Demonstration of calmodulin-stimulated phosphatase isozymes by monoclonal antibodies

A procedure combining immunoprecipitation and immunotransblot employing subunit-specific monoclonal antibodies of the brain phosphatase, VJ6 and VA1, was used on tissues including heart, muscle, lung, spleen, pancreas, uterus, and liver. The various tissue extracts were subjected to immunoprecipitation by the beta subunit-specific VA1-immunoabsorbant, the immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunotransblot, using both the alpha and beta subunit-specific antibodies VJ6 and VA1, respectively. Protein bands corresponding to alpha and beta subunits and the immunostain of beta subunit were detected in all samples, whereas alpha subunit was strongly stained only in the brain extract, weakly in heart and muscle extracts, and essentially negatively in all the other samples. In contrast, a polyclonal antiserum of bovine brain calmodulin-stimulated phosphatase could immunostain both alpha and beta subunits from all tissues. Calmodulin-binding protein fractions from a number of bovine tissues were all shown to contain the immunoprecipitable alpha subunit, as well as calmodulin-stimulated p-nitrophenylphosphatase activity. Micropeptide mapping showed that alpha subunits of bovine brain and bovine lung calmodulin-stimulated phosphatase isozymes were distinct molecular species. These results provide direct evidences for the existence of calmodulin-stimulated phosphatase isozymes in mammalian tissues.     

Demonstration of human alpha-L-fucosidase polymorphism by means of monoclonal antibodies

Conventional rabbit antibodies and mouse monoclonal antibodies were raised to alpha-L-fucosidase purified from human placenta. Four monoclonal antibodies were studied, of which only one (A) was able to immunoprecipitate the fucosidase activity completely. Two antibodies (B and C) precipitated 65% and one (D) 35% of the activity. The enzyme precipitated by the monoclonal antibodies remained fully active, whereas the enzyme precipitated by conventional antibodies was partly inactivated. As shown by the method of successive immunoprecipitations, the monoclonal antibodies B and C recognized the same set of placental fucosidase molecules, and D a subset thereof. The purified fucosidase also yielded two components after gel electrophoresis in nondenaturing conditions, and the slower component corresponded to the set recognized by antibodies B and C. The fucosidase extracted from different tissues and serum was studied by immunoprecipitation. In all cases, the enzyme was completely precipitated by monoclonal antibody A. Two patterns were found with B, C and D: either part of the activity was precipitated by these antibodies (leucocytes, placenta, brain, liver, spleen, thymus) or B, C and D failed to precipitate any of the enzyme (serum, heart, kidney, testes).     

Pseudomonas aeruginosa outer membrane: peptidoglycan-associated proteins.

The Pseudomonas aeruginosa outer membrane was isolated with attached peptidoglycan and fractionated with Triton X-100, ethylenediaminetetraacetate, and lysozyme. The data suggest that major outer membrane proteins F, H2, and I are noncovalently associated with the peptidoglycan.     

Demonstration of carbohydrate- and protein-determined Ia antigens by monoclonal antibodies

Ten monoclonal alloantibodies were examined by submitting each antibody to five independent tests in order to determine whether they reacted primarily with the glycoprotein or glycolipid class of Ia antigens. The tests employed were as follows: (1) the ability to precipitate an la-like protein from the cell surface as detected by SDS-PAGE; (2) inhibition by protein-la extracts free of CHO-Ia; (3) inhibition by CHO-Ia extracts free of protein-la; (4) neuraminidase sensitivity of the antigen and (5) inhibition by simple sugars. Using these tests, three of the ten monoclonal antibodies were shown to recognize a CHO-Ia antigen while seven recognized the protein class of Ia antigens. The three CHO-Ia-specific monoclonal antibodies recognized Ia specificities 2,9 and 17. Monoclonal antibodies recognizing protein-defined Ia.2 and 17 specificities were also characterized. These results imply that some Ia specificities, as defined by genetic testing, can occur both as carbohydrate-defined and protein-defined determinants.— Sugar inhibition studies showed that CHO-Ia.2 has D-glucosamine as its immunodominant sugar while CHO-Ia. 17 shows preference for a- linked galactose. Furthermore, studies with neuraminidase demonstrated that sialic acid plays a role in the antigenic determinants of CHO-Ia.9 and CHO-Ia.17. Finally, it is noteworthy that CHO-Ia.2, the private specificity of thek haplotype, appears to be expressed only on cells and not in serum. These studies clearly demonstrate the existence of the two Ia antigen classes and emphasize the complexity of the murineI region.     

Protection mediated by antibodies to iron-regulated outer-membrane proteins of S. typhi in a mouse peritonitis model

Iron limitation induces the expression of iron-regulated outer-membrane proteins, which are not expressed under iron sufficient growth conditions. In the present study, these proteins were purified in order to evaluate their protective potential in the experimental model. Anti IROMPs antiserum was raised in rabbits. In mice, passively transferred anti-IROMPs antibodies provided 60% protection against the serovar Typhi challenge dose (9.6 LD₅₀). The hyperimmune serum containing anti-IROMPs antibodies were also found to be bactericidal in the presence of complement whereas no bacterial killing was observed with pre-immunized serum. Bactericidal titre of anti-IROMPs serum was fond to be 2000 as more than 50% killing was observed with serum diluted to 1:2000. The role of IROMPs was assessed in actively-immunized mice followed by challenge with serovar Typhi. These proteins provided protection in 90% mice against challenge (480 LD₅₀) with the pathogen. The levels of isotypes of antibodies (IgG, IgM & IgA) in the sera and secretory antibodies (sIgA) in the gut fluid of immunized mice correlated with the protection. This study, thus indicates that anti IROMPs antibodies may play an important role in providing protection at systemic as well as at mucosal level.     

Binding to native proteins by antipeptide monoclonal antibodies

mAb raised against synthetic peptides derived from cholera toxin, myohemerythrin, and sickle hemoglobin were analyzed by both solid-phase and solution-phase methods. Antipeptide mAb against cholera toxin (mAb TE32 and TE33), against myohemerythrin (mAb B13I2, B13C2, and B13F2), and against sickle hemoglobin (mAb HuS-1 and HuS-2), had been previously described and used for vaccine development, structural characterization, or identification of a specific antigenic determinant, and each was apparently capable of binding both peptide and native Ag. In this study, all were found to bind whole protein when tested against immobilized Ag in a standard solid-phase assay (ELISA), yet none of the antibodies recognized the Ag in its true native form, failing to bind when tested in several solution-phase assay systems, including size exclusion HPLC. This discrepancy may be the result of modifications of the epitope created by interaction and possible denaturation of the protein on the solid-phase matrix. As a consequence, binding of these antibodies to peptides, either immobilized or in solution, or to immobilized protein, cannot be used to infer that the peptide has assumed a conformation that corresponds to that of the cognate sequence in the native protein. A re-evaluation of binding data that relates antipeptide mAb to native structural characteristics may be necessary.     

Regulation of peptidoglycan synthesis by outer-membrane proteins

Growth of the mesh-like peptidoglycan (PG) sacculus located between the bacterial inner and outer membranes (OM) is tightly regulated to ensure cellular integrity, maintain cell shape, and orchestrate division. Cytoskeletal elements direct placement and activity of PG synthases from inside the cell, but precise spatiotemporal control over this process is poorly understood. We demonstrate that PG synthases are also controlled from outside of the sacculus. Two OM lipoproteins, LpoA and LpoB, are essential for the function, respectively, of PBP1A and PBP1B, the major E.?coli bifunctional PG synthases. Each Lpo protein binds specifically to its cognate PBP and stimulates its transpeptidase activity, thereby facilitating attachment of new PG to?the sacculus. LpoB shows partial septal localization, and our data suggest that the LpoB-PBP1B complex contributes to OM constriction during cell?division. LpoA/LpoB and their PBP-docking regions are restricted to γ-proteobacteria, providing models for niche-specific regulation of sacculus growth.     

The use of monoclonal antibodies to study the proteins specified by the transforming region of human adenoviruses.

Monoclonal antibodies against two of the proteins specified by one of the transforming genes (early region 1B) of human adenovirus type 2 have been produced and characterized. Two clones (RA1 and PA6), generated by fusion of mouse myeloma NSO cells with splenocytes from rats immunized with whole-cell lysates of an adenovirus-transformed rat cell line (F19), secreted antibodies against a 58 kDa protein. Another clone (DC1) produced antibodies against the same protein, and resulted from fusion of immune rat splenocytes with the rat myeloma Y3.Ag.1.2.3. Immunoprecipitation studies showed that all three antibodies recognized [35S]-methionine-labelled 58 kDa protein, and phosphorylated derivatives of the 58 kDa protein labelled with [32P]orthophosphate present in infected human cells. One clone (EC3) produced antibody against a 19 kDa protein also encoded by early region 1B, but not sharing sequence homology with 58 kDa. The identity of the 19 kDa protein recognized by the EC3 antibody was established by immunoprecipitation from lysates of labelled-infected cells and from products of cell-free translation directed by mRNA isolated from adenovirus 2-infected cells. Indirect immunofluorescent-antibody staining of infected human cells using the RA1 and EC3 antibodies revealed a nuclear location of the 58 kDa protein and a mainly cytoplasmic location of the 19 kDa protein.     

Demonstration of carbohydrate- and protein determined Ia antigens by monoclonal antibodies

Ten monoclonal alloantibodies were examined by submitting each antibody to five independent tests in order to determine whether they reacted primarily with the glycoprotein or glycolipid class of Ia antigens. The tests employed were as follows: (1) the ability to participate an Ia-like protein from the cell surface as detected by SDS-PAGE; (2) inhibition by protein-Ia extracts free of CHO-Ia; (3) inhibition by CHO-Ia extracts free of protein-Ia; (4) neuraminidase sensitivity of the antigen and (5) inhibition by simple sugars. Using these tests, three of the ten monoclonal antibodies were shown to recognize a CHO-Ia antigen while seven recognized the protein class of Ia antigens. The three CHO-Ia-specific monoclonal antibodies recognized Ia specificities 2, 9 and 17. Monoclonal antibodies recognizing protein-defined Ia.2 and 17 specificities were also characterized. These results imply that some Ia specificities, as defined by genetic testing, can occur both as carbohydrate-defined and protein-defined determinants.--Sugar inhibition studies showed that CHO-Ia.2 has D-glucosamine as its immunodominant sugar while CHO-Ia.17 shows preference for a beta-linked galactose. Furthermore, studies with neuraminidase demonstrated that sialic acid plays a role in the antigenic determinants of CHO-Ia.9 and CHO-Ia.17. Finally, it is noteworthy that CHO-Ia.2, the private specificity of the k haplotype, appears to be expressed only on cells and not in serum. These studies clearly demonstrate the existence of the two Ia antigen classes and emphasize the complexity of the murine I region.     

Screening for Drosophila proteins with distinct expression patterns during development by use of monoclonal antibodies

Kc 167 is a cell line established from Drosophila embryonic hemocytes and has been shown to express many extracellular matrix (ECM) and other proteins important during development. We have screened monoclonal antibodies (mAbs) raised against heparin affinity purified proteins from conditioned medium of Kc 167 cells to identify novel proteins with important roles for development. One mAb recognized a protein expressed with temporary and tissue specific patterns during Drosophila embryogenesis and larval development. This approach is an alternative to screening of Expression Sequence Tag (EST) clones by in situ hybridization to initiate reverse genetics. In addition, a number of mAbs recognizing ECM proteins were also identified. These mAbs will be useful for biochemical and cell biological analyses of Drosophila ECM proteins.     

Characterization of lipopolysaccharide from Myxococcus xanthus by use of monoclonal antibodies.

Lipopolysaccharide is a major constituent of the cell surface of the gram-negative procaryote Myxococcus xanthus. We have purified lipopolysaccharide from M. xanthus and have shown by silver staining that the lipopolysaccharide contains a heterogeneous population of molecules which migrate as a broad low-molecular-mass band (approximately 5 kilodaltons) and as a stepladder of about 30 higher-molecular-mass bands (15- to 70-kilodalton range). The broad band consists of lipopolysaccharide molecules with just lipid A and core regions. The stepladder bands contain lipopolysaccharide molecules with lipid A, core regions, and various numbers of O-antigen units. Monoclonal antibodies generated against the cell surface of developing M. xanthus cells (J. S. Gill and M. Dworkin, Proc. Natl. Acad. Sci. USA 84:4505-4508, 1987) were used to help characterize the lipopolysaccharide molecules. Five monoclonal antibodies bound to carbohydrate epitopes on the stepladder but not to the broad band, indicating that these monoclonal antibodies recognize carbohydrates on the O antigen of the lipopolysaccharide molecules. Four of these five monoclonal antibodies bound to doublet bands in the stepladder, while the other monoclonal antibody bound to singlet bands in the stepladder. One monoclonal antibody bound to a carbohydrate epitope on both the broad band and the stepladder, indicating that it bound to the core of the lipopolysaccharide.     

Demonstration of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes by monoclonal antibodies

Calmodulin-dependent cyclic nucleotide phosphodiesterase from bovine brain is found to be composed of two distinct subunits, 60,000- and 63,000-dalton polypeptides. Peptide mapping of the subunits by partial proteolysis demonstrated that the 60-kDa polypeptide is not derived from the 63-kDa species. The interaction of the enzyme with three monoclonal antibodies, A6, C1, and A2, and the analysis of immunocomplexes by sucrose density gradient centrifugation revealed that calmodulin-dependent cyclic nucleotide phosphodiesterase exists in three different forms, i.e. (a) homodiamer of 60-kDa, (b) heterodimer of 60- and 63-kDa, and (c) homodimer of 63-kDa. A6 antibody reacts with both 60- and 63-kDa polypeptides indicating that they are immunologically related. C1 and A2 antibodies react with only 60-kDa polypeptide species. By using C1 Sepharose 4B affinity column chromatography, the 63-kDa homodimer which did not bind to the column (Fraction I) was separated from the 60-kDa polypeptide containing isozymes (the heterodimer and the 60-kDa homodimer) which were retained on the column and later eluted as a mixture (Fraction II). Fraction I, the 63-kDa homodimer enzyme, has higher Vmax toward cGMP as substrate than cAMP whereas the opposite was found with Fraction II. The specific activity of Fraction II enzyme toward cAMP was approximately 500 mumol/min/mg, the highest value ever reported for brain calmodulin-dependent cyclic nucleotide phosphodiesterase preparations.     

Demonstration of human kidney differentiation antigens with monoclonal antibodies

Six human differentiation antigens (EE24.6, EG9.11, EG14.1, EI16.1, EK8.1, EK17.1) have been defined using monoclonal antibodies obtained from mice immunized with embryonic kidney cells. Their histologic distribution was determined on frozen sections of embryonic, fetal, and adult human kidneys by immunofluorescence assay. EE24.6, an ureteral bud marker, was detected only on the germ layer of mature kidney urothelium. EG9.11 and EG14.1 were detected on the S-shaped bodies and also on the adult proximal convoluted tubule for the former and the glomerular basement membrane for the latter. EI16.1, a marker of condensed mesenchyme, was detected only on epithelial cells of adult proximal convoluted tubule. EK8.1 was found in the mesangium, connective tissue, and with particularly dense labeling in the basement membranes. This labeling pattern was present throughout renal organogenesis. EK17.1 recognized both cell and plasma human fibronectins. Staining for all antibodies was nearly identical in mesonephros and metanephros. These results demonstate that some antigens follow their embryonic destiny. They indicate an antigenic similarity between the mesonephros and the metanephros and, therefore, a very early appearance of these antigens. During differentiation, these antigens concentrate on more defined structures, and staining became increased with an increased degree of differentiation.     

Mapping nucleolar proteins with monoclonal antibodies

Using monoclonal antibodies as probes, we have characterized three antigens with respect to localization in the nucleolus, molecular weight and solubility. Two proteins, of 110,000 and 94,000 apparent molecular weight, were found associated with the ribonucleoprotein fibers. A third protein, with a molecular weight of 40,000, was accumulated at the nucleolar periphery, was present in the nucleoplasm, and may be involved in pre-ribosome maturation and transport.     

Analysis of herpesvirus saimiri structural proteins with monoclonal antibodies.

Herpesvirus saimiri is a lymphotrophic virus isolated from squirrel monkeys that causes highly malignant lymphomas in owl monkeys, marmosets, and rabbits, but not in its natural host. We have been interested in exploring immunological and biological aspects of this phenomenon and describe in this report the isolation of 27 monoclonal antibodies (MCAs) to herpesvirus saimiri which were grouped into 11 distinct sets based on the proteins they immunoprecipitated. In total, these 11 groups of MCAs identify the majority of the proteins present in the virion. Immunofluorescence was used to study how the viral antigens are compartmentalized in infected cells. One antibody produced intense nuclear staining and immunoprecipitated both the largest protein seen in gels (150,000 daltons) and one of the smallest (13,000 daltons). All of the other groups of MCAs principally stained the cytoplasm of infected cells. One of the unexpected results of this study was the observation that a majority of the MCAs precipitated more than one protein (15 of 27 antibodies, 7 of 11 groups). Whereas one group of MCAs, which normally precipitated four proteins, identified only single polypeptides after dithiothreitol pretreatment, the other sets of antibodies continued to recognize two or more viral antigens under reducing conditions. Immunoprecipitation of viral proteins with polyvalent sera obtained from virus-infected rabbits and monkeys was carried out to verify and extend previously published reports on the proteins of herpesvirus saimiri.     

The use of monoclonal antibodies for detecting the common antigenic determinants of Brucella spp. and Yersinia enterocolitica O:9

Monoclonal antibodies (McAb) 2AH10, specifically reacting with protein preparations having mol. wt. of 18 and 38 kD and not interacting with brucellar lipopolysaccharide (LPS) and protein-polysaccharide antigen, have been obtained. As shown with the use of McAb 2AH10, Brucella spp. and Yersinia enterocolitica O:9 possess common antigenic determinants, localized not only in the area of their LPS, which is generally known, but also in the area of their outer cell-wall proteins with mol. wt. 18 and 38 kD. The sensitivity of the solid-phase enzyme immunoassay and the latex agglutination test with the use of McAb 2AH10 is essentially higher in the detection of B. abortus and B. suis, than B. melitensis and B. rangiferi, as well as Y. enterocolitica O:9. Essential differences observed in the detected concentrations of different Brucella species and Y. enterocolitica O:9 are seemingly linked with different expression of specific antigenic determinants, detected with the use of McAb 2AH10 in the corpuscular antigens under study.     

Gonococcal outer-membrane protein PIB: comparative sequence analysis and localization of epitopes which are recognized by type-specific and cross-reacting monoclonal antibodies.

Comparison of the inferred amino acid sequence of outer-membrane protein PIB from gonococcal strain P9 with those from other serovars reveals that sequence variations occur in two discrete regions of the molecule centred on residues 196 (Var1) and 237 (Var2). A series of peptides spanning the amino acid sequence of the protein were synthesized on solid-phase supports and reacted with a panel of monoclonal antibodies (mAbs) which recognize either type-specific or conserved antigenic determinants on PIB. Four type-specific mAbs reacted with overlapping peptides in Var1 between residues 192-198. Analysis of the effect of amino acid substitutions revealed that the mAb specificity is generated by differences in the effect of single amino acid changes on mAb binding, so that antigenic differences between strains are revealed by different patterns of reactivity within a panel of antibodies. The variable epitopes in Var1 recognized by the type-specific mAbs lie in a hydrophilic region of the protein exposed on the gonococcal surface, and are accessible to complement-mediated bactericidal lysis. In contrast, the epitope recognized by mAb SM198 is highly conserved but is not exposed in the native protein and the antibody is non-bactericidal. However, the conserved epitope recognized by mAb SM24 is centred on residues 198-199, close to Var1 , and is exposed for bactericidal killing.     

Preparation of monoclonal antibodies to phosphotyrosine and their use for identification of phosphotyrosine-containing proteins

Protein kinases phosphorylating proteins at tyrosine residues play an essential role in the cell growth regulation and neoplastic transformation. However, the functions of the majority of tyrosine protein kinases are still obscure, thus creating hindrances in the identification and isolation of phosphotyrosine-containing proteins. The use of the phosphotyrosine structural analog, aminobenzyl phosphonate, as a hapten group enabled the preparation of monoclonal antibodies capable of reacting to phosphotyrosine. The phosphotyrosine specificity of six clones of monoclonal antibodies was tested by a competitive solid phase immunoenzymatic assay. Using fluorescence quenching, the values of constants of binding for antibodies of four clones to phosphotyrosine (2.5-4.0 x 10(6) M-1) were determined. Using two independent methods, it was shown that clone B4 antibodies reveal the highest specificity towards phosphotyrosine. An immunoadsorbent based on clone B4 antibodies was obtained; this immunoadsorbent possessed an ability to selectively interact with an EFR receptor phosphorylated at tyrosine residue. Using eluate acid hydrolysis from the immunoadsorbent, it was demonstrated that clone B4 antibodies interact only with the phosphotyrosine-containing proteins. The experimental results are suggestive of clone B4 monoclonal antibody specificity to phosphotyrosine and of the feasibility of their application for the isolation and identification of tyrosine protein kinases and their substrates.     

Two-dimensional SDS-polyacrylamide gel electrophoresis of heat-modifiable outer-membrane proteins.

An examination has been made of the effect which temperature of solubilization has upon the subsequent migration in SDS-polyacrylamide gel electrophoresis of proteins from the cell envelopes of Escherichia coli K12 and Neisseria sicca ATCC 9913. Conventional electrophoresis in tubes revealed substantial differences in the staining patterns of gels, depending upon whether the envelope samples were solubilized at 37 degrees C or 100 degrees C; in the case of N. sicca at least 6 of 13 discernible bands displayed heat-modifiable behavior. The relationship of the bands produced by each of the two temperatures was investigated by a two-dimensional electrophoresis procedure, in which a sample was solubilized at 37 degrees C and run in a usual cylindrical gel; the entire gel was then resolubilized at 100 degrees C, and laid along an acrylamide slab for electrophoresis in the second dimension. It was found that "free endotoxin" of both organisms examined contained the same major proteins as the total envelope fraction, and that these free endotoxin proteins showed the same heat-modifiable properties as when present in total envelopes.     

Industrial production of monoclonal antibodies and therapeutic proteins by dialysis fermentation

A novel and powerful fermentation method is reported for the large-scale growth of mammalian cells and their secreted products. The system described illustrates many of the advantages of conventional batch fermentation processes but in addition has been shown to yield cell densities in excess of 1×10⁷ cells/ml with concomitant increase in product concentration.Abbreviations MAb Monoclonal Antibody - STR Stirred Tank Reactor - FBS Foetal Bovine Serum