Generation and characterization of murine monoclonal antibodies against immunoreactive trypsinogen for newborn screening of cystic fibrosis.

Cystic fibrosis (CF) is a multisystem disorder that reduces quality of life and survival in affected individuals. In newborns, the release of pancreatic enzymes into the blood raises the levels of immunoreactive trypsinogen (IRT), the main marker for CF screening, which is detected in dried blood samples on filter paper by immunoenzymatic assays. In Cuba, CF has an estimated incidence of 1/9862 live births and should be included in the national basic newborn screening (NBS) panel given its benefits in terms of nutrition, lung function and survival. The Immunoassay Center develops and produces diagnostic kits allowing the establishment of large-scale NBS programs for inherited metabolic disorders in Cuba and other Latin American countries. IRT-specific monoclonal antibodies (MAbs) obtained at the Immunoassay Center are essential for developing an affordable immunoassay for IRT to support CF NBS in our low-income country. An immunization scheme with trypsinogen-1 originated two IgG1-producing murine hybridomas. 4C9C9 and 4C9E11 MAbs recognized different determinants on both trypsin-1 and trypsin-2 molecules. Both antibodies identified conformational epitopes on the molecule of trypsin-1 and of its zymogen. As 4C9E11 MAb cross-reacted with proteins structurally and functionally related to trypsinogen, it was used as revealing antibody in a sandwich-type UMELISA® assay for IRT determination with 4C9C9 MAb for capture. This combination, aside from detecting several commercially available trypsins, adequately quantified IRT from dried blood samples on filter paper of newborns. The evaluation of the assay's accuracy yielded percentage recoveries ranging 93.3-109.2% for commercial controls. The properties of the studied MAbs demonstrate their suitability for being used in a sandwich-type UMELISA® assay for the CF NBS in Cuba.

Rapid production of a chimeric antibody-antigen fusion protein based on 2A-peptide cleavage and green fluorescent protein expression in CHO cells.

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.

Enhancement of monoclonal antibody production after single and combination treatment of the hybridoma cells with all-trans retinoic acid and docosahexaenoic acid: An in vitro and in vivo study.

Murine hybridoma cells can produce monoclonal antibody (MAb) and the production of these antibodies in culture and peritoneum can be affected by different factors, including stimulants, inhibitors and supplements. Among these factors, the impact of micronutrients on the production of MAbs by mouse hybridoma cells has not fully been explored. In this study the murine hybridoma cells, M3C5, were cultured and treated with different concentrations of ATRA and DHA, alone, in combinations, and at different time of exposure. Then, changes in the production of MAb in culture medium were evaluated using ELISA. The hybridoma cells after single and combined treatment with ATRA, DHA and vehicles were IP injected to Balb/c mice and the changes in production of MAb in ascites were determined by ELISA. The results showed that single and combined treatment of ATRA and DHA elevated the production of MAb by hybridoma cells in both in vivo and in vitro. The production of MAb following in vitro single treatment with 1 µM of ATRA and 10 µM of DHA for 2 days was significantly increased. The in vitro effects of ATRA on increase of MAb production was obtained more than DHA. The MAb productions in combined treatment with 0.5 µΜ of ATRA plus 5 µΜ of DHA were significantly increased in in vivo and in vitro. However, the effect of DHA was obtained more significant in in vivo conditions. The results of this study showed for the first time that in vitro and in vivo treatments of ATRA and DHA could increase the production of MAb in mouse M3C5 hybridoma cells.

Energy-based culture medium design for biomanufacturing optimization: A case study in monoclonal antibody production by GS-NS0 cells.

Demand for high-value biologics, a rapidly growing pipeline, and pressure from competition, time-to-market and regulators, necessitate novel biomanufacturing approaches, including Quality by Design (QbD) principles and Process Analytical Technologies (PAT), to facilitate accelerated, efficient and effective process development platforms that ensure consistent product quality and reduced lot-to-lot variability. Herein, QbD and PAT principles were incorporated within an innovative in vitro-in silico integrated framework for upstream process development (UPD). The central component of the UPD framework is a mathematical model that predicts dynamic nutrient uptake and average intracellular ATP content, based on biochemical reaction networks, to quantify and characterize energy metabolism and its adaptive response, metabolic shifts, to maintain ATP homeostasis. The accuracy and flexibility of the model depends on critical cell type/product/clone-specific parameters, which are experimentally estimated. The integrated in vitro-in silico platform and the model's predictive capacity reduced burden, time and expense of experimentation resulting in optimal medium design compared to commercially available culture media (80% amino acid reduction) and a fed-batch feeding strategy that increased productivity by 129%. The framework represents a flexible and efficient tool that transforms, improves and accelerates conventional process development in biomanufacturing with wide applications, including stem cell-based therapies.

Generation of murine monoclonal antibodies with specificity against conventional camelid IgG1 and heavy-chain only IgG2/3.

Camelids possess antibodies with a conventional four-chain structure consisting of two heavy and two light chains (of subclass IgG1) but further they also generate heavy-chain only antibodies (of subclass IgG2 and 3) which are fully functional in antigen binding. In this study subclass-specific murine monoclonal antibodies specific to conventional camelid IgG1 and heavy-chain only IgG2/3 were generated and validated for the use as potent secondary detection reagents. The monoclonal antibodies are able to differentiate between all camelid IgGs, conventional four-chain camelid antibodies (of subclass IgG1) and exclusively heavy chain-only antibodies (of subclasses IgG2 and IgG3). Further these antibodies were used to detect specific immune responses after vaccination of Camelids against bovine corona- and rotavirus strains and different E.coli and Clostridia - antigens and to identify Erysipelothrix rhusiopathiae infected animals within a herd. The described antibodies are suitable as new secondary agents for the detection of different camelid subclasses and the validation of camelid immune reactions.

Production of a Mouse Monoclonal Antibody Against Mortalin by Whole Cell Immunization.

Pancreatic carcinoma is the fourth leading cause of cancer death and is characterized by early invasion and metastasis. Advances in molecular biology directed new strategies in targeted therapy using monoclonal antibodies. To identify new biomarkers, we generated a panel of monoclonal antibodies against the newly established cell line, Faraz-ICR, from a patient with acinar cell carcinoma. After immunization of BALB/c female mice with Faraz-ICR cell line and fusion of splenocytes with SP2/0 myeloma cell line, high reactive hybridoma producing antibodies to Faraz-ICR were detected using enzyme-linked immunosorbent assay, immunofluorescence staining and flow cytometry. Western blot and two-dimensional immunoblot were used for further characterization of the targets antibodies. Among high reactive clones, the reactivity of 1C11 clone was assessed with other epithelial tumors. The isotype of the antibody was revealed to be IgM, and the antibody reacted to a protein with a molecular weight of about 70 kDa in Western blot analysis. To further characterization of the target antigen, immunoproteome of the Faraz-ICR cell line was performed. By liquid chromatography-mass spectrometry (LC-MS) analysis, we identified that the target of 1C11 clone was HSP70. In conclusion, pancreatic cancer is a fatal malignancy with no reliable biomarker for early screening and diagnosis. In this study, by establishing a pancreatic cell line, a panel of monoclonal antibodies was generated aiming to explore specific or associated cancer targets. We then introduced 1C11 monoclonal antibody that can specifically recognize mortalin as a main tumor marker and may serve as a new tool for diagnostic kit and therapeutic strategies targeting this molecule.

Production a monoclonal antibody specific to granulocytes of swimming crab (Portunus trituberculatus) and its cross reactivity with other crustaceans.

In this study, a monoclonal antibody (mAb) 3F4 specific to granulocytes of swimming crab, Portunus trituberculatus, was obtained by immunizing mice with whole haemocytes. mAb 3F4 showed strong immunofluorescent reaction with granulocytes, but no reaction with hyalinocytes. The positive cell percentage of granulocytes was 86.3% detected by Flow cytometry (FCM). A special antigen with molecular weight of about 26kDa was further recognized by mAb 3F4 in haemocytes of P. trituberculatus. mAb 3F4 also showed strong cross-reactivity with haemocytes of Eriocheir sinensis and Petalomera japonica, but no reaction with other crustaceans tested. In E. sinensis, the positive cell percentage was 73.4% for granulocytes and 59.8% for hyalinocytes; while in P. japonica, the positive cell percentage was 81.2% for granulocytes and 7.1% for hyalinocytes. There was also a special antigen with molecular weight of about 31kDa identified by mAb 3F4 in haemocytes of E.sinensis, but no corresponding protein band in P. japonica haemocytes. These results demonstrated that mAb 3F4 can be used as a marker for granulocytes of crabs.

Capto MMC mixed-mode chromatography of murine and rabbit antibodies.

Murine antibodies have weak affinity for Protein-A. Here, we have tested binding of murine monoclonal antibody (mAb) to Protein-A or Protein-A/Protein-G mixture under salting-out conditions. The addition of ammonium sulfate to HEK conditioned medium (CM) expressing murine mAb resulted in complete binding, leading to its elution by low pH or neutral arginine solution. Alternatively, a mixed-mode chromatography using Capto MMC resin was developed as a capture step. Binding of murine mAb occurred at neutral pH. The bound mAb was eluted with a gradient from 0.3 M NaCl to 0.3 M arginine/0.3 M NaCl at pH 7.0. The Capto MMC-purified murine mAb was further purified by hydroxyl apatite chromatography. Similarly, rabbit mAb was processed with some modifications. Binding of rabbit mAb to Capto MMC required a lower pH. Elution of the bound rabbit mAb was achieved by a gradient to 0.3 M NaCl, pH 7.0.

CHO cell production and sequence improvement in the 13C6FR1 anti-Ebola antibody.

From March 2014 through February 2015, the Ebola virus spread rapidly in West Africa, resulting in almost 30,000 infections and approximately 10,000 deaths. With no approved therapeutic options available, an experimental antibody cocktail known as ZMapp™ was administered to patients on a limited compassionate-use basis. The supply of ZMapp™ was highly constrained at the time because it was in preclinical development and a novel production system (tobacco plants) was being used for manufacturing. To increase the production of ZMapp™ for an uncertain future demand, a consortium was formed in the fall of 2014 to quickly manufacture these anti-Ebola antibodies in Chinese hamster ovary (CHO) cells using bioreactors for production at a scale appropriate for thousands of doses. As a result of the efforts of this consortium, valuable lessons were learned about the processing of the antibodies in a CHO-based system. One of the ZMapp™ cocktail antibodies, known as c13C6FR1, had been sequence-optimized in the framework region for production in tobacco and engineered as a chimeric antibody. When transfected into CHO cells with the unaltered sequence, 13C6FR1 was difficult to process. This report describes efforts to produce 13C6FR1 and the parental murine hybridoma sequence, 13C6mu, in CHO cells, and provides evidence for the insertion of a highly conserved framework amino acid that improved the physical properties necessary for high-level expression and purification. Furthermore, it describes the technical and logistical lessons learned that may be beneficial in the event of a future Ebola virus or other pandemic viral outbreaks where mAbs are considered potential therapeutics.

Development of monoclonal antibodies and immunochromatographic lateral flow device for rapid test of alanine aminotransferase isoenzyme 1.

BACKGROUND: Alanine aminotransferase (ALT) has been used as a sensitive marker for liver injury in people and in preclinical toxicity studies. But measurement of ALT isoenzymes, ALT1 and ALT2, was reported to be of more diagnostic value. The aim of this study is to develop an ideal pair of anti-ALT1 monoclonal antibodies (MAbs) of high specificity and affinity, and subsequently prepare a Immunochromatographic lateral flow device (LFD) for rapid test of ALT1 in human serums. METHODS: The complete coding sequence of ALT1 gene (1500 bp) was cloned from human hepatoma G2 cells (HepG2) and inserted into the expression vector pET-32a(+). ALT1 recombinant protein was routinely prepared by E. coli BL21 (DE3) expression and Ni(2+) affinity purification. Balb/c mice were immunized with purified ALT1 and the splenocytes were fused with Sp2/0 myeloma cells. The positive clones, verified by indirect enzyme-linked immunosorbent assay (ELISA) using purified ALT1, were subcloned to single clones by limiting dilution process. A MAb pair was selected from the obtained MAbs according the sandwich ELISA pairing results and then used for lateral flow device (LFD) production. After evaluation of the sensitivity and specificity, the LFD strips were employed to test human serum samples with known ALT activity levels. RESULTS: ALT1 recombinant protein was expectedly prepared by expression and purification. A total of 8 stable clones that produced antibodies specifically recognizing ALT1 protein were developed. After sandwich ELISA pairing, an ideal pair of anti-ALT1 MAbs, designated as BD7 and DG3, were selected and proved to be of high specificity, titer and affinity. Based on the MAb pair, LFD strips specifically for ALT1 rapid test were subsequently prepared. The detection threshold of the LFD strips was 12 U/L. No cross reaction was found. CONCLUSIONS: The ALT1 LFD with high sensitivity and specificity was successfully developed. It is valuable for testing ALT1 protein in human sera and can be a beneficial complement for traditional ALT test.

Impact on monoclonal antibody production in murine hybridoma cell cultures of adenosine receptor antagonists and phosphodiesterase inhibitors.

The effects of different adenosine receptor antagonists and cyclic nucleotide phosphodiesterase (PDE) inhibitors on monoclonal antibody (mAb) titer and cell viability of murine hybridoma cells in culture were measured as part of our investigations to discover additives that enhance mAb production. Specific adenosine receptor antagonists and PDE inhibitors were found to enhance or decrease the titer of immunoglobulin G1 (IgG1) mAbs relative to negative controls, depending on the specific compound and cell line employed. The observed enhancements or decreases in IgG1 mAb titer appeared to be mainly due to an increase or decrease in specific productivity rates (ngmAb/cell), respectively. The different effects of the selective adenosine antagonists suggest that antagonism at the level of the adenosine A2A and A1 or the adenosine A3 receptors result in either enhancement or suppression of IgG1 mAb production by hybridoma cells. Overall, these studies have identified hitherto unknown activities of specific adenosine antagonists and PDE inhibitors which indicate they may have valuable roles as cell culture additives in industrial biomanufacturing processes designed to enhance the yields of mAbs or other recombinant proteins produced by mammalian cell culture procedures.

[PRODUCTION AND CHARACTERIZATION OF THE PANEL OF MONOCLONAL ANTIBODIES AGAINST HUMAN ENDOGLIN].

Endoglin (CD105) is the marker of endothelial and mesenchymal stem cells and the component of TGF-ß, BMP-9 and BMP-10-binding receptor complexes. Its expression is significantly increased on blood vessels endothelium of ischemic tissues and growing tumors. Measurement of concentration of the soluble endoglin in the serum or urine is used as a method for diagnosing cancer and pregnancy disorders. The aim of this work was to create a novel family of monoclonal antibodies recognizing endoglin on the cell surface and in biological fluids. Murine myeloma cells' derived recombinant protein representing the whole extracellular part of endoglin was used as an antigen. F1(SJL/JxBALB/c) mice were the donors of immune splenocytes. Hybridoma screening procedures were performed using E. coli-produced copies of the antigen, endoglin-expressing immortalized human cell lines, and primary cultures of human mesenchymal stromal cells. Ten novel monoclonal antibodies recognizing at least eight distinct epitopes were produced. Eight antibodies bind membrane form of endoglin on the surface of normal and transformed human cells derived from different tissue sources. Two antibodies recognize linear antigenic determinants of the molecule and can be used to detect endoglin by western blot. Sandwich ELISA system was designed in order to measure soluble endoglin in cell culture medium.

Generation of a novel monoclonal antibody that recognizes the alpha (α)-amidated isoform of a valine residue.

BACKGROUND: Alpha (α)-amidation of peptides is a mechanism required for the conversion of prohormones into functional peptide sequences that display biological activities, receptor recognition and signal transduction on target cells. Alpha (α)-amidation occurs in almost all species and amino acids identified in nature. C-terminal valine amide neuropeptides constitute the smallest group of functional peptide compounds identified in neurosecretory structures in vertebrate and invertebrate species. METHODS: The α-amidated isoform of valine residue (Val-CONH2) was conjugated to KLH-protein carrier and used to immunize mice. Hyperimmune animals displaying high titers of valine amide antisera were used to generate stable hybridoma-secreting mAbs. Three productive hybridoma (P15A4, P17C11, and P18C5) were tested against peptides antigens containing both the C-terminal α-amidated (-CONH2) and free α-carboxylic acid (-COO(-)) isovariant of the valine residue. RESULTS: P18C5 mAb displayed the highest specificity and selectivity against C-terminal valine amidated peptide antigens in different immunoassays. P18C5 mAb-immunoreactivity exhibited a wide distribution along the neuroaxis of the rat brain, particularly in brain areas that did not cross-match with the neuronal distribution of known valine amide neuropeptides (α-MSH, adrenorphin, secretin, UCN1-2). These brain regions varied in the relative amount of putative novel valine amide peptide immunoreactive material (nmol/µg protein) estimated through a fmol-sensitive solid-phase radioimmunoassay (RIA) raised for P18C5 mAb. CONCLUSIONS: Our results demonstrate the versatility of a single mAb able to differentiate between two structural subdomains of a single amino acid. This mAb offers a wide spectrum of potential applications in research and medicine, whose uses may extend from a biological reagent (used to detect valine amidated peptide substances in fluids and tissues) to a detoxifying reagent (used to neutralize exogenous toxic amide peptide compounds) or as a specific immunoreagent in immunotherapy settings (used to reduce tumor growth and tumorigenesis) among many others.

Overexpression of Vascular Endothelial Growth Factor A in Invasive Micropapillary Colorectal Carcinoma.

BACKGROUND: Invasive micropapillary carcinoma (IMPC) is a rare variant of colorectal cancer with an adverse prognosis. "Retraction artifact" around tumor cells is a feature of IMPC. The aim of this study was to assess the nature of the retractions around the tumor cells and to describe the histopathological features of a group of 18 cases of IMPC. METHODS: A pathology review of 128 consecutive colorectal cancers identified 18 cases of histologically proven IMPC using 5% of the total tumor volume comprised of a micropapillary component as the diagnostic criterion. Immunostains for D2-40, CD31, CD34, vascular endothelial growth factor A (VEGF-A), and mucin 1 (MUC-1) were performed using the avidin-biotin complex method. RESULTS: Cases of IMPC were characterized by pseudomicropapillae surrounded by lacunar-like clear spaces. These structures exhibited the inside-out growth pattern as highlighted by MUC-1 staining. The lining of the lacunar spaces was immunoreactive to CD31 but not CD34 or D2-40, indicating that they are neovascular structures. Furthermore, the tumor cells strongly and diffusely expressed VEGF-A. CONCLUSIONS: The strong coexpression of VEGF-A and CD31 suggests a prominent role of neoangiogenesis in these tumors.

Monoclonal Antibodies Against NS2B of Japanese Encephalitis Virus.

Japanese encephalitis (JE) is one of the most important viral encephalitis, caused by the Japanese encephalitis virus (JEV). The function of non-structural protein 2B (NS2B) mostly remains unclear. In our study, NS2B of Japanese encephalitis virus (JEV) was expressed in Escherichia coli and purified by dialysis. After fusing mouse myeloma cell line SP2/0 with spleen lymphocytes from NS2B protein immunized mice, three clones of monoclonal antibodies (MAbs), named 1B9, 3E12, and 4E6, were generated. The specificity and sensitivity of MAbs were demonstrated by ELISA, indirect immunofluorescence assay, and Western blot. These MAbs will be useful in further exploration of the functions of NS2B and the pathogenesis of Japanese encephalitis virus.

Monoclonal Antibodies Against the Human Respiratory Syncytial Virus Obtained by Immunization with Epitope Peptides and CpG-DNA-liposome Complex.

Respiratory syncytial virus (RSV) is the major cause of pulmonary inflammation in infants, young children, and immunocompromised adults. However, the RSV vaccine is not yet available commercially. The RSV-F glycoprotein mediates virus-host cell fusion, leading to syncytial formation; therefore, the RSV-F glycoprotein has been a treatment target for prevention and therapy of RSV infection. To produce the RSV-F-protein epitope-specific monoclonal antibody (MAb), BALB/c mice were immunized with a complex consisting of epitope peptide and MB-ODN 4531(O), encapsulated in a phosphatidyl-ß-oleoyl-γ-palmitoyl ethanolamine (DOPE):cholesterol hemisuccinate (CHEMS) complex (Lipoplex(O)). Using conventional hybridoma technology, we obtained two clones able to produce antibodies reactive to two B-cell epitopes of RSV-F protein. Each anti-RSV-F glycoprotein MAb efficiently binds to each epitope. The F7-1A9D10 clone showed specific binding with RSV-F protein. There was no specific protein detected by Western blot analysis using F9 epitope-specific anti-RSV-F glycoprotein MAb (clone F9-1A6C8). However, based on confocal-image analysis, the antibody from the F9-1A6C8 clone showed specific binding with RSV-F protein. It is important that further study on possible applications for passive immunotherapy against RSV infection, such as therapeutic antibody production, is carried out.

Monoclonal antibody against M protein of spring viremia of carp virus.

M protein of spring viremia of carp virus (SVCV) was expressed in Escherichia coli and then used to immunize BALB/c mice. One monoclonal antibody (5A1) against M protein was generated by fusion of mouse myeloma cell line SP2/0 and spleen lymphocytes from immunized mice. The characterizations of this MAb were confirmed by ELISA (enzyme linked immunosorbent assay), IFA (immunofluorescent assay), and Western blot analysis. All results indicate that MAb 5A1 was specific to SVCV M protein.

Preparation and Characterization of a Monoclonal Antibody Against the Core Protein VP7 of the 25th Serotype of Bluetongue Virus.

Bluetongue virus (BTV) is a member of the genus Orbivirus, within the family Reoviridae. The VP7 protein of BTV is used for developing group-specific serological assays. To prepare monoclonal antibody (MAb) against VP7 of the 25th serotype BTV, the RNA S7 encoding VP7 was cloned into prokaryotic expression vectors pET-28a (+) and pGEX-6P-1 to generate recombinant plasmids. The recombinant protein VP7 was expressed in Escherichia coli BL21 (DE3), respectively. The results of SDS-PAGE revealed that the VP7 was expressed and the molecular mass of recombinant fusion protein pET-28a (+)/VP7 and pGEX-6P-1/VP7 was approximately 44 kDa and 64 kDa, respectively. The Western blot analysis indicated that the recombinant VP7 possessed good immunoreactivity. After purification, pET-28a (+)/VP7 was used to immunize BALB/c mice, while pGEX-6P-1/VP7 was used to screen for well-to-well MAb-secreting hybridomas. The hybridoma cell line 3H7 against recombinant VP7 that secreted MAbs was obtained. The isotype of 3H7 was identified as IgG1. The purification of recombinant VP7 protein and the monoclonal antibody will have potential applications on competitive ELISA format for BT-specific serum detection method.

Generation and Characterization of a New Monoclonal Antibody Against CXCL4.

CXCL4 plays important roles in numerous disease processes, which makes the CXCL4 signaling pathway a potential therapeutic target. In this study, we aimed to develop a neutralizing antibody against both human and mouse CXCL4. Rats were immunized with recombinant human CXCL4 (rhCXCL4). Hybridoma clones were created by fusion of the immunized rat spleen cells with mouse myeloma SP2/0 cells and screened using recombinant mouse CXCL4 (rmCXCL4) and rhCXCL4. The CXCL4 monoclonal antibody (CXCL4 MAb) produced by the 16D6-3 hybridoma clone was sequenced and characterized by Western blot and Biacore assays. It recognized both human and mouse CXCL4 with high affinity and neutralized the effect of rhCXCL4 in vitro. Thus, the antibody may be used in the studies of CXCL4 in murine disease models and as a template in the antibody humanization for clinical developments.