High-level expression of Mycobacterium tuberculosis protein EspB in E. coli and preparation of novel anti-EspB monoclonal antibodies

The ESX-1 secretion system plays a critical role in the virulence of Mycobacterium tuberculosis. The ESX-1 secreted protein EspB is cleaved close to its C-terminus during secretion and is necessary for inhibiting phagosome maturation. In this study, the EspB gene of M. tuberculosis H37Rv was cloned into the expression vector of pET21a(+) and was effectively expressed in Escherichia coli BL21(DE3). The expressed fusion protein existed as a soluble form in cell lysate and was first purified by a column packed with Ni-NTA resin and then a column packed with DEAE-Sepharose Fast Flow matrix. Using the purified protein to immunize BALB/c mice, five monoclonal antibodies were produced. As shown by ELISA and immunoblotting, the five respective antibodies could recognize the EspB protein. These monoclonal antibodies will provide powerful reagents for further investigation of EspB protein functions.     

Polyclonal and monoclonal anti-CEA antibodies in immunolocalization of colorectal cancer

Antibodies to carcinoembryonic antigen (CEA) from sheep and monkey were immunoadsorbent purified. Mouse monoclonal antibody (MAb) anti-CEA I-38S1 and Fab fragments of this antibody were prepared from mouse ascitic fluid. The IgG preparations were labelled with 123I or 131I, the Fab fragments with 131I. The antibody reactivity was unchanged after labelling. Patients with advanced colorectal carcinomas received an intravenous injection of 50-200 MBq 123I or 30-160 MBq 131I coupled to 250-500 micrograms antibody or antibody fragment. Patient examinations were performed using emission tomography (SPECT) and/or conventional gamma camera scintigraphy. The specific localization of labelled anti-CEA to tumor was compared to known tumor localized by CAT-scan, other x-ray methods or laparotomy, 50% of known tumors were accurately localized with sheep anti-CEA. In contrast, 70-80% of known tumor sites were correctly localized with polyclonal monkey anti-CEA antibodies, with monoclonal anti-CEA antibodies or with Fab fragments of the latter. A few previously unknown tumors were detected.     

Generation and characterization of monoclonal antibodies to prostate secretory protein

Monoclonal antibodies (MAbs) were produced against a highly purified preparation of prostate secretory protein (PSP) isolated from normal seminal plasma. Fifteen antibodies were selected for further evaluation based on their strong reactivity and specificity for PSP. All the MAbs had a specificity for prostate epithelial cells and none reacted to any of a variety of normal tissues as determined by immunoperoxidase staining. Six of the MAbs were selected for further immunohistochemical evaluation based on their ability to recognize different antigenic determinants. Using competitive binding immunoassays, a variety of overlapping specificities were observed with at least 2 distinct epitopes identified. Although some staining variability was noted, the 6 antibodies, in general, gave the same pattern of tissue reactivity. Both the normal prostate and the benign prostate hyperplastic ductal epithelial cells stained intensely, with 78 to 100% and 50-100% of the cells staining, respectively. The number and often the staining intensity of the tumor cells decreased as the tumor became more undifferentiated. Approximately 40 to 100% and 15 to 70% of the tumor cells stained in the moderately-differentiated and well-differentiated carcinoma tissues, respectively, whereas either no staining was observed or less than 20% of the tumor cells stained in the poorly-differentiated and undifferentiated tumors. Most of the metastatic prostate tumors showed either no staining or scattered staining in a few cells (i.e., less than 20%).     

Generation and characterization of monoclonal antibodies to TDRD7 protein

TDRD7 is a scaffold protein whose specific function is unknown. It has been identified in complexes with proteins that regulate cytoskeleton dynamics and centrosomal movements, mRNA transport, and protein translation apparatus. Here we report the generation and characterization of monoclonal antibodies against TDRD7 protein. Bacterially expressed His-tagged fragments of TDRD7 were used as antigens. Spleen cells from immunized mice were collected and fused with SP2/0 myeloma cells using PEG 2000. High titer anti-TDRD7 antibody-producing hybridoma cell lines were identified by enzyme-linked immunosorbent assay (ELISA) and then subcloned by limiting dilution. Antibodies produced by E6 clone were further tested for their reactivity with the TDRD7 recombinant proteins. The results obtained clearly indicate that E6 anti-TDRD7 antibodies recognize specifically recombinant 6His-tagged TDRD7 proteins and endogenous TDRD7 in Western blotting, immunoprecipitation, and immunocytochemistry. In summary, these antibodies will be useful for researchers investigating TDRD7 and molecular complexes involving this protein.     

Diversity of cellular molecules in human cells detected by monoclonal antibodies reactive with c-myc proteins produced in Escherichia coli

Six clones of monoclonal antibodies, MYC-1 to -6, were prepared by using two kinds of truncated c-myc proteins, p23 and p42, produced in Escherichia coli as immunogens. Analysis with enzyme-linked immunosorbent assays and immunoblotting assays with peptides produced in Escherichia coli showed that 5 clones of monoclonal antibodies, MYC-1 to -4 and -6, were reactive with c-myc protein encoded by exon 2. The remaining one clone, MYC-5, was reactive with the portion of c-myc protein encoded by exon 3. All monoclonal antibodies were also reactive with phosphorylated c-myc protein produced by insect cells infected by the baculovirus expression vector with the human c-myc gene. With immunoblotting assays using cellular lysates, MYC-1 and -3 detected bands at the levels of 58 kDa and 60 kDa, MYC-5 detected a band at 56 kDa and MYC-6 detected bands at 68 kDa and 75 kDa. All of these bands were detectable in nuclear extracts of HL-60 and Colo320, both of which have amplified c-myc genes, and also the extract of RmycYl which is the c-myc gene transfectant into 3Yl rat cells. None of them was detectable in peripheral blood mononuclear cells and 3Yl, both of which lacked activated c-myc genes. This indicates that these nuclear proteins are either c-myc gene products or molecules closely related to the c-myc gene. The remaining two clones, MYC-2 and -4, detected a band at the level of 85 kDa in cytoplasmic extracts of all the above-mentioned cells independent of the presence of the c-myc gene. This suggests that 85 kDa protein might be irrelevant to the c-myc gene. The 56 kDa protein was detectable by MYC-5 in phytohemagglutinin-stimulated peripheral blood mononuclear cells as well as leukemic cells of some patients.     

Human monoclonal antibodies to lung-cancer antigens.

Lymphocytes obtained from hilar and bronchial lymph nodes from 23 patients undergoing radical surgery for carcinoma of the bronchus were fused with established rat or mouse myeloma lines. 62% of the resultant hybrids were found to be secreting human Ig detected by a sensitive staphylococcal Protein A-coupled SRBC assay. Immunoglobulins synthesized by such hybrids were internally labelled with 3H-lysine and their antibody activity against a variety of membrane preparations determined. Nine monoclonal antibodies were found which bound to molecules on lung-cancer membranes and not on normal lung membranes from the same patient.     

Characterization of monoclonal antibodies to human soluble MD-2 protein

Toll-like receptors (TLRs) are mammalian innate immune recognition receptors that are activated by pathogen associated molecular patterns (PAMPs). TLR4 is the signaling molecule of the lipopolysaccharide (LPS) receptor complex. TLR4 associates with its adapter molecule, MD-2, which is absolutely required for LPS-induced activation of TLR4. MD-2 exists as a cell surface protein in association with TLR4 and as secreted forms consisting of MD-2 monomers and multimers. To facilitate the studies of MD-2 distribution, abundance, and function, we produced monoclonal antibodies (MAbs) to baculovirally expressed soluble MD-2 (sMD-2). Eleven MAbs were characterized by enzyme-linked immunosorbent assay (ELISA) with soluble TLR4/MD-2 complex (sTLR4/MD-2) and sMD-2, Western blotting against sMD-2 monomer and multimers, and inhibition of direct LPS binding to sMD-2. Four MAbs preferentially recognized mainly MD-2 oligomers, not monomers, as judged by Western blotting and ELISA. Anti-MD-2 MAbs useful for indirect immunofluorescent staining of cells expressing TLR4 and MD-2 were identified. One MAb that recognized all forms of MD-2 was used in an ELISA to measure sMD-2 in normal human sera as well as sera from intensive care patients with and without sepsis. Serum levels of sMD-2 were undetectable or very low in normal and in nonsepsis patients but significantly (p < 0.05) increased in sepsis patients. These MAbs should therefore be very useful new tools for studies of MD-2 expression and function in health and disease.     

Antibody responses to HPV6b E polyproteins and production of monoclonal antibodies

A range of fusion constructs (expressed in Escherichia coli) were produced that contained two or more HPV6b E proteins, producing a single continuous amino acid sequence corresponding to the sequences of the individual E proteins. The constructs also included a C-terminal hexahistidine tag fused in-frame to aid purification. The fusion proteins (polyproteins) were semipurified by Ni(++) metal affinity chromatography under denaturing conditions. Immunization of BALB/c mice with these polyproteins resulted in the production of specific E protein antibodies. The draining lymph nodes from these mice were used to produce monoclonal antibodies (MAbs). The specificity of the polyclonal and MAbs was confirmed by immunoblotting and by screening for reaction with a series of synthetic peptides of E proteins. HPV E polyproteins were found to be immunogenic and immunization with the polyproteins resulted in specific antibody responses to the component E proteins.     

Mouse monoclonal immunoglobulin E antibodies specific for the microsporidian Encephalitozoon cuniculi polar tube protein 1

The microsporidian Encephalitozoon cuniculi is a spore-forming, obligate, intracellular parasitic pathogen with a unique organelle called a polar tube, the extrusion of which is essential for invading a host cell. The polar tube consists of three proteins: polar tube protein 1 (PTP1), PTP2, and PTP3. We established three mouse monoclonal antibodies (MAb1, MAb2, and MAb4) against E. cuniculi PTP1. An enzyme-linked immunosorbent assay (ELISA) indicated that all three MAbs reacted with the outer surface of extruded polar tubes and that they also strongly bound to an intracellular antigen in infected host cells. Two-dimensional (2-D) immunoblot analysis showed that MAb1 and MAb2 recognized PTP1 spots at 52 kDa and some spotty smears at molecular weights of less than 50 kDa, whereas MAb4 recognized only PTP1 spots at 52 kDa. Interestingly, all three MAbs were of the immunoglobulin (Ig) E class, suggesting that, in addition to the highly immunogenic or antigenic nature, the PTP1 antigen may have the potential to induce specific IgE antibody production in mice. These antibodies may be useful in the study of allergenic PTP1 as well in the purification and detection of the PTP1 antigen.     

Rat monoclonal antibodies produced against rat colorectal adenocarcinomas define tumor- and colon-associated, auto-immunogenic antigens.

Monoclonal antibodies (MAbs) were derived from rats immunized against allo- and syngeneic rat colon carcinomas. Screening was performed by immunohistochemistry modified for MAbs of the same species as the tissue used for frozen sections. From 2 fusions, several MAbs were found that bound to syngeneic tumor tissue but showed little or no staining of normal adult tissues. Another group of MAbs demonstrated an intracellular staining of tumor cells and staining of mucus and goblet cells in the distal 2/3 of the normal colon. A third group of MAbs showed staining of both tumor and normal colon tissue. Staining patterns were reproduced with 6 MAbs selected from the first 2 groups after purification and biotinylation. One MAb, 10B12, recognized an antigen expressed in 10/11 colon carcinomas, the lowest parts of colonic crypts, intracellularly in a subpopulation of pancreatic acinar cells and mucus in antrum. It was used for affinity purification of a high-molecular-weight antigen, to which 3 of the other rat MAbs also bound. This antigen was also bound by antibodies to blood group A and isogloboseries carbohydrate determinants. Competition with the labelled 10B12 MAb for binding to the purified antigen was demonstrated in sera of tumor-bearing and immune rats. Thus, this high-molecular-weight glycoprotein expressed both auto-antigenic, tumor-associated and tissue-type-restricted epitopes. The 10B12 MAb homogeneously stained the majority of colorectal carcinomas and the antigenic determinant was expressed on the cell surface of 12/13 tissue-cultured colon-carcinoma clones. Modulation of the cell-surface antigen phenotype by recombinant rat interferon-gamma markedly increased expression of this determinant.     

Diagnostic application of immunoperoxidase monolayer assay using monoclonal antibodies produced against equine arteritis virus 14-kDa nucleocapsid protein

Two monoclonal antibodies against the Bucyrus strain of equine arteritis virus (EAV) were produced, and according to immunoperoxidase reaction following Western blot of electrophoresed EAV structural proteins, they recognized the nucleocapsid (N) protein antigen (14-kDa protein). Besides reacting with the blotted polypeptide, the antibodies of the two clones (designated 1H1 and 4G6) selected from 576 have shown high affinity and specificity to intracellular virus antigen as well. Both antibodies reacted with the representatives of the different subtypes of equine arteritis virus providing a suitable general tool for diagnostic purposes using immunoperoxidase monolayer assay (IPMA). Isotypes of the antibodies were examined by Ouchterlony immundiffusion assay. The subtyping of the two examined MAbs proved that the light chains are of the kappaisotype, whereas the heavy chains were identified as IgG 1 isotype.     

Vincristine-resistant human leukemia cell line: new monoclonal antibodies to a 65kDa membrane protein.

A vincristine-resistant human myelomonocytic leukemic cell line (KY-VCR) was established. KY-VCR exhibited approximately a 2.5 x 10(6)-fold increase in resistance to vincristine compared to the parental cell line. KY-VCR showed a decreased uptake and, an increased efflux of vincristine, and cross-resistance to Adriamycin and Actinomycin D. The M(r) 200,000 membrane glycoprotein was overexpressed in KY-VCR. Furthermore, two antibodies, designated TO73 and TO77, preferentially reacting with KY-VCR were obtained. Enzyme linked immunosorbent study indicated that both antibodies recognized the same epitope and TO77 the wide portion. Immunoprecipitation analysis demonstrated that the antibodies recognized M(r) 65,000 membrane protein, which was distinct from overexpressed glycoprotein in KY-VCR. The induction of membrane protein identified by the antibodies may play a role in drug resistance. KY-VCR cells and two antibodies to them may be very useful for the study of drug resistance and prediction of drug efficacy.     

Probing structure and function of the raf protein kinase domain with monoclonal antibodies.

Five monoclonal antibodies were generated against the raf kinase domain. All antibodies react with different isozymes of the raf family, as well as Raf proteins from different species, albeit with differential affinities. Epitope mapping showed all five epitopes clustered in the vicinity of the conserved APE sequence. Although thought to be an essential part of the catalytic site, antibody binding to that domain does not affect kinase activity in vitro or the capability to specifically associate with other cellular proteins. Based on a detailed dissection of the epitopes, a comparative analysis of secondary structure predictions indicates a common structural motif in that region, which is highly conserved amongst protein kinases of the serine/threonine as well as the tyrosine class.     

Production and characterization of monoclonal antibodies to E1 Tor toxin co-regulated pilus of Vibrio cholerae

Murine monoclonal antibodies (MAbs) against Vibrio cholerae toxin co-regulated pilus (TCP) were generated using conventional hybridoma procedures. Four hybridomas were obtained and two characterized. Hybridomas 10E10E1 and 4D6F9 secreted antibodies of the IgG2a and IgG1 isotypes, respectively, that reacted with a 24-kDa antigen corresponding to the product of the El Tor tcpA gene fused to a six Histidine tail. Additionally, MAbs produced by 4D6F9 selectively recognized the major pilin subunit (TcpA) of El Tor and O139 vibrios in western immunoblot, while MAbs from 10E10E1 also cross-reacted with classical TcpA. Furthermore, vibrios expressing TCP on their surface selectively inhibited binding of the antibodies secreted by both hybridomas to TcpA-coated microtiter plates. Thus, the MAbs reported in this work detected the structural subunit of the pilus either denatured or assembled on the bacterial surface.     

Successful challenges using native E. coli asparaginase after hypersensitivity reactions to PEGylated E. coli asparaginase

Purpose

Asparaginase is an essential component of pediatric acute lymphoblastic leukemia (ALL) therapy. However, asparaginase-induced hypersensitivity reactions can compromise its efficacy either by directly influencing the pharmacokinetics of asparaginase or by leading to a discontinuation of asparaginase treatment. Here, we report successful challenges using native Escherichia coli asparaginase after previous hypersensitivity reactions to both PEGylated E. coli asparaginase and Erwinia asparaginase.

Patients and methods

The two patients included in this case report were diagnosed with B-precursor ALL at St. Jude Children’s Research Hospital and were treated with a common regimen. Both patients developed hypersensitivity reactions to PEGylated E. coli asparaginase and Erwinia asparaginase early in treatment, and they were challenged with native E. coli asparaginase. Serum samples were collected for estimating the pharmacokinetic parameters of each patient during native E. coli asparaginase therapy.

Results

Challenges with native E. coli asparaginase were successful, and asparaginase serum concentrations above therapeutic levels were attained in both patients.

Conclusions

These two cases suggest that some patients can be given native E. coli asparaginase after hypersensitivity reactions to PEGylated asparaginase and achieve therapeutic concentrations of the drug in serum.     

Preparation and characterization of monoclonal antibodies to thrombomodulin

Thrombomodulin (TM) is an endothelial cell surface molecule, capable of specific binding for thrombin. The thrombin/TM complex promotes activation of plasma anticoagulant protein C (PC) and negatively regulates blood coagulation. Along with anticoagulant function, TM has been shown to have additional physiological functions such as regulation of fibrinolysis, cell adhesion, tumor growth, and embryonic development. The extracellular region of TM contains a lectin domain and six epidermal growth factor (EGF)-like domains, which are required for the various functions. To analyze the functions, we established a panel of monoclonal antibodies (MAbs) reactive to each functional domain. We obtained MAbs that react to the lectin domain or the front half of EGF domains from the first to the third using the antigen of a transfected cell line expressing full-length TM. We also obtained MAbs that reacted to the bottom half of the EGF domain from the fourth to the sixth using the antigen of a transfected cell line expressing truncated form of TM lacking the lectin domain and the EGF domains from the first to the third. All obtained MAbs could be used for Western blotting. Endothelial cell function for PC activation can be mimicked by transfected cells positive for TM and the endothelial cell protein C receptor (EPCR). Effects of the established MAbs on thrombin-dependent PC activation on the transfected cells were examined. Strong inhibition was demonstrated by three MAbs, which reacted to the fourth or fifth EGF domain, but not by MAbs to the other domains. The fourth EGF domain is known as the interaction site for PC, and the fifth domain is known to be required for thrombin binding. The sixth EGF domain also has been shown to be required for thrombin binding. An MAb against the domain strongly inhibited thrombin-binding. However, the MAb demonstrated little effect on thrombin dependent PC activation. The contradictory results demonstrated with the MAb to the sixth EGF domain suggest an unknown molecular mechanism for PC activation on the cell surface. A panel of MAbs reactive to each domain could be useful for analyzing the multifunctional molecule thrombomodulin.     

Adenovirus E4orf4 protein interacts with both Balpha and B' subunits of protein phosphatase 2A, but E4orf4-induced apoptosis is mediated only by the interaction with Balpha

Adenovirus E4orf4 protein is a multifunctional viral regulator, which is involved in down regulation of virally-modulated signal transduction, in control of alternative splicing of viral mRNAs, and in induction of apoptosis in transformed cells. It has been previously shown that E4orf4 interacts with protein phosphatase 2A through the phosphatase Balpha subunit. It was further shown that PP2A is required for performing the various E4orf4 functions. We report here that E4orf4 interacts with multiple isoforms of the PP2A-B' subunit, as well as with Balpha. We map the interaction sites of the B subunits on E4orf4 and show that they overlap but are not identical. We identify a dominant negative E4orf4 mutant, which disrupts the PP2A holoenzyme. We show that induction of apoptosis by E4orf4, which we previously reported to require the interaction with Balpha, is not affected by the interaction with B'. Our results suggest that the interaction of E4orf4 with various PP2A subpopulations may mediate the different E4orf4 functions.