Glypican-3 (GPC3) has been reported as a novel serum and histochemical marker for hepatocellular carcinoma (HCC) by several groups. As an oncofetal protein, it is expressed abundantly in the fetal liver, inactive in the normal adult liver, and frequently reactivated in HCC. Immunology reagents are urgently needed to proceed with mechanism-related research, clinical validation, and application. In this report, monoclonal antibodies (MAbs) against GPC3 were made from hyperimmune BALB/c mice by injecting 100 μg of purified antigen intraperitoneally. Hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) using purified protein. Finally 13 mouse hybridomas producing MAbs to GPC3 were established. The MAbs obtained were fully characterized using Western blot analysis, immunofluorescence, and immunohistochemistry. The results showed that these antibodies could be used for preliminary application of the next step mechanism-related research and GPC3 expression level analysis.
B and T lymphocyte attenuator (BTLA) is recently identified as the third co-inhibitory receptor with similarities to CTLA-4 and PD-1. Previous reports have shown that BTLA is associated with autoimmune diseases, viral infections and tumor immune evasion. However, the possibility of the existence and role of a soluble form of human BTLA (sBTLA) has not been revealed. Based on our previously generated mouse anti-BTLA monoclonal antibodies, we intend here to develop a novel enzyme-linked immunosorbent assay (ELISA) for detecting sBTLA. Using monoclonal (MAb) 8H9 as coated antibody and the biotin-labeled MAb 7D7 for detection, a sandwich ELISA was developed with good sensibility, line reliability, and specificity. With the established ELISA, the existence and concentration of sBTLA were demonstrated for the first time. It was found that soluble sBTLA existed in the sera of healthy donors and the quantitation of sBTLA climbed along with increasing age, indicating sBTLA may be correlated with immune dysregulation resulting from the aging. Hence, a sandwich ELISA for detecting sBTLA was successfully established and soluble BTLA existing in human serum was first identified, which might play an important role in immunoregulation.
Morbidity from allergic diseases is increasing. Basophils play a critical role in systemic anaphylaxis and chronic allergic inflammation. The prenatal environment must be regarded as a possible early risk factor for allergic diseases in children. Our objective was to determine if basophils harvested from neonates genetically predisposed to atopic disease had different levels of CD63 expression and IL-4 release properties in response to various stimuli (peptidoglycan, Dermatophagoides farinae, hyperosmotic mannitol). Blood samples were collected from 16 asthmatic and 18 healthy women and their newborns. Peripheral blood basophil histamine was measured using the human basophil degranulation test (HBDT), whereas activation was assessed by flow cytometric measurement of CD63 expression on the cord blood basophil surface. IL-4 levels were quantified by ELISA following allergen stimulation. The basophil degranulation index (DI) in granulocytes harvested from the peripheral blood of asthmatic women was assessed following stimulation with peptidoglycan (PGN), Dermatophagoides farinae (Df ) extract, or hyperosmotic mannitol. The DI was significantly higher in atopic women than in healthy controls. Upregulation of CD63 on the cord blood basophil surface was also detected in response to these stimuli. Basophils purified from the cord blood of neonates born to atopic mothers produced more IL-4 compared to basophils purified from the controls. These data suggested that various stimuli play a role in augmenting allergic reactions via modulation of activated basophil cytokine secretion. It may require new methods to stabilize the basophils in allergic diseases.
Protein tyrosine phosphatase 1B (PTP1B), a member of the protein tyrosine phosphatase (PTP) family, plays a crucial role in metabolic signaling, with insulin and leptin signaling being well studied. New evidence indicates that PTP1B is also involved in cancer. In the present study, we report on the establishment of a monoclonal antibody specific for catalytic domain of PTP1B (PTP1Bc) generated through the hybridoma method. The monoclonal antibody is measured to have a titer of 4.1×10(6) against PTP1Bc in indirect ELISA. Western blot and immunofluorescent analyses indicated that this antibody can specifically combine native PTP1B in MDA-MB-231 and MDA-MB-453 cells. This monoclonal antibody against PTP1Bc can help enhance the understanding of PTP1B-related physiological and pathological mechanisms and may act as a therapeutic agent for diabetes, obesity, and cancer in the future.
B7-H3, a novel member of the B7 superfamily, plays a critical role during T cell activation; its functions are still unclear. In this study we obtained a novel anti-mouse B7-H3 monoclonal antibody (MAb) and characterized its biological functions. Our results demonstrated that this MAb could be used for flow cytometry and Western blot and immunohistochemistry analyses, suggesting that the performance of this MAb is much better than a commercial MAb (M3.2D7). Furthermore, data showed different expression profiles of mouse B7-H3 on various immune cells. We further showed that mouse B7-H3 protein was not expressed on normal tissues except for bladder epithelial cells using this MAb. Interestingly, the MAb could stimulate the proliferation and cytokine secretion of T cells. Taken together, this MAb might be of great value for further investigation of B7-H3 molecule.
The G10P[15] rotavirus CC0812-1 isolated from a diarrheal woman in Wuhan, China, in 2008 is phylogenetically close to the Lanzhou lamb rotavirus (LLR) of a monovalent human rotavirus vaccine produced by the Lanzhou Institute of Biological Products, China, and rotavirus Lamb-NT. This rotavirus can be used as the backbone of the attenuated rotavirus reassortant as a rotavirus vaccine candidate. In this study, rotavirus CC0812-1 was purified from the culture supernatant of CC0812-1-infected MA104 cells and used as antigen to immunize BALB/c mice. Four hybridoma clones were developed secreting antibodies that reacted with CC0812-1, designated as 1B1, 1B8, 1F11, and 1G10, respectively. Western blot analysis indicated that the four monoclonal antibodies (MAbs) were all specific for VP4 of rotavirus CC0812-1. Isotyping revealed that MAbs 1B1, 1B8, and 1G10 belonged to the IgM class, while MAb 1F11 belonged to the IgG1 subclass. A neutralization test demonstrated that the four MAbs all had the capacity to neutralize rotavirus CC0812-1. The neutralizing titers of the BALB/c mice ascites were 1:2048, 1:1024, 1:512, and 1:512 for MAbs 1B1, 1B8, 1F11, and 1G10, respectively.
Developing assays for detecting HCV antigens could be beneficial because viral proteins appear earlier than antibodies and are more stable than RNA in the serum. Monoclonal antibody was prepared by immunization and cell fusion. Subclass, specificity, and efficiency of monoclonal antibodies were determined by ELISA. Epitope specificity of monoclonal antibodies was analyzed by ELISA additivity test. HCV antigen in serum of hepatitis patients was examined by double monoclonal antibody sandwich ELISA. Five hybridoma cell lines were screened and named HCV(1), HCV(2), HCV(3), HCV(4), and HCV(5). These five monoclonal antibodies had high specificity and efficiency. The additivity test showed that HCV(2), HCV(4), and HCV(5) recognized different epitopes, which can be matched in ELISA. Of 173 anti-HCV positive patients, 37 (21.4%) were positive for HCV antigen. Of 1498 anti-HCV negative patients, 10 (0.67%) were positive for HCV antigen. Fifty normal controls were negative for HCV antigen. HCV antigen detection had moderate agreement and correlation with HCV RNA detection (kappa=0.577, p<0.01; r=0.59, p<0.01). This result indicates that the monoclonal antibody against HCV NS(3) may be a potential diagnostic reagent, which would provide a foundation for developing a sandwich ELISA of HCV antigen detection.
Renalase is generated mainly by the kidneys, and renalase's expression in chronic kidney disease patients is reduced due to renal dysfunction. In this study, human renalase recombinant protein with prokaryotic expression was used for immunization of male New Zealand rabbits, and polyclonal antibodies against human renalase were obtained. To prepare and verify renalase polyclonal antibody, renalase recombinant protein was used as antigen and male New Zealand rabbits were immunized to obtain anti-serum for identification. On the basis of renalase antibody, the expression of renalase in renal tubular epithelial cells and renal tissue was detected. Two anti-renalase polyclonal antibodies were obtained. Renalase was constitutively expressed in human renal tubular epithelial cells, with the maximum expression in proximal convoluted tubules in renal tissue, and a small amount of expression in the glomeruli. Anti-renalase polyclonal antibodies were successfully prepared, which lays a foundation for further studies.
Porcine circovirus type 2 (PCV2) is associated with postweaning multisystemic syndrome in pigs, whereas the ubiquitous related porcine circovirus type 1 (PCV1) is nonpathogenic. Corroborating an earlier observation in PCV2, Rep and Rep' proteins encoded by ORF1 are essential for the initiation of PCV2 replication. Cap protein encoded by ORF2 has a potential causative role in the initiation of PCV2 replication and contains a type-specific epitope. The putative ORF3 of PCV2 oriented in the opposite direction within ORF1 is unknown. In this study, ORF3-encoding protein of PCV2 was expressed in vitro as a fusion protein (GST-ORF3 protein), and monoclonal antibodies (MAbs) to the PCV2-ORF3-encoding protein were generated and biologically characterized. The mRNA transcript of ORF3 was characterized during a productive infection in PK-15 cells, and the PCV2 infectious DNA clone lacking ORF3 was constructed. GST-ORF3 protein, with an approximate molecular weight of 37.7 kDa, was obtained from the Escherichia coli transformed with the recombinant vector pGEX-4T-1-F3 after codon optimization of ORF3 DNA sequence. Four MAbs reacted strongly to the ORF3-encoding protein expressed in PK-15 cells in immunohistochemical staining. The mRNA transcript of ORF3 was confirmed in RT-PCR, Northern blot, and sequencing analyses. The progeny PCV2 virions were not revealed in the PK-15 cells transfected by the PCV2 infectious DNA clone without ORF3. These results demonstrate that the ORF3 of PCV2 can be transcribed and expressed and that ORF3-encoding protein plays a pivotal role in viral replication.
The UL16-binding proteins (ULBPs) are a novel family of human MHC class I-related, cell surface proteins that function as ligands for NKG2D. In this study, the gene encoding human ULBP3 was cloned into prokaryotic expression vector pQE30, resulting in a recombinant plasmid pQE30-ULBP3. The pQE30-ULBP3 was transformed into Escherichia coli M15 and induced the expression of recombinant protein ULBP3 (rec-ULBP3). The purified rec-ULBP3 as an antigen was used to immunize BALB/c mice. Through cell fusion, sub-cloning, and screening approach, three hybridoma cell clones expressing monoclonal antibodies (MAb) were acquired. The results from Western blot analysis, flow cytometry, and enzyme-linked immunosorbent assay showed that the hybridoma clones B2-F1-F1 and B4-C5-D11, and not G2-A4-A12, reacted with rec-ULBP3 and nature ULBP3 expressed on the cell surface of the tumor cells. In conclusion, the new MAb described here provides a valuable tool for further investigating ULBP3 function and clinical application.
The cannabinoid receptor 1 (CBR1) is being widely investigated because of its specific structure and functions compared with other cannabinoid receptors. In this study, we immunized BALB/c mice with synthesized human CBR1 polypeptide and obtained a novel monoclonal antibody (MAb) against human CBR1. Analysis through enzyme-linked immunosorbent assay (ELISA), spot-ELISA, Western blot, and immunohistochemistry revealed that the MAb was specifically against recombinant human CBR1 protein, and its subtype and affinity constant (Kaff) were IgG2b/k and 7.85 × 10(8) M/L, respectively. Using this MAb we found that CBR1 is expressed on HL-7702 cells and lipid tissue, raising the possibility that the CBR1 may take a role in glucose and lipid metabolism. Thus, this antibody might facilitate studies for pathophysiology of diseases associated with glucose and lipid metabolism abnormality.
VP7 is a major group-specific protein of the bluetongue virus (BTV), and is therefore a candidate for use as a diagnostic reagent. In this study, BALB/c mice were immunized with BTV16, and the lymphocyte hybridoma technique and indirect ELISA screening method were employed to obtain two strains of hybridoma cells secreting specific monoclonal antibodies (MAbs) to BTV16. Eukaryotic recombinant plasmids coding for 10 segments of BTV16 separately were transfected into BHK-21 cells, respectively, followed by immunofluorescence, showing that two MAbs only reacted with BTV-VP7. Western blot analysis showed the same result. Indirect immunofluorescence results indicated that two of the MAbs present different response spectrums with BTV1~24 serotypes. These results indicate that these MAbs may be good candidates for a specific diagnostic method and functional exploration of the VP7 protein.
Calreticulin (CRT) has various versatile functions. It is one of the family of heat shock proteins (HSPs), and is mainly located in the lumen of the endoplasmic reticulum (ER). In order to elucidate the functional and structural properties of CRT, we expressed and purified CRT protein; we then developed a monoclonal antibody (MAb) against mouse CRT by immunizing BALB/c mice with a specific region of the N and P domains of CRT (dCRT) as antigen, which was expressed in Escherichia coli. A stable hybridoma cell line was established by enzyme linked immunosorbent assay (ELISA) screening. The MAb was then prepared from mouse ascites after inoculating the hybridoma cells. Different methods were used to analyze the characterization of the MAb: ELISA, Western blot analysis, immunofluorescence, and flow cytometry. The dCRT protein was expressed and purified and a MAb cell line for CRT was established through immunization, fusion, and screening. ELISA and Western blot analysis indicated that the MAb specifically recognized CRT. In addition, immunofluorescence and flow cytometry demonstrated that the MAb exhibits excellent reactivity to the ecto-CRT when the cells were induced to apoptosis. This CRT MAb will be a valuable tool for further investigation of calreticulin functions.
Prions, or infectious proteins, cause a class of uniformly fatal neurodegenerative diseases. Prions are composed solely of an aberrantly folded isoform (PrPSc) of a normal cellular protein (PrPC). Shared sequence identity of PrPSc with PrPC has limited the detection sensitivity of immunochemical assays, as antibodies specific for the disease-causing PrPSc isoform have not been developed. Here we report the generation of three new monoclonal antibodies (MAbs) to PrP, which were isolated following immunization of Prnp(0/0) Balb/cJ mice with highly purified PrPSc isolated from brain lipid rafts. Epitope mapping using synthetic PrP peptides revealed that the three MAbs bind different epitopes of PrP. The DRM1-31 MAb has a conformational epitope at the proposed binding site for the putative prion conversion co-factor "protein X.'' The DRM1-60 MAb binds a single linear epitope localized to the beta 2-alpha 2 loop region of PrP, whereas DRM2-118 binds an epitope that includes sequences within the octarepeat region and near the site of N-terminal truncation of PrPSc by proteinase K. Our novel anti-PrP MAbs with defined PrP epitopes may be useful in deciphering the conformational conversion of PrPC into PrPSc.
TonB is known to be a bacterial periplasmic protein that transduces proton from the inner membrane to the outer membrane receptor in complex with the ExbB and ExbD proteins. Actinobacillus pleuropneumoniae TonB2 protein is the second TonB protein that is important for iron acquisition and virulence. The TonB2 protein was verified to be immunogenic and could afford partial protection for animals from lethal infection. In the present study, the recombinant TonB2 (rTonB2) was overexpressed in Escherichia coli BL21(DE3) and purified. The rTonB2 was then used as antigen to immunize BALB/c mice for the production of monoclonal antibodies (MAb). Four clones of TonB2-specific MAb secretion hybridomas—2F2, 2G8, 3D2, and 6F10—were selected. The MAbs 2F2, 3D2, and 6F10 were classified as IgG1 isotype and 2G8 was of IgG2a isotype. Western blot and ELISA results indicated that MAbs had specific binding activity to rTonB2. The MAbs generated here will be used for further functional analyses of the TonB2 protein.
The conserved domain of bacteria-derived flagellin coupling Toll-like receptor 5 (TLR5) activates NF-κB and MAPK signaling transductions, which subsequently regulate the transcription and expression of genes encoding immune mediators. However, whether the flagellin binding monoclonal antibody (MAb) obstructs TLR5-associated signaling is unclear. Here we report on the production and characterization of MAb 5G10 that specifically recognizes flagellin. The MAb 5G10 was produced by the hybridization of mouse myeloma cell SP2/0 with splenocyte from a flagellin immunized BALB/c mouse. We observed that deletion of the conserved amino acid residues 89-96 made flagellin lose its capacity for binding 5G10. Additionally, MAb 5G10 remarkably suppressed the expression of cytokine IL8 of Caco-2 cell by blocking the flagellin-TLR5 signaling. Furthermore, this MAb would be useful for cytosolic localization of flagellin and would facilitate the elucidation of the physiological function of specific pathogen-associated molecular patterns.
Non-structural proteins NS3 and NS5 of Japanese encephalitis virus (JEV) were expressed in Escherichia coli and purified by dialysis. Two monoclonal antibodies (MAbs) named 1H7 and 2D4 against NS3 protein and three MAbs named 3C4, 3H7, and 3F10 against NS5 protein were generated by fusing mouse myeloma cell line SP2/0 with spleen lymphocytes from NS3 or NS5 protein immunized mice. Then activity of MAbs was characterized by enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and indirect immunofluorescent assays (IFA). Our results demonstrated that all the MAbs showed high specificity and sensitivity in IFA at 1:100 dilution and in Western blot analysis at 1:500 dilution, which indicated that these MAbs against NS3 and NS5 proteins of JEV may be used as valuable tools for analysis of the protein functions and pathogenesis of JEV.
Emerging evidence suggests that the catalytically inactive ErbB3 (HER3) protein plays a fundamental role in normal tyrosine kinase receptor signaling as well as in aberrant functioning of these signaling pathways, resulting in several forms of human cancers and some mental disorders. Here we report the generation of a specific anti-ErbB3 antibody intended for use in diagnosing disease or therapeutic application. By using the hybridoma technique, one cell line (2E(12)C(3)) stably producing anti-ErbB3 antibody was obtained. Its molecular weight was about 185 kDa and its isotype was IgG 2a and κ, respectively. The affinity constant (Kaff) of the anti-ErbB3 MAb was 5.83×10(10) M(-1). This antibody may become a useful tool for diagnostic and therapeutic targeting of ErbB3-expressing cancers or helpful in highlighting the etiology of schizophrenia.
PEX14 is an integral membrane protein essential for protein docking onto the peroxisomes and is a bi-functional protein capable of acting as a transcriptional co-repressor and a polypeptide transport modulator. Further studies showed that PEX14 is the sole peroxin that has a unique dual function in peroxisome formation and selective degradation. Its RNA transcripts are ubiquitously expressed; there is, however, no data on the expression profiles of PEX14 at the protein level due to a lack of MAbs suitable for immunohistochemical staining, thus hindering studies on its functions. In the present study, we generated one MAb that could be used in immunocytochemistry and immunohistochemistry and investigated PEX14 expression in normal and malignant human tissues. In contrast to the ubiquitous expression of its RNA transcripts, there is no PEX14 protein expression in normal human tissue, including liver, spleen, lung, rectum, brain, prostate, breast, ovary, and stomach. Protein expression of PEX14 was dramatically upregulated in some malignancies. Presented here are the first data on the expression profiles of PEX14 at the protein level, which will further our understanding of its functions.
Human ubiquitin-conjugating enzyme E2, also known as UbcH10, is defined as a cyclin-selective ubiquitin carrier protein and is essential for selective degradation of many short-lived proteins in eukaryotic cells. Recently more and more data show that UbcH10 could be a potential cancer biomarker. In this study, we have developed a monoclonal antibody (MAb) against UbcH10 using an expression recombinant protein. Hybridomas F001, F007, and F008 with high affinities belong to IgG1 subclass with κ light and are highly specific for UbcH10. Further experimentation showed that MAbs F001, F007, and F008 are suitable for the development of immunoassay core agents with sufficient sensitivity and specificity in vitro by Western-blot, immunofluorescence, and immunohistochemistry. These MAbs can be used as a tool for further investigation on functions related to the role of UbcH10 in tumorigenesis and development.
