Production Of Mouse Monoclonal Antibodies Research Articles

Mouse Monoclonal Antibody Specific To Human Ena Blood Group Antigen

Abstract The aim of this study was to produce mouse monoclonal antibodies (mAbs) specific to human blood group antigens. Two percent solution of normal human red blood cells (RBCs) O, RhD+ in normal saline was freshly prepared, and 100 uL was immunized weekly into BALB/mice, intraperitoneal route for 3 weeks. Prior to each immunization, 100 uL of mouse blood was collected and serum was separated. The rising of anti-RBCs antibody titer was performed by standard hemagglutination compared to pre-immunized serum. Mouse was then sacrificed, and the spleen was collected. The fusion between mouse spleen cells and mouse myeloma (X63Ag8.653) cell lines was performed according to the standard hybridoma technique. Hybrid clones were grown in DMEM high glucose supplemented with 20% FCS at 3 °C, 5% CO2 incubator with 5% humidity. The anti-RBCs in hybrid cell culture supernatant were tested by standard hemagglutination and a monoclonal antibody-producing single clone of the hybrid cell was generated by limiting dilution technique. Antibody screening and identification were performed using screening cells, panel cells, and rare blood type RBCs, followed by enzyme treatment technique. The Isotype of mAb was also identified. The results showed that amongst those obtained, mAb clone 1E10-2B2, was confirmed to be anti-Ena characterized as ficin resistant (anti-EnaFR) and isotype IgG2b, kappa. The anti-Ena mAb is very helpful and can be applied as specific mAb for the phenotyping of Ena which is the high-frequency blood group antigens in all populations. Keywords: Glyphorin, MNS blood group, Ena, alloantibody, HDFN, HTR

Subcellular localization of the porcine deltacoronavirus nucleocapsid protein.

Porcine deltacoronavirus (PDCoV) has been recently identified as an emerging enteropathogenic coronavirus that mainly infects newborn piglets and causes enteritis, diarrhea and high mortality. Although coronavirus N proteins have multifarious activities, the subcellular localization of the PDCoV N protein is still unknown. Here, we produced mouse monoclonal antibodies against the PDCoV N protein. Experiments using anti-haemagglutinin antibodies and these monoclonal antibodies revealed that the PDCoV N protein is shuttled into the nucleolus in both ectopic PDCoV N-expressing cells and PDCoV-infected cells. The results of deletion mutagenesis experiments demonstrated that the predicted nucleolar localization signal at amino acids 295–318 is critical for nucleolar localization. Cumulatively, our study yielded a monoclonal antibody against the PDCoV N protein and revealed a mechanism by which the PDCoV N protein translocated into the nucleolus. The tolls and findings from this work will facilitate further investigations on the functions of the PDCoV N protein.

Development of a rapid immunochromatographic assay for detection of surface array protein (Sap), a potent biomarker of Bacillus anthracis

Bacillus anthracis, the causative agent of anthrax poses serious threat to human health. Genetically, B. anthracis is very close to the other species of this group. Hence, it is important to develop a rapid screening test for B. anthracis. Surface array protein (Sap), a surface layer protein produced by B. anthracis is considered an important biomarker for its detection as it is excreted in the growth media. In the present study, we have developed an Immunochromatography test (ICT) strip for detection of Sap, for rapid screening of B. anthracis strains. The recombinant Sap was produced and used for production of mouse monoclonal antibodies. The antibodies were conjugated with colloidal gold for preparation of ICT. For determination of sensitivity and specificity, different Bacillus species were grown in culture broth, heat inactivated to kill the live bacteria and tested on ICT. The B. anthracis Sterne could be detected after 3 h of growth. Sample testing by ICT takes only 2 min in comparison to 4–5 h in sandwich plate ELISA. The results corroborated that the ICT can be used for rapid screening of B. anthracis isolates. The proposed method is rapid, safe and user friendly for detection of B. anthracis culture.

Generation of single-chain Fv antibody fragments against Mu-2-related death-inducing gene in Escherichia coli.

Mu-2-related death-inducing (MuD) gene is involved in apoptosis in tumor cells. Although we have previously produced mouse monoclonal antibodies (MAbs) that specifically recognize human MuD, the application scope of MuD MAbs was restricted due to their mouse origin. Therefore, we attempted the generation of single-chain variable fragment (scFv) against MuD. The heavy- and light-chain variable region genes from two MuD hybridomas were isolated by PCR and joined by DNA encoding a (Gly4Ser1)3 linker. These scFv fragments were cloned into a phagemid vector and expressed as E-tagged fusion proteins in Escherichia coli HB2151. The reactivity of selected Abs was evaluated using ELISA. Selected MuDscFv Abs specifically recognized human MuD, retaining ~ 50% potency of the parent MAbs. MuDscFv-M3H9 recognized the middle region of MuD, while MuDscFv-C22B3 recognized a broad region. Intracellular expression of MuDscFvs-C22B3 protected cells from TRAIL-induced apoptosis. These MuDscFv Abs may help in the study of intracellular signaling pathway centered on MuD and of drug use target and points.

Isolation of Antigen-Specific, Antibody-Secreting Cells Using a Chip-Based Immunospot Array.

Antigen-specific monoclonal antibodies are useful tools to detect very small amounts of antigenic materials and are applicable for antibody therapeutics. To produce mouse monoclonal antibodies, a hybridoma between B lymphocytes and myeloma cells is used to produce antigen-specific monoclonal antibodies. However, a good hybridoma system is not available to obtain human monoclonal antibodies. To produce antigen-specific human monoclonal antibodies, transformation of B lymphocytes with Epstein-Barr viruses or a phage-display system is used. Here, we describe the screening of antigen-specific, antibody-secreting cells using microwell array chips to obtain antigen-specific human monoclonal antibodies. The system can be applied to screen antigen-specific, antibody-secreting cells from any animal species.

Monoclonal antibodies against recombinant GAPDH of Edwardsiella tarda reveal the conserved nature of the protein

ABSTRACTThe outer membrane protein, encoded by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, of Edwardsiella tarda is a highly conserved immunogenic protein. The GAPDH was cloned and expressed in Escherichia coli. The purified protein was used to produce mouse monoclonal antibodies (MAbs). Four stable hybridomas producing MAbs (3G12, 4E9, 5A11 and 9G1) against rGAPDH were obtained. The heavy chains of antibodies produced by the hybridomas were of the isotypes IgG1 and IgM. Cross reactivity of MAbs (3G12 and 9G1) was observed with GAPDH of Aeromonas hydrophila and Micrococcus luteus. MAbs 3G12 and 4E9 reacted with Vibrio cholerae, Salmonella enterica and Penaeus monodon tissues but not with vertebrate GAPDH. None of the MAbs reacted with Staphylococcus aureus. The results indicate that the level of conservation of GAPDH is high among evolutionarily close species. The MAbs developed will be a useful tool to study the evolutionarily conserved and functionally diverse GAPDH.

P2.01-076 Drebrin: A New Targetable Molecular Marker of Lung Adenocarcinoma

Embryonic antigens, such as carcinoembryonic antigen (CEA) and alfa-fetoprotein (AFP), are routinely employed as serum and immunohistochemical tumor markers in clinical medicine. Since they are not expressed in adult human tissues, it is reasonable to conclude that embryonic antigens are extremely specific tumor markers. However, no systematic studies have yet identified clinically useful embryonic antigens for tumor diagnosis. In the present study, we developed a strategy for systematic identification of lung adenocarcinoma markers using monoclonal antibodies generated from embryonic swine tissue. Swine mRNA shows more than 80% homology with human mRNA, and embryonic swine tissue is thought to be a useful material for detection of human embryonic markers. In order to produce mouse monoclonal antibodies, we immunized BALB/c mice by injection of fetal swine lung nuclear fraction into the hock, and used a human lung adenocarcinoma nuclear fraction for the second immunization. We recovered lymph nodes from the mice, and fused mouse B lymphocytes with the murine myeloma cell line SP2/0 using polyethylene glycol. The resulting hybridomas were then selectively cultured. For selection of interesting hybridoma clones, we performed immunohistochemical staining using the supernatant from each one, with tissue microarray loading swine fetal and adult lung, human lung cancer and normal lung tissue. Immunohistochemical screening of 284-clones showed that the antibodies derived from four of them were strongly reactive with the cytoplasm and cytomembrane of fetal swine lung and human lung cancer. We then focused on one clone (B246) whose antibody reacted most clearly with human lung adenocarcinoma cells. Protein microarray analysis confirmed that B246 reacted specifically with “drebrin”, one of the actin binding proteins, originally identified in neuronal cells. There are two drebrin isoforms in human tissue: drebrin E (embryonic) is abundant in the developing neurons, and drebrin A (adult) is expressed in adult brain. We then examined the association of the drebrin expression pattern with the pathological features and prognosis of lung adenocarcinoma using 200 selected cases for which formalin-fixed and paraffin-embedded samples were available. Drebrin immunohistochemistry delineated the samples into those with strong (n=85) and weak (n=115) drebrin expression. Kaplan-Meier analysis demonstrated a significant difference in disease-free survival (DFS) between the groups with strong and weak drebrin expression (p=0.033). Drebrin is expressed specifically in lung adenocarcinoma and is associated with outcome. The present findings indicate that drebrin is a new marker of lung adenocarcinoma and indicative of prognosis.

Novel Monoclonal Antibodies for Studies of Human and Rhesus Macaque Secretory Component and Human J-Chain.

Immunoglobulin A (IgA) antibodies exist in monomeric, dimeric, and secretory forms. Dimerization of IgA depends on a 15-kD polypeptide termed "joining (J) chain," which is also part of the binding site for an epithelial glycoprotein called "secretory component (SC)," whether this after apical cleavage on secretory epithelia is ligand bound in secretory IgA (SIgA) or in a free form. Uncleaved membrane SC, also called the "polymeric Ig receptor," is thus crucial for transcytotic export of SIgA to mucosal surfaces, where it interacts with and modulates commensal bacteria and mediates protective immune responses against exogenous pathogens. To evaluate different forms of IgA, we have produced mouse monoclonal antibodies (MAbs) against human J-chain and free SC. We found that J-chain MAb 9A8 and SC MAb 9H7 identified human dimeric IgA and SIgA in enzyme-linked immunoassay and western blot analysis, as well as functioning in immunohistochemistry to identify cytoplasmic IgA of intestinal lamina propria plasmablasts/plasma cells and crypt epithelium of distal human intestine. Finally, we demonstrated that SC MAb 9H7 cross-reacted with rhesus macaque SIgA. These novel reagents should be of use in the study of the biology of various forms of IgA in humans and SIgA in macaques, as well as in monitoring the production and/or isolation of these forms of IgA.

Immunocapture-Selected Reaction Monitoring Screening Facilitates the Development of ELISA for the Measurement of Native TEX101 in Biological Fluids*

Monoclonal antibodies that bind the native conformation of proteins are indispensable reagents for the development of immunoassays, production of therapeutic antibodies and delineating protein interaction networks by affinity purification-mass spectrometry. Antibodies generated against short peptides, protein fragments, or even full length recombinant proteins may not bind the native protein form in biological fluids, thus limiting their utility. Here, we report the application of immunocapture coupled with selected reaction monitoring measurements (immunocapture-SRM), in the rapid screening of hybridoma culture supernatants for monoclonal antibodies that bind the native protein conformation. We produced mouse monoclonal antibodies, which detect in human serum or seminal plasma the native form of the human testis-expressed sequence 101 (TEX101) protein-a recently proposed biomarker of male infertility. Pairing of two monoclonal antibodies against unique TEX101 epitopes led to the development of an ELISA for the measurement of TEX101 in seminal plasma (limit of detection: 20 pg/ml) and serum (limit of detection: 40 pg/ml). Measurements of matched seminal plasma samples, obtained from men pre- and post-vasectomy, confirmed the absolute diagnostic specificity and sensitivity of TEX101 for noninvasive identification of physical obstructions in the male reproductive tract. Measurement of male and female serum samples revealed undetectable levels of TEX101 in the systemic circulation of healthy individuals. Immunocapture-SRM screening may facilitate development of monoclonal antibodies and immunoassays against native forms of challenging protein targets.

Prion-Specific Antibodies Produced in Wild-Type Mice.

Peptide-specific antibodies produced against synthetic peptides are of high value in probing protein structure and function, especially when working with challenging proteins, including not readily available, non-immunogenic, toxic, and/or pathogenic proteins. Here, we present a straightforward method for production of mouse monoclonal antibodies (MAbs) against peptides representing two sites of interest in the bovine prion protein (boPrP), the causative agent of bovine spongiform encephalopathy ("mad cow disease") and new variant Creutzfeldt-Jakob's disease (CJD) in humans, as well as a thorough characterization of their reactivity with a range of normal and pathogenic (misfolded) prion proteins. It is demonstrated that immunization of wild-type mice with ovalbumin-conjugated peptides formulated with Freund's adjuvant induces a good immune response, including high levels of specific anti-peptide antibodies, even against peptides very homologous to murine protein sequences. In general, using the strategies described here for selecting, synthesizing, and conjugating peptides and immunizing 4-5 mice with 2-3 different peptides, high-titered antibodies reacting with the target protein are routinely obtained with at least one of the peptides after three to four immunizations with incomplete Freund's adjuvant.

Production of mouse monoclonal antibody against Streptococcus dysgalactiae GapC protein and mapping its conserved B-cell epitope

Streptococcus dysgalactiae (S. dysgalactiae) GapC protein is a protective antigen that induces partial immunity against S. dysgalactiae infection in animals. To identify the conserved B-cell epitope of S. dysgalactiae GapC, a mouse monoclonal antibody 1E11 (mAb1E11) against GapC was generated and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12). Eleven positive clones recognized by mAb1E11 were identified, most of which matched the consensus motif TGFFAKK. Sequence of the motif exactly matched amino acids 97–103 of the S. dysgalactiae GapC. In addition, the epitope 97TGFFAKK103 showed high homology among different streptococcus species. Site-directed mutagenic analysis further confirmed that residues G98, F99, F100 and K103 formed the core of 97TGFFAKK103, and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1E11. Collectively, the identification of conserved B-cell epitope within S. dysgalactiae GapC highlights the possibility of developing the epitope-based vaccine.

Identification and characterization of a late gene encoded by grouper iridovirus 2L (GIV-2L).

Grouper iridovirus (GIV) belongs to the Ranavirus genus and is one of the most important viral pathogens in grouper, particularly at the fry and fingerling stages. In this study, we identified and characterized the GIV-2L gene, which encodes a protein of unknown function. GIV-2L is 1242bp in length, with a predicted protein mass of 46.2kDa. It displayed significant identity only with members of the Ranavirus and Iridovirus genera. We produced mouse monoclonal antibodies against the GIV-2L protein by immunizing mice with GIV-2L-His-tag recombinant protein. By inhibiting de novo protein and DNA synthesis in GIV-infected cells, we showed that GIV-2L was a late gene during the viral replication. Finally, immunofluorescence microscopy revealed that GIV-2L protein accumulated in both the nucleus and cytoplasm of infected cells. These results offer important insights into the pathogenesis of GIV.

Production and Characterization of Monoclonal Antibodies against Aflatoxin B1

In this article, we embarked on production of mouse monoclonal antibodies against aflatoxin B1 which is the most commonly occurring fungal toxin in food and feed products. After immunization and fusion with myloma cells, two stable clones (A218 and B319) were selected. Isotyping showed that these monoclonal antibodies (mAbs) were IgG2b with kappa light chains. The affinity of A218 and B319 clons were 5×1011 M−1 and 6×109 M−1, respectively. Competitive indirect ELISA results indicated these mAbs had complete (100%) cross-reaction with four major types of aflatoxins: B1, B2, G1, and G2. These mAbs could be used for immunoassay measurement of aflatoxins with high affinity and low detection limits.

Auto-vaccins

Auto-vaccination is a procedure that recently attracted the interest of a growing number of investigators as an alternative to gene inactivation for functional studies of cytokines or other mediators. It is based on the observation that autologous cytokines cross-linked to a foreign protein or peptide are recognized by self-reactive B cells that present foreign peptides, and by doing so attract illicit help from helper T cells that recognize the foreign peptide on the self-reactive B cell MHC Class II complex. This leads to the production of antibodies reacting with self-proteins and thus to neutralization of the targeted factor. Here, we summarize the different techniques that were successful in breaking this self-tolerance and provide several examples of the functional consequences of these auto-vaccines. An additional output of auto-vaccination is the production of mouse monoclonal antibodies against mouse factors. Such antibodies have obvious advantages for long-term use invivo.

Production of mouse monoclonal antibodies against human HLA-A3 for use in microlymphocytotoxicity technique

Monoclonal antibody production using the hybridoma technology is one of the attractive areas of research. The aim of this study was to investigate the production of monoclonal antibodies to human HLA-A3 antigen for use in microlymphocytotoxicity technique. HLA-A3 antigen was purified from human leukocytes and injected to several mice. The spleen-derived cells of immunized animal were fused with P3-X63-Ag8 myeloma cells. Obtained hybridoma cells were grown in HAT medium and the supernatant of the cells were screened for antibodies using ELISA and microlymphocytotoxicity technique. Produced monoclonal antibody from the hybridoma clone, B1F10 showed the specificity for human HLA-A3. Furthermore, in this study, we encountered with clones of immortal hybrid cells with completely different features, they were adherent and non-producers for antibodies. This observation could imply the possible role of the antigen (HLA) and its nature in the expansion of cell types other than B cells in the spleen of mouse during immunization.

Determination of Trace Amount of Cyanobacterial Toxin in Water by Microchip Based Enzyme-Linked Immunosorbent Assay

Routine monitoring of microcystin in natural waters is difficult because the concentration of the toxin is usually lower than the detection limits. As a more sensitive detection method for microcystin, we developed a microchip based enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies. New monoclonal antibodies against the microcystin leucine-arginine variant (MCLR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. We used keyhole limpet hemocyanin(KLH)-conjugated MCLR as an immunogen for the production of mouse monoclonal antibody. The immunization, cell fusion, and screening of hybridoma cells producing anti-MCLR antibody were conducted. Since the ELISA test was highly sensitive, the newly developed microchip based ELISA can be suitable for the trace analysis of cyanobacterial hepatotoxins, microcystins in water. The linear responses of monoclonal antibodies with different concentrations of microcystin LR were established between 0.025 and 0.3 ng/mL.

Production of mouse monoclonal antibodies against Helicobacter pylori Catalase and mapping the antigenic epitope by phage display library

The Catalase of Helicobacter pylori (H. pylori) helps bacteria to protect themselves from oxygen toxicity and damage and have been identified an immunodominant antigen. To obtain mouse monoclonal antibodies (mAbs) against Catalase and to map its antigenic epitope is potentially to develop a vaccine for prevention and treatment of H. pylori infection. In our study, MAbs were produced by the hybridoma technique using recombinant Catalase--GST as the immunogen and were immunoscreened against phage-displayed random dodecapeptide library (Ph.D.-12). After three rounds of biopanning, 34 phage clones were randomly selected and their specificity to mAb was verified by sandwich and competitive inhibition ELISA. Fifteen phage clones were sequenced and their amino acids were deduced. One mimotope (SVSLPYANLATH) showed good match with Catalase protein at 394-405aa and the serum of mice induced by the phage clone clearly recognized Catalase protein. Our work suggests that the antigenic epitope could be mapped through screening the phage-displayed peptide libraries with mAb and a mimotope of Catalase would provide an alternative approach for the development of a vaccine for H. pylori.

Generation and Characterization of Mouse Monoclonal Antibody 5E1 Against Human Transcription Factor GLI3

GLI3 is a transcriptional effector of the developmentally important hedgehog (Hh) signaling pathway. Here we report the production of mouse monoclonal antibody (MAb) against putative repressive motif in GLI3 (GLI3pRM). BALB/c mice were immunized with purified recombinant human GLI3pRM protein, and the splenocytes from these mice were fused with myeloma cell line (SP2/0) by using standard hybridoma production techniques. Resulting hybridomas producing anti-GLI3pRM antibodies were screened by enzyme-linked immunosorbent assay (ELISA) and isotyped. The specificity of MAb 5E1 was determined based on its activity in Western blot and immunofluorescence analyses of the human NT2/D1 cell line. The results showed that MAb 5E1 was immunoglobulin IgM/kappa, recognizing recombinant human GLI3pRM specifically. In addition, MAb 5E1 bound to the full-length (FL-GLI3) as well as a short protein (GLI3R) and did not cross-react with a similar region in GLI2. MAb 5E1 could also be used to detect the expression of Gli3 in mouse cell lines and embryonic tissues.

Production of mouse monoclonal antibodies against Helicobacter pylori Lpp20 and mapping the antigenic epitope by phage display library

Lpp20, an outer membrane protein of Helicobacter pylori ( H. pylori), has been identified as an immunodominant antigen. To obtain mouse monoclonal antibodies (mAbs) against it and to map its antigenic epitope is potentially to develop a vaccine for prevention and treatment of H. pylori infection. In our study, the Lpp20 gene was obtained from H. pylori genomic DNA by PCR (GenBank accession no. DQ106902), cloned into pGEX-4T-1 vector and expressed in Escherichia coli ( E. coli) as a recombinant fusion protein with glutathione- S-transferase (GST), which was purified by GST-affinity chromatography. mAbs were produced by the hybridoma technique using Lpp20-GST as the immunogen. Using mAb as the target molecule and immunoscreening phage-displayed random dodecapeptide library (Ph.D.-12), the positive phage clones were sequenced and analyzed. Phage clones were chosen to immunize mice to evaluate the potential of phagotopes as effective vaccines. One mimotope (SWPLYSDASGLG) showed a good match with the Lpp20 proteins at 114–117aa (DASG) and the serum of mice induced by the phage clone clearly recognized Lpp20 protein. Our work suggests that the antigenic epitope could be mapped through screening the phage-displayed peptide libraries with mAb and a mimotope of Lpp20 providing an alternative approach for the diagnosis and development of a vaccine for H. pylori.

Generation and Characterization of Mouse Monoclonal Antibody 5E1 Against Human Transcription Factor GLI3

GLI3 is a transcriptional effector of the developmentally important hedgehog (Hh) signaling pathway. Here we report the production of mouse monoclonal antibody (MAb) against putative repressive motif in GLI3 (GLI3pRM). BALB/c mice were immunized with purified recombinant human GLI3pRM protein; the splenocytes from these mice were fused with myeloma cell line (SP2/0) by using standard hybridoma production techniques. Resulting hybridomas producing anti-GLI3pRM antibodies were screened by enzyme-linked immunosorbent assay (ELISA) and isotyped. The specificity of MAb 5E1 was determined based on its activity in Western blot and immunofluorescence analysis of human NT2/D1 cell line. The results showed that MAb 5E1 was immunoglobulin IgM/ê and it recognized recombinant human GLI3pRM specifically. In addition, MAb 5E1 bound to the full-length (FL-GLI3) as well as a short protein (GLI3R) and did not cross-react with a similar region in GLI2. MAb 5E1 could also be used to detect the expression of GLI3 in mouse cell lines and embryonic tissues.