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.

Expression of soluble recombinant human matrix metalloproteinase 9 and generation of its monoclonal antibody.

We have successfully produced a recombinant human matrix metalloproteinase 9 (hMMP9) antigen with high yield and purity and used it to generate a hybridoma cell-culture-based monoclonal anti-hMMP9 antibody. We selected the most effective antibody for binding antigens and successfully identified its nucleotide sequence. The entire antigen and antibody developmental procedures described herein can be a practical approach for producing large amounts of monoclonal antibodies against hMMP9 and other antigens of interest. Additionally, the nucleotide sequence information of the anti-hMMP9 monoclonal antibody revealed herein will be useful for the generation of recombinant antibodies or antibody fragments against hMMP9.

Rapid and reliable hybridoma screening method that is suitable for production of functional structure-recognizing monoclonal antibody.

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.

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

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

Model-Based Optimization of a Fed-Batch Bioreactor for mAb Production Using a Hybridoma Cell Culture.

Production of monoclonal antibodies (mAbs) is a well-known method used to synthesize a large number of identical antibodies, which are molecules of huge importance in medicine. Due to such reasons, intense efforts have been invested to maximize the mAbs production in bioreactors with hybridoma cell cultures. However, the optimal control of such sensitive bioreactors is an engineering problem difficult to solve due to the large number of state-variables with highly nonlinear dynamics, which often translates into a non-convex optimization problem that involves a significant number of decision (control) variables. Based on an adequate kinetic model adopted from the literature, this paper focuses on developing an in-silico (model-based, offline) numerical analysis of a fed-batch bioreactor (FBR) with an immobilized hybridoma culture to determine its optimal feeding policy by considering a small number of control variables, thus ensuring maximization of mAbs production. The obtained time stepwise optimal feeding policies of FBR were proven to obtain better performances than those of simple batch operation (BR) for all the verified alternatives in terms of raw material consumption and mAbs productivity. Several elements of novelty (i-iv) are pointed out in the "conclusions" section (e.g., considering the continuously added biomass as a control variable during FBR).

Liquid Nitrogen Storage of Hybridoma Cells.

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 (LN2). Cells stored in LN2 will remain viable for years. Once a frozen vial has been removed from LN2 storage, it should be thawed as described, grown out into log phase, and refrozen.

Collecting and Storing Hybridoma Tissue Culture Supernatants.

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.

Preparation of Monoclonal Antibody Against EMA-1 and Development of Rapid Serological Detection Method for Theileria equi Infection, Xinjiang, China.

The erythrocytic-stage surface protein equi merozoite antigen 1 (EMA-1) of Theileria equi is a major candidate for the development of a diagnostic antigen for equine piroplasmosis. In this study, BALB/c mice were immunized with purified recombinant EMA-1 to prepare monoclonal antibody (mAb) against T. equi EMA-1, and 1 mAb 5H2 was obtained that showed good reaction with infected red blood cells (RBC) in the indirect immunofluorescence assay (IFA). To develop a rapid serological detection method for T. equi infection in Xinjiang Uygur Autonomous Region, China, recombinant EMA-1 originating from the local T. equi strain and the mAb to EMA-1 were employed to develop an immunochromatographic test (ICT) to detect antibodies to T. equi in horse sera. The ICT showed high sensitivity and specificity and no cross-reaction with Babesia caballi. Ninety-two horse serum samples collected from Ili, Xinjiang, were tested by ICT and compared with the detection results of a commercial ELISA kit. The results showed that 56 of 92 (61%) serum samples were seropositive according to the ICT assay, and 50 (54%) samples were seropositive according to the ELISA kit. The ICT had a high coincidence (91.3%) but was more sensitive than the reference ELISA kit. To confirm whether the horses were infected by T. equi, 30 blood DNA samples from 92 horses were examined by PCR. The results showed that 14 of 30 (47%) horses were confirmed to be infected with T. equi by PCR, while 16 of 30 (53%) horses were seropositive by ICT. All PCR-positive horses were ICT-positive. The findings indicate that T. equi is endemic in Ili, Xinjiang, and that the ICT is reliable as a serological diagnosis method. The ICT developed in this study could be an efficient diagnostic tool to detect T. equi infection in horses in the Xinjiang area.

A novel selection strategy for antibody producing hybridoma cells based on a new transgenic fusion cell line.

The use of monoclonal antibodies is ubiquitous in science and biomedicine but the generation and validation process of antibodies is nevertheless complicated and time-consuming. To address these issues we developed a novel selective technology based on an artificial cell surface construct by which secreted antibodies were connected to the corresponding hybridoma cell when they possess the desired antigen-specificity. Further the system enables the selection of desired isotypes and the screening for potential cross-reactivities in the same context. For the design of the construct we combined the transmembrane domain of the EGF-receptor with a hemagglutinin epitope and a biotin acceptor peptide and performed a transposon-mediated transfection of myeloma cell lines. The stably transfected myeloma cell line was used for the generation of hybridoma cells and an antigen- and isotype-specific screening method was established. The system has been validated for globular protein antigens as well as for haptens and enables a fast and early stage selection and validation of monoclonal antibodies in one step.

Nanodisc technology facilitates identification of monoclonal antibodies targeting multi-pass membrane proteins.

Multi-pass membrane proteins are important targets of biologic medicines. Given the inherent difficulties in working with membrane proteins, we sought to investigate the utility of membrane scaffold protein nanodiscs as a means of solubilizing membrane proteins to aid antibody discovery. Using a model multi-pass membrane protein, we demonstrate how incorporation of a multi-pass membrane protein into nanodiscs can be used in flow cytometry to identify antigen-specific hybridoma. The use of target protein-loaded nanodiscs to sort individual hybridoma early in the screening process can reduce the time required to identify antibodies against multi-pass membrane proteins.

Preparing Feeder Cell Cultures to Support Hybridoma Growth.

Feeder layer plates are prepared with cells that provide the appropriate growth factors to support the growth of hybridoma cells until they can expand in number and provide their own. Good feeder cells should have properties that allow them to be selected against during the future growth of the hybridomas. Peritoneal macrophages, myeloma cells, splenocytes, and MRC-5 cells (a human lung fibroblast line) are the most common feeder layer cells used in hybridoma fusions. Methods for their preparation are described here.

Single-Cell Cloning of Hybridoma Cells by Limiting Dilution.

Isolating a stable clone of hybridoma cells that all secrete the correct antibody is the most time-consuming step in the production of hybridomas. Single-cell cloning ensures that cells that produce the antibody of interest are truly monoclonal and that the secretion of this antibody can be stably maintained. The original positive well will often contain more than one clone of hybridoma cells, and many hybrid cells have an unstable assortment of chromosomes. Both of these problems may lead to the desired cells being outgrown by cells that are not producing the antibody of interest. Cloning hybridoma cells by limiting dilution is the easiest of the single-cell-cloning techniques. Two approaches are presented here, one rapid technique for generating cultures that are close to being single-cell-cloned and one for single-cell cloning directly. Even though every attempt is made to ensure that the cells are in a single-cell suspension before plating, there is no way to guarantee that the colonies do not arise from two cells that were stuck together. Therefore, limiting dilution cloning should be performed at least twice to generate a clonal population.

Single-Cell Cloning of Hybridoma Cells by Growth in Soft Agar.

Single-cell cloning during hybridoma production ensures that cells that produce the antibody of interest are truly monoclonal and that the secretion of this antibody can be stably maintained. Cloning of hybridoma cells in semisolid medium is one of the most commonly used methods for producing single-cell clones. The technique is easy, but, because it is performed in two stages, it does take longer than other methods. Not all cells will grow in soft agar, and there may be a bias on the type of colony that appears. However, most of the commonly used myeloma fusion partners have relatively good cloning efficiencies in soft agar, and, consequently, so do most hybridomas. Even though every attempt is made to ensure that the cells are in a single-cell suspension before plating, there is no way to guarantee that the colonies do not arise from two cells that were stuck together. Therefore, single-cell cloning in soft agar should be repeated at least twice before the cells are considered clonal.

Myeloma and Hybridoma Cell Counts and Viability Checks.

For most purposes, the number of hybridoma or myeloma cells can be estimated simply by observing the cells under the microscope. When an exact cell count is needed, the number can be determined by using a hemocytometer (improved Neubauer counting chambers are the most commonly used). This is a simple device in which a special coverslip rests on supports that hold it 0.1 mm above the base of the slide. The slide is engraved with a series of lines that form 1 × 1-mm squares. By counting the number of cells within the 0.1-mm3 chamber formed by the 1 × 1-mm square and the height of the coverslip, an accurate quantitation of cells per milliliter can be calculated. To determine the percentage of viable cells within a population, the cell suspension is mixed with a vital dye and observed under the microscope. Vital dyes are excluded from living cells but stain dead cells. The most common dye used for these stains is Trypan Blue.

In situ microscopy as online tool for detecting microbial contaminations in cell culture.

Microbial contamination in mammalian cell cultures causing rejected batches is costly and highly unwanted. Most methods for detecting a contamination are time-consuming and require extensive off-line sampling. To circumvent these efforts and provide a more convenient alternative, we used an online in situ microscope to estimate the cell diameter of the cellular species in the culture to distinguish mammalian cells from microbial cells depending on their size. A warning system was set up to alert the operator if microbial cells were present in the culture. Hybridoma cells were cultured and infected with either Candida utilis or Pichia stipitis as contaminant. The warning system could successfully detect the introduced contamination and alert the operator. The results suggest that in situ microscopy could be used as an efficient online tool for early detection of contaminations in cell cultures.

Electrofusion by a bipolar pulsed electric field: Increased cell fusion efficiency for monoclonal antibody production.

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.

Development of T cell lines sensitive to antigen stimulation.

Immortalized T cells such as T cell hybridomas, transfectomas, and transductants are useful tools to study tri-molecular complexes consisting of peptide, MHC, and T cell receptor (TCR) molecules. These cells have been utilized for antigen discovery studies for decades due to simplicity and rapidness of growing cells. However, responsiveness to antigen stimulation is typically less sensitive compared to primary T cells, resulting in occasional false negative outcomes especially for TCRs having low affinity to a peptide-MHC complex (pMHC). To overcome this obstacle, we genetically engineered T cell hybridomas to express additional CD3 molecules as well as CD4 with two amino acid substitutions that increase affinity to MHC class II molecules. The manipulated T cell hybridomas that were further transduced with retroviral vectors encoding TCRs of interest responded to cognate antigens more robustly than non-manipulated cells without evoking non-antigen specific reactivity. Of importance, the manipulation with CD3 and mutated human CD4 expression was effective in increasing responsiveness of T cell hybridomas to a wide variety of TCR, peptide, and MHC combinations across class II genetic loci (i.e. HLA-DR, HLA-DQ, HLA-DP, and murine H2-IA) and species (i.e. both humans and mice), and thus will be useful to identify antigen specificity of T cells.

Development of Mouse Monoclonal Antibodies Against Human Amyloid Fibril Proteins for Diagnostic and Research Purposes.

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.

Evaluation of an ATP-Bioluminescence Rapid Microbial Screening Method for In-Process Biologics.

This study compared an adenosine triphosphate (ATP)-based bioluminescence rapid microbial method (RMM) with a conventional sterility method for biologics sample testing. The RMM is based on a comparison of ATP levels in inoculated and uninoculated microbiological growth medium samples following growth enrichment incubation. The biologics samples qualified in this study were recombinant monoclonal antibodies and hybridoma cell culture supernatants. Initially, the lot-to-lot variation in background ATP of these samples posed significant challenges. Two strategies to increase the signal-to-noise ratio (positive result/background ATP) were evaluated: enzyme-based signal amplification and reduction of the broth-based noise through broth selection. Following qualification of the RMM for antibody and cell culture samples, the RMM was also utilized for rapid screening of several sources of purified water. This ATP-based RMM has proved invaluable in routine testing of diverse biologics samples at our discovery research site and plays a key role in the investigation of contaminated samples.LAY ABSTRACT: Biologics research laboratories routinely conduct sterility testing of products in development. However, the lengthy turnaround time for detection of microbial contaminants when using a conventional sterility test is a bottleneck in this fast-paced environment. This study investigated an adenosine triphosphate-based bioluminescence rapid microbial method (RMM) for biologics samples, including monoclonal antibodies and hybridoma cell cultures. The results showed that the RMM allowed detection of antibody sample contaminants after only three days of incubation. In addition to being faster than the standard method, the RMM proved more reliable in detecting contaminants in cell culture samples with antibiotics. Since its initial evaluation, this RMM has accelerated biologics sterility testing across multiple projects at our site.

Screening for Good Batches of Fetal Bovine Serum for Myeloma and Hybridoma Growth.

Not all lots of fetal bovine serum (FBS) are good at supporting hybridoma growth at low density. The key constituents that distinguish good batches of serum from bad are not known. It is necessary to order test batches from several suppliers and screen them as described here or purchase prescreened serum directly from the distributor.