Hybridoma Cell Research Articles

Cell Surface Vimentin Detection in Cancer Cells by Peptide-Based Monoclonal Antibody

Vimentin is a prominent Intermediate Filaments (IFs) protein expressed in different mesenchymal origin cell types. Besides a wide range of cellular function roles associated with vimentin expression, its dysregulation and cell surface expression in the induction of malignancy properties have been reported extensively, making it a promising cancer-specific target. Therefore, this study aimed to generate and characterize anti-vimentin monoclonal antibodies. A 14-mer synthetic peptide from vimentin was conjugated to Keyhole Limpet Hemocyanin (KLH) and used for immunization of Blab/C mice and monoclonal production by conventional hybridoma technology. The monoclonal antibody was purified using affinity chromatography of supernatants from the selected hybridoma cells. ELISA, Immunoprecipitation-Western blotting (IP-WB), Immunocytochemistry (ICC), and flow cytometry were employed to characterize the produced monoclonal antibody in terms of interaction with vimentin immunizing peptide as well as vimentin protein. Amid the several obtained producing anti-vimentin antibody hybridomas, the 7C11-D9 clone (IgG1 isotype with kappa light chain) showed higher reactivity with the immunizing peptide, and led to its selection for purification and characterization. The purified antibody could detect vimentin protein in IP-WB, ICC and flow cytometry of the normal and cancerous cells with different origin. No vimentin expression was found in normal healthy Peripheral Blood Mononuclear Cell (PBMC). Taken together, 7C11-D9 anti-vimentin monoclonal antibody might be used as immune diagnostic or immune therapeutic tool where detection or targeting of vimentin in a wide range of organisms is required.

A Novel Hybridoma Cell Segmentation Method Based on Multi-Scale Feature Fusion and Dual Attention Network

The hybridoma cell screening method is usually done manually by human eyes during the production process for monoclonal antibody drugs. This traditional screening method has certain limitations, such as low efficiency and subjectivity bias. Furthermore, most of the existing deep learning-based image segmentation methods have certain drawbacks, due to different shapes of hybridoma cells and uneven location distribution. In this paper, we propose a deep hybridoma cell image segmentation method based on residual and attention U-Net (RA-UNet). Firstly, the feature maps of the five modules in the network encoder are used for multi-scale feature fusion in a feature pyramid form and then spliced into the network decoder to enrich the semantic level of the feature maps in the decoder. Secondly, a dual attention mechanism module based on global and channel attention mechanisms is presented. The global attention mechanism (non-local neural network) is connected to the network decoder to expand the receptive field of the feature map and bring more rich information to the network. Then, the channel attention mechanism SENet (the squeeze-and-excitation network) is connected to the non-local attention mechanism. Consequently, the important features are enhanced by the learning of the feature channel weights, and the secondary features are suppressed, hence improving the cell segmentation performance and accuracy. Finally, the focal loss function is used to guide the network to learn the hard-to-classify cell categories. Furthermore, we evaluate the performance of the proposed RA-UNet method on a newly established hybridoma cell image dataset. Experimental results show that the proposed method has good reliability and improves the efficiency of hybridoma cell segmentation compared with state-of-the-art networks such as FCN, UNet, and UNet++. The results show that the proposed RA-UNet model has improvements of 0.8937%, 0.9926%, 0.9512%, and 0.9007% in terms of the dice coefficients, PA, MPA, and MIoU, respectively.

Adjuvant Vaccination with Allogenic Dendritic Cells Significantly Prolongs Overall Survival in High-Grade Gliomas: Results of a Phase II Trial

Immunotherapy for cancer treatment has gained increased attention in recent years. Recently, our group reported the case of a patient with glioblastoma who underwent vaccination based on dendritic cells and experienced a strong Th1 immune response together with near-complete tumor remission. Here we report the results of a phase I/II prospective, non-controlled clinical trial with 37 patients harboring glioblastoma or grade 4 astrocytomas. At the time of first recurrence after surgery, patients began receiving monthly intradermal injections of allogenic DC-autologous tumor cell hybridomas. Overall survival, quality of life, and immunological profiles were assessed prospectively. Compared with patients in the Genomic Data Commons data bank, overall survival for vaccinated patients with glioblastoma was 27.6 ± 2.4 months (vs. 16.3 ± 0.7, log-rank p < 0.001, hazard ratio 0.53, 95%CI 0.36-0.78, p < 0.01), and it was 59.5 ± 15.9 for vaccinated astrocytoma grade 4 patients (vs. 19.8 ± 2.5, log-rank p < 0.05, hazard ratio 0.18, 95%CI 0.05-0.62, p < 0.01). Furthermore, seven vaccinated patients (two IDH-1-mutated and five wild type) remain alive at the time of this report (overall survival 47.9 months, SD 21.1, range: 25.4-78.6 months since diagnosis; and 34.2 months since recurrence, range: 17.8 to 40.7, SD 21.3). We believe that the data reported here can foster the improvement of treatment protocols for high-grade gliomas based on cellular immunotherapy.

Enhancement of polyethylene glycol-cell fusion efficiency by novel application of transient pressure using a jet injector.

Cell-cell fusion involves the fusion of somatic cells into a single hybrid cell. It is not only a physiological process but also an important cell engineering technology which can be applied to various fields, such as regenerative medicine, antibody engineering, genetic engineering, and cancer therapy. There are three major methods of cell fusion: electrical cell fusion, polyethylene glycol (PEG) cell fusion, and virus-mediated cell fusion. Although PEG cell fusion is the most economical approach and does not require expensive instrumentation, it has a poor fusion rate and induces a high rate of cell cytotoxicity. To improve the fusion rate of the PEG method, we combined it with the pyro-drive jet injector (PJI). PJI provides instant pressure instead of cell agitation to increase the probability of cell-to-cell contact and shorten the distance between cells in the process of cell fusion. Here, we report that this improved fusion method not only decreased cell cytotoxicity during the fusion process, but also increased fusion rate compared with the conventional PEG method. Furthermore, we tested the functionality of cells fused using the PJI-PEG method and found them to be comparable to those fused using the conventional PEG method in terms of their application for dendritic cell (DC)-tumor cell fusion vaccine production; in addition, the PJI-PEG method demonstrated excellent performance in hybridoma cell preparation. Taken together, our data indicate that this method improves cell fusion efficiency as compared to the PEG method and thus has the potential for use in various applications that require cell fusion technology.

Preparation and characterization of a neutralizing murine monoclonal antibody against tetanus toxin

After Clostridium tetani infects the human body, it propagates under anaerobic conditions and produces tetanus neurotoxin (TeNT). TeNT can affect the central nervous system, inhibit the release of neurotransmitters, and result in respiratory failure, which are the root causes of death in tetanus patients. Identifying monoclonal antibodies (mAbs) targeting TeNT with neutralizing activity is urgently needed for the prevention and treatment of tetanus infection. In this study, through immunizing BALB/c mice with tetanus toxoid (TT), we obtained six positive hybridoma cell lines (1A7, 2C7, 3A7, 3H4, 4C1, and 4E12). Antibody isotyping showed that the antibodies are all of the IgG1/κ subclass. Ascites fluid was prepared by allogeneic ascites induction and the antibodies were purified through protein G affinity chromatography columns. Purities of the produced murine mAbs were all greater than 95%. All six antibodies bound to linear epitopes, among which 3A7 bound to the TeNT/L domain and the other five antibodies bound to the TeNT/Hc domain. Moreover, the affinity constants of these six antibodies against the antigen were all in the nanomolar range, and the affinity of 4E12 antibody reached the picomolar range. Results from toxin-neutralization assays in mice showed that 2C7 antibody delayed animal death, while 1A7, 3A7, 3H4, and 4E12 antibodies conferred partial protection. Additionally, 4C1 antibody offered complete protection, as 200 μg of 4C1 antibody fully protected against toxin challenge with 10 LD50 of TeNT and had a window period of 1 h. Antibody epitope grouping results revealed that the binding epitopes of 4C1 antibody were different from those of the other five antibodies. When 4C1 antibody was used in combination with another antibody, the neutralizing activities of antibodies were all evidently enhanced. Specifically, 4C1 combined with 3A7 antibody led to the greatest improvement in neutralizing activities, and 20 μg antibodies total (10 + 10 μg) fully protected against toxin challenge with 10 LD50. When 4E12, 3A7, and 4C1 antibodies were used in combination, 18 μg antibodies total (6 + 6 + 6 μg) completely neutralized 10 LD50 toxin. The present study derived murine mAbs with neutralizing activities and laid the foundation for follow-up therapeutic drug development for TeNT poisoning as well as establishment of TeNT detection methods.

CRISPR/Cas9-based Engineering of Immunoglobulin Loci in Hybridoma Cells.

Development of the hybridoma technology by Köhler and Milstein (1975) has revolutionized the immunological field by enabling routine use of monoclonal antibodies (mAbs) in research and development efforts, resulting in their successful application in the clinic today. While recombinant good manufacturing practices production technologies are required to produce clinical grade mAbs, academic laboratories and biotechnology companies still rely on the original hybridoma lines to stably and effortlessly produce high antibody yields at a modest price. In our own work, we were confronted with a major issue when using hybridoma-derived mAbs: there was no control over the antibody format that was produced, a flexibility that recombinant production does allow. We set out to remove this hurdle by genetically engineering antibodies directly in the immunoglobulin (Ig) locus of hybridoma cells. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and homology-directed repair (HDR) to modify antibody's format [mAb or antigen-binding fragment (Fab')] and isotype. This protocol describes a straightforward approach, with little hands-on time, leading to stable cell lines secreting high levels of engineered antibodies. Parental hybridoma cells are maintained in culture, transfected with a guide RNA (gRNA) targeting the site of interest in the Ig locus and an HDR template to knock in the desired insert and an antibiotic resistance gene. By applying antibiotic pressure, resistant clones are expanded and characterized at the genetic and protein level for their ability to produce modified mAbs instead of the parental protein. Finally, the modified antibody is characterized in functional assays. To demonstrate the versatility of our strategy, we illustrate this protocol with examples where we have (i) exchanged the constant heavy region of the antibody, creating chimeric mAb of a novel isotype, (ii) truncated the antibody to create an antigenic peptide-fused Fab' fragment to produce a dendritic cell-targeted vaccine, and (iii) modified both the constant heavy (CH)1 domain of the heavy chain (HC) and the constant kappa (Cκ) light chain (LC) to introduce site-selective modification tags for further derivatization of the purified protein. Only standard laboratory equipment is required, which facilitates its application across various labs. We hope that this protocol will further disseminate our technology and help other researchers. Graphical abstract.

Rapid and sensitive detection of quizalofop-p-ethyl by gold nanoparticle-based lateral flow immunoassay in agriproducts and environmental samples.

Quizalofop-p-ethyl is a widely used herbicide that poses a threat to human health and environmental safety. In this study, anti-quizalofop-p-ethyl monoclonal antibodies (mAbs) were prepared and used to develop a gold nanoparticle-based lateral flow immunoassay (AuNP-LFIA) for the detection of quizalofop-p-ethyl in agriproducts and environmental samples. Four hybridoma cell lines were obtained, among which 5B6D10E11 secreted mAb with the highest sensitivity, with a 50 % inhibition concentration of 4.57 ng/mL in the indirect competitive enzyme-linked immunosorbent assay. After optimization, the AuNP-LFIA strip based on the mAb (5B6D10E11) showed a visual detection limit of 10 ng/mL, and the results could be directly determined by the naked eye within 8 min. The cross-reactivity of the AuNP-LFIA for analogs of quizalofop-p-ethyl was negligible except for quizalofop-p-acid. The established AuNP-LFIA was proven to be accurate and precise based on the recovery test. Furthermore, the detection results of AuNP-LFIA were consistent with those of ultra-high-performance liquid chromatography tandem mass spectrometry.

Preparation and Biochemical Characteristics of a New IgG-Type Monoclonal Antibody against K Subgroup Avian Leukosis Virus.

This study focused on preparing a new IgG-type monoclonal antibody (MAb) against subgroup K avian leukosis virus (ALV-K) and identifying its biochemical characteristics. A specific gene fragment of ALV-K was amplified by polymerase chain reaction and expressed in E. coli. The purified expressed products were inoculated into BALB/c mice to prepare antibody-secreting spleen lymphocytes, and hybridoma cells were obtained after cell fusion of spleen lymphocytes and myeloma cells. A new hybridoma cell line named 30B9, which stably secreted IgG2b-antibody against ALV-K, was screened and contained 98 chromosomes. The MAb secreted by the 30B9 cells could recognize the ALV-K strain but not the ALV-A/B/J strains in an indirect immunofluorescence assay. Seventeen overlapping truncated ALV-K gp85 protein fragments were expressed, and eight peptides were artificially synthesized to analyze the MAb's antigen epitope by Western blot or enzyme-linked immunosorbent assay, and the results showed that the linear epitope was located on the 217-RRNYT-221 of ALV-K gp85 protein. A bioinformatics analysis showed that the epitope has a high antigenicity index, hydrophilicity, and surface accessibility and forms a unique linear spatial structure. Its five amino acids are highly conserved in all published ALV-K strains but are very low in ALV-A/B/J/C/D/E strains. This study provides a new biomaterial for developing specific detection methods against ALV-K.

Identification of Unique and Conserved Neutralizing Epitopes of Vestigial Esterase Domain in HA Protein of the H9N2 Subtype of Avian Influenza Virus.

The H9N2 subtype of avian influenza virus (AIV) has been reported to infect not only birds, but also humans. The hemagglutinin (HA) protein is the main surface antigen of AIV and plays an important role in the viral infection. For treatment strategies and vaccine development, HA protein has been an important target for the development of broadly neutralizing antibodies against influenza A virus. To investigate the vital target determinant cluster in HA protein in this work, HA gene was cloned and expressed in the prokaryotic expression vector pET28a. The spleen lymphocytes from BALC/c mice immunized with the purified recombinant HA protein were fused with SP2/0 cells. After Hypoxanthine-Aminopterin-Thymidine (HAT) medium screening and indirect ELISA detection, six hybridoma cell lines producing anti-HA monoclonal antibodies were screened. The gradually truncated HA gene expression and western blotting were used to identify their major locations in epitopes specific to these monoclonal antibodies. It was found that the epitopes were located in three areas: 112NVENLEEL119, 117EELRSLFS124, and 170PIQDAQ175. Epitope 112NVENLEEL119 has a partial amino acid crossover with 117EELRSLFS124, which is located in the vestigial esterase domain "110-helix" of HA, and the monoclonal antibody recognizing these epitopes showed the neutralizing activity, suggesting that the region 112NVENLEELRSLFS124 might be a novel neutralizing epitope. The results of the homology analysis showed that these three epitopes were generally conserved in H9N2 subtype AIV, and will provide valuable insights into H9N2 vaccine design and improvement, as well as antibody-based therapies for treatment of H9N2 AIV infection.

A recombinant Der p 1-specific allergen-toxin demonstrates superior killing of allergen-reactive IgG+ hybridomas in comparison to its recombinant allergen-drug conjugate.

Current treatments for asthma help to alleviate clinical symptoms but do not cure the disease. In this study, we explored a novel therapeutic approach for the treatment of house dust mite allergen Der p 1induced asthma by aiming to eliminate specific population of B-cells involved in memory IgE response to Der p 1. To achieve this aim, we developed and evaluated two different proDer p 1-based fusion proteins; an allergen-toxin (proDer p 1-ETA) and an allergen-drug conjugate (ADC) (proDer p 1-SNAP-AURIF) against Der p 1 reactive hybridomas as an in vitro model for Der p 1 reactive human B-cells. The strategy involved the use of proDer p 1 allergen as a cell-specific ligand to selectively deliver the bacterial protein toxin Pseudomonas exotoxin A (ETA) or the synthetic small molecule toxin Auristatin F (AURIF) into the cytosol of Der p 1 reactive cells for highly efficient cell killing. As such, we demonstrated recombinant proDer p 1 fusion proteins were selectively bound by Der p 1 reactive hybridomas as well as primary IgG1+ B-cells from HDM-sensitized mice. The therapeutic potential of proDer p 1-ETA' and proDer p 1-SNAP-AURIF was confirmed by their selective cytotoxic activities on Der p 1 reactive hybridoma cells. The allergen-toxin demonstrated superior cytotoxic activity, with IC50 values in the single digit nanomolar value, compared to the ADC. Altogether, the proof-of-concept experiments in this study provide a promising approach for the treatment of patients with house dust mite-driven allergic asthma.

Production, characterization, and application of phage-derived PK34 recombinant anti-microbial peptide.

PK34 is a D29 mycobacteriophage-derived anti-microbial peptide (AMP) with anti-Mycobacterium tuberculosis activity. It is expected to become an auxiliary drug for the treatment of M. tuberculosis infection, or as a template for the development of anti-M. tuberculosis drugs. The focus of this paper is to obtain recombinant PK34 by a novel method of prokaryotic expression and purification by affinity chromatography. The minimum inhibitory concentration (MIC) of recombinant PK34 was better than that of synthetic PK34 as measured by the microplate-based Alamar Blue assay (MABA). In order to further compare the different anti-bacterial effects of PK34 obtained by the two methods on M. tuberculosis, the bacterial changes after drug incubation were observed at the microscopic level by transmission electron microscopy (TEM). In order to apply PK34 to clinical treatment earlier in the future, this paper tested the maximum non-toxic concentration of recombinant PK34 to the two most studied immune cells, RAW264.7 and THP-1, through cytotoxicity experiments. The maximum non-toxic concentration was the same as the MIC of recombinant PK34 to M. tuberculosis H37Rv, and both were 12.5μg/mL. The monoclonal antibodies against PK34 and their hybridoma cell lines were prepared using recombinant PK34 as the antigen. Next, we obtained the gene sequence of the monoclonal antibody, which was prepared for the basic research of PK34 in M. tuberculosis treatment. In addition, the possible molecular docking mode between PK34 and trehalose-6,6-dimycolate (TDM) was predicted by AI simulation. To sum up, this paper provides a new idea for the birth of more new AMPs of the same type as PK34 in the future. KEY POINTS: • Design and prepare a novel recombinant PK34 anti-microbial peptide. • Recombinant PK34 has higher purity and anti-bacterial activity than synthetic PK34. • The monoclonal antibody against recombinant PK34 was prepared and sequenced.

The Investigation of Antitumor Effect of Humanized Bispecific BCMA/CD38 CAR-T Cells on Multiple Myeloma

Background Multiple myeloma (MM) is an incurable disease that will eventually deteriorate. Chimeric antigen receptor (CAR) T cell therapy has been widely used in the treatment of hematological malignancy and showed a promising effect. BCMA (CD269) is exclusively expressed on the MM cells, making it an ideal target for immunotherapy. CD38 is highly expressed on the surface of MM cells, but is also expressed in immune cells and a minority of hematopoietic stem/ progenitor cells. Humanization reshaped antibody may reduce the potential risk of immunogenicity associated with the animal-derived antibodies. In this study, we constructed humanized BCMA CAR-T, CD38 CAR-T and bispecific BCMA/CD38 CAR-T, and explored their killing efficiency towards MM cells, providing the basis for clinical application. Methods Mouse anti-human BCMA and CD38 single chain fragment variable (scFv) sequences were obtained from previously established monoclonal antibody hybridoma cells (69G8 and HIT2) in our institution, respectively. The sequences were then humanized. Single-target CAR (BCMA CAR, CD38 CAR) and dual-target BCMA/CD38 CAR (LoopBC38 CAR) plasmids were constructed. Phenotype of CAR-T cells was detected. CAR-T cells and tumor cell lines were co-cultured in vitro, followed by the detection of the percentage of residual tumor cells, the degranulation and cytokine release level of T cells. Bone marrow mononuclear cells (BMMNC) from MM patients were co-cultured with CAR-T cells. MM cells were intravenously injected into immunodeficient mice, followed by CAR-T cell therapy. After co-culturing with peripheral blood mononuclear cells (PBMC) from healthy donors and CD34+ umbilical cord blood (UCB) stem cells, the proportion, cell subsets of residual cells and colony formation ability were assessed to verify the safety of CAR-T cells. Results The proliferation ability of CD38 CAR-T and LoopBC38 CAR-T was weaker than that of VEC-T and BCMA CAR-T. The residual H929 cells (BCMA+CD38+) after 48h cocultured with VEC-T and LoopBC38 CAR-T at E:T ratio of 1:1 was (20.82±9.773) % vs. (0±0) % (p<0.0001). After 48h at E:T ratio of 1:1, residual K562-BCMA cells (BCMA+CD38-) cocultured with CD38 CAR-T were (49.53±9.773) %, while that of LoopBC38 CAR-T were (8.49±4,766) % (p<0.0001). After 48h at E:T ratio of 1:4, residual Raji cells (BCMA-CD38+) were (62.69±22.2) % in BCMA CAR-T and (19.81±12.22) % in LoopBC38 group (p<0.01). The secretion level of cytokines such as IFN-γ, TNF-α, IL-2 was significantly higher in CAR-T cells than that of VEC-T cells. After co-cultured with H929, the IL-6 secretion level of LoopBC38 CAR-T cells was (7.28±0.4558) %, which was lower than that of CD38 CAR-T cells [(259.4±3.039) %, p<0.0001]. When cocultured with BMMNC derived from MM patients, BCMA CAR-T could kill BCMA+ cells, CD38 CAR T could kill CD38+ cells, while LoopBC38 CAR-T has both effects. In H929 xenograft NSG mice, the median survival time of MM mice was 35 days for VEC-T, 82 days for CD38 CAR-T. Until now, more than 50% mice are alive in BCMA CAR-T group and LoopBC38 CAR-T group, so they didn't reach the median survival time yet. LoopBC38 CAR-T did not significantly kill PBMC form healthy donors, with the residual PBMC cells after 24h co-culturing was (48.26±1.708) % in VEC-T and (49.0±2.517%) in LoopBC38 CAR-T (p>0.05), but had killing efficiency towards mononuclear-macrophages and NK cell. A slight killing effect on UCB CD34+ cells was observed in LoopBC38 CAR-T. The residual CD34+ cells in LoopBC38 CAR-T were (29.47±4.077) %, while that of VEC-T was (48.5±11.88) % (p>0.05). The number of CFU-GEMM in LoopBC38 CAR-T was (3.0±1.512) %, which was lower than that of VEC-T [(4.667±3.386) %, (p<0.001)]. Conclusion All CAR-T cells showed strong killing efficiency toward MM cells both in vitro and in vivo. Humanized BCMA CAR-T and CD38 CAR-T can specifically kill multiple myeloma cell lines and primary cells of patients. Bispecific CAR-T was able to target both antigens. CD38 CAR-T and bispecific CAR-T cells showed self-killing ability and altered their phenotypes. They can kill NK cells, mononuclear-macrophages in peripheral blood of healthy donor and have certain effects on the formation of CFU-GEMM, which may be a safety problem that requires further improvement for the application of CD38 CAR-T and bispecific BCMA/CD38 CAR-T cells.

Advanced hybridoma technology for selective production of high-affinity monoclonal antibodies through B-cell receptors

In general, it is difficult to raise novel monoclonal antibodies against relatively low-molecular weight antigen, and particularly those with high homology for the mouse protein. The optimized B-cell targeting (BCT) technique can overcome this limitation. The point of this advanced technology is the selection of sensitized B lymphocytes by the antigen through B-cell receptors (BCRs). This strict selection by specific and strong interaction between antigen and antibody enables the efficient production of monoclonal antibodies with high specificity and affinity. It also offers the condensation of sensitized target B lymphocytes to selectively generate hybridoma cells secreting desired monoclonal antibodies. In this study, several kinds of biotinylated human myoglobin (hMyo) were prepared to select sensitized B lymphocytes via BCRs. Biotinylated hMyo prepared by a 3.75- and 7.5-fold molar excess of N-hydroxysuccinimide (NHS)-biotin provided high antigenicity of 68–88%. B lymphocytes selected by these biotinylated antigens had an ELISA-positive rate >17 times higher than that with usual biotinylated antigen. Monoclonal antibodies generated by the optimized BCT technology by preselecting sensitized B lymphocytes with the target antigen were identified to specifically recognize lower antigenic epitopes in hMyo with high affinity, while this would be impossible by the polyethylene glycol (PEG) method. Furthermore, combination of these high-affinity monoclonal antibodies gave the best binding rate in an epitope binning assay. These outcomes could be attributed to the unique characteristic that BCRs on sensitized B lymphocytes themselves can select the target epitopes in the antigen. The BCRs may act as a strict sensor of B lymphocytes to precisely select the target epitopes, even though the number of immunized B lymphocytes is low.

The Simultaneous Determination of Chlorpyrifos-Ethyl and -Methyl with a New Format of Fluorescence-Based Immunochromatographic Assay.

The improper and excessive use in agriculture of chlorpyrifos-methyl (CPSM) and chlorpyrifos-ethyl (CPSE) may affect the health of human beings. Herein, a fluorescence-based immunochromatographic assay (FICA) was developed for the simultaneous determination of CPSM and CPSE. A monoclonal antibody (mAb) with equal recognition of CPSM and CPSE was generated by the careful designing of haptens and screening of hybridoma cells. Instead of labeling fluorescence with mAb, the probe was labeled with goat-anti-mouse IgG (GAM-IgG) and pre-incubated with mAb in the sample. The complex could compete with CPS by coating antigen in the test line. The new format of FICA used goat-anti-rabbit IgG (GAR-IgG) conjugated with rabbit IgG labeled with fluorescence microspheres as an independent quality control line (C line). The novel strategy significantly reduced nonspecific reactions and increased assay sensitivity. Under the optimal conditions, the proposed FICA showed a linear range of 0.015-64 mg/L and limit of detection (LOD) of 0.015 mg/L for both CPSE and CPSM. The average recoveries of CPS from spiked food samples by FICA were 82.0-110.0%. The accuracy was similar to the gas chromatography-tandem mass spectrometry (GC-MS/MS) results. The developed FICA was an ideal on-site tool for rapid screening of CPS residues in foods.

Production and characterization of a murine anti-dal monoclonal antibody for blood typing in dogs

Considering the strong immunogenicity of the Dal antigen, and that > 98% of dogs, including blood donors, are Dal-positive, finding compatible blood for a previously transfused Dal-negative patient may be challenging. This is exacerbated by limited access to typing reagents, which currently rely on polyclonal antibodies (PAb) produced following sensitization of dogs. Therefore, the objective of this study was to produce and characterize an anti-Dal murine monoclonal antibody (MAb). Conventional hybridoma technology was used to produce MAb directed against canine red blood cells (cRBC). Briefly, female BALB/c mice were immunized via repeated intraperitoneal injections of washed Dal-positive cRBC (DEA 1,3,7 negative; DEA 4,5 positive) until serologic titers were sufficient (>1:1000). Following fusion with myeloma cells, 573 hybridoma cell culture supernatants were obtained and screened for MAb of interest using a gel column agglutination technique and known Dal-negative and Dal-positive cRBC. Fifteen supernatants led to cRBC agglutination, but only one had the desired pattern (i.e. anti-Dal). To assess its specificity and sensitivity, Dal blood typing of 62 canine EDTA-blood samples was performed using the anti-Dal MAb and two canine PAb: 45 Dal-positive and 17 Dal-negative were identified with 100% agreement between reagents (kappa =1). The anti-Dal MAb produced was further determined to be an IgG1. Conventional hybridoma technology, aided by a gel column technique, has enabled the production of a murine MAb specific against the canine Dal antigen. This will ensure long-term perennity of Dal blood typing, facilitate clinical management and research, as well as avoid resorting to repeat dog sensitization.

Isolation and characterization of full-length genes encoding the anti-human CD45 antibody from the hybridoma cell line 16E8-F2

Though the hybridoma technology has been widely applied in the production of monoclonal antibodies, it has existed some disadvantages including low yield and genetic instability. Therefore, an alternative approach should be taken into account. Recently, recombinant monoclonal antibody technology has emerged as the best choice to cure hybridoma related drawbacks. However, recombinant antibodies require known genes for their generation. The purpose of this study is to collect the full-length genes encoding the anti-human CD45 antibody derived from the hybridoma cell line 16E8-F2. In this research, we designed specific primer pairs to amplify the light and heavy chain genes of the antibody through the PCR method. Afterward, the genes were separately cloned into a cloning vector called pJET1.2/blunt. The generated recombinant pJET1.2 vectors will serve as the main material source to manufacture the recombinant monoclonal antibody recognizing human CD45 protein tomorrow.

Preparation of monoclonal antibodies against Pinellia ternata lectin protein and establishment of double-antibody sandwich ELISA

To determine the content of endogenous toxic substance Pinellia ternata lectin(PTL) protein in Pinelliae Rhizoma and the related processed products, this study prepared specific monoclonal antibodies against PTL by hybridoma cell technology, and established a quantitative double-antibody sandwich enzyme linked immunosorbent assay(ELISA) for PTL antigen. The detection conditions were 2.5 μg·mL~(-1) working concentration of the captured antibody and 1∶450 of the dilution multiple of detected antibody. The coating condition was staying overnight at 4 ℃. The blocking time and incubation times of antigen and detected antibody were all 90 minutes. The incubation time of horseradish peroxidase conjugated streptavidin-horseradish peroxidase(SA-HRP) was 15 minutes. The quantitative limit of the method for PTL antigen was 0.375 ng·mL~(-1). The linear range was 75.000-4 800.000 pg·mL~(-1), and R~2=0.997 1. The recovery rate was 90.0%-110.0%, and the variation coefficients of intra-test and inter-test precision were 2.0%-3.0% and 2.0%-8.5%.The content of PTL in three batches of Pinelliae Rhizoma and the related processed products was determined by the method, and the average content of PTL in Pinelliae Rhizoma was 35.42 mg·g~(-1). The average content of PTL in Pinelliae Rhizoma Praeparatum Cum Alumine, Pinelliae Rhizoma Praeparatum, and Pinelliae Rhizoma Praeparatum Cum Zingibere Et Alumine were 1.15 mg·g~(-1), 16.53 μg·g~(-1), and 122.63 ng·g~(-1), respectively, indicating that the content of PTL decreased significantly after processing. The quantitative double-antibody sandwich ELISA for PTL antigen established in this paper had good linearity, sensitive response, and high accuracy, which provided a simple and effective monitoring method for the detection of PTL content in the processing of Pinelliae Rhizoma.

Single-cell sorting using integrated pneumatic valve droplet microfluidic chip

Single-cell analysis and sorting have been increasingly prevalent in scientific study and industry in recent years. Droplet-based microfluidic systems are significant tools for single cell separation due to the advantages of high-throughput screening flux and relatively simple chip manufacturing. Herein, this work integrated single-cell droplet generation, incubation, and sorting functions into a single microfluidic chip using pneumatic microvalve for single-cell screening of hybridoma cells. The chip realized the generation, storage, incubation, and assay sorting of droplets in order by adjusting the switch of the microvalve. Numerical methods were used to simulate the effects of membrane size and pressure on valve deformation. Incubation results showed that fluorescent signals were generated in the droplets containing both hybridoma cells and microspheres under the control of pneumatic microvalves. In addition, the purity of target droplets was increased from 4.87% before sorting to 89.70% after sorting using fluorescence-activated droplet sorting platform, demonstrating the feasibility of the integrated chip with a pneumatic valve for single-cell screening. The integrated chip is proved to be operated efficiently with minimum cell loss and human labor through reducing the risk of droplet fusion and facilitating the development of automated devices. So, the present study provides an integrated design for microdroplet-based single-cell sorting with various applications, such as single-cell sequencing, antibody screening, and directed evolutionary studies.

Modulating the microenvironment during FVIII uptake influences the nature of FVIII-peptides presented by antigen-presenting cells.

Hemophilia A is a severe bleeding disorder caused by the deficiency of functionally active coagulation factor VIII (FVIII). The induction of neutralizing anti-drug antibodies is a major complication in the treatment of hemophilia A patients with FVIII replacement therapies. Why some patients develop neutralizing antibodies (FVIII inhibitors) while others do not is not well understood. Previous studies indicated that the induction of FVIII inhibitors requires cognate interactions between FVIII-specific B cells and FVIII-specific CD4+ T cells in germinal center reactions. In this study, we investigated the FVIII peptide repertoire presented by antigen-presenting cells (APCs) under different microenvironment conditions that are expected to alter the uptake of FVIII by APCs. The aim of this study was to better understand the association between different microenvironment conditions during FVIII uptake and the FVIII peptide patterns presented by APCs. We used a FVIII-specific CD4+ T cell hybridoma library derived from humanized HLA-DRB1*1501 (human MHC class II) hemophilic mice that were treated with human FVIII. APCs obtained from the same mouse strain were preincubated with FVIII under different conditions which are expected to alter the uptake of FVIII by APCs. Subsequently, these preincubated APCs were used to stimulate the FVIII-specific CD4+ T cell hybridoma library. Stimulation of peptide-specific CD4+ T-cell hybridoma clones was assessed by analyzing the IL-2 release into cell culture supernatants. The results of this study indicate that the specific microenvironment conditions during FVIII uptake by APCs determine the peptide specificities of subsequently activated FVIII-specific CD4+ T cell hybridoma clones. Incubation of APCs with FVIII complexed with von Willebrand Factor, FVIII activated by thrombin or FVIII combined with a blockade of receptors on APCs previously associated with FVIII uptake and clearance, resulted in distinct peptide repertoires of subsequently activated hybridoma clones. Based on our data we conclude that the specific microenvironment during FVIII uptake by APCs determines the FVIII peptide repertoire presented on MHC class II expressed by APCs and the peptide specificity of subsequently activated FVIII-specific CD4+ T cell hybridoma clones.

Hybrid Dynamic Models of Bioprocesses Based on Elementary Flux Modes and Multilayer Perceptrons

The derivation of minimal bioreaction models is of primary importance to develop monitoring and control strategies of cell/microorganism culture production. These minimal bioreaction models can be obtained based on the selection of a basis of elementary flux modes (EFMs) using an algorithm starting from a relatively large set of EFMs and progressively reducing their numbers based on geometric and least-squares residual criteria. The reaction rates associated with the selected EFMs usually have complex features resulting from the combination of different activation, inhibition and saturation effects from several culture species. Multilayer perceptrons (MLPs) are used in order to undertake the representation of these rates, resulting in a hybrid dynamic model combining the mass-balance equations provided by the EFMs to the rate equations described by the MLPs. To further reduce the number of kinetic parameters of the model, pruning algorithms for the MLPs are also considered. The whole procedure ends up with reduced-order macroscopic models that show promising prediction results, as illustrated with data of perfusion cultures of hybridoma cell line HB-58.