Identification and primary application of hybridomas cell secreting monoclonal antibodies against mink (Neovison vison) interferon-gamma.

Due to their susceptibility to several human viruses, the mink has been proposed as potential animal models for the study of human viral infections. However, there are no specific monoclonal antibody (mAbs) currently available for the detection of mink-specific interferon-gamma (miIFN-γ). The BALB/c mice were immunized intraperitoneally with purified recombinant miIFN-γ protein. The splenocytes were obtained and fused with murine myeloma cells. Five of 24 hybridoma clones were obtained to produce mAbs steadily with the strongest affinity to recombinant miIFN-γ protein. The isotype of the 31A, 31B and 31G were lgG 2b. The isotype of 44 and 46 were lgG 2a and 1. All five mAbs were κ light chains. Western blotting and indirect ELISA method showed that 5 mAbs were positive to miIFN-γ. Immunofluorescence showed that 2 mAbs (44 and 46) had a positive reaction to miIFN-γ. The hybridoma clone 46 had the highest sensitivity for the detection of miIFN-γ. Most importantly, our primary sandwich ELISA system (mAbs 46 and polyclonal antiserum) detected endogenous IFN-γ in mink lymphocytes infected with canine distemper virus (CDV). We have thus developed a novel mAbs could recognize miIFN-γ, and have demonstrated the first ELISA-based measurement of IFN-γ in lymphocyte of the mink.

Production of single-chain fragment variable (scFv) antibodies specific to plasma membrane epitopes on bull Y-bearing sperm.

Distinguishing between bull Y- and X-bearing sperm populations is advantageous for techniques using sexed bull semen. The aim of this study was to produce a single-chain fragment variable (scFv) antibody against plasma membrane epitopes on bull Y-bearing sperm. Variable heavy (VH)- and variable light (VL)-region genes generated from a hybridoma cell secreting a specific Y-bearing sperm monoclonal antibody (mAb-1F9) were cloned and expressed. The expected sizes of the DNA bands were ∼350 bp for the VH gene and ∼318 bp for the VL gene. The VH and VL genes were generated and used to construct an scFv gene (∼650 bp), which was expressed in E.coli TG1 cells and produced the corresponding soluble scFv antibody. Compared with the parent mAb-1F9, the scFv antibodies presented a high affinity for Y-bearing sperm and low cross-reactivity with X-bearing sperm. An immunofluorescence analysis confirmed that the scFv antibodies and mAb-1F9 recognize epitopes on the Y-bearing sperm surface. The fluorescence signal was strong on the plasma membrane of Y-bearing sperm but very weak for X-bearing sperm. This study aids the application and production of engineered scFv antibodies specific to Y-bearing sperm to distinguish between Y- and X-bearing sperm populations for techniques involving sexed bull semen.

Detection of Myosin 1g Overexpression in Pediatric Leukemia by Novel Monoclonal Antibodies.

Myosin 1g (Myo1g) is a mechanoenzyme associated with actin filaments, expressed exclusively in hematopoietic cells, and involved in various cellular functions, including cell migration, adhesion, and membrane trafficking. Despite the importance of Myo1g in distinct functions, there is currently no monoclonal antibody (mAb) against Myo1g. mAbs are helpful tools for the detection of specific antigens in tumor cells and other tissues. The development of mAbs against targeted dysregulated molecules in cancer cells remains a crucial tool for aiding in the diagnosis and the treatment of patients. Using hybridoma technology, we generated a panel of hybridomas specific for Myo1g. ELISA, immunofluorescence, and Western blot assay results revealed the recognition of Myo1g by these novel monoclonal antibodies in normal and transformed T and B cells. Here, we report the development and application of new monoclonal antibodies against Myo1g for their potential use to detect its overexpression in acute lymphoblastic leukemia (ALL) patients.

[A monoclonal antibody-based icELISA for puerarin].

Puerarin was conjugated with bovine serum albumin(BSA) and ovalbumin(OVA) by periodate oxidation to serve as the immunogen and coating antigen, respectively. BALB/c mice were immunized with puerarin-BSA according to the routine immunization procedure, and the titer and specificity of serum were detected after three immunization. After booster immunization, mouse spleen lymphocytes were fused with mouse myeloma cells, and 24 hybridoma cell lines of the monoclonal antibodies against puerarin were screened by monoclonal antibody screening technique. Ascites was prepared and purified. The cross-reactivity of monoclonal antibody(mAb) M1 with 4'-methoxy puerarin, daidzin, puerarin-6″-O-xyloside, daidzein, mirificin, 3'-methoxy puerarin, and 3'-hydroxy puerarin was 239.84%, 112.18%, 67.89%, 58.28%, 22.37%, 0.40%, and 0.20%, respectively, and those with other analogs such as baicalein and baicalin were all less than 0.10%. The IC_(50) and the working range of the indirect competitive enzyme-linked immunosorbent assay(icELISA) for puerarin were 44.80 ng·mL~(-1) and 8.20-292.30 ng·mL~(-1), respectively. The average recovery was 91.95%-98.20% with an RSD in the range of 0.70%-2.60%. The content of puerarin in different Puerariae Lobatae Radix samples was determined with icELISA and validated by UPLC-MS. The correlation between data obtained from icELISA and UPLC-MS was 0.999 0, indicating that icELISA is suitable for the rapid detection of puerarin in Puerariae Lobatae Radix samples.

Characterization of glycoprotein IIb/IIIa-specific alloantibodies induced by cross-strain platelet immunization in mice.

BACKGROUND: We previously described a mouse model in which platelet immunization between selected strains leads to production of alloantibodies and severe autoimmune thrombocytopenia and mimics the human condition posttransfusion purpura (PTP). This report describes studies defining epitopes recognized by these alloantibodies. STUDY DESIGN: Hybridomas were produced from spleen cells of immunized mice. Glycoprotein (GP) targets of resulting monoclonal antibodies were characterized by immunoprecipitation using platelets from the immunizing strains. Antigens defined by single amino acid (AA) polymorphisms recognized by monoclonal antibodies were identified by mutagenizing target glycoproteins expressed in Chinese hamster ovary cells and observing the effects on antibody binding. RESULTS: Three monoclonal antibodies (417.1, 417.3, 425.1) were produced that recognized GPIIb on immunizing platelets. Monoclonal antibodies 417.1 and 417.3 both required G111 and 425.1 required V37, located on the beta propeller domain of GPIIb, for binding to platelets from the immunizing strains C57 and PWK, respectively. Injection of 417.3 and 425.1 into mice caused platelet destruction only in mice with GPIIb containing the targeted AAs. CONCLUSIONS: Findings made provide evidence that alloantibodies produced by mice experiencing thrombocytopenia in a mouse model of PTP are specific for single AA polymorphisms that differ in GPIIb/IIIa integrin of the immunizing and immunized strains and therefore closely resemble the potent alloantibodies found in patients with PTP. The observations show that naturally occurring single AA differences in GPIIb/IIIa integrin of various mouse strains are highly immunogenic in the mouse strains studied and readily induce antibodies comparable to human platelet antigen-specific antibodies found in transfused and pregnant humans.

A new reliable and highly specific monoclonal antibody to detect the C-terminal region of silencer of cytokine signaling 1.

INTRODUCTION: SOCS1, a negative regulator of JAK/STAT signaling, is among the most frequently mutated genes in DLBCL and classical Hodgkin lymphoma. The C-terminal SOCS box domain, mediating the degradation of phospho-JAK2, is often affected or even lacking. The analysis of such variants is hampered by the lack of a SOCS1-specific monoclonal antibody recognizing the C-terminus of SOCS1. As this C-terminus is often lost or mutated in B-cell lymphomas, staining with amino-terminal targeting antibodies in a lymphoma setting might be misleading. METHODS: BALB/c mice were immunized with a truncated SOCS1 C-terminal protein. The supernatant of generated hybridoma cells was screened by ELISA and, immunohistochemically, on formalin-fixed and paraffin-embedded tonsil. After antibody purification by affinity chromatography, epitope mapping and cross-reactivity check followed via substitution scans. SOCS1 protein expression was investigated on cell cultures and cytoblocks of SOCS1WT stably transfected HEK293T cells, lymphoma cell lines and lymphoid tissues. RESULTS: Procedures resulted in one monoclonal IgG1 anti-SOCS1 antibody, 424C, that recognizes and strongly binds to the C-terminal region of SOCS1 in immunoblot and immunohistochemistry analyses. CONCLUSION: This new anti-SOCS1 monoclonal antibody is a valuable tool to detect SOCS1 expression dependent on an existing SOCS1 box and, therefore, indicating a full-length SOCS1 protein.

Characterization of Variable Region Genes and Discovery of Key Recognition Sites in the Complementarity Determining Regions of the Anti-Thiacloprid Monoclonal Antibody.

Sequence-defined recombinant antibodies (rAbs) have emerged as alternatives to hybridoma-secreted monoclonal antibodies (mAbs) for performing immunoassays. However, the polyploidy nature of hybridomas often leads to the coexistence of aberrant or non-specific functional variable region (VR) gene transcripts, which complicates the identification of correct VR sequences. Herein, we introduced the use of LC-MS/MS combined with next-generation sequencing to characterize VR sequences in an anti-thiacloprid mAb, which was produced by a hybridoma with genetic antibody diversity. The certainty of VR sequences was verified by the functional analysis based on the recombinant antibody (rAb) expressed by HEK293 mammalian cells. The performance of the rAb was similar to that of the parental mAb, with IC50 values of 0.73 and 0.46 µg/L as measured by ELISAs. Moreover, molecular docking analysis revealed that Ser52 (H-CDR2), Trp98, and Trp93 (L-CDR3) residues in the complementarity determining regions (CDRs) of the identified VR sequences predominantly contributed to thiacloprid-specific recognition through hydrogen bonds and the CH-π interaction. Through single-site-directed alanine mutagenesis, we found that Trp98 and Trp93 (L-CDR3) showed high affinity to thiacloprid, while Ser52 (H-CDR2) had an auxiliary effect on the specific binding. This study presents an efficient and reliable way to determine the key recognition sites of hapten-specific mAbs, facilitating the improvement of antibody properties.

Establishment of Murine Hybridoma Cells Producing Antibodies against Spike Protein of SARS-CoV-2.

In 2020 the world faced the pandemic of COVID-19 severe acute respiratory syndrome caused by a new type of coronavirus named SARS-CoV-2. To stop the spread of the disease, it is crucial to create molecular tools allowing the investigation, diagnoses and treatment of COVID-19. One of such tools are monoclonal antibodies (mAbs). In this study we describe the development of hybridoma cells that can produce mouse mAbs against receptor binding domain of SARS-CoV-2 spike (S) protein. These mAbs are able to specifically detect native and denatured S proteins in all tested applications, including immunoblotting, enzyme-linked immunosorbent assay, immunofluorescence staining of cells and immunohistochemical staining of paraffin embedded patients' tissue samples. In addition, we showed that the obtained mAbs can efficiently block SARS-CoV-2 infection in in vitro experiments. Finally, we determined the amino acid sequence of light and heavy chains of the mAbs. This information will allow the use of corresponding peptides to establish genetically engineered therapeutic antibodies. To date multiple mAbs against SARS-CoV-2 proteins have been established, however, bigger sets of various antibodies will allow the detection and neutralization of SARS-CoV-2, even if the virus acquires novel mutations.

Discovery and bio-optimization of human antibody therapeutics using the XenoMouse® transgenic mouse platform.

Since the late 1990s, the use of transgenic animal platforms has transformed the discovery of fully human therapeutic monoclonal antibodies. The first approved therapy derived from a transgenic platform--the epidermal growth factor receptor antagonist panitumumab to treat advanced colorectal cancer--was developed using XenoMouse(®) technology. Since its approval in 2006, the science of discovering and developing therapeutic monoclonal antibodies derived from the XenoMouse(®) platform has advanced considerably. The emerging array of antibody therapeutics developed using transgenic technologies is expected to include antibodies and antibody fragments with novel mechanisms of action and extreme potencies. In addition to these impressive functional properties, these antibodies will be designed to have superior biophysical properties that enable highly efficient large-scale manufacturing methods. Achieving these new heights in antibody drug discovery will ultimately bring better medicines to patients. Here, we review best practices for the discovery and bio-optimization of monoclonal antibodies that fit functional design goals and meet high manufacturing standards.

Development and applications of a monoclonal antibody against caprine interferon-gamma.

BACKGROUND: Interferon-gamma (IFN-γ) is an important mediator of type I immune response and has antiviral, immunoregulatory and anti-tumor properties, plays a wide range of roles in inflammation and autoimmune diseases. The aim of this study was to obtain monoclonal antibody (mAb) against caprine IFN-γ by immunizing of BALB/c mice with the purified rIFN-γ. RESULTS: Recombinant caprine IFN-γ was expressed in Escherichia coli strain BL21 (DE3) and monoclonal antibodies against caprine IFN-γ were produced by immunizing of BALB/c mice with rIFN-γ. One hybridoma secreting mAb was screened by enzyme-linked immunosorbent assay (ELISA) which was designated as 2C. MAb secreted by this cell line were analyzed through ELISA, western blot and application of the mAb was evaluated by immunofluorescence analysis using goat lip tissues infected with Orf virus. ELISA analysis revealed that mAb 2C can specifically recognize rIFN-γ protein and culture supernatant of goat peripheral blood mononuclear cells (PBMCs) stimulated by concanavalin A (Con A) but cannot recognize the fusion tag protein of pET-32a. Western blot analysis showed that mAb 2C can specifically react with the purified 34.9 kDa rIFN-γ protein but does not react with the fusion tag protein of pET-32a. Immunofluorescence results demonstrated that mAb 2C can detect IFN-γ secreted in histopathological sites of goats infected with Orf virus. CONCLUSIONS: A caprine IFN-γ-specific mAb was successfully developed in this study. Further analyses showed that the mAb can be used to detect IFN-γ expression level during contagious ecthyma in goats.

Identification of specific B cell linear epitopes of mycoplasma hyorhinis P37 protein using monoclonal antibodies against baculovirus-expressed P37 protein.

BACKGROUND: Mycoplasma hyorhinis (Mhr) is the etiologic agent of lameness and polyserositis in swine. P37 is a membrane protein of Mhr that may be an important immunogen and is a potential target for diagnostic development. However, there is little information concerning Mhr P37 protein epitopes. A precise analysis of the P37 protein epitopes should extend our understanding of the antigenic composition of the P37 protein and the humoral immune responses to Mhr infection. Investigating the epitopes of Mhr P37 will help to establish a detection method for Mhr in tissue and provide an effective tool for detecting Mhr infection. RESULTS: Western blot and indirect immunofluorescence assays (IFA) confirmed that the expressed P37 protein was recognized by Mhr-positive porcine and mouse sera. Furthermore, the P37 protein was purified using affinity chromatography and used to immunize mice for hybridoma cell fusion. Four monoclonal antibodies (mAbs) found to be positive for Mhr were detected in infected lung tissue. A panel of truncated P37 proteins was used to identify the minimal B cell linear epitopes of the protein based on these mAbs. The core epitope was determined to be 206KIKKAWNDKDWNTFRNF222. CONCLUSIONS: In this study, we identified 17 critical amino acids that determine the epitope of the P37 protein of Mhr. This study identified mAbs that could provide useful tools for investigating the Mhr P37 antigenic core epitope (amino acids 206-222) and detecting Mhr-specific antigens in infected tissue.

Multivariate analysis of metabolic parameters and optimization of antibody production using high cell density hybridoma in hollow fiber bioreactors.

OBJECTIVES: The relationships of manipulation of culture temperature and medium circulation rate on the metabolic parameters were regressed by multiple linear regression analysis in hollow fiber bioreactors (HFB). RESULTS: The high circulation rate could significantly enhance the oxygen consumption of the hybridoma cells and the medium's oxidation-reduction potential. A mildly hypothermic condition of 36 °C and a circulation rate of 182.5 mL/min could support the hybridoma had the maximal antibody titer of 60.75 µg/mL for 20 days. When the ammonium ion was 65 ppm or lactate close to 2.6 g/L, the medium was replaced to maintain the stable and healthy cells at the high cell concentration of 3.33 × 108/mL for continuous antibody production. Two serum-free media could be successfully applied to this perfusion system and maintain hybridoma growth and antibody production. CONCLUSION: The single-use HFBs could provide the advantages including high cell density, low shear stress, and continuous antibody production.

Human single-chain variable fragment antibody expressed in E. coli with optimal in vitro cross-neutralizing and no enhancing activity.

Single chain fragment variable (scFv) is a small molecule antibody comprising of only the variable region of heavy and light chain responsible for antigen binding. For dengue disease, the Fc region of antibody molecule was reported to be involved with dengue complication caused by Antibody-dependent enhancement (ADE). We attempted to produce small molecule scFv human monoclonal antibody (HuMAb), which lacking the Fc portion to eliminate the ADE effect of the IgG. This scFv antibody was produced in Escherichia coli. The biologically active form of scFv antibody was successfully generated. 23-1C2D2-scFv showed neutralizing activity similar to the IgG obtained from parental hybridoma, but lacked enhancing activity in all studied concentrations. This antibody was targeted to the 101WXN103 motif of dengue envelop protein domain II, studied by western blot analysis with truncated E protein and random peptide phage display. This scFv is verified as a candidate for further development as therapeutic candidate for DENV infection.

Targeting Anti-TGF-ß Therapy to Fibrotic Kidneys with a Dual Specificity Antibody Approach.

Targeted delivery of a therapeutic agent to a site of pathology to ameliorate disease while limiting exposure at undesired tissues is an aspirational treatment scenario. Targeting diseased kidneys for pharmacologic treatment has had limited success. We designed an approach to target an extracellular matrix protein, the fibronectin extra domain A isoform (FnEDA), which is relatively restricted in distribution to sites of tissue injury. In a mouse unilateral ureteral obstruction (UUO) model of renal fibrosis, injury induced significant upregulation of FnEDA in the obstructed kidney. Using dual variable domain Ig (DVD-Ig) technology, we constructed a molecule with a moiety to target FnEDA and a second moiety to neutralize TGF-ß After systemic injection of the bispecific TGF-ß + FnEDA DVD-Ig or an FnEDA mAb, chemiluminescent detection and imaging with whole-body single-photon emission computed tomography (SPECT) revealed significantly higher levels of each molecule in the obstructed kidney than in the nonobstructed kidney, the ipsilateral kidney of sham animals, and other tissues. In comparison, a systemically administered TGF-ß mAb accumulated at lower concentrations in the obstructed kidney and exhibited a more diffuse whole-body distribution. Systemic administration of the bispecific DVD-Ig or the TGF-ß mAb (1-10 mg/kg) but not the FnEDA mAb attenuated the injury-induced collagen deposition detected by immunohistochemistry and elevation in Col1a1, FnEDA, and TIMP1 mRNA expression in the obstructed kidney. Overall, systemic delivery of a bispecific molecule targeting an extracellular matrix protein and delivering a TGF-ß mAb resulted in a relatively focal uptake in the fibrotic kidney and reduced renal fibrosis.

Dynamic optimization of fed-batch bioprocesses using flower pollination algorithm.

There exist several optimization strategies such as sequential quadratic programming (SQP), iterative dynamic programing (IDP), stochastic-based methods such as differential evolution (DE), genetic algorithm (GA), particle swarm optimization (PSA), and ant colony optimization (ACO) for finding optimal feeding profile(s) during fed-batch fermentations. Here in the present study, flower pollination algorithm (FPA) which is inspired by the pollination process in terrestrial flowering plants has been used for the first time to find the optimal feeding profile(s) during fed-batch fermentations. Single control variable, two control variables and state variable bounded problems were chosen to test the robustness of the FPA for optimal control problems. It was observed that FPA is computationally less intensive in comparison with other stochastic strategies. Thus, obtained results were compared to other studies and it has been found that the FPA converged either to newer optima or closer to the established global optimum for the cases studied.

Development and Characterization of Monoclonal Antibodies to the 32 kDa Viral Attachment Protein of Lymphocystis Disease Virus and Their Neutralizing Ability in Vitro.

In previous research, a 32 kDa protein in lymphocystis disease virus (LCDV) was identified as viral attachment protein (VAP) that specifically interacted with the 27.8 kDa cellular receptor from flounder Paralichthys olivaceus gill (FG) cells, and the recombinant VAP (rVAP) was expressed in Escherichia coli strain BL21 (DE3). In this study, monoclonal antibodies (MAbs) against 32 kDa VAP are produced by immunization of BALB/c mice with the rVAP. Seven hybridoma secreting MAbs were screened by enzyme-linked immunosorbent assay, five of which designated as 1C6, 1C8, 3B5, 3D11 and 3H10 are cloned by the limiting dilution method, depending on the strongly positive results of ELISA. Western blotting analysis shows that the five MAbs can specifically react with the 32 kDa protein of LCDV and the purified 50 kDa rVAP, and the subtype of the MAbs is identified as IgG. Immunofluorescence results demonstrate that the specific fluorescence signals for LCDV appear in the cytoplasm of FG cells at 24 h post LCDV infection. Neutralization assay results indicate that pre-incubations of LCDV with the five MAbs can significantly decrease the LCDV copy numbers and delay the development of the cytopathic effect in FG cells, revealing that the five MAbs can neutralize the LCDV particles and block viral infection in vitro. The neutralizing MAbs against 32 kDa VAP would be useful for the study on the LCDV⁻host interaction and might be promising inhibitors of LCDV infection in fish.

Metabolically inactive insulin analogue does not prevent autoimmune diabetes in NOD mice.

AIMS/HYPOTHESIS: Insulin is widely considered to be a driver antigen in type 1 diabetes in humans and in mouse models of the disease. Therefore, insulin or insulin analogues are candidates for tolerogenic drugs to prevent disease onset in individuals with risk of diabetes. Previous experiments have shown that autoimmune diabetes can be prevented in NOD mice by repeated doses of insulin administered via an oral, nasal or parenteral route, but clinical trials in humans have not succeeded. The hypoglycaemic activity of insulin is dose-limiting in clinical studies attempting tolerance and disease prevention. Here, we aimed to investigate the therapeutic potential of metabolically inactive insulin analogue (MII) in NOD mice. METHODS: The tolerogenic potential of MII to prevent autoimmune diabetes was studied by administering multiple i.v. or s.c. injections of MII to non-diabetic 7-12-week-old female NOD mice in three geographical colony locations. The incidence of diabetes was assessed from daily or weekly blood glucose measurements. The effect of MII on insulin autoantibody levels was studied using an electrochemiluminescence-based insulin autoantibody assay. The effect on the number of insulin-reactive CD8+ and CD4+ T lymphocytes in peripheral lymphoid tissue was studied with MHC class I and MHC class II tetramers, respectively. RESULTS: We found that twice-weekly s.c. administration of MII accelerates rather than prevents diabetes. High-dose i.v. treatment did not prevent disease or affect insulin autoantibody levels, but it increased the amount of insulin-reactive CD4+ T lymphocytes in peripheral lymphoid tissue. CONCLUSIONS/INTERPRETATION: Our data suggest that parenteral MII, even when used in high doses, has little or no therapeutic potential in NOD mice and may exacerbate disease.

A novel monoclonal antibody suitable for the detection of leukotriene B4.

Leukotriene B4 as an inflammatory mediator is an important biomarker for different respiratory diseases like asthma, chronic obstructive pulmonary disease or cystic lung fibrosis. Therefore the detection of LTB4 is helpful in the diagnosis of these pulmonary diseases. However, until now its determination in exhaled breath condensates suffers from problems of accuracy. Reasons for that could be improper sample collection and preparation methods of condensates and the lack of consistently assay specificity and reproducibility of the used immunoassay detection system. In this study we describe the development and the characterization of a specific monoclonal antibody (S27BC6) against LTB4, its use as molecular recognition element for the development of an enzyme-linked immunoassay to detect LTB4 and discuss possible future diagnostic applications.

Immunological characterization of a rigid α-Tn mimetic on murine iNKT and human NK cells.

The ability of a rigid α-Tn mimetic (compound 1) to activate murine invariant natural killer T (iNKT) and human natural killer (NK) cells, two subsets of lymphocytes involved in cancer immunesurveillance, was investigated. For this purpose, the mimetic 1 was properly conjugated to a stearic acid containing glycerol-based phospholipid (compound 5) to be presented, in the context of the conserved non polymorphic major histocompatibility complex class I-like molecules (CD1d), to iNKT cells. On the contrary, the mimetic 1 was conjugated to a multivalent peptide-based scaffold (compound 6) to induce NK cell activation.