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The antigen-mediated B cell isolation method, based on the detection of surface IgG (sIgG), has increased the efficiency of therapeutic antibody (Ab) discovery. However, the reduction in sIgG expression on B cells during plasma cell differentiation presents challenges as it enables Ab production from only a small subset of B cells (e.g., memory B cells). The present study aimed to addressed this problem by developing a workflow to isolate human-IgG-secreting hybridoma cells produced by cell fusion, the majority of which express sIgG. We showed that our sIgG-based antigen-coated bead separation method efficiently enriched hybridoma cells expressing antigen-specific Abs with a yield of 83.5% (from the cell fusion pool) and a positive rate of 73.2%. Furthermore, because the separation could be performed after only a short (1-2-day) culture period following cell fusion, diverse hybridoma clones could be obtained, minimizing clonal selection and the incidence of duplicates. Given that the expression of membrane-bound IgG and sIgG are regulated by different splicing mechanisms, we speculate that the cell fusion step potentially attenuated the suppression of human sIgG expression. Overall, our proposed method is expected to markedly improve the efficiency of therapeutic Ab candidate production, which will have important clinical implications., (© 2024. The Author(s).)

In order to generate monoclonal antibodies against the akabane virus (AKAV) N protein, this study employed a prokaryotic expression system to express the AKAV N protein. Following purification, BALB/c mice were immunized, and their splenocytes were fused with mouse myeloma cells (SP2/0) to produce hybridoma cells. The indirect ELISA method was used to screen for positive hybridoma cells. Two specific hybridoma cell lines targeting AKAV N protein, designated as 2C9 and 5E9, were isolated after three rounds of subcloning. Further characterization was conducted through ELISA, Western blotting, and indirect immunofluorescence assay (IFA). The results confirmed that the monoclonal antibodies specifically target AKAV N protein, exhibiting strong reactivity in IFA. Subtype analysis identified the heavy chain of the 2C9 mAb's as IgG2b and its light chain as κ-type; the 5E9 mAb's heavy chain was determined to be IgG1, with a κ-type light chain. Their ELISA titers reached 1:4 096 000. This study successfully developed two monoclonal antibodies targeting AKAV N protein, which lays a crucial foundation for advancing diagnostic methods for akabane disease prevention and control, as well as for studying the function of the AKAV N protein.

Droplet-based high-throughput screening systems are an emerging technology that provides a quick test to screen millions of cells with distinctive characteristics. Biopharmaceuticals, specifically therapeutic proteins, are produced by culturing cells that secrete heterologous recombinant proteins with different populations and expression levels; therefore, a technology to discriminate cells that produce more target proteins is needed. Here, we present a droplet-based microfluidic strategy for encapsulating, screening, and selecting target cells with redox-responsive hydrogel beads (HBs). As a proof-of-concept study, we demonstrate the enrichment of hybridoma cells with enhanced capability of antibody secretion using horseradish peroxidase (HRP)-catalyzed hydrogelation of tetra-thiolate poly(ethylene glycol); hybridoma cells were encapsulated in disulfide-bonded HBs. Recombinant protein G or protein M with a C-terminal cysteine residue was installed in the HBs via disulfide bonding to capture antibodies secreted from the cells. HBs were fluorescently stained by adding the protein L-HRP conjugate using a tyramide signal amplification system. HBs were then separated by fluorescence-activated droplet sorting and degraded by reducing the disulfide bonds to recover the target cells. Finally, we succeeded in the selection of hybridoma cells with enhanced antibody secretion, indicating the potential of this system in the therapeutic protein production.

The rise of biological therapeutics in the global pharmaceuticals market has escalated the demand for quality monoclonal antibodies for healthcare and scientific applications. Reducing costs while enhancing production yields without compromising quality are the main challenges to the growth of this industry today. Over the last two decades non-ionizing radiation has been demonstrated to elicit targeted biological responses in a frequency and dose dependent manner. We hypothesize and design a millimeter wave radiation procedure to enhance the yields of antibody-producing hybridoma cell lines. We demonstrate this method enhances the production of IgA and IgG antibodies from MOPC315.BM and U13.6 cells by a factor of 24.05 ± 3.32 and 1.41 ± 0.03 respectively relative to untreated cells. No treatment associated cytotoxicity was observed in either cell line corroborating physiological viability of irradiated cells. Our results demonstrate proof-of-concept of a novel technique to significantly enhance antibody yields from hybridoma cells which could lead to a reduction in antibody production costs. Further studies will focus on scaling up of this technology and employment of non-contact, tuned electromagnetic stimulation of biological systems for targeted responses., (© 2023 Wiley-VCH GmbH.)

Monoclonal antibodies (mAbs) have been used in the development of immunotherapies that target a variety of diseases, such as cancer, autoimmune diseases, and even viral infections; they play a key role in immunization and are expected after vaccination. However, some conditions do not promote the development of neutralizing antibodies. Production and use of mAbs, generated in biofactories, represent vast potential as aids in immunological responses when the organism cannot produce them on their own, these convey unique specificity by recognizing and targeting specific antigen. Antibodies can be defined as heterotetrametric glycoproteins of symmetric nature, and they participate as effector proteins in humoral responses. Additionally, there are different types of mAbs (murine, chimeric, humanized, human, mAbs as Antibody-drug conjugates and bispecific mAbs) discussed in the present work. When these molecules are produced in vitro as mAbs, several common techniques, such as hybridomas or phage display are used. There are several preferred cell lines that function as biofactories, for the production of mAbs, the selection of which rely on the variation of adaptability, productivity and both phenotypic and genotypic shifts. After the cell expression systems and culture techniques are used, there are diverse specialized downstream processes to achieve desired yield and isolation as well as product quality and characterization. Novel perspectives regarding these protocols represent a potential improvement for mAbs high-scale production., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The rise of antimicrobial-resistant bacterial infections is a crucial health concern in the 21st century. In particular, antibiotic-resistant Pseudomonas aeruginosa causes difficult-to-treat infections associated with high morbidity and mortality. Unfortunately, the number of effective therapeutic interventions against antimicrobial-resistant P. aeruginosa infections continues to decline. Therefore, discovery and development of alternative treatments are necessary. Here, we present pre-clinical efficacy studies on an anti- P. aeruginosa therapeutic monoclonal antibody. Using hybridoma technology, we generated a monoclonal antibody and characterized its binding to P. aeruginosa in vitro using ELISA and fluorescence correlation spectroscopy. We also characterized its function in vitro and in vivo against P. aeruginosa . The anti- P. aeruginosa antibody (WVDC-5244) bound P. aeruginosa clinical strains of various serotypes in vitro , even in the presence of alginate exopolysaccharide. In addition, WVDC-5244 induced opsonophagocytic killing of P. aeruginosa in vitro in J774.1 murine macrophage, and complement-mediated killing. In a mouse model of acute pneumonia, prophylactic administration of WVDC-5244 resulted in an improvement of clinical disease manifestations and reduction of P. aeruginosa burden in the respiratory tract compared to the control groups. This study provides promising pre-clinical efficacy data on a new monoclonal antibody with therapeutic potential for P. aeruginosa infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Horspool, Sen-Kilic, Malkowski, Breslow, Mateu-Borras, Hudson, Nunley, Elliott, Ray, Snyder, Miller, Kang, Blackwood, Weaver, Witt, Huckaby, Pyles, Clark, Al Qatarneh, Lewis, Damron and Barbier.)

The coronavirus disease 2019 pandemic has caused more than 532 million infections and 6.3 million deaths to date. The reactive and neutralizing fully human antibodies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are effective detection tools and therapeutic measures. During SARS-CoV-2 infection, a large number of SARS-CoV-2 reactive and neutralizing antibodies will be produced. Most SARS-CoV-2 reactive and neutralizing fully human antibodies are isolated from human and frequently encoded by convergent heavy-chain variable genes. However, SARS-CoV-2 viruses can mutate rapidly during replication and the resistant variants of neutralizing antibodies easily survive and evade the immune response, especially in the face of such focused antibody responses in humans. Therefore, additional tools are needed to develop different kinds of fully human antibodies to compensate for current deficiency. In this study, we utilized antibody humanized CAMouse HG mice to develop a rapid antibody discovery method and examine the antibody repertoire of SARS-CoV-2 RBD-reactive hybridoma cells derived from CAMouse HG mice by using high-throughput single-cell V(D)J sequencing analysis. CAMouse HG mice were immunized by 28-day rapid immunization method. After electrofusion and semi-solid medium screening on day 12 post-electrofusion, 171 hybridoma clones were generated based on the results of SARS-CoV-2 RBD binding activity assay. A rather obvious preferential usage of IGHV6-1 family was found in these hybridoma clones derived from CAMouse HG mice, which was significantly different from the antibodies found in patients with COVID-19. After further virus neutralization screening and antibody competition assays, we generated a noncompeting two-antibody cocktail, which showed a potent prophylactic protective efficacy against SARS-CoV-2 in cynomolgus macaques. These results indicate that humanized CAMouse HG mice not only provide a valuable platform to obtain fully human reactive and neutralizing antibodies but also have a different antibody repertoire from humans. Thus, humanized CAMouse HG mice can be used as a good complementary tool in discovery of fully human therapeutic and diagnostic antibodies., Competing Interests: Author MW is part-time employed by Chongqing Jinmaibo Bio. MW, LG and XL are shareholders of Chongqing Jinmaibo Bio. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yang, Chi, Wu, Wang, Lang, Han, Wang, Liu, Li, Wang, Huang, Bi, Liang, Gao, Zhao, Feng, Yang, Wang, Xia and Ge.)

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.

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.

Zearalenone (ZEN) contamination in food and feed is prevalent and has severe effects on humans and animals post-consumption. Therefore, a sensitive, specific, rapid, and reliable method for detecting a single residue of ZEN is necessary. This study aimed to establish a highly sensitive and specific ZEN monoclonal antibody (mAb) and an indirect competitive enzyme-linked immunosorbent assay (icELISA) for the detection of ZEN residues in food and feed. The immunogen ZEN-BSA was synthesized via the amino glutaraldehyde (AGA) and amino diazotization (AD) methods and identified using 1H nuclear magnetic resonance (1H NMR), a high-resolution mass spectrometer (HRMS), and an ultraviolet spectrometer (UV). The coating antigens ZEN-OVA were synthesized via the oxime active ester (OAE), formaldehyde (FA), 1,4-butanediol diglycidyl ether (BDE), AGA, and AD methods. These methods were used to screen the best antibody/antigen combination of a heterologous icELISA. Balb/c mice were immunized with a low ZEN-BSA dose at long intervals and multiple sites. Suitable cell fusion mice and positive hybridoma cell lines were screened using a homologous indirect non-competitive ELISA (inELISA) and an icELISA. The ZEN mAbs were prepared by inducing ascites in vivo. The immunological characteristics of ZEN mAbs were then assessed. The standard curves of the icELISA for ZEN were constructed under optimal experimental conditions, and the performance of the icELISA was validated. The two ZEN-BSA immunogens (conjugation ratios, 11.6:1 (AGA) and 9.2:1 (AD)) were successfully synthesized. Four hybridoma cell lines (2B6, 4D9, 1A10, and 4G8) were filtered, of which 2B6 had the best sensitivity and specificity. The mAb 2B6-based icELISA was then developed. The limit of detection (LOD), the 50% inhibitive concentration (IC50), and the linear working range (IC20 to IC80) values of the icELISA were 0.76 μg/L, 8.69 μg/L, and 0.92-82.24 μg/L, respectively. The cross-reactivity (CR) of the icELISA with the other five analogs of ZEN was below 5%. Three samples were spiked with different concentrations of ZEN and detected using the icELISA. The average intra-assay recoveries, inter-assay recoveries, intra-assay coefficients of variations (CVs), and inter-assay CVs were 93.48-99.48%, 94.18-96.13%, 12.55-12.98%, and 12.53-13.58%, respectively. The icELISA was used to detect ZEN in various samples. The results were confirmed using high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) (correlation coefficient, 0.984). The proposed icELISA was highly sensitive, specific, rapid, and reliable for the detection of ZEN in food and feed samples.

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., (Copyright © 2021. Published by Elsevier Ltd.)

The CRISPR/Cas9 system, a defense mechanism naturally occurring in prokaryotes, has been recently repurposed as an RNA-guided DNA targeting platform and widely used as a powerful tool for genome editing. Here we describe how to modify the carboxy-terminal region, called Fragment crystallizable (Fc) region, of a murine monoclonal antibody by replacing the heavy chain constant exons with those from a teleost fish antibody by the CRISPR/Cas9 system. We outline optimal conditions for knockout and knockin mechanisms to edit the Immunoglobulin heavy chain (IgH) constant region gene locus in a murine hybridoma cell line. A chimeric mouse-fish monoclonal antibody can be successfully produced by hybridoma cell lines engineered according to this protocol., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

Despite their common use in research, monoclonal antibodies are currently not systematically sequenced. This can lead to issues with reproducibility and the occasional loss of antibodies with loss of cell lines. Hybridoma cell lines have been the primary means of generating monoclonal antibodies from immunized animals, including mice, rats, rabbits, and alpacas. Excluding therapeutic antibodies, few hybridoma-derived antibody sequences are known. Sanger sequencing has been "the gold standard" for antibody gene sequencing, but this method relies on the availability of species-specific degenerate primer sets for amplification of light and heavy antibody genes and it requires lengthy and expensive cDNA preparation. Here, we leveraged recent improvements in long-read Oxford Nanopore Technologies (ONT) sequencing to develop Nanopore Antibody sequencing (NAb-seq): a three-day, species-independent, and cost-effective workflow to characterize paired full-length immunoglobulin light- and heavy-chain genes from hybridoma cell lines. When compared to Sanger sequencing of two hybridoma cell lines, long-read ONT sequencing was highly accurate, reliable, and amenable to high throughput. We further show that the method is applicable to single cells, allowing efficient antibody discovery in rare populations such as memory B cells. In summary, NAb-seq promises to accelerate identification and validation of hybridoma antibodies as well as antibodies from single B cells used in research, diagnostics, and therapeutics.

Fast-dissociating, highly specific monoclonal antibodies (FDSAs) are single-molecule imaging probes useful for many biological assays including consecutive, multiplexable super-resolution microscopy. We developed a screening assay to characterize the kinetics of antibody-antigen interactions using single-molecule microscopy and established a pipeline to identify FDSAs from thousands of monoclonal candidates. Provided here are detailed protocols to prepare multi-well glass-bottom plates necessary for our assay to identify hybridoma clones secreting FDSAs. Synthesis of fluorescently labeled Fab fragments (Fab probes) from FDSAs is also described. For complete details on the use and execution of this protocol, please refer to Miyoshi et al. (2021)., Competing Interests: The authors declare no competing interests.

For drug-selective media to work for hybridoma selection, myeloma cells expressing a mutation abrogating the function of their HGPRT gene (and subsequently unable to produce purines for DNA biosynthesis) are used. HGPRT will recognize 8-AG as a substrate and convert it to the monophosphate nucleotide. The 8-AG-containing nucleotide is then processed further and incorporated into DNA and RNA, where it is toxic. Therefore, cells with a functional HGPRT enzyme grown in the presence of 8-AG will die. Cells that are deficient in HGPRTase cannot incorporate 8-AG in vivo and thus continue to grow. Cells that have been selected for resistance to 8-AG should be checked periodically to ensure that they maintain sensitivity to drugs that block the de novo synthesis of DNA. In addition, all myeloma cell lines should be checked periodically for reversion of their drug selection markers. Any line that is not killed completely by drug selection should either be reselected or replaced with a new line., (© 2021 Cold Spring Harbor Laboratory Press.)

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 SOCS1 WT 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., (© 2021 The Authors. European Journal of Haematology published by John Wiley & Sons Ltd.)

Monoclonal antibodies are extremely valuable functional biomaterials that are widely used not only in life science research but also in antibody drugs and test drugs. There is also a strong need to develop high-quality neutralizing antibodies as soon as possible in order to stop the rapid spread of new infectious diseases such as the SARS-CoV-2 virus. This study has developed a membrane-type immunoglobulin-directed hybridoma screening (MIHS) method for obtaining high-quality monoclonal antibodies with high efficiency and high speed. In addition to these advantages, this paper demonstrates that the MIHS method can selectively obtain monoclonal antibodies that specifically recognize the functional structure of proteins. The MIHS method is a useful technology that greatly contributes to the research community because it can be easily introduced in any laboratory that uses a flow cytometer., (Copyright © 2021 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

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., (© 2021 AABB.)

Fast-dissociating, specific antibodies are single-molecule imaging probes that transiently interact with their targets and are used in biological applications including image reconstruction by integrating exchangeable single-molecule localization (IRIS), a multiplexable super-resolution microscopy technique. Here, we introduce a semi-automated screen based on single-molecule total internal reflection fluorescence (TIRF) microscopy of antibody-antigen binding, which allows for identification of fast-dissociating monoclonal antibodies directly from thousands of hybridoma cultures. We develop monoclonal antibodies against three epitope tags (FLAG-tag, S-tag, and V5-tag) and two F-actin crosslinking proteins (plastin and espin). Specific antibodies show fast dissociation with half-lives ranging from 0.98 to 2.2 s. Unexpectedly, fast-dissociating yet specific antibodies are not so rare. A combination of fluorescently labeled Fab probes synthesized from these antibodies and light-sheet microscopy, such as dual-view inverted selective plane illumination microscopy (diSPIM), reveal rapid turnover of espin within long-lived F-actin cores of inner-ear sensory hair cell stereocilia, demonstrating that fast-dissociating specific antibodies can identify novel biological phenomena., Competing Interests: Declaration of interests The authors declare no conflict of interest associated with this manuscript., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Cisplatin is a platinum-based chemotherapy compound effective against a variety of cancers. However, it can cause increased reactive oxygen species (ROS) production in auditory and vestibular tissue leading to permanent hearing and balance loss. The amino acid, L-serine, has been shown to reduce ROS in some tissue types. In this project, we first investigated whether L-serine could reduce cisplatin-mediated ROS generation in zebrafish utricular tissue culture using spectrophotometry and the fluorescent ROS detector dye, H 2 DCFDA. Then, we examined whether L-serine could prevent the effect of cisplatin against cellular viability in the mouse auditory hybridoma cell line, HEI-OC1, using the spectrophotometric (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. As a final step, we used H 2 DCFDA dye and flow cytometry analysis to determine if L-serine could counteract the effect of cisplatin on ROS production in this cell line. We found that cisplatin and L-serine treatment may influence ROS production in utricular tissue. Further, although L-serine did not counteract the effect of cisplatin against HEI-OC1 cellular viability, the amino acid did prevent the platinum compound's effect to increase ROS in these cells. These results suggest that L-serine may act in auditory and vestibular tissues as an effective protectant against cisplatin-mediated toxicity.

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.

For most immunochemical methods, tissue culture supernatants will be the most useful source of monoclonal antibodies. The supernatants are not contaminated with high levels of other antibodies, and the concentration is high enough for most assays if used undiluted. This protocol describes the procedure of collecting tissue culture supernatants. When collecting supernatants for antibodies, allow the individual cultures to grow until the hybridomas die. This will allow collection of higher-titer supernatants. In general, antibodies are resistant to the proteases that are released from dying cells, so allowing the cells to die should not affect the quality of the antibodies. If extraneous IgG molecules will alter any of the assays for which the supernatants are being prepared, use medium with fetal bovine serum or use serum-free medium. The yield of this method is ∼20-50 µg of antibody/mL of supernatant. The most common problem encountered in storage of tissue culture supernatants after collection is contamination with bacteria or fungi. This can be prevented by the addition of sodium azide as described., (© 2020 Cold Spring Harbor Laboratory Press.)

Hybridoma and myeloma cell lines can be stored by slowly freezing cells in an appropriate solution of nutrients and a cryoprotectant such as glycerol or dimethyl sulfoxide (DMSO). In this protocol, cells are centrifuged at 4°C, resuspended in cold freezing solution (10% DMSO in FBS), and then transferred to an appropriate freezing vial. The vials are slowly frozen to -70°C in Styrofoam racks and then stored in liquid nitrogen (LN 2 ). Cells stored in LN 2 will remain viable for years. Once a frozen vial has been removed from LN 2 storage, it should be thawed as described, grown out into log phase, and refrozen., (© 2020 Cold Spring Harbor Laboratory Press.)

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 IC 50 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.

Background: The lack of facile methods for the specific characterization of malignant thyroid nodules makes the diagnosis of thyroid cancer (TC) challenging. Due to its restricted expression in such nodules, the cell-associated lectin galectin-3 (Gal3) has emerged as a marker for TC with growing interest for in vivo imaging as well as targeted radionuclide therapy. To accelerate translation into clinical application, we have developed a cognate chimeric human antigen-binding fragment (Fab) derived from the rat anti-Gal3 monoclonal antibody M3/38. Methods: The variable immunoglobulin (Ig) light and heavy chain sequences were cloned from the hybridoma cell line, and the corresponding Fab carrying human IgG1/κ constant genes was functionally produced in the periplasm of Escherichia coli and purified to homogeneity. To moderately prolong its plasma half-life and, thus, increase tumor uptake, the recombinant Fab was fused with a long disordered amino acid chain comprising in total 200 Pro, Ala, and Ser residues (PASylation). This novel tracer was subjected to in vitro characterization and in vivo validation by using two thyroid cancer orthotopic murine models. To this end, the αGal3-Fab-PAS 200 was conjugated with deferoxamine (Dfo), labeled with 89 Zr under mild conditions and tested for binding on TC cell lines. Athymic nude mice were inoculated either with FRO82-1 or with CAL62 tumor cells into the left thyroid lobe. After intravenous injection with ∼3.0 MBq of 89 Zr-Dfo-PAS 200 -Fab, these mice were subjected to positron emission tomography (PET)/computed tomography imaging followed by quantification of tumor accumulation and immunohistochemical analysis. Results: The αGal3-Fab-PAS 200 revealed high affinity toward the recombinant Gal3 antigen, with a dissociation constant ≤1 nM as measured via enzyme-linked immunosorbent assay, surface plasmon resonance spectroscopy, and radioactive cell binding assay. The in vivo Gal3-targeting by the 89 Zr(IV)-labeled protein tracer, as investigated by immuno-PET, demonstrated highly selective and fast accumulation in orthotopically implanted tumors, with strong contrast images achieved 24 hours postinjection, and no uptake in the tumor-free thyroid lobe, as also confirmed by biodistribution studies. Conclusions: The chimeric αGal3 89 Zr-Dfo-PAS 200 -Fab tracer exhibits selective accumulation in the tumor-bearing thyroid lobe of xenograft mice. Thus, this novel radioactive probe offers potential to change TC management, in addition to current diagnostic procedures, and to reduce unnecessary thyroidectomies.

To investigate the fine epitope(s) of anti-C1q A08 antibodies and their roles in complement activation in lupus nephritis, C1q A08 and related peptides with various amino acid sequences around A08 were synthesized. Anti-C1q A08 antibodies from 10 lupus nephritis patients were purified from plasmapheresis samples, and four monoclonal antibodies against C1q A08 were screened and identified from mouse hybridoma cells, to study the fine epitope(s) of C1q A08 using ELISA and Biolayer Interferometry (BLI). The biofunction of anti-C1q A08 antibodies for complement classical pathway activation was investigated by C3 activation assay. Anti-C1q A08 antibodies and anti-C1q antibodies were also detected in the sera of female BALB/C mice immunized by C1q A08 peptides. None of the anti-C1q A08 antibodies, which were affinity purified from the 10 lupus nephritis patients, could bind intact C1q coated on microtitre plates, neither could the anti-C1q antibodies bind to C1q A08 peptides coupled on resin, indicating that the human anti-C1q antibodies and anti-C1q A08 antibodies may recognize different epitopes of C1q. One of the four C1q A08 mAbs (32-4) bound to the six amino acids of N-terminus of C1q A08, while another C1q A08 mAb (17-9) bound to eight or 10 amino acids of C-terminus of A08. The third and fourth C1q A08 mAb (1A12 and 4B11) bound to the whole sequence of A08. Only 32-4 mAb bound to the intact C1q coating on an ELISA plate, whereas 17-9 mAb, 1A12 mAb, and 4B11 mAb could not. However, using a BLI assay, 17-9 mAb, 1A12 mAb, and 4B11 mAb, but not 32-4 mAb, could bind to intact C1q. Furthermore, 1A12 mAb and 4B11 mAb, but not 32-4 and 17-9 mAb, could inhibit the activation of complement classical pathway. Anti-C1q A08 antibodies were detected in all the female BALB/C mice in the experimental group but not in the control group. Two out of six in the experimental group developed anti-C1q antibodies. C1q A08 is a half-cryptic epitope of C1q involving N-terminal six amino acids of C1q A08, and this is important to the activation of a complement classical pathway, and some anti-C1q A08 antibodies were able to prevent this process. Epitope spreading of C1q occurred in the mice immunized with C1q A08 peptides., (Copyright © 2020 Wu, Tan, Liu, Yu and Zhao.)

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.

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 206 KIKKAWNDKDWNTFRNF 222 . 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.

Antibody engineering in mammalian cells offers the important advantage of expression and screening of libraries in their native conformation, increasing the likelihood of generating candidates with more favorable molecular properties. Major advances in cellular engineering enabled by CRISPR-Cas9 genome editing have made it possible to expand the use of mammalian cells in biotechnological applications. Here, we describe an antibody engineering and screening approach where complete variable light (V L ) and heavy (V H ) chain cassette libraries are stably integrated into the genome of hybridoma cells by enhanced Cas9-driven homology-directed repair (HDR), resulting in their surface display and secretion. By developing an improved HDR donor format that utilizes in situ linearization, we are able to achieve >15-fold improvement of genomic integration, resulting in a screening workflow that only requires a simple plasmid electroporation. This proved suitable for different applications in antibody discovery and engineering. By integrating and screening an immune library obtained from the variable gene repertoire of an immunized mouse, we could isolate a diverse panel of >40 unique antigen-binding variants. Additionally, we successfully performed affinity maturation by directed evolution screening of an antibody library based on random mutagenesis, leading to the isolation of several clones with affinities in the picomolar range.

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 × 10 8 /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.

ND7/23 cells are gaining traction as a host model to express peripheral sodium channels such as NaV1.8 and NaV1.9 that have been difficult to express in widely utilized heterologous cells, like CHO and HEK293. Use of ND7/23 as a model cell to characterize the properties of sodium channels requires clear understanding of the endogenous ion channels. To define the nature of the background sodium currents in ND7/23 cells, we aimed to comprehensively profile the voltage-gated sodium channel subunits by endpoint and quantitative reverse transcription-PCR and by whole-cell patch clamp electrophysiology. We found that untransfected ND7/23 cells express endogenous peak sodium currents that average -2.12nA (n = 15) and with kinetics typical of fast sodium currents having activation and inactivation completed within few milliseconds. Furthermore, sodium currents were reduced to virtually nil upon exposure to 100nM tetrodotoxin, indicating that ND7/23 cells have essentially null background for tetrodotoxin-resistant (TTX-R) currents. qRT-PCR profiling indicated a major expression of TTX-sensitive (TTX-S) NaV1.6 and NaV1.7 at similar levels and very low expression of TTX-R NaV1.9 transcripts. There was no expression of TTX-R NaV1.8 in ND7/23 cells. There was low expression of NaV1.1, NaV1.2, NaV1.3 and no expression of cardiac or skeletal muscle sodium channels. As for the sodium channel auxiliary subunits, β1 and β3 subunits were expressed, but not the β2 and β4 subunits that covalently associate with the α-subunits. In addition, our results also showed that only the mouse forms of NaV1.6, NaV1.7 and NaV1.9 sodium channels were expressed in ND7/23 cells that was originally generated as a hybridoma of rat embryonic DRG and mouse neuroblastoma cell-line. By molecular profiling of auxiliary β- and principal α-subunits of the voltage gated sodium channel complex, our results define the background sodium channels expressed in ND7/23 cells, and confirm their utility for detailed functional studies of emerging pain channelopathies ascribed to mutations of the TTX-R sodium channels of sensory neurons., Competing Interests: The authors have declared that no competing interests exist.

Tools to control protein-protein interactions by external stimuli have been extensively developed. For this purpose, thermal stimulation can be utilized in addition to light. In this study, we identify a monoclonal antibody termed C13 mAb, which shows an approximately 480-fold decrease in the affinity constant at 37 °C compared to that at 4 °C. Next, we apply this temperature-dependent protein-peptide interaction for one-step protein purifications. We term this THermal-Elution-based TAg system as the THETA system, in which gel-immobilized C13 mAb-derived single-chain variable fragment (scFv) (termed THETAL) is able to bind with proteins tagged by C13 mAb-epitope(s) (THETAS) at 4 °C and thermally release at 37-42 °C. Moreover, to reveal the temperature-dependent interaction mechanism, molecular dynamics simulations are performed along with epitope mapping experiments. Overall, the high specificity and reversibility of the temperature-dependent features of the THETA system will support a wide variety of future applications such as thermogenetics., Competing Interests: Competing interestsThe authors declare no competing interests.

A 110-kDa type II transmembrane glycoprotein with dipeptidyl peptidase IV (DPPIV) activity in its extracellular region, CD26 has a multitude of biological functions and plays an important role in the regulation of inflammatory responses and tumor biology. Our work has focused on CD26 as a novel therapeutic target for various tumors and immune disorders, and we have recently developed a humanized anti-CD26 monoclonal antibody (mAb), YS110, which has promising safety profile and clinical activity in patients with malignant pleural mesothelioma. The development of an anti-human CD26 mAb that can clearly and reliably detect the denatured CD26 molecule in formalin-fixed paraffin-embedded (FFPE) tissues in the clinical setting is therefore of the utmost importance. To develop novel anti-CD26 mAbs capable of binding to denatured CD26, we immunized mice with urea-treated CD26 protein. Hybridoma supernatants were screened for specific reactivity with human CD26 by immunostaining through the use of a set of FFPE human CD26-positive or negative tumor cell lines. This screening method enables us to develop novel anti-human CD26 mAbs suitable for immunohistochemical staining of CD26 in FFPE non-tumor and tumor tissue sections with reliable clarity and intensity. Specifically, these mAbs display strong binding affinity to denatured human CD26 rather than undenatured human CD26, and are capable of detecting denatured human CD26 in decalcified specimens. These novel anti-CD26 mAbs are potentially useful for the analysis of CD26 expression in cancer patients with bony metastasis, and may help decide the appropriateness of YS110 therapy for future cancer patients., Competing Interests: Chikao Morimoto is an inventor of the humanized anti-CD26 mAb YS110 (US Patent #7402698). Y’s AC Co., Ltd. (Tokyo, Japan) owns this patent, and Taketo Yamada, Nam H. Dang, Kei Ohnuma and Chikao Morimoto are founding members and shareholders of this company. Kissei Pharmaceutical Co., Ltd. (Tokyo, Japan) is the industry sponsor of a phase I/II clinical trial of YS110 for malignant pleural mesothelioma which is currently being conducted in Japan. Nichirei Biosciences, Inc. (Tokyo Japan) is involved in the collaborative development of the companion diagnostic kit utilizing the anti-human CD26 mAb 19-32. Ryo Hatano, Taketo Yamada, Kei Ohnuma and Chikao Morimoto are inventors of the novel anti-human CD26 mAbs U16-3 and U38-8, and are now applying for a patent regarding their invention (Anti-human CD26 monoclonal antibody. Application 2018-03-16 JP2018049308). This does not alter our adherence to PLOS ONE policies on sharing data and materials. Other authors declare no competing financial interests associated with this manuscript, and all authors have approved the manuscript for submission.

The excessive cell death rate caused by electrofusion with unipolar pulses (UPs) has been a bottleneck to increasing cell fusion efficiency in monoclonal antibody technology. Several studies have confirmed that compared with UPs, bipolar pulses (BPs) with microsecond pulse widths can increase electropermeabilization while reducing cell death. Given these characteristics, BPs were used to increase cell fusion efficiency in this study. Cell staining and hybridoma culture experiments were performed using SP2/0 mouse myeloma cells and lymphocytes. Based on the equal energy principle, UPs and BPs were delivered to electrodes at a distance of 3.81 mm, with electric field intensities ranging from 2 kV/cm to 3 kV/cm and pulse duration of 40 μs for the UPs and 20-20 μs for the BPs. The results of cell staining experiments showed that cell fusion efficiency was 3-fold greater with BPs than with UPs. Similarly, the results of the hybridoma culture experiments showed that the hybridoma yields were 0.26‰ and 0.23‰ (2.5 kV/cm and 3 kV/cm, respectively) in the UP groups and increased to 0.46‰ and 0.35‰ in the BP groups. Taken together, the results show that the efficiency of heterologous cell fusion can be greatly increased if BPs are used instead of the commonly applied UPs. This study may provide a promising method for monoclonal antibody technology., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The carcinoembryonic antigen, glypican‑3 (GPC3), is a putative therapeutic target and diagnostic marker of hepatoma. In the present study, a monoclonal antibody (mAb) specifically against GPC3 was obtained via cloning the sequence of GPC3 via polymerase chain reaction and inserting it into a pET16b vector prior to transfection into Escherichia coli (E. coli) BL21. BALB/c mice were immunized with 20 µg purified antigen by intrasplenic embedding. Splenocytes and mouse myeloma cells SP2/0 were fused; then, the hybridoma cells were screened by an indirect ELISA. The properties of the mAb were examined by western blotting and immunofluorescence analysis against the purified protein. The results revealed that the prokaryotic expression vector of GPC3 had been successfully generated and GPC3 was stably expressed in E. coli BL21. A stable hybridoma cell line, 2F3, was generated in the present study, which produced mAbs against GPC3. The mAb 2F3 had a high antibody titer and the isotype was identified as IgG1/κ; 2F3 hybridomas had a median chromosome number of 98. Western blot and immunofluorescence analyses revealed that 2F3 specifically recognized recombinant and native GPC3. The 2F3 clone was proposed as a stable secretor of this mAb against GPC3. The results of present study indicated that the successful preparation of recombinant GPC3 protein and an anti‑human GPC3 mouse mAb may be provide a basis for developments in the diagnosis and treatment of liver cancer.

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (<1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

Hybridoma screening is a key step for the successful generation of high-affinity analyte-specific monoclonal antibodies (MAbs). This work presents an innovative screening method, known as a bacterial macroarray, generated by contact printing of hybridoma cell supernatant samples on a nitrocellulose (NC) membrane initially coated with fluorescein isothiocyanate (FITC)-labeled bacteria. Given that bacterial fixation will be influenced by complex bacterial surface structures, we selected both gram-positive bacteria ( Staphylococcus aureus and Listeria monocytogenes) and gram-negative bacteria ( Escherichia coli O157:H7 and Cronobacter sakazakii) to optimize the fixation conditions for binding to the NC membrane, such as the aperture of the NC membrane, the concentration of bacteria, the dosage of glycerin in the spotting buffer, and the fixation time and temperature. As a result, we found that a better bacterial macroarray could be developed when the spotting buffer, containing 10 11 CFU mL -1 of FITC-labeled bacteria and 15% (V/V) glycerol, was spotted onto a 0.45 µm NC membrane with an incubation of 2 h at 37 °C. Finally, we verified the stability and specificity of the prepared bacterial macroarray by detecting cell cultures with the addition of two MAbs ( Escherichia coli O157:H7 MAb E7 and Cronobacter sakazakii MAb 1E9) to simulate the screening experiments. Here, we describe a bacterial macroarray to efficiently screen the targeted antibody-secreted hybridomas.

C-X-C motif ligand 10 (CXCL10) is a pro-inflammatory chemokine and has been extensively evaluated in people and mice. In horses, CXCL10 and its involvement in host immunity has rarely been analyzed due to the lack of specific antibodies. We generated a mAb specific for the equine chemokine CXCL10 using hybridoma technology. Antibody specificity was confirmed by intracellular staining and flow cytometric analysis of Chinese Hamster Ovary (CHO) cells expressing equine rCXCL10, while CHO cells expressing equine rCXCL9 were not detected. Native CXCL10 expression in PBMC from horses of different age groups was analyzed by flow cytometry after in vitro stimulation. CXCL10 expressing PBMC were characterized by triple staining of CXCL10 combined with cell-surface markers. Stimulation with IFN-γ for 5 h similarly induced CXCL10 production in cluster of differentiation (CD)14 + CD16 - MHCII high monocytes of adult horses and weanlings. The newly generated mAb enables the quantitative intracellular analysis of CXCL10 by flow cytometry and provides a new valuable tool to improve the evaluation of inflammatory responses in horses., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The major histocompatibility complex (MHC) class I restricted pathway of antigen processing allows the presentation of intracellular antigens to cytotoxic T lymphocytes. The proteasome is the main protease in the cytoplasm and the nucleus, which is responsible for the generation of most peptide ligands of MHC-I molecules. Peptides produced by the proteasome can be further trimmed or destroyed by numerous cytosolic or endoplasmic reticulum (ER) luminal proteases. Small molecule inhibitors are useful tools for probing the role of proteases in MHC class I antigen processing. Here, we describe different methods to test the impact of protease inhibitors in antigen presentation assays.

Most of clinically used serum biomarkers for cancer detection were established in early 1980s when the Nobel Prize in physiology or medicine was awarded for the "discovery of the principle for the production of monoclonal antibodies." Using this "Nobel" technology, various monoclonal antibodies were obtained when different types of cancer cells were injected into mice and the ligands on the cancer cell surface were characterized. Both aberrant glycan structures and aberrant glycan-associated glycoproteins were revealed as a common feature of cancer cell surfaces through the specific interactions with the monoclonal antibodies. These results indicate that the biosynthesis of the environment-sensitive glycan structures goes awry in cancer cells, which is beyond genetic mutations. Later on, the glycan-related biomarkers were detected in the sera of cancer patients and then developed into serum biomarkers, such as CA125, CA153, CA195, CA199, CA242, CA27.29, CA50, and CA724, which are still in clinical use as of today. During the past 30 years, even with the advancement of different OMICS technologies not limited to genomics, epigenomics, proteomics, glycomics, lipidomics, and metabolomics, very few serum biomarkers have been introduced into clinical practice. The reason is that most of the newly discovered cancer biomarkers are inferior in terms of sensitivity and specificity to these biomarkers. We will summarize the reported sensitivity and specificity of currently used cancer biomarkers, especially the glycan-related biomarkers, in the forms of tables and radar plots and discuss the pros and cons of currently used cancer biomarkers., (© 2019 Elsevier Inc. All rights reserved.)

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., (Copyright © 2018 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

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.

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.

Monoclonal antibodies (mAbs) are a valuable biomaterial for basic life sciences and industrial purposes. The production of the mAb is time and effort intensive. In this report, we established a time- and labor-saving method for the mAb production. Because membrane-type immunoglobulin on a hybridoma cell surface and its secreted form, called as antibody, share the same binding property to the antigen, the fluorescence-labeled antigen bound to membrane-type immunoglobulin can be used as a screening marker. In the method, a hybridoma labeled by a fluorescent antigen was selected and sorted singly into 96-well plate using flow cytometer. Model experiments indicated that the method is highly efficient to obtain good mAbs suitable for Western blotting and immunofluorescence. Notably, most mAbs established by this method belonged to the IgG isotype, which is preferred over the IgM counterpart. Using a high-throughput flow cytometer, the method avoids tedious repeated screening and cloning processes. Because the method uses conventional myeloma for cell fusion and all reagents required in this method are commercially available, all research laboratories can apply the method to obtain mAbs efficiently., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Monoclonal antibodies are a main player in modern drug discovery. Many antibody screening formats exist, each with specific advantages and limitations. Nonetheless, it remains challenging to screen antibodies for the binding of cell-surface receptors (the most important class of all drug targets) or for the binding to target cells rather than purified proteins. Here, we present a high-throughput droplet microfluidics approach employing dual-color normalized fluorescence readout to detect antibody binding. This enables us to obtain quantitative data on target cell recognition, using as little as 33 fg of IgG per assay. Starting with an excess of hybridoma cells releasing unspecific antibodies, individual clones secreting specific binders (of target cells co-encapsulated into droplets) could be enriched 220-fold after sorting 80,000 clones in a single experiment. This opens the way for therapeutic antibody discovery, especially since the single-cell approach is in principle also applicable to primary human plasma cells., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective To prepare monoclonal antibodies (mAb) against RecQ helicase, characterize their biological properties, and then investigate their activity against RecQ helicase in tumor cells. Methods BALB/c mice were immunized with purified recombinant RecQ helicase from E.coli. Hybridoma technique was used to generate the mAb against RecQ helicase. The chromosome karyotype analysis of the hybridoma cells was performed by colchicine blocking. The Ig subtype and titer of mAb in ascitic fluid were determined by ELISA. The biological properties of mAb were detected via Western blotting and fluorescence polarization. Immunofluorescence was used to detect the interactions of mAb with BLM, RecQ4 and RecQ5 helicases in human breast cancer MDA-MB-231 cells. Immunohistochemistry was applied to detect the binding of mAb and RecQ helicase in K562 tumor cells and the corresponding stem cells. Results One stable hybridoma cell line expressing anti-RecQ mAb was obtained and named 6H5. The number of chromosomes was from 94 to 104, and Ig subtype was IgG1. The titer of 6H5 in ascitic fluid was 1×10 -7 . The mAb could specifically recognize E. coli RecQ helicase and thus inhibit its DNA binding activity. Besides, the mAb could also recognize BLM, RecQ4 and RecQ5 in MDA-MB-231 cells, and sensitively detect the expression of the RecQ helicase in K562 tumor cells and the corresponding stem cells. Conclusion The mAb against RecQ helicase was successfully prepared with a high titer and specificity.

Commercial antibodies against varying proteins are often not optimal for identification of proteins in their amyloid fibril forms. Reasons can be the different conformation but also a variety of modifications like N- or C-terminal truncation. Therefore, development of own monoclonal antibodies against amyloid fibril proteins may be advantageous. This chapter gives suggestions of how to be successful in such approaches.

From the perspective of academic and small research laboratories, the most common and practical strategy for recombinant expression of full-length monoclonal antibodies is to perform transient plasmid transfection of mammalian cells, resulting in small-scale and limited protein production. The generation of stable antibody producing mammalian cell lines enables larger-scale and consistent expression, however this approach is rarely pursued due to the time-consuming and expensive process of single colony screening and characterization. In order to bridge the gap between the simplicity of transient transfection and consistent production by stable cell lines, we describe a method to stably integrate antibody genes into the endogenous immunogenomic loci of hybridoma cells using CRISPR/Cas9 genome editing. Initially, the antibody variable light (VL) chain is deleted by multiplexed Cas9 cleavage; subsequently, the variable heavy (VH) chain is replaced by a fluorescent reporter gene (mRuby) by Cas9-assisted homology-directed repair (HDR). This cell line is customized by replacing mRuby with a synthetic antibody (consisting of a VL, light constant region and VH) by once again using Cas9-assisted HDR. Due to hybridomas' inherent ability to surface display and secrete antibodies, they provide a suitable host for both the selection and the production process, substantially streamlining the process for stable cell line generation, and thus we refer to this platform as plug-and-(dis)play (PnP) hybridomas.