypes specific to human DNA topoisomerase I, to 170- and 180-kDa DNA topoisomerase II isozymes, were produced and characterized. The specificity of monoclonal antibodies was confirmed by comparison with polyclonal antibodies by Western blot, by immunoprecipitation of enzyme activity, and by immunoprecipitation of DNA topoisomerases with characterized polyclonal antisera. Morphological studies performed by immunofluorescence indicate that the three groups of monoclonal antibodies (MoAbs) stain the nucleus with characteristic patterns, which can be compared with those obtained with polyclonal antibodies. In particular the MoAbs to the 100-kDa DNA topoisomerase I stain the nucleolus and the nucleoplasm; the MoAbs to 170- and 180-kDa DNA topoisomerase II give completely distinct intranuclear patterns: those to the 170-kDa protein stain mainly the nucleoplasm, whereas those to the 180-kDa protein stain only the nucleolus. The two DNA topoisomerase II isozymes clearly exhibit fluctuations in their expression during cell growth: the 170-kDa isozyme is more abundant during the logarithmic phase of growth, while the 180-kDa isozyme is mainly present during the plateau phase of growth.     

Monoclonal antibodies prepared against heparin lyase I and their reactivity toward heparin lyase I,II and III

• 1.1. Six different monoclonal IgG mouse antibodies to heparin lyase I from Flavobacterium heparinum were prepared.
• 2.2. The monoclonal antibodies were used to detect heparin lyases I, II and III by dot-blotting immunoassay and by Western blotting.
• 3.3. Individual antibodies showed different reactivity toward the three heparin lyases.
• 4.4. The reactivity of two of the monoclonal antibodies was destroyed by exposing heparin lyases to sodium dodecyl sulfate.
• 5.5. The antibodies can be used to rapidly distinguish between the three heparin lyases.

     

Type I and II insulin-like growth factor receptors on human phytohemagglutinin-activated T lymphocytes

Human T cells activated with mitogens, antigens, or antibodies to the T-cell receptor complex acquire a cascade of new receptors, including the receptors for interleukin-2, transferrin, and insulin. We investigated whether receptors for insulin-like growth factors (IGF) also were expressed on activated T cells. Based on competitive binding studies, immunoprecipitation of labeled cell surface receptors and blocking of radiolabeled peptide binding by a specific monoclonal antibody (alpha IR-3) to the type I IGF receptor, as well as affinity crosslinking of radiolabeled peptides to their receptors, we concluded that both type I and type II IGF receptors are expressed on activated T cells. A specific binding site for IGF-II also was observed on the type I IGF receptor which was not inhibited by alpha IR-3. Receptors for IGF were more numerous on activated T cells than on resting T cells, and their peak expression appeared by the peak of DNA synthesis. Thus, human activated T cells were shown to express both type I and II IGF receptors which could potentially play a role in the regulation of T-cell proliferation, differentiation, and function.     

Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique alpha and common beta subunits

Using monoclonal antibody technology and affinity chromatography we have identified four distinct classes of cell surface receptors for native collagen on a cultured human fibrosarcoma cell line, HT-1080. Two classes of monoclonal antibodies prepared against HT-1080 cells inhibited adhesion to extracellular matrix components. Class I antibodies inhibited cell adhesion to collagen, fibronectin, and laminin. These antibodies immunoprecipitated two noncovalently linked proteins (subunits) with molecular masses of 147 and 125 kD, termed alpha and beta, respectively. Class II antibodies inhibited cell adhesion to native collagen only and not fibronectin or laminin. Class II antibodies immunoprecipitated a single cell surface protein containing two noncovalently linked subunits with molecular masses of 145 and 125 kD, termed alpha and beta, respectively. The two classes of antibodies did not cross-react with the same cell surface protein and recognized epitopes present on the alpha subunits. Pulse-chase labeling studies with [35S]methionine indicated that neither class I nor II antigen was a metabolic precursor of the other. Comparison of the alpha and beta subunits of the class I and II antigens by peptide mapping indicated that the beta subunits were identical while the alpha subunits were distinct. In affinity chromatography experiments HT-1080 cells were extracted with Triton X-100 or octylglucoside detergents and chromatographed on insoluble fibronectin or native type I or VI collagens. A single membrane protein with the biochemical characteristics of the class I antigen was isolated on fibronectin-Sepharose and could be immunoprecipitated with the class I monoclonal antibody. The class I antigen also specifically bound to type I and VI collagens, consistent with the observation that the class I antibodies inhibit cell adhesion to types VI and I collagen and fibronectin. The class II antigen, however, did not bind to collagen (or fibronectin) even though class II monoclonal antibodies completely inhibited adhesion of HT-1080 cells to types I and III-VI collagen. The class I beta and II beta subunits were structurally related to the beta subunit of the fibronectin receptor described by others. However, none of these receptors shared the same alpha subunits. Additional membrane glycoprotein(s) with molecular mass ranges of 80-90 and 35-45 kD, termed the class III and IV receptors, respectively, bound to types I and VI collagen but not to fibronectin. Monoclonal antibodies prepared against the class III receptor had no consistent effect on cell attachment or spreading, suggesting that it is not directly involved in adhesion to collagen-coated substrates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Possible role of calpain I and calpain II in differentiating muscle

The variable distribution of the 80-kD subunit of two calcium-activated proteases, calpain I and calpain II, has been examined in L8 and L6 myoblasts, and their non-fusing variants, fu-1 and M3A using non-cross-reacting monoclonal antibodies to both subunits. Immunofluorescence results have shown that while the 80-kD subunit of calpain I is localized in the cytoplasm of all the myoblasts, the 80-kD subunit of calpain II appears to be predominantly associated with the plasma membranes of L8 and L6 myoblasts. The distribution of the 80-kD subunit of calpain II in non-fusing myoblasts, fu-1 and M3A, is generally cytoplasmic and diffuse. Immunoblot analysis of membrane and cytosol fractions of all the myoblasts using the monoclonal antibodies described above essentially confirms the immunofluorescence findings. Because calpain II exhibits a peripheral distribution in cells which are fusion-competent, L6 and L8 myoblasts, but not in fu-1 and M3A myoblasts, we suggest that calpain II may play a role in the Ca2+-mediated fusion events of differentiating (prefusion) myoblasts.     

Involvement of major histocompatibility complex class II antigen in Epstein-Barr virus-mediated B cell proliferation.

Five MHC class II monoclonal antibodies costimulated proliferation of cord blood leukocytes with Epstein-Barr virus. These agonistic antibodies were of different isotypes, but all of them were either specific for or cross-reacting with HLA-DR. The other MHC class II antibodies, including three that were specific for HLA-DQ and one that was specific for HLA-DP and also those that were specific for MHC class I or leukocyte common antigen, were not costimulatory. The agonistic effect of different MHC class II antibodies was additive, such that costimulation by different antibodies combined significantly exceeded that achieved by either of these antibodies alone. Spent culture media of B cell lines also costimulated B cell proliferation with the virus. Although MHC class II antibodies augmented the effects of suboptimal concentration of the conditioned media, their combined effects did not exceed the maximum costimulation achieved by either the antibodies or the spent culture media alone. These results raised the possibility that MHC class II antigen may contain distinct functional domains involved in the regulation of B cell progression.     

Monoclonal antibodies for a comparative serological study of strains of Vibrio anguillarum

Murine monoclonal antibodies against characteristic determinants of heat stable somatic antigens of typical serovar I (820/15/8 Kop) and serovar II (134/82/1 Kiel) of European Vibrio anguillarum strains were produced. Three stable hybridoma cell lines, called aVaI/1F4, aVaI/6F4 and aVaI/2H5, produced monoclonal antibodies each against a typical serovar I determinant of strain 820/15/8 Kop. One hybridoma cell line (aVaII/5A4) producing monoclonal antibodies against Vibrio anguillarum strain 134/82/1 Kiel (serovar. II) was established. Fourteen Vibrio strains and Aeromonas salmoniáda strains were serologically compared by Enzyme-Linked Immunosorbent Assay (Elisa ).     

Antibody-induced association between discrete regions of HLA class I and II antigens

The anti-HLA-DR + DP monoclonal antibody (MoAb) CR11-462 was unexpectedly found to cross-inhibit the binding to B lymphoid cells of the anti-HLA Class I MoAb CR10-215 and CR11-115. The latter two antibodies recognized the same or spatially close antigenic determinant. The cross-blocking of anti-HLA Class I MoAb CR10-215 and CR11-115 by MoAb CR11-462 reflects neither its contamination by anti-HLA Class I antibodies nor its cross-reactivity with HLA Class I antigens. On the other hand, the cross-blocking appears to reflect redistribution of HLA Class II antigens by the MoAb CR11-462, since the MoAb CR10-215 and CR11-115 are not susceptible to blocking when lymphoid cells are treated with 0.025% glutaraldehyde or are coated with Fab' fragments of the MoAb CR11-462. Furthermore, immunoprecipitates from B lymphoid cells preincubated with the MoAb CR11-462 before solubilization contain HLA Class I antigens. Therefore, these results have shown for the first time an antibody-induced association between discrete regions of HLA Class I and Class II antigens on the membrane of B lymphoid cells.     

Adsorption of Trichoderma reesei CBH I and EG II and their catalytic domains on steam pretreated softwood and isolated lignin

The presence of lignin has shown to play an important role in the enzymatic degradation of softwood. The adsorption of enzymes, and their constituent functional domains on the lignocellulosic material is of key importance to fundamental knowledge of enzymatic hydrolysis. In this study, we compared the adsorption of two purified cellulases from Trichoderma reesei, CBH I (Cel7A) and EG II (Cel5A) and their catalytic domains on steam pretreated softwood (SPS) and lignin using tritium labeled enzymes. Both CBH I and its catalytic domain exhibited a higher affinity to SPS than EG II or its catalytic domain. Removal of cellulose binding domain decreased markedly the binding efficiency. Significant amounts of CBH I and EG II also bound to isolated lignin. Surprisingly, the catalytic domains of the two enzymes of T. reesei differed essentially in the adsorption to isolated lignin. The catalytic domain of EG II was able to adsorb to alkaline isolated lignin with a high affinity, whereas the catalytic domain of CBH I did not adsorb to any of the lignins tested. The results indicate that the cellulose binding domain has a significant role in the unspecific binding of cellulases to lignin.