RESUMEN
We previously reported a chromatography system for purifying immunoglobulin M (IgM) using N,N,N',N'-ethylenediaminetetrakis(methylenephosphonic acid)-modified zirconia particles that selectively absorb immunoglobulins. Here, we report a simple procedure for preparing biotinylated IgM from hybridoma culture medium using this zirconia-based chromatography system. The culture medium of an IgM-producing hybridoma cell line was used as the starting sample solution, and the IgM in the medium was concentrated and partially purified by zirconia chromatography. Next, 9-(biotinamido)-4,7-dioxanonanoic acid N-succinimidyl ester was added to react with the proteins in the sample. Subsequently, only the biotinylated IgM was isolated by Capto Core 400 polishing column chromatography. The entire process was easy to perform, could be completed within 2 h, and provided highly pure biotin-labeled IgM. This procedure is expected to be applicable to the labeling of IgM with various compounds and drugs.
Asunto(s)
Biotinilación , Medios de Cultivo , Hibridomas , Inmunoglobulina M , Inmunoglobulina M/química , Inmunoglobulina M/aislamiento & purificación , Animales , Medios de Cultivo/química , Ratones , Circonio/química , Biotina/químicaRESUMEN
Introduction: Tularemia, caused by the bacterium Francisella tularensis, poses health risks to humans and can spread through a variety of routes. It has also been classified as a Tier 1 Select agent by the CDC, highlighting its potential as a bioterrorism agent. Moreover, it is difficult to diagnose in a timely fashion, owing to the non-specific nature of tularemia infections. Rapid, sensitive, and accurate detection methods are required to reduce mortality rates. We aimed to develop antibodies directed against the outer membrane protein A of F. tularensis (FopA) for rapid and accurate diagnosis of tularemia. Methods: We used a baculovirus insect cell expression vector system to produce the FopA antigen and generate anti-FopA antibodies through immunization of BALB/c mice. We then employed hybridoma and phage display technologies to screen for antibodies that could recognize unique epitopes on FopA. Result: Two monoclonal antibodies, 6B12 and 3C1, identified through phage display screening specifically bound to recombinant FopA in a dose-dependent manner. The binding affinity of the anti-FopA 6B12 and 3C1 antibodies was observed to have an equilibrium dissociation constant of 1.76 × 10-10 M and 1.32 × 10-9 M, respectively. These antibodies were used to develop a sandwich ELISA system for the diagnosis of tularemia. This assay was found to be highly specific and sensitive, with detection limits ranging from 0.062 ng/mL in PBS to 0.064 ng/mL in skim milk matrices. Discussion: Our findings demonstrate the feasibility of a novel diagnostic approach for detecting F. tularensis based on targeting FopA, as opposed to existing tests that target the bacterial lipopolysaccharide.
Asunto(s)
Anticuerpos Antibacterianos , Anticuerpos Monoclonales , Proteínas de la Membrana Bacteriana Externa , Francisella tularensis , Ratones Endogámicos BALB C , Proteínas Recombinantes , Tularemia , Tularemia/diagnóstico , Animales , Francisella tularensis/inmunología , Francisella tularensis/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/genética , Anticuerpos Monoclonales/inmunología , Ratones , Inmunoensayo/métodos , Sensibilidad y Especificidad , Femenino , Técnicas de Visualización de Superficie Celular , Epítopos/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/genética , Hibridomas , Baculoviridae/genéticaRESUMEN
Mpox (formerly known as monkeypox) is a zoonotic disease caused by monkeypox virus (MPXV), a DNA virus belonging to the Orthopoxvirus genus, in the Poxviridae family. The disease constitutes a moderate risk to public health at the global level. The MPXV A29L protein plays a crucial role in coordinating virion assembly and facilitating important virus-host interactions. This study focused on the expression, purification, and recombinant protein synthesis of the A29L protein of MPXV using prokaryotic systems. Using hybridoma technology, we successfully generated the monoclonal antibodies (mAbs) 1E12 and 4B2, which specifically recognize the A29L protein. These mAbs were found to be suitable for use in indirect immunofluorescence assays (IFA), Western blotting, and immunoprecipitation (IP). Our investigation also revealed that mAbs 1E12 and 4B2 could detect the A27L protein, a homologous protein found in the vaccinia virus Western Reserve (VACV WR) strain, using IFA, Western blotting, and immunoprecipitation (IP). Using mAbs 1E12 and 4B2 as primary immunological probes, A27L protein expression was detected as early as 6 h postinfection with VACV WR, with increasing protein levels being observed throughout the infection. This study enhances our understanding of the protein structure and function of MPXV and contributes to the development of specific MPXV detection methods.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Antivirales , Monkeypox virus , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/aislamiento & purificación , Animales , Ratones , Anticuerpos Antivirales/inmunología , Monkeypox virus/inmunología , Monkeypox virus/genética , Ratones Endogámicos BALB C , Proteínas Virales/inmunología , Proteínas Virales/genética , Humanos , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/genética , Femenino , Virus Vaccinia/inmunología , Virus Vaccinia/genética , HibridomasRESUMEN
Monoclonal antibodies (mAbs) hold significant potential as therapeutic agents and are invaluable tools in biomedical research. However, the lack of efficient high-throughput screening methods for single antibody-secreting cells (ASCs) has limited the diversity of available antibodies. Here, we introduce a novel, integrated workflow employing self-seeding microwells and an automated microscope-puncher system for the swift, high-throughput screening and isolation of single ASCs. The system allows for the individual screening and isolation of up to 6,400 cells within approximately one day, with the opportunity for parallelization and efficient upscaling. We successfully applied this workflow to both hybridomas and human patient-derived B cells, enabling subsequent clonal expansion or antibody sequence analysis through an optimized, single-cell nested reverse transcription-polymerase chain reaction (RT-PCR) procedure. By providing a time-efficient and more streamlined single ASC screening and isolation process, our workflow holds promise for driving forward progress in mAb development.
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Ensayos Analíticos de Alto Rendimiento , Hibridomas , Análisis de la Célula Individual , Humanos , Análisis de la Célula Individual/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Células Productoras de Anticuerpos/inmunología , Células Productoras de Anticuerpos/metabolismo , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/biosíntesisRESUMEN
The hybridoma method for production of monoclonal antibodies has been a cornerstone of biomedical research for several decades. Here we convert the monoclonal antibody sequence from mouse-derived hybridomas into a "devilized" recombinant antibody with devil IgG heavy chain and IgK light chain. The chimeric recombinant antibody can be used in functional assays, immunotherapy, and to improve understanding of antibodies and Fc receptors in Tasmanian devils. The process can be readily modified for other species.
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Hibridomas , Inmunoglobulina G , Marsupiales , Animales , Ratones , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Hibridomas/inmunología , Marsupiales/inmunología , Marsupiales/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Hybridoma technology is a well-established and indispensable tool for generating high-quality monoclonal antibodies and has become one of the most common methods for monoclonal antibody production. In this process, antibody-producing B cells are isolated from mice following immunization of mice with a specific immunogen and fused with an immortal myeloma cell line to form antibody-producing hybridoma cell lines. Hybridoma-derived monoclonal antibodies not only serve as powerful research and diagnostic reagents but have also emerged as the most rapidly expanding class of therapeutic biologicals. In spite of the development of new high-throughput monoclonal antibody generation technologies, hybridoma technology still is applied for antibody production due to its ability to preserve innate functions of immune cells and to preserve natural cognate antibody paring information. In this chapter, an overview of hybridoma technology and the laboratory procedures used for hybridoma production and antibody screening of peptide-specific antibodies are presented.
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Anticuerpos Monoclonales , Hibridomas , Péptidos , Hibridomas/inmunología , Animales , Ratones , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/biosíntesis , Péptidos/inmunología , Humanos , Ensayo de Inmunoadsorción Enzimática , Formación de Anticuerpos/inmunologíaRESUMEN
Objective: To construct a novel chimeric antigen receptor T (CAR-T) cell targeting CD138 and to investigate its cytotoxicity against myeloma cells. Methods: The hybridoma strain that can stably secrete the CD138 monoclonal antibody (mAb) was prepared and obtained through monoclonal antibody screening technology. The hybridoma strain cells were intraperitoneally injected into mice to produce ascites containing monoclonal antibodies, which were then collected and purified to obtain pure CD138 mAb. Further examinations were performed to assess the biological characteristics of CD138 mAb. The variable region sequence of this antibody was amplified through reverse transcription polymerase chain reaction and was used as the antigen recognition domain of CD138 CAR, which was subsequently expressed on the surface of T cells by lentiviral infection. Flow cytometry was employed to assess the phenotype of CD138 CAR-T cells. In vitro cytotoxicity and degranulation assays were performed to evaluate their antitumor effects. Results: â We successfully prepared anti-human CD138 antibody hybridoma cell lines and screened a hybridoma cell strain, 5G2, which could persistently and stably secrete the anti-CD138 antibody. â¡ The purified CD138 (5G2) mAb can especially recognize CD138(+) cells with a binding affinity constant (K(D)) of 6.011×10(-9) mol/L and showed no significant binding activity with CD138(-) cells. â¢The variable region sequence of the CD138 (5G2) antibody was obtained using molecular cloning technology, and CD138 (5G2) CAR was successfully constructed and expressed on T cells through lentivirus infection and, concurrently, demonstrated effective binding to recombinant human CD138 protein.⣠The proliferation of T cells transduced with the CD138 (5G2) CAR was highly efficient. The phenotype analysis revealed that CD138 (5G2) CAR-T cells exhibited a greater tendency to differentiate into central memory T cells and memory stem T cells, with a reduced proportion of terminally differentiated effector memory subsets. â¤CD138 (5G2) CAR-T cells demonstrated specific cytotoxicity against CD138(+) myeloma cell line H929, whereas CD138(-) cell line K562 remained unaffected. The percentage of residual H929 cells was (12.92±8.02) % after co-culturing with CD138 (5G2) CAR-T cells, while (54.25±15.79) % was left in the Vector-T group (Eâ¶T=1â¶2; P<0.001). â¥Results of degranulation assays demonstrated a significant activation of CD138 (5G2) CAR-T cells after co-culture with the H929 cell line, whereas no significant activation was observed in Vector-T cells [ (25.78±3.35) % vs (6.13±1.30) %, P<0.001]. â¦After co-culturing with CD138(+) cells, CD138 (5G2) CAR-T cells exhibited a significant increase in cytokine secretion compared to the Vector-T group [interleukin-2: (1 697.52±599.05) pg/ml vs (5.07±1.17) pg/ml, P<0.001; interferon-γ: (3 312.20±486.38) pg/ml vs (9.28±1.46) pg/ml, P<0.001; and tumor necrosis factor-α: (1 837.43±640.49) pg/ml vs (8.75±1.65) pg/ml, P<0.001]. However, no significant difference was observed in cytokine secretion levels between the two groups after co-culturing with CD138(-) cells. Conclusion: This study successfully prepared a novel monoclonal antibody against CD138, and CAR-T cells constructed with the antigen recognition domain derived from this 5G2 mAb demonstrated effective antitumor activity against myeloma cells. This can be used as a new option for the detection of the CD138 antigen and proposes a novel strategy for multiple myeloma immunotherapy.
Asunto(s)
Mieloma Múltiple , Receptores Quiméricos de Antígenos , Sindecano-1 , Linfocitos T , Mieloma Múltiple/terapia , Mieloma Múltiple/inmunología , Receptores Quiméricos de Antígenos/inmunología , Ratones , Animales , Humanos , Sindecano-1/inmunología , Linfocitos T/inmunología , Hibridomas , Inmunoterapia Adoptiva/métodos , Línea Celular Tumoral , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/genética , Anticuerpos Monoclonales/inmunologíaRESUMEN
The aim of this study was to prepare a mouse monoclonal antibody against the nonstructural protein 1 (NS1) of respiratory syncytial virus (RSV) to analyze its expression and distribution during transfection and infection. Additionally, we aimed to evaluate the antibody's application in immunoprecipitation assay. Firstly, the NS1 gene fragment was cloned into a prokaryotic plasmid and expressed in Escherichia coli. The resulting NS1 protein was then purified by affinity chromatography, and used to immunize the BALB/c mice. Subsequently, hybridoma cells capable of stably secreting the NS1 monoclonal antibody were selected using indirect enzyme linked immunosorbent assay (ELISA). This monoclonal antibody was employed in both indirect immunofluorescence assay (IFA) and Western blotting to analyze the expression and distribution of RSV NS1 in overexpressed and infected cells. Finally, the reliability of this monoclonal antibody was evaluated through the immunoprecipitation assay. The results showed that the RSV NS1 protein was successfully expressed and purified. Following immunization of mice with this protein, we obtained a highly specific RSV NS1 monoclonal antibody, which belonged to the IgG1 subtype with an antibody titer of 1:15 360 000. Using this monoclonal antibody, the RSV NS1 protein was identified in both transfected and infected cells. The IFA results revealed predominant distribution of NS1 in the cytoplasm and nucleus. Moreover, we confirmed that this monoclonal antibody could effectively bind specifically to NS1 protein in cell lysates, making it suitable as a capture antibody in immunoprecipitation assay. In conclusion, our study successfully achieved production of the RSV NS1 protein through a prokaryotic expression system and prepared a specific monoclonal antibody against NS1. This antibody demonstrates the ability to specifically identify the NS1 protein and can be used in the immunoprecipitation assay, thereby laying a foundation for the functional studies of the NS1 protein.
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Anticuerpos Monoclonales , Proteínas no Estructurales Virales , Animales , Femenino , Ratones , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Anticuerpos Antivirales/inmunología , Escherichia coli/genética , Escherichia coli/metabolismo , Hibridomas/inmunología , Ratones Endogámicos BALB C , Virus Sincitiales Respiratorios/inmunología , Virus Sincitiales Respiratorios/genética , Proteínas no Estructurales Virales/inmunología , Proteínas no Estructurales Virales/genéticaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales , Animales , Femenino , Ratones , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Escherichia coli/genética , Escherichia coli/metabolismo , Hibridomas/inmunología , Hibridomas/metabolismo , Ratones Endogámicos BALB C , Proteínas de la Nucleocápside/inmunología , Proteínas de la Nucleocápside/genética , Orthobunyavirus/inmunología , Orthobunyavirus/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
The antibodies to the microtubule-associated protein tau play a role in basic and clinical studies of Alzheimer's disease (AD) and other tauopathies. With the recombinant human tau441 as the immunogen, the hybridoma cell strains secreting the anti-human tau N-terminal domain (NTD-tau) monoclonal antibodies were generated by cell fusion and screened by limiting dilution. The purified monoclonal antibodies were obtained by inducing the mouse ascites and affinity chromatography. The sensitivity and specificity of the monoclonal antibodies were examined by indirect ELISA and Western blotting, respectively. A double antibody sandwich ELISA method for detecting human tau protein was established and optimized. The results showed that the positive cloning rate of hybridoma cells was 83.6%. A stable cell line producing ZD8F7 antibodies was established, and the antibody titer in the supernatant of the cell line was 1:16 000. The antibody titer in the ascitic fluid was higher than 1:256 000; and the titer of purified ZD8F7 monoclonal antibodies was higher than 1:128 000. The epitope analysis showed that the ZD8F7 antibody recognized tau21-37 amino acid in the N-terminal domain. The Western blotting results showed that the ZD8F7 antibody recognized the recombinant human tau protein of 50-70 kDa and the human tau protein of 50 kDa in the brain tissue of transgenic AD model mice (APP/PS1/tau). With ZD8F7 as a capture antibody, a quantitative detection method for human tau protein was established, which showed a linear range of 7.8-500.0 pg/mL and could identify human tau protein in the brain tissue of AD transgenic mice and human plasma but not recognize the mouse tau protein. In conclusion, the human NTD-tau-specific monoclonal antibody and the double antibody sandwich ELISA method established in this study are highly sensitive and can serve as a powerful tool for the detection of tau protein in neurodegenerative diseases.
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Enfermedad de Alzheimer , Anticuerpos Monoclonales , Proteínas tau , Proteínas tau/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/biosíntesis , Humanos , Ratones , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/sangre , Ensayo de Inmunoadsorción Enzimática , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Hibridomas/inmunología , Ratones Endogámicos BALB C , Especificidad de Anticuerpos , Dominios Proteicos , Epítopos/inmunologíaRESUMEN
The importance of humoral immunity to fungal infections remains to be elucidated. In cryptococcosis, patients that fail to generate antibodies against antigens of the fungus Cryptococcus neoformans are more susceptible to the disease, demonstrating the importance of these molecules to the antifungal immune response. Historically, antibodies against C. neoformans have been applied in diagnosis, therapeutics, and as important research tools to elucidate fungal biology. Throughout the process of generating monoclonal antibodies (mAbs) from a single B-cell clone and targeting a single epitope, several immunization steps might be required for the detection of responsive antibodies to the antigen of interest in the serum. This complex mixture of antibodies comprises the polyclonal antibodies. To obtain mAbs, B-lymphocytes are harvested (from spleen or peripheral blood) and fused with tumor myeloma cells, to generate hybridomas that are individually cloned and specifically screened for mAb production. In this chapter, we describe all the necessary steps, from the immunization to polyclonal antibody harvesting, hybridoma generation, and mAb production and purification. Additionally, we discuss new cutting-edge approaches for generating interspecies mAbs, such as humanized mAbs, or for similar species in distinct host backgrounds.
Asunto(s)
Anticuerpos Antifúngicos , Anticuerpos Monoclonales , Cryptococcus neoformans , Hibridomas , Cryptococcus neoformans/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Animales , Humanos , Hibridomas/inmunología , Anticuerpos Antifúngicos/inmunología , Anticuerpos Antifúngicos/aislamiento & purificación , Ratones , Linfocitos B/inmunología , Criptococosis/inmunología , Criptococosis/diagnóstico , Antígenos Fúngicos/inmunología , InmunizaciónRESUMEN
Objective To prepare a monoclonal antibody (mAb) against mouse NOD-like receptor family pyrin domain-containing 3 (NLRP3) and assess its specificity. Methods A gene fragment encoding mouse NLRP3 exon3 (Ms-N3) was inserted into the vector p36-G3-throhFc to construct a recombinant plasmid named Ms-N3-throhFc. This plasmid was then transfected into HEK293F cells for eukaryotic expression. NLRP3-/- mice were immunized with Ms-N3 protein purified using a protein A chromatography column, and splenocytes from the immunized mice were fused with SP2/0 myeloma cells to generate hybridoma cells. Specific mAbs against murine NLRP3 from hybridoma cells were screened using ELISA and immunofluorescence assay(IFA). Results The Ms-N3-throhFc recombinant plasmid was successfully constructed and exhibited stable expression in HEK293F cells. Twelve hybridoma cell lines were initially screened using ELISA. IFA revealed that the mAb secreted by the 9-B8-3-2-C5 cell line specifically recognized the native form of mouse NLRP3 protein. The heavy and light chain subtypes of this mAb were identified as IgM and κ, respectively. Conclusion A monoclonal antibody against mouse NLRP3 has been successfully prepared.
Asunto(s)
Anticuerpos Monoclonales , Proteína con Dominio Pirina 3 de la Familia NLR , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/aislamiento & purificación , Animales , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Humanos , Células HEK293 , Hibridomas , Transfección , Exones , Clonación Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Ensayo de Inmunoadsorción EnzimáticaRESUMEN
BACKGROUND: Middle East Respiratory Syndrome Coronavirus is a highly pathogenic virus that poses a significant threat to public health. OBJECTIVE: The purpose of this study is to develop and characterize novel mouse monoclonal antibodies targeting the spike protein S1 subunit of the Middle East Respiratory Syndrome Corona Virus (MERS-CoV). METHODS: In this study, three mouse monoclonal antibodies (mAbs) against MERS-CoV were generated and characterized using hybridoma technology. The mAbs were evaluated for their reactivity and neutralization activity. The mAbs were generated through hybridoma technology by the fusion of myeloma cells and spleen cells from MERS-CoV-S1 immunized mice. The resulting hybridomas were screened for antibody production using enzyme-linked immunosorbent assays (ELISA). RESULTS: ELISA results demonstrated that all three mAbs exhibited strong reactivity against the MERS-CoV S1-antigen. Similarly, dot-ELISA revealed their ability to specifically recognize viral components, indicating their potential for diagnostic applications. Under non-denaturing conditions, Western blot showed the mAbs to have robust reactivity against a specific band at 116 KDa, corresponding to a putative MERS-CoV S1-antigen. However, no reactive bands were observed under denaturing conditions, suggesting that the antibodies recognize conformational epitopes. The neutralization assay showed no in vitro reactivity against MERS-CoV. CONCLUSION: This study successfully generated three mouse monoclonal antibodies against MERS-CoV using hybridoma technology. The antibodies exhibited strong reactivity against MERS-CoV antigens using ELISA and dot ELISA assays. Taken together, these findings highlight the significance of these mAbs for potential use as valuable tools for MERS-CoV research and diagnosis (community and field-based surveillance and viral antigen detection).
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Ensayo de Inmunoadsorción Enzimática , Hibridomas , Coronavirus del Síndrome Respiratorio de Oriente Medio , Glicoproteína de la Espiga del Coronavirus , Animales , Glicoproteína de la Espiga del Coronavirus/inmunología , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Anticuerpos Monoclonales/inmunología , Ratones , Anticuerpos Antivirales/inmunología , Hibridomas/inmunología , Humanos , Anticuerpos Neutralizantes/inmunología , Ratones Endogámicos BALB C , Epítopos/inmunología , Pruebas de Neutralización , Especificidad de Anticuerpos/inmunología , Infecciones por Coronavirus/inmunología , FemeninoRESUMEN
Avian influenza (AI), caused by H9N2 subtype avian influenza virus (AIV), poses a serious threat to poultry farming and public health due to its transmissibility and pathogenicity. The PB2 protein is a major component of the viral RNA polymerase complex. It is of great importance to identify the antigenic determinants of the PB2 protein to explore the function of the PB2 protein. In this study, the PB2 sequence of H9N2 subtype AIV, from 1090 to 1689 bp, was cloned and expressed. The recombinant PB2 protein with cutting gel was used to immunize BALB/c mice. After cell fusion, the hybridoma cell lines secreting monoclonal antibodies (mAbs) targeting the PB2 protein were screened by indirect ELISA and western blotting, and the antigenic epitopes of mAbs were identified by constructing truncated overlapping fragments in the PB2 protein of H9N2 subtype AIV. The results showed that three hybridoma cell lines (4B7, 4D10, and 5H1) that stably secreted mAbs specific to the PB2 protein were screened; the heavy chain of 4B7 was IgG2α, those of 4D10 and 5H1 were IgG1, and all three mAbs had kappa light chain. Also, the minimum B-cell epitope recognized was 475LRGVRVSK482 and 528TITYSSPMMW537. Homology analysis showed that these two epitopes were conserved among the different subtypes of AIV strains and located on the surface of the PB2 protein. The above findings provide an experimental foundation for further investigation of the function of the PB2 protein and developing monoclonal antibody-based diagnostic kits.
Asunto(s)
Anticuerpos Monoclonales , Epítopos de Linfocito B , Subtipo H9N2 del Virus de la Influenza A , Gripe Aviar , Ratones Endogámicos BALB C , Proteínas Virales , Subtipo H9N2 del Virus de la Influenza A/inmunología , Subtipo H9N2 del Virus de la Influenza A/genética , Animales , Anticuerpos Monoclonales/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunología , Proteínas Virales/metabolismo , Ratones , Gripe Aviar/virología , Gripe Aviar/inmunología , Epítopos de Linfocito B/inmunología , Hibridomas , ARN Polimerasa Dependiente del ARN/genética , Anticuerpos Antivirales/inmunología , Pollos/virología , FemeninoRESUMEN
Antibodies that specifically bind to individual human fragment crystallizable γ receptors (FcγRs) are of interest as research tools in studying immune cell functions, as well as components in bispecific antibodies for immune cell engagement in cancer therapy. Monoclonal antibodies for human low-affinity FcγRs have been successfully generated by hybridoma technology and are widely used in pre-clinical research. However, the generation of monoclonal antibodies by hybridoma technology that specifically bind to the high-affinity receptor FcγRI is challenging. Monomeric mouse IgG2a, IgG2b, and IgG3 bind human FcγRI with high affinity via the Fc part, leading to an Fc-mediated rather than a fragment for antigen binding (Fab)-mediated selection of monoclonal antibodies. Blocking the Fc-binding site of FcγRI with an excess of human IgG or Fc during screening decreases the risk of Fc-mediated interactions but can also block the potential epitopes of new antibody candidates. Therefore, we replaced hybridoma technology with phage display of a single-chain fragment variable (scFv) antibody library that was generated from mice immunized with FcγRI-positive cells and screened it with a cellular panning approach assisted by next-generation sequencing (NGS). Seven new FcγRI-specific antibody sequences were selected with this methodology, which were produced as Fc-silent antibodies showing FcγRI-restricted specificity.
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Anticuerpos Monoclonales , Receptores de IgG , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Animales , Ratones , Humanos , Anticuerpos Monoclonales/inmunología , Inmunoglobulina G/inmunología , Inmunización , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/genética , Biblioteca de Péptidos , Técnicas de Visualización de Superficie Celular , Hibridomas , Especificidad de Anticuerpos , Femenino , Ratones Endogámicos BALB CRESUMEN
When purifying mAb from serum-containing hybridoma culture supernatant, it is essential that mouse IgG remains free from contaminations of bovine IgG. However, the broadly used Protein A resin cannot achieve this goal due to binding between both mouse and bovine IgG. Here, a novel nanobody-based affinity purification magnetic beads that discriminates mouse IgG from bovine IgG was developed. To bind all subtypes of mouse IgG (IgG1, IgG2a, IgG2b and IgG3) that contain the kappa light chain, mCK (mouse kappa constant region)-specific nanobody binders were selected from an immune phage display VHH library; this library was constructed with peripheral blood mononuclear cells (PBMCs), which were collected from Bactrian camels immunized with a mix of intact mouse IgGs (IgG1, IgG2a, IgG2b and IgG3). A novel clone that exhibited a higher expression level and a higher binding affinity was selected (4E6). Then, the 4E6 nanobody in the format of VHH-hFC (human Fc) was conjugated on magnetic beads with a maximal binding capacity of 15.41±0.69 mg mouse IgG/mL beads. Furthermore, no bovine IgG could be copurified from hybridoma culture supernatant with immunomagnetic beads. This approach is valuable for the large-scale in vitro production of highly pure antibodies by hybridoma cells.
Asunto(s)
Anticuerpos Monoclonales , Animales , Bovinos , Humanos , Ratones , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Camelus , Cromatografía de Afinidad/métodos , Hibridomas , Regiones Constantes de Inmunoglobulina/química , Inmunoglobulina G/aislamiento & purificación , Inmunoglobulina G/inmunología , Cadenas kappa de Inmunoglobulina/inmunología , Cadenas kappa de Inmunoglobulina/química , Biblioteca de Péptidos , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/aislamiento & purificaciónRESUMEN
Monoclonal antibody (mAb) discovery plays a prominent role in diagnostic and therapeutic applications. Droplet microfluidics has become a standard technology for high-throughput screening of antibody-producing cells due to high droplet single-cell confinement frequency and rapid analysis and sorting of the cells of interest with their secreted mAbs. In this work, a new method is described for on-demand co-encapsulation of cells that eliminates the difficulties associated with washing in between consecutive steps inside the droplets and enables the washing and addition of fresh media. The new platform identifies hybridoma cells that are expressing antibodies of interest using antibody-characterization assays to find the best-performing or rare-cell antibody candidates.
Asunto(s)
Anticuerpos Monoclonales , Microfluídica , Anticuerpos Monoclonales/química , Microfluídica/métodos , Animales , Hibridomas/citología , Análisis de la Célula Individual/métodos , Ratones , Humanos , Automatización , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodosRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales , Proteína S100A12 , Porcinos , Animales , Hibridomas , Calgranulina A , Calgranulina B , TecnologíaRESUMEN
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.