Generation and Application of Monoclonal Antibodies against Porcine S100A8, S100A9, and S100A12 Proteins Using Hybridoma Technology.

S100A8, S100A9, and S100A12 proteins are important members of the S100 protein family, act primarily as congenital immunomodulators, and are closely related to the occurrence of infectious diseases. There have been few reports on the functional properties of S100A8, S100A9, and S100A12 proteins in swine, but it is certain that porcine S100A8, S100A9, and S100A12 proteins are highly expressed in diseased swine. To address the current lack of reliable and timely detection tools for these three proteins, we generated monoclonal antibodies specific to the porcine S100A8, S100A9, and S100A12 proteins using hybridoma technology. The results of serum sample testing showed that the above monoclonal antibodies specifically recognize the proteins S100A8, S100A9, and S100A12 in the serum and were able to evaluate the content change of these proteins during the infection process. This provides the basis for the use of porcine S100A8, S100A9, and S100A12 in the surveillance and diagnosis of swine diseases and laid a foundation for further understanding their roles in infection, immunity, and inflammation, as well as their potential applications in preventing or treating gastrointestinal tract or inflammatory diseases in swine.

A Functional Hydrogel Bead-Based High-Throughput Screening System for Mammalian Cells with Enhanced Secretion of Therapeutic Antibodies.

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 non-ionizing electromagnetic stimulation enhanced antibody production (NESEAP) effect - Discovery and technological applications.

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.

Deep in situ microscopy for real-time analysis of mammalian cell populations in bioreactors.

An in situ microscope based on pulsed transmitted light illumination via optical fiber was combined to artificial-intelligence to enable for the first time an online cell classification according to well-known cellular morphological features. A 848 192-image database generated during a lab-scale production process of antibodies was processed using a convolutional neural network approach chosen for its accurate real-time object detection capabilities. In order to induce different cell death routes, hybridomas were grown in normal or suboptimal conditions in a stirred tank reactor, in the presence of substrate limitation, medium addition, pH regulation problem or oxygen depletion. Using such an optical system made it possible to monitor real-time the evolution of different classes of animal cells, among which viable, necrotic and apoptotic cells. A class of viable cells displaying bulges in feast or famine conditions was also revealed. Considered as a breakthrough in the catalogue of process analytical tools, in situ microscopy powered by artificial-intelligence is also of great interest for research.

Recent Advances in the Development of Monoclonal Antibodies and Next-Generation Antibodies.

mAbs are highly indispensable tools for diagnostic, prophylactic, and therapeutic applications. The first technique, hybridoma technology, was based on fusion of B lymphocytes with myeloma cells, which resulted in generation of single mAbs against a specific Ag. Along with hybridoma technology, several novel and alternative methods have been developed to improve mAb generation, ranging from electrofusion to the discovery of completely novel technologies such as B cell immortalization; phage, yeast, bacterial, ribosome, and mammalian display systems; DNA/RNA encoded Abs; single B cell technology; transgenic animals; and artificial intelligence/machine learning. This commentary outlines the evolution, methodology, advantages, and limitations of various mAb production techniques. Furthermore, with the advent of next-generation Ab technologies such as single-chain variable fragments, nanobodies, bispecific Abs, Fc-engineered Abs, Ab biosimilars, Ab mimetics, and Ab-drug conjugates, the healthcare and pharmaceutical sectors have become resourceful to develop highly specific mAb treatments against various diseases such as cancer and autoimmune and infectious diseases.

[Preparation of HSV-IgM human-mouse chimeric antibody and development of stable recombinant cell line].

In order to achieve large-scale production of HSV-IgM (HSV1, HSV2) human-mouse chimeric antibody in vitro, the gene sequence of the corresponding hybridoma cell was harvested by RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) technique to clone the chimeric antibody into eukaryotic expression vectors, and express the target proteins in CHO-S cells. At the same time, the screening process of stable cell lines was optimized, and the pressure conditions of pool construction stage and monoclonal screening stage were explored. Finally, the target protein was purified by protein L affinity purification method and the biological activity was detected. The recombinant IgM antibodies, HSV1 and HSV2, weighted at 899 kDa and 909 kDa respectively, were prepared. The optimal screening pressure was 20P200M (the first phase of pressure) and 50P1000M (the second phase of pressure). The final titer for the monoclonal expression of HSV1-IgM and HSV2-IgM was 1 620 mg/L and 623 mg/L, respectively. This study may facilitate the development of quality control products of HSV1 and HSV2 IgM series recombinant antibodies as well as efficient expression of IgM subtype antibodies in vitro.

Antibody Phage Display.

The application of antibodies has transcended across many areas of work but mainly as a research tool, for diagnostic and for therapeutic applications. Antibodies are immunoproteins from vertebrates that have the unique property of specifically binding foreign molecules and distinguish target antigens. This property allows antibodies to effectively protect the host from infections. Apart from the hybridoma technology using transgenic animals, antibody phage display is commonly considered the gold standard technique for the isolation of human monoclonal antibodies. The concept of antibody phage display surrounds the ability to display antibody fragments on the surface of M13 bacteriophage particles with the corresponding gene packaged within the particle. A repetitive in vitro affinity based selection process permits the enrichment of target specific binders. This process of recombinant human monoclonal antibody generation also enables additional engineering for various applications. This makes phage display an indispensable technique for antibody development and engineering activities.

Impaired proliferation and migration of HUVEC and melanoma cells by human anti-FGF2 mAbs derived from a murine hybridoma by guided selection.

Disadvantages of using murine monoclonal antibodies (mAb) in human therapy, such as immunogenicity response, led to the development of technologies to transform murine antibodies into human antibodies. The murine anti-FGF2 3F12E7 mAb was proposed as a promising agent to treat metastatic melanoma tumors; once it blocks the FGF2, responsible for playing a role in tumor growth, angiogenesis, and metastasis. Considering the therapeutic potential of anti-FGF2 3F12E7 mAb and its limited use in humans due to its origin, we used this antibody as the template for a guided selection humanization technique to obtain human anti-FGF2 mAbs. Three Fab libraries (murine, hybrid, and human) were constructed for humanization. The libraries were phage-displayed, and the panning was performed against recombinant human FGF2 (rFGF2). The selected human variable light and heavy chains were cloned into AbVec vectors for full-length IgG expression into HEK293-F cells. Surface plasmon resonance analyses showed binding to rFGF2 of seven mAbs out of 20 expressed. Assays performed with these mAbs resulted in two that showed proliferation reduction and cell migration attenuation of HUVEC and SK-Mel-28 melanoma cells. In-silico analyses predicted that these two human anti-FGF2 mAbs interact with FGF2 at a similar patch of residues than the chimeric anti-FGF2 antibody, comprehending a region within the heparin-binding domains of FGF2, essential for its function. These results are comparable to those achieved by the murine anti-FGF2 3F12E7 mAb and showed success in the humanization process and selection of two human mAbs with the potential to inhibit undesirable FGF2 roles.

The guided selection humanization process enabled the production of 20 human mAbs anti-FGF2;Seven human anti-FGF2 mAbs showed binding to the rFGF2 antigen in the SPR binding assay;Two human anti-FGF2 mAbs inhibited the proliferation and migration of HUVEC and SK-Mel-28 cells and were predicted to contact the FGF2 at a similar patch of residues than the original mAb.

Comment on "Monoclonal Antibody Discovery Based on Precise Selection of Single Transgenic Hybridomas with an On-Cell-Surface and Antigen-Specific Anchor".

In the original paper, Li and co-workers [ACS Appl. Mater. Interfaces 2022, 14, 17128-17141] described their approach to select specific hybridoma cells from a polyclonal hybridoma pool by using a cell surface anchor to catch the secreted antibody. The antigen-specific detection was performed with streptavidin-labeled antigen and a PE-labeled anti-F(ab')2 antibody. The present comment offers a clearer description of the selection system originally published by Listek et al. in 2020 and provides further information about the importance of controls and recent adaptations made by our lab.

High-volume hybridoma sequencing on the NeuroMabSeq platform enables efficient generation of recombinant monoclonal antibodies and scFvs for neuroscience research.

The Neuroscience Monoclonal Antibody Sequencing Initiative (NeuroMabSeq) is a concerted effort to determine and make publicly available hybridoma-derived sequences of monoclonal antibodies (mAbs) valuable to neuroscience research. Over 30 years of research and development efforts including those at the UC Davis/NIH NeuroMab Facility have resulted in the generation of a large collection of mouse mAbs validated for neuroscience research. To enhance dissemination and increase the utility of this valuable resource, we applied a high-throughput DNA sequencing approach to determine immunoglobulin heavy and light chain variable domain sequences from source hybridoma cells. The resultant set of sequences was made publicly available as a searchable DNA sequence database (neuromabseq.ucdavis.edu) for sharing, analysis and use in downstream applications. We enhanced the utility, transparency, and reproducibility of the existing mAb collection by using these sequences to develop recombinant mAbs. This enabled their subsequent engineering into alternate forms with distinct utility, including alternate modes of detection in multiplexed labeling, and as miniaturized single chain variable fragments or scFvs. The NeuroMabSeq website and database and the corresponding recombinant antibody collection together serve as a public DNA sequence repository of mouse mAb heavy and light chain variable domain sequences and as an open resource for enhancing dissemination and utility of this valuable collection of validated mAbs.

Functionalized Parylene Films for Enhancement of Antibody Production by Hybridoma Cells.

In this study, the influence of microenvironments on antibody production of hybridoma cells was analyzed using six types of functionalized parylene films, parylene-N and parylene-C (before and after UV radiation), parylene-AM, and parylene-H, and using polystyrene as a negative control. Hybridoma cells were cultured on modified parylene films that produced a monoclonal antibody against the well-known fungal toxin ochratoxin-A. Surface properties were analyzed for each parylene film, such as roughness, chemical functional groups, and hydrophilicity. The proliferation rate of the hybridoma cells was observed for each parylene film by counting the number of adherent cells, and the total amount of produced antibodies from different parylene films was estimated using indirect ELISA. In comparison with the polystyrene, the antibody-production by parylene-H and parylene-AM was estimated to be observed to be as high as 210-244% after the culture of 24 h. These results indicate that the chemical functional groups of the culture plate could influence antibody production. To analyze the influence of the microenvironments of the modified parylene films, we performed cell cycle analysis to estimate the ratio of the G0/G1, S, and G2/M phases of the hybridoma cells on each parylene film. From the normalized proportion of phases of the cell cycle, the difference in antibody production from different surfaces was considered to result from the difference in the proliferation rate of hybridoma cells, which occurred from the different physical and chemical properties of the parylene films. Finally, protein expression was analyzed using an mRNA array to determine the effect of parylene films on protein expression in hybridoma cells. The expression of three antibody production-related genes (CD40, Sox4, and RelB) was analyzed in hybridoma cells cultured on modified parylene films.

Mapping the T cell repertoire to a complex gut bacterial community.

Certain bacterial strains from the microbiome induce a potent, antigen-specific T cell response1-5. However, the specificity of microbiome-induced T cells has not been explored at the strain level across the gut community. Here, we colonize germ-free mice with complex defined communities (roughly 100 bacterial strains) and profile T cell responses to each strain. The pattern of responses suggests that many T cells in the gut repertoire recognize several bacterial strains from the community. We constructed T cell hybridomas from 92 T cell receptor (TCR) clonotypes; by screening every strain in the community against each hybridoma, we find that nearly all the bacteria-specific TCRs show a one-to-many TCR-to-strain relationship, including 13 abundant TCR clonotypes that each recognize 18 Firmicutes. By screening three pooled bacterial genomic libraries, we discover that these 13 clonotypes share a single target: a conserved substrate-binding protein from an ATP-binding cassette transport system. Peripheral regulatory T cells and T helper 17 cells specific for an epitope from this protein are abundant in community-colonized and specific pathogen-free mice. Our work reveals that T cell recognition of commensals is focused on widely conserved, highly expressed cell-surface antigens, opening the door to new therapeutic strategies in which colonist-specific immune responses are rationally altered or redirected.

NIH/3T3 Fibroblasts Selectively Activate T Cells Specific for Posttranslationally Modified Collagen Type II.

It has been shown that synovial fibroblasts (SF) play a key role in the initiation of inflammation and joint destruction, leading to arthritis progression. Fibroblasts may express major histocompatibility complex class II region (MHCII) molecules, and thus, they could be able to process and present antigens to immunocompetent cells. Here we examine whether different types of fibroblasts (synovial, dermal, and thymic murine fibroblasts, destructive LS48 fibroblasts, and noninvasive NIH/3T3 fibroblasts) may be involved in the initiation of rheumatoid arthritis (RA) pathogenesis and can process and present type II collagen (COL2)-an autoantigen associated with RA. Using a panel of MHCII/Aq-restricted T-cell hybridoma lines that specifically recognize an immunodominant COL2 epitope (COL2259-273), we found that NIH/3T3 fibroblasts activate several T-cell clones that recognize the posttranslationally glycosylated or hydroxylated COL2259-273 epitope. The HCQ.3 hybridoma, which is specific for the glycosylated immunodominant COL2 epitope 259-273 (Gal264), showed the strongest response. Interestingly, NIH/3T3 cells, but not destructive LS48 fibroblasts, synovial, dermal, or thymic fibroblasts, were able to stimulate the HCQ.3 hybridoma and other COL2-specific T-cell hybridomas. Our experiments revealed that NIH/3T3 fibroblasts are able to activate COL2-specific T-cell hybridomas even in the absence of COL2 or a posttranslationally modified COL2 peptide. The mechanism of this unusual activation is contact-dependent and involves the T-cell receptor (TCR) complex.

Development of an anti-Pseudomonas aeruginosa therapeutic monoclonal antibody WVDC-5244.

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.

Production of monoclonal antibodies for therapeutic purposes: A review.

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.

High-throughput spatiotemporal monitoring of single-cell secretions via plasmonic microwell arrays.

Methods for the analysis of cell secretions at the single-cell level only provide semiquantitative endpoint readouts. Here we describe a microwell array for the real-time spatiotemporal monitoring of extracellular secretions from hundreds of single cells in parallel. The microwell array incorporates a gold substrate with arrays of nanometric holes functionalized with receptors for a specific analyte, and is illuminated with light spectrally overlapping with the device's spectrum of extraordinary optical transmission. Spectral shifts in surface plasmon resonance resulting from analyte-receptor bindings around a secreting cell are recorded by a camera as variations in the intensity of the transmitted light while machine-learning-assisted cell tracking eliminates the influence of cell movements. We used the microwell array to characterize the antibody-secretion profiles of hybridoma cells and of a rare subset of antibody-secreting cells sorted from human donor peripheral blood mononuclear cells. High-throughput measurements of spatiotemporal secretory profiles at the single-cell level will aid the study of the physiological mechanisms governing protein secretion.

Generation of a novel monoclonal antibody against inflammatory biomarker S100A8 using hybridoma technology.

OBJECTIVES: S100A8 is highly expressed in several inflammatory and oncological conditions. To address the current lack of a reliable and sensitive detection method for S100A8, we generated a monoclonal antibody with a high binding affinity to human S100A8 to enable early disease diagnosis. RESULTS: A soluble recombinant S100A8 protein with a high yield and purity was produced using Escherichia coli. Next, mice were immunized with recombinant S100A8 to obtain anti-human S100A8 monoclonal antibodies using hybridoma technology. Lastly, the high binding activity of the antibody was confirmed and its sequence was identified. CONCLUSIONS: This method, including the production of antigens and antibodies, will be useful for the generation of hybridoma cell lines that produce anti-S100A8 monoclonal antibodies. Moreover, the sequence information of the antibody can be used to develop a recombinant antibody for use in various research and clinical applications.

Production and characterization of monoclonal antibodies to Xenopus proteins.

Monoclonal antibodies are powerful and versatile tools that enable the study of proteins in diverse contexts. They are often utilized to assist with identification of subcellular localization and characterization of the function of target proteins of interest. However, because there can be considerable sequence diversity between orthologous proteins in Xenopus and mammals, antibodies produced against mouse or human proteins often do not recognize Xenopus counterparts. To address this issue, we refined existing mouse monoclonal antibody production protocols to generate antibodies against Xenopus proteins of interest. Here, we describe several approaches for the generation of useful mouse anti-Xenopus antibodies to multiple Xenopus proteins and their validation in various experimental approaches. These novel antibodies are now available to the research community through the Developmental Study Hybridoma Bank (DSHB).

Hybridoma-derived neutralizing monoclonal antibodies against Beta and Delta variants of SARS-CoV-2 in vivo.

Neutralizing monoclonal antibodies (mAb) are a major therapeutic strategy for the treatment of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. The continuous emergence of new SARS-CoV-2 variants worldwide has increased the urgency for the development of new mAbs. In this study, we immunized mice with the receptor-binding domain (RBD) of the SARS-CoV-2 prototypic strain (WIV04) and screened 35 RBD-specific mAbs using hybridoma technology. Results of the plaque reduction neutralization test showed that 25 of the mAbs neutralized authentic WIV04 strain infection. The 25 mAbs were divided into three categories based on the competitive enzyme-linked immunosorbent assay results. A representative mAb was selected from each category (RD4, RD10, and RD14) to determine the binding kinetics and median inhibitory concentration (IC50) of WIV04 and two variants of concern (VOC): B.1.351 (Beta) and B.1.617.2 (Delta). RD4 neutralized the B.1.617.2 variant with an IC50 of 2.67 â€‹ng/mL; however, it completely lost neutralizing activity against the B.1.351 variant. RD10 neutralized both variants with an IC50 exceeding 100 â€‹ng/mL; whereas RD14 neutralized two variants with a higher IC50 (>1 â€‹mg/mL). Animal experiments were performed to evaluate the protective effects of RD4 and RD10 against various VOC infections. RD4 could protect Adv-hACE2 transduced mice from B.1.617.2 infection at an antibody concentration of 25 â€‹mg/kg, while RD10 could protect mice from B.1.351 infection at an antibody concentration of 75 â€‹mg/kg. These results highlight the potential for future modifications of the mAbs for practical use.

Development of an indirect competitive enzyme-linked immunosorbent assay for formononetin and its application in a cell-based assay using MC3T3-E1 cells.

Formononetin (FMN) is a methoxy isoflavone found abundantly in leguminous plants and associated foods. Several analytical methods have been developed to detect FMN. However, they are costly, complicated, and time-consuming. This study describes an indirect competitive enzyme-linked immunosorbent assay (icELISA) to determine FMN content in food samples using a monoclonal antibody (mAb) against FMN produced by a newly established hybridoma cell line. Validation studies were conducted, and this assay was found to be sufficiently reliable, with an analytical measurement range of 19.53-1250 ng/mL and a detection limit of 17.42 ng/mL. Furthermore, icELISA was successfully applied for a cell-based assay in which the amount of FMN and ononin uptake was quantified in MC3T3-E1 cells. Hence, icELISA is a simple and reliable method for the detection and quantification of FMN, as well as elucidation of its functions and underlying mechanisms of action.