Showing papers in "Hybridoma and Hybridomics in 2002"

Caloric restriction and experimental carcinogenesis.

Abstract: Research marches on the feet of methodology Advances are made when we have acquired the means to utilize the accrued information In this way, investigation into the influence of energy restriction in cancer has gone through three distinct periods After the initial observation by Moreschi in 1909, there was about a decade of active research in this area Then interest waned, possibly because the field had gone as far as it could considering the knowledge and methodology available at the time Interest was rekindled in 1940 due, principally, to the work coming from the laboratories of Tannenbaum at the Michael Reese Hospital in Chicago and Baumann at the University of Wisconsin Another decade of active research followed In this period we learned how to design experimental diets and interest was expressed in dietary constituents By 1950 publications on this type of research had dwindled and the field lay virtually dormant for 30 years Since the early 1980s research on this topic has blossomed and we now know enough about physiology and molecular biology to probe the mechanisms underlying the phenomenon Energy flux, as in exercise, also inhibits carcinogenesis Energy restriction modulates oxidative DNA damage and enhances DNA repair It is now apparent that energy restriction affects adrenal metabolism, insulin metabolism, and various aspects of gene expression Understanding the basic mechanisms should provide important insights into control of tumor proliferation

Monoclonal antibody-based enzyme immunoassay for detection of ricin.

Abstract: A sensitive and specific enzyme-linked immunoadsorbent assay (ELISA) was developed to detect ricin in biological fluids. The assay is based on the sandwich format using monoclonal antibodies (MAbs) of two distinct specificities. An affinity-purified anti-ricin B chain MAb (1G7) is utilized to adsorb ricin from solution and the second anti-ricin A chain MAb (5E11) conjugated with peroxidase is then used to form a sandwich, and peroxidase allows color development and measurement of optical density at 450 nm. Standard curves were linear over the range of 2.5–100 ng/mL ricin. The limit of detection was below 5 ng/mL in assay buffer as well as in a 1:10 dilution of urine or 1:50 dilution of human serum spiked with ricin.

Monoclonal antibodies selective for low molecular weight neurofilaments.

Abstract: Neurofilaments are necessary for the maintenance of axonal caliber and structural organization of nerve cells. The low molecular weight form of neurofilament, the neurofilament light protein, which serves as the core of the filament, was used as immunogen for generation of hybridomas with selective reactivity with this form of the filament. Six hybridomas, out of approximately 100 tested clones, were highly discriminatory. All involved epitopes were localized to the rod region of the antigen, as determined by alpha-chymotrypsin digestion of the purified filament and enzymatic peptide mapping. Synthetic peptides (20 mers) covering the entire rod region did not react with the antibodies. A phage display peptide library was used to identify four consensus sequences for the antibodies. The results indicate that all epitopes were of conformational type. Pair-wise BIAcore data furthermore indicated that the epitopes were independent. The access to such specific reagents is a prerequisite for further elucidation of the biology of the low molecular weight form of neurofilaments proteins.

A Panel of Monoclonal Antibodies against Human Polycomb Group Proteins

Abstract: Polycomb-group (PcG) proteins are chromatin-associated proteins that heritably repress gene activity in many organisms, including man. Two distinct human PcG complexes have been identified. The HPC/HPH PcG complex I contains the HPC, HPH, RING1, and BMI1 proteins, the EED/EZH2 PcG complex II contains the EED, EZH2, and YY1 proteins. Previously we found that the relative expression levels of proteins of the human PcG complexes I and II are severely deregulated in human tumors. These findings signify an important role for antibodies against human PcG proteins as diagnostic tools. To be able to produce standardized anti-human PcG antibodies, we developed a panel of five mouse monoclonal antibodies (MAbs) against the human PcG proteins HPC2, BMI1, RING1A, EED, and EZH2. All MAbs can be used for Western blot analysis and immunofluorescence labeling of tissue culture cells. With the exception of the MAb against HPC2, all MAbs can also be used in immunoprecipitation experiments and immunohistochemistry of human tissues. The novel MAbs are therefore valuable tools for the cell biological, biochemical, and pathological analysis of human PcG proteins.

In vivo and in vitro anti-tumor effect of 14F7 monoclonal antibody.

Abstract: The 14F7 monoclonal antibody (MAb) is an IgG(1) antibody that reacts specifically with GM3 (NeuGc) and with tissue sections of human tumors. We demonstrated here that this MAb is agglutinin that specifically agglutinated horse erythrocytes. Additionally, the capacity of 14F7 MAb to mediate cytotoxicity against GM3 (NeuGc)-positive murine myeloma cells, in vitro and in vivo, was evaluated. High concentrations of 14F7 MAb were needed to induce complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) against the murine myeloma cells. The most relevant finding was the ability of this MAb to directly kill the target cells without participation of complement. This cytotoxicity was dependent on the temperature and MAb concentration and the number of the target cells. In vivo, the passive treatment with 14F7 MAb produced a strong anti-tumor activity, similar to the anti-tumoral response obtained with standard chemotherapy treatment.

SA-Lea and Tumor Metastasis: The Old Prediction and Recent Findings

Abstract: Several in vivo studies demonstrated that tumor metastasis depend on the expression of carbohydrate Lewis structures. Lewis antigens and their derivatives such as Lewis b (Leb), Lewis X (LeX), sialyl Lewis X (SA-LeX), sialyl Lewis a (SA-Lea), and Lewis Y (LeY) were identified as tumor-associated structures approximately 20 years ago by Koprowski et al. using hybridoma technology and showed that upregulation and/or de novo expression of these determinants on the tumor cell surface is associated with a poor prognosis. LeX and SA-LeX are ligands for selectin adhesion molecules; E- and P-selectins are vascular receptors expressed on activated endothelial cells (ECs) and L-selectin is expressed on leukocytes. Leukocytes also express on their surface LeX and SA-LeX determinants, which are involved in the initial steps of extravasation, that is, rolling, which is alpha step mediated by interaction with E-selectin on ECs. We hypothesized that the tumor cells transmigration from the bloodstream to metastatic sites is similar to lymphocyte extravasation and that adhesion of cancer cells in analogy with the lymphocyte rolling is mediated by interaction of carbohydrate determinants on tumor cells with selectins on ECs. To assess the role of interaction of carbohydrate structures with E-selectin in metastatic process in vivo, we demonstrated that the peptides mimicking SA-Lea blocked colonization of tumor cells in experimental model of lung metastasis in vivo. Furthermore, the metastases formation was completely attenuated in E-selectin-knock out (KO) mice demonstrating the importance of selectin-mediated interaction in this process. We also showed that a peptide mimicking SA-Lea E-selectin ligand has an ability to significantly reduce neutrophil recruitment into peritoneal cavity in acute inflammatory conditions. These studies support the hypothesis that the interaction of tumor cells via the carbohydrate SA-Lea determinant and E-selectin constitutes the important step in the metastatic process in analogy with lymphocyte extravasation and that carbohydrate antigen mimics have a potential as anti-inflammatories and anti-adhesive tumor therapeutics.

Development of humanized monoclonal antibody TMA-15 which neutralizes Shiga toxin 2.

Abstract: A murine monoclonal antibody (MAb), VTm1.1, specifically recognizing and neutralizing Shiga toxin 2 (Stx2), was obtained. To prevent a humoral response against murine antibody when used clinically, a humanized antibody was constructed by combining the complementarity-determining regions of VTm1.1 with human framework and constant regions. In addition, several amino acids in the framework were changed to improve the binding affinity of the antibody and further reduce its potential immunogenicity. The humanized antibody, TMA-15, recognized the B-subunit of Stx2 and had affinity for Stx2 of 3.3 x 10(-9) M, within two-fold of that of the original murine antibody. TMA-15 neutralized the cytotoxicity of Stx2 and several different Stx2 variants in vitro, and it completely protected mice from death in a Stx2-challenged mice model. These results suggest that TMA-15 will have clinical potency in Stx-producing Escherichia coli infections, including E. coli O157 infections.

Comparative assessment expression of the inhibitor of growth 1 gene (ING1) in normal and neoplastic tissues.

Abstract: Studies have indicated that the tumor suppressor p33ING1b (13q33-34) interact with p53. Moreover, the association of functional protein forms of each member of the p33ING1b/p53 complex is essential for optimum activity of p53. The present report describes the sequencing of cDNAs corresponding to the p33ING1b mRNAs in a series of normal and tumor cell lines, and the production of monoclonal antibodies (MAbs) reactive with p33ING1b. These antibodies were subsequently used to analyze p33ING1b expression in normal and tumor cell lines and tissues. No evidence of mutation of p33ING1b was found in any of the 15 tumor cell lines cDNAs studied. Our investigation of a wide range of normal tissues have shown that expression of the nuclear epitope is highly ubiquitous, whereas expression of the cytoplasmic form could be detected in only 50% of tissues studied. Considering neoplastic tissues, loss of nuclear p33ING1b was observed in melanoma, seminoma, papillary thyroid carcinoma, ductal breast carcinoma, and acute l...

Generation of monoclonal antibodies specific for desmoglein family members.

Abstract: Desmosomes are the most prominent cell-cell junctions in most epithelial cells and serve to link the intermediate filament cytoskeletons of adjacent cells. Desmogleins and desmocollins are the transmembrane core of the desmosome and both are members of the cadherin family of cell-cell adhesion molecules. In the skin, the three desmoglein gene products (Dsg 1, 2, and 3) are expressed in a stratification dependent manner, and therefore contribute to compositionally different desmosomes throughout the differentiating layers. In this study we generated a panel of monoclonal antibodies (MAbs) specific for each of the desmoglein gene products and evaluated their usefulness in a number of immunological procedures including immunoblotting, immunoprecipitation, and immunofluorescence. In addition, we showed that these antibodies are useful for immunoprecipitating desmogleins from cell extracts prepared in 0.1% Empigen BB, a zwitterionic detergent capable of solubilizing the desmosomal structure. Identification of conditions that solubilize the desmosome and allow the use of immunological reagents will help facilitate an increased understanding of desmosome assembly and regulation.

Analyses of Streptococcus mutans in saliva with species-specific monoclonal antibodies.

Abstract: Three species-specific monoclonal antibodies (MAbs) against Streptococcus mutans were used to detect and quantify S. mutans levels in saliva. This study shows that MAb-based salivary S. mutans tests exhibit significantly higher specificity and sensitivity than the commonly used selective culture method. Examination of nearly 2000 human saliva samples shows that S. mutans counts in human saliva vary from less than 10,000 to a high 36 million cells/mL. Over 15% of the saliva samples examined have salivary S. mutans counts over 500,000 cells/mL. When saliva samples were collected at different time points during a day, the number of salivary S. mutans in the same human subject varied, especially before and after sugar uptake. Additionally, data obtained from stimulated versus unstimulated saliva in the same human subjects differed greatly and appear to be completely uncorrelated. This study provides useful information and tools for analyzing the role of S. mutans in human dental caries.

Identification of human mannose binding lectin (MBL) recognition sites for novel inhibitory antibodies.

Abstract: Mannose binding lectin (MBL) binding initiates activation of the lectin complement pathway. Recent studies from our laboratory have demonstrated that MBL-dependent complement activation mediates cellular injury following oxidative stress in vivo and in vitro. A panel of novel inhibitory monoclonal antibodies (MAbs) against MBL (e.g., MAb 3F8, 2A9, and hMBL1.2) has been developed that inhibit MBL binding and lectin pathway activation. Here, we further characterized the interactions of these MAbs and their Fab fragments to MBL. Whole MAbs or their Fab fragments bound to MBL with relatively high affinity. Fab fragments of 3F8 were functionally effective in inhibiting MBL-dependent complement activation, however, steric hindrance of MAb 2A9 was essential for inhibition of MBL-dependent complement activation. We identified the hinge region, and residues EDCVLLL within the carbohydrate recognition domain of MBL as the recognition sites for MAb 3F8 and 2A9, respectively. The interaction of MAbs (e.g., 3F8 and 2A9) to MBL was dependent on the conformation of their recognition sites. These findings demonstrate that MBL binding can be inhibited by at least two separate and independent mechanisms.

Generation of Human Interferon Gamma and Tumor Necrosis Factor Alpha Chimeric TNT-3 Fusion Proteins

Abstract: Studies have shown that cytokines can effectively treat solid tumors by a direct cytotoxic effect as well as by immunomodulation. Both human interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) have been used to treat a variety of colon carcinoma cell lines and tumors in patients. These cytokines, however, are dose limited by their toxicity and fast clearance rates when given intravenously. To improve their therapeutic value, we now report on the generation of two new fusion proteins consisting of human IFNgamma and TNFalpha genetically linked to the C-terminal portion of chTNT-3, a monoclonal antibody (MAb), which targets human solid tumors by binding to intracellular antigens exposed in degenerating cells associated with tumor necrosis. In vitro characterization studies demonstrate that both the IFNgamma and TNFalpha fusion proteins are able to maintain their binding affinity to antigen as well as their direct cytotoxic effect and immunomodulatory functions. When both fusion proteins are combined at optimal doses, they demonstrate a 30% direct cellular cytotoxicity of human colon carcinoma cells of which approximately 14% can be attributed to apoptosis. In vivo, these agents were studied for their pharmakocinetic clearance rates and their ability to target human colon carcinomas heterotransplanted in nude mice. The results of these studies show that, compared with chTNT-3 parental antibody, both fusion proteins have a substantially shorter whole body half-life, yet are able to target tumor in a similar manner. As each of these fusion proteins are cleared from the circulation and normal tissues, tumor-to-normal-tissues ratios rise demonstrating the retention of these reagents in tumor. The generation of long-acting and targeted human IFNgamma and TNFalpha antibody fusion proteins will enable investigators to study the role of these potent immunostimulatory cytokines in the treatment of human solid tumors.

New approaches to the development of live attenuated rabies vaccines.

Abstract: In the United States, extensive reservoirs of the rabies virus exist in many diverse wild animal species, which continue to pose a serious risk of lethal infection of humans and cause an economic burden exceeding $1 billion annually. Previous experience with rabies control in foxes in Europe has clearly demonstrated that oral immunization with live vaccines is the only practical approach to eradicate rabies in free-ranging animals. However, unlike Europe where vulpine rabies was the only major reservoir, the Americas harbor a variety of species including raccoons, skunks, coyotes, and bats that serve as the primary reservoirs of rabies. Each of these animal reservoirs carries an antigenically distinct virus variant. The currently available modified-live rabies virus vaccines have either safety problems or do not induce sufficient protective immunity in particular wildlife species. Therefore, there is a need for the development of new live rabies virus vaccines that are very safe and highly effective in particular wildlife species. Based on previous observations indicating that the potency of a vaccine is significantly increased if the G protein of the vaccine strain is identical to that of the target virus, we have used a reverse genetics approach to engineer viruses that contain G proteins from virus strains associated with relevant wildlife species. Furthermore, because our recent data also indicate that the pathogenicity of a particular rabies virus strain is inversely proportional to its ability to induce apoptosis and that low-level apoptosis-inducing ability is associated with low anti-viral immune responses, we inserted genes encoding pro-apoptotic proteins to stimulate immunity or otherwise interfere with viral pathogenesis into these recombinant viruses to enhance their efficacy and safety.

Monoclonal antibodies to molluskan hemocyanin from Concholepas concholepas demonstrate common and specific epitopes among subunits.

Abstract: We studied the reactivity of mouse monoclonal antibodies (MAbs) against the hemocyanin from the Chilean marine gastropod Concholepas concholepas (CCH). This protein has been successfully used as a carrier to produce antibodies to haptens and peptides. All MAbs (13) belonging to IgG subclass exhibit dissociation constants (K(d)) from 1 x 10(-7) M to 1 x 10(-9) M. MAbs were characterized by enzyme-linked immunosorbant assay (ELISA) using CCH treated with different procedures, including dissociation into CCH-A and CCH-B subunits, Western blot, enzymatic digestion, chemical deglycosylation, and thermal denaturation. MAbs were classified into three categories, according to subunit specificity by ELISA. The epitope distribution shows that CCH subunits display common epitopes (group I, 5 MAbs, 1H5, 2A8, 3A5, 3B3, and 3E3), as well as specific epitopes for CCH-A subunits (group II, 3 MAbs, 1B8, 4D8, and 8E5) and for CCH-B subunits (group III, 5 MAbs, 1A4, 1E4, 2H10, 3B7, and 7B4). The results can be summarized as follows: (1). six antibodies react with thermal denatured CCH, suggesting that they recognize linear epitopes, whereas seven recognize conformational epitopes; (2). oxidation of carbohydrate moieties does not affect the binding of the MAbs; (3). enzymatic digestion of CCH decreases the reactivity of all antibodies irrespective of the protease used (elastase or trypsin); (4). bringing together the above data, in addition to epitopic complementarity analysis, we identified 12 different epitopes on the CCH molecule recognized by these MAbs. The anti-CCH MAbs presented here can be useful tools to understand the subunit organization of the CCH and its complex structure, which can explain its immunogenic and immunostimulating properties in mammals.

Rabies vaccines: from the past to the 21st century.

Abstract: Since the first development of a rabies vaccine by Pasteur in the late 19th century, second- and third-generation vaccines with improved efficacy and less reactogenicity have been developed for use in humans and animals. Despite the availability of safe but rather expensive vaccines based on inactivated virus propagated in diploid cell cultures, much of the human vaccinations worldwide are still carried out with nerve tissue-containing vaccines, which have various side effects. A number of experimental vaccines are under development that may provide alternative safe and potent but less expensive vaccine options. These include DNA vaccines, recombinant viral vaccines, and recombinant protein vaccines. Further testing is needed to determine if and which one of these novel vaccines will make their way into mass production and application in the future.

Isolation of native human monoclonal autoantibodies to breast cancer.

Abstract: Using a unique fusion partner cell line, MFP-2, and B-lymphocytes from breast cancer patients, we developed a set of fully human monoclonal antibodies (MAbs) that bind with high specificity and sensitivity to breast cancer cells. Immunofluorescent staining of normal tissues, primary tumors, and metastatic lymph nodes demonstrates that these antibodies are specific for breast cancer of autologous and allogeneic origin. We have also determined that many of the antibodies selected based on specific binding to breast cancer cells and tissue also bind prostate cancer cells and tissue with high specificity and sensitivity. The targets of these antibodies have been localized to the cytoplasm and membrane. Biological assays for internalization and cytotoxicity demonstrated the ability of three antibodies to rapidly internalize. Our study demonstrates that isolation of native human MAbs from the natural antibody repertoire, targeted to cancer cells, is feasible and may provide a source of tools for immunotherapy.

Stable, genetically engineered F(ab')(2) fragments of chimeric TNT-3 expressed in mammalian cells.

Abstract: F(ab′)2 fragments are desirable structural derivatives of monoclonal antibodies (MAbs) because of their pharmacokinetic properties and bivalent binding to antigen. Production of these fragments, however, has proven difficult because of the variable sensitivity of intact antibodies to proteolytic enzymes, which can result in very low yields and unstable product. To circumvent these problems, we attempted to apply genetic engineering methods to generate stable F(ab′)2 fragments in NSO murine myeloma cells using the glutamine synthase expression system. For these studies, the chimeric MAb, chTNT-3, directed against necrotic regions of solid tumors, was used to generate several F(ab′)2 variants, which contained between one and three cysteine residues at the end of the hinge region. In addition, two different affinity tags (his tag, streptactin tag) were used with each variant to determine the best tag for purification procedures. Stability was measured by sodium dodecyl sulfate-polyacrylamide gel electrophore...

Production of antibodies by single DNA immunization: comparison of various immunization routes.

Abstract: DNA immunization is a recent vaccination method that induces humoral and cellular immune responses in a range of hosts. Different immunization routes induce a different degree of the immune response. In the present report, we demonstrate that multiple intramuscular immunizations of plasmid DNA encoding various leukocyte surface molecules induced a specific antibody response. In contrast, a single intramuscular immunization could not induce antibody production. To study the induction of antibody response after a single immunization of plasmid DNA, mice were single-dose intramuscularly, intraperitoneally, intravenously and intrasplenically immunized, simultaneously, with the same preparation of plasmid DNA encoding CD147 membrane protein. We observed that only the intrasplenic route induced specific antibody production. The induction of antibody by intrasplenic immunization was confirmed by using plasmid DNA encoding CD54 molecule. By this single-dose DNA intrasplenic immunization, the generated antibodies could be detected in mice up to 6 months. These results suggest that the injected DNA is expressing the relevant protein antigen in the spleen for several months after injection. Our results demonstrate that direct immunization of antigen-encoding DNA into the spleen is a more effective method for induction of antibody production. This finding may support future investigations of DNA vaccination strategies that specifically promote the uptake of plasmid by splenocytes. Intrasplenic immunization may also be helpful in the understanding of the early events of the immune response to DNA vaccine and be useful as an effective route for the induction of immune responses.

Selection and characterization of human antibodies against hepatitis B virus surface antigen (HBsAg) by phage-display.

Abstract: Hepatitis B virus (HBV) is one of the main pathogens of hepatitis and hepatocarcinoma. Human plasma-derived antibody to HBV is being used as a prophylactic for postexposure to HBV and liver transplantation currently. However, it is required to replace the plasma-derived anti-HBs antibody (Ab) to a recombinant antibody because of limited availability of human plasma with high anti-HBs Ab titer and possible contamination of human pathogens. We constructed an anti-HBs Ab-enriched phage-display library from peripheral blood B cells of vaccinated volunteers and the size of library was approximately 1.0 x 10(7). The library was panned against hepatitis B surface antigen (HBsAg) and five different clones were isolated. All five clones exhibited the same heavy chain sequence; in contrast, light-chain exhibited one lambda and four different kappa sequences. The Fabs were expressed soluble in E. coli and exhibited affinities of 2.1 x 10(8) approximately 7.7 x 10(8) M(-1).

Protective, anti-tumor monoclonal antibody recognizes a conformational epitope similar to melibiose at the surface of invasive murine melanoma cells.

Abstract: Polyclonal and monoclonal antibodies (MAbs) have been raised against B16F10 cells collected from growing tumors in vivo or grown in culture media supplemented with normal mouse serum to avoid xenogeneic reactivity. Antibody binding to glutaraldehyde-fixed melanoma cells and Melan A melanocytes was assayed using chemiluminescent-enzyme-linked immunosorbent assay (CL-ELISA) for increased sensitivity. Most of the reactivity of antitumor polyclonal IgG (92%) was inhibitable by a carbohydrate pool consisting of melibiose, mannose, lactose, and sialic acid. Two monoclonal IgG(2a) antibodies, A4 and B11, had their reactivity to melanoma cells completely and specifically inhibited by melibiose. MAb A4 did not bind to alpha-galactosyl residues abundantly expressed in a protozoan mucin used as substrate, and its binding to the tumor cells was not affected by alpha-galactosidase treatment or addition of alpha-methyl-galactopyranoside or raffinose. Recognition of a mimotope similar to melibiose is suggested. MAb is cytotoxic in vitro in a complement-mediated reaction and effectively neutralizes melanoma cells protecting syngeneic mice against tumor development in vivo. This MAb is thus an important tool for further studies on antitumor adjuvant therapy combined with other agents associated with immuno- and chemotherapy of invasive melanoma.

Identification of Ewing's sarcoma gene product as a glycoprotein using a monoclonal antibody that recognizes an immunodeterminant containing O-linked N-acetylglucosamine moiety.

Abstract: The Ewing's sarcoma (EWS) oncogene is fused to a variety of cellular transcription factors in various forms of human cancers. Although EWS fusion proteins have been extensively studied, the normal function of EWS remains poorly characterized. We previously reported that a monoclonal antibody, referred to as MY95, recognized nucleoporins such as p62, Nup98, and CAN/Nup214 and an uncharacterized polypeptide with an apparent molecular mass of 83 kDa. In the present study, an amino acid sequence analysis of this 83-kDa protein revealed that it is, in fact, EWS, which is not known to belong to the nucleoporins. We further demonstrated that the immunodeterminant of MY95 contains an N-acetylglucosamine moiety, indicating that EWS is a glycoprotein. Interestingly, the glycosylation level of EWS changes during the neural differentiation of P19 cells. MY95 will be quite useful in further studies of the glycosylated form of EWS in terms of understanding the normal cellular function of this oncogene product.

Monoclonal antibodies to gonadotropin-releasing hormone (GnRH) inhibit binding of the hormone to its receptor.

Abstract: Monoclonal antibodies (MAbs) specific to gonadotropin-releasing hormone (GnRH) were obtained using different strategies of conjugation of the peptide to carrier protein and immunization. Of several antibodies obtained, two, namely F1D3C5 and E2D2 bound GnRH in solution phase. Though the epitopes corresponding to the two overlapped, there was a one amino acid shift in the core epitope. These two antibodies were characterized with respect to inhibition of GnRH induced responses in rat pituitary cultures and \alpha-T3.1 mouse gonadotrope cell line.

In vivo expression of a GST-fusion protein mediates the rapid generation of affinity matured monoclonal antibodies using DNA-based immunizations.

Abstract: Previously we demonstrated the rapid generation of affinity matured monoclonal antibody (MAb) producing cell lines following gene gun delivery of DNA using a mammalian expression vector (pAlpha/hFc), which enables the expression of human Fc-chimera proteins in vivo. Here we compare the pAlpha/hFc vector to modified vectors that replace human IgG(1) with either a Glutathione-S-Transferase (GST) fusion protein or a mouse IgG(2c) (mFc) fusion protein. We report that in vivo expression of a GST-chimera results in the rapid generation of affinity matured MAbs, comparable with antibodies raised using the pAlpha/hFc vector, that were reactive with annexin V. The mFc vector failed to induce early antigen-specific B-cell responses suitable for MAb development.

Preparation and Identification of Specific and High-Affinity Monoclonal Antibodies against Morphine

Abstract: A C6-hemisuccinate derivative of morphine was prepared and conjugated to bovine serum albumin and ovalbumin. High titer antibody producing spleen cells were removed and fused with myeloma cells of SP2/0 origin. Using a conventional immunization protocol generated twenty-six stable murine monoclonal antibodies (MAbs) producing cell lines to morphine. The donor mouse produced antiserum with a high titer of 1/640,000. Twelve MAbs were selected for further characterization since they showed high sensitivities (53 pg/well to inhibit 50% of the tracer) in improved group-selective immunoassay (IGSI). The assay, which maintains high sensitivity, high precision, and a wide range of optical density (OD) values, was developed using the conjugate M-6-S-OVA to screen and characterize the anti-morphine MAbs. After four successive limiting dilutions, antibodies produced by 12 clones had high affinities ranging from 109 to 1010 M-1. These clones were found to be of IgG class and IgM class with κ and λ light chain. Subcla...

Borrelia burgdorferi-specific monoclonal antibodies derived from mice primed with Lyme disease spirochete-infected Ixodes scapularis ticks.

Abstract: We have generated a panel of IgG monoclonal antibodies (MAbs) directed against Borrelia burgdorferi strain B31 antigens, using a method whereby mice were primed with organisms naturally inoculated by Ixodes scapularis nymphal ticks. Western blot analysis showed that these MAbs recognized several B. burgdorferi B31 antigens, including the complement inhibitor factor H-binding proteins ErpA/I/N and ErpC. Two other MAbs were specific for the RevA protein, and have enabled characterization of that previously unknown protein. The data presented here suggest that the production of MAbs from animals infected by tick-bite is a potentially useful tool for the identification of novel proteins synthesized by B. burgdorferi during mammalian infection.

Preparation and characterization of monoclonal antibody against digoxin

Abstract: Mouse-mouse hybridoma cell lines producing stable, highly specific and with good affinity monoclonal antibody (MAb) against the cardiac glycoside digoxin were established. Balb/c mice were immunized via injection of digoxin-3'-bovine serum albumin (BSA). The spleens of which were fused with myeloma cells of SP2/0 origin. Three clones designated as BBA, MBE, and BMG producing good antibodies displayed different patterns of fine specificity for digoxin and low cross-reaction with several digoxin analogues as elucidated by inhibition enzyme-linked immunosorbant assay (ELISA). All three MAbs were of the same class and subclass (IgG(1)). Affinity purification was performed for the selected clone BBA displaying the highest affinity and nearly no cross-reactivity with any of the structurally related molecules. Ultrafiltered concentrated hybrid cell supernatant was also purified by polyethylene glycol (PEG) 6000 precipitation for large-scale preparation and coated onto the wells of microtiter plates. The standard curve was constructed with a sensitivity of 10 pg/well covering up to 10 ng/well.

Anti-Idiotypic Antibody as the Surrogate Antigen for Cloning scFv and its Fusion Proteins

Abstract: Single-chain variable fragment (ScFv) is a versatile building block for novel targeting constructs. However, a reliable screening and binding assay is often the limiting step for antigens that are ...

MAb17-1A and cytokines for the treatment of patients with colorectal carcinoma.

Abstract: CO17-1A/GA73-3/EpCam/KSA is a cellular adhesion molecule expressed on the majority of tumor cells in most patients with colorectal carcinoma. One of the first mouse monoclonal antibodies (MAbs) for therapeutic use was produced against this particular tumor associated antigen (MAb17-1A). MAb17-1A has served as a model for the development of antibody therapy. It exerts therapeutic effects through antibody dependent cellular cytotoxicity (ADCC), induction of an idiotypic network cascade and maybe also by complement activation. Addition of cytokines that augment these functions, mainly granulocyte macrophage-cerebrospinal fluid (GM-CSF), seemed to improve the clinical efficacy as well as chemotherapeutic agents (5-Fu). In advanced disease the clinical effect is, however, modest while the most beneficial clinical situation seems to be the adjuvant setting. Twenty years have passed since the EpCam antigen was identified as a target structure for immunotherapy, but still we do not know how to optimally use this ...

Generation and characterization of polyclonal antibodies specific for human p110 sEGFR.

Abstract: The EGFR/ERBB family of receptor tyrosine kinases mediates intracellular signal transduction pathways important in the regulation of cell growth, differentiation, and transformation We previously have reported the cloning and expression of a 3 kb alternative EGFR transcript which encodes a 110 kDa form of the receptor (p110 sEGFR) This receptor isoform is identical to the extracellular region of the full-length 170 kDa EGFR through amino acid 603; in addition, p110 sEGFR contains 78 unique carboxy-terminal amino acids Here, we report the generation and characterization of polyclonal antisera specific for the unique carboxy-terminal sequence of p110 sEGFR Polyclonal antisera were generated by immunizing rabbits with synthetic peptides corresponding to peptides contained within the unique carboxy-terminal sequence of p110 sEGFR Immunoglobulin fractions from antisera which tested positive for immune reactivity to these peptides by ELISA were affinity-purified by protein G and peptide-based chromatography This affinity-purified immunoglobulin fraction specifically recognizes p110 sEGFR by ELISA, immunoprecipitation, immunoblot analysis, and immunocytochemical methods No cross-reactivity with full-length p170 EGFR is observed using any of these detection methods These new polyclonal antibodies will be useful in determining the expression, localization, and function of p110 sEGFR, and importantly will allow us to distinguish between the expression of this receptor isoform and p170 EGFR

Partial characterization of endothelial FGF receptor functional domain by monoclonal antibody VBS-1.

Abstract: Polypeptide growth factors mediate their cellular responses by binding to and activating specific cell surface receptors. Monoclonal antibody (MAb) VBS-1, produced against native fibroblast growth factor receptor-1 (FGFR-1), inhibited the binding of fibroblast growth factor-2 (FGF-2) to its receptor on coronary venular endothelial cells (CVECs) as determined by 125I-FGF-2 Scatchard analysis and [3H]thymidine uptake assays (ED50 = 80 ng/mL). Enzyme studies demonstrated that MAb VBS-1 binds to a protein epitope. Proteolytic mapping of the CVEC-FGFR established that a 52 kDa doublet contained the FGF binding site and the MAb VBS-1 antigenic epitope. N-glycanase digestion suggested the presence of a 50 kDa core protein for the CVEC-FGFR. Tunicamycin treatment resulted in the loss of expression of the core protein and the mature receptor, indicating the importance of CVEC-FGFR n-linked glycosylation. By Northern blot analysis, it was determined that CVECs express fgfr-1 and not fgfr-2. VBS-1 recognized FGFR-1 ...