scholarly journals Hybridoma technology; advancements, clinical significance, and future aspects

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Sanchita Mitra ◽  
Pushpa Chaudhary Tomar
Keyword(s):  
Monoclonal Antibodies ◽  
Clinical Significance ◽  
Cell Lines ◽  
Tumor Antigens ◽  
Specific Antigen ◽  
Hybridoma Cells ◽  
Main Body ◽  
Highly Sensitive ◽  
Hybridoma Technology

Abstract Background Hybridoma technology is one of the most common methods used to produce monoclonal antibodies. In this process, antibody-producing B lymphocytes are isolated from mice after immunizing the mice with specific antigen and are fused with immortal myeloma cell lines to form hybrid cells, called hybridoma cell lines. These hybridoma cells are cultured in a lab to produce monoclonal antibodies, against a specific antigen. This can be achieved by an in vivo or an in vitro method. It is preferred above all the available methods to produce monoclonal antibodies because antibodies thus produced are of high purity and are highly sensitive and specific. Main body of the abstract Monoclonal antibodies are useful in diagnostic, imaging, and therapeutic purposes and have a very high clinical significance. Once hybridoma cells become stable, these cell lines offer limitless production of homogenized antibodies. This method is also cost-effective. The antibodies produced by this method are highly sensitive and specific to the targeted antigen. It is an important tool used in various fields of research such as in toxicology, animal biotechnology, medicine, pharmacology, cell, and molecular biology. Monoclonal antibodies are used extensively in the diagnosis and therapeutic applications. Radiolabeled monoclonal antibodies are used as probes to detect tumor antigens in the living system; also radioisotope coupled antibodies are used for therapeutic target specific action on oncogenic cells. Short conclusion Presently, the monoclonal antibodies used are either raised in mice or rats; this poses a risk of disease transfer from mice to humans. There is no guarantee that antibodies thus created are entirely virus-free, despite the purification process. Also, there are some immunogenic responses observed against the antibodies of mice origin. Technologically advanced techniques such as genetic engineering helped in reducing some of these limitations. Advanced methods are under development to make lab-produced monoclonal antibodies as human as possible. This review discusses the advantages and challenges associated with monoclonal antibody production, also enlightens the advancement, clinical significance, and future aspects of this technique.

scholarly journals Innovative and Highly Sensitive Detection of Clostridium perfringens Enterotoxin Based on Receptor Interaction and Monoclonal Antibodies

Toxins ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 266
Author(s):  
Thea Neumann ◽  
Maren Krüger ◽  
Jasmin Weisemann ◽  
Stefan Mahrhold ◽  
Daniel Stern ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Clostridium Perfringens ◽  
Food Poisoning ◽  
Receptor Interaction ◽  
Clostridium Perfringens Enterotoxin ◽  
Fast Detection ◽  
Highly Sensitive ◽  
Basic And Applied Research ◽  
Highly Sensitive Detection

Clostridium perfringens enterotoxin (CPE) regularly causes food poisoning and antibiotic-associated diarrhea; therefore, reliable toxin detection is crucial. To this aim, we explored stationary and mobile strategies to detect CPE either exclusively by monoclonal antibodies (mAbs) or, alternatively, by toxin-enrichment via the cellular receptor of CPE, claudin-4, and mAb detection. Among the newly generated mAbs, we identified nine CPE-specific mAbs targeting five distinct epitopes, among them mAbs recognizing CPE bound to claudin-4 or neutralizing CPE activity in vitro. In surface plasmon resonance experiments, all mAbs and claudin-4 revealed excellent affinities towards CPE, ranging from 0.05 to 2.3 nM. Integrated into sandwich enzyme-linked immunosorbent assays (ELISAs), the most sensitive mAb/mAb and claudin-4/mAb combinations achieved similar detection limits of 0.3 pg/mL and 1.0 pg/mL, respectively, specifically detecting recombinant CPE from spiked feces and native CPE from 30 different C. perfringens culture supernatants. The implementation of mAb- and receptor-based ELISAs into a mobile detection platform enabled the fast detection of CPE, which will be helpful in clinical laboratories to diagnose diarrhea of assumed bacterial origin. In conclusion, we successfully employed an endogenous receptor and novel high affinity mAbs for highly sensitive and specific CPE-detection. These tools will be useful for both basic and applied research.

scholarly journals C57BL/6 congenic mouse NRASQ61Kmelanoma cell lines are highly sensitive to the combination of Mek and Akt inhibitors in vitro and in vivo

2019 ◽  
Vol 32 (6) ◽  
pp. 829-841 ◽  
Author(s):  
Valérie Petit ◽  
Jeremy Raymond ◽  
Christophe Alberti ◽  
Marie Pouteaux ◽  
Stuart J. Gallagher ◽  
...  
Keyword(s):  
Cell Lines ◽  
Congenic Mouse ◽  
Highly Sensitive

The expression of nonagglutinating fimbriae and its role inProteus mirabilisadherence to epithelial cells

1997 ◽  
Vol 43 (8) ◽  
pp. 709-717 ◽  
Author(s):  
Douglas L. Tolson ◽  
Blair A. Harrison ◽  
Roger K. Latta ◽  
Kok K. Lee ◽  
Eleonora Altman
Keyword(s):  
Monoclonal Antibodies ◽  
Urinary Tract ◽  
Cell Lines ◽  
Proteus Mirabilis ◽  
Growth Conditions ◽  
Structural Abnormalities ◽  
Uroepithelial Cell ◽  
Uroepithelial Cells ◽  
Tract Infections

Proteus mirabilis is a common causative agent of human urinary tract infections, especially in catheterized patients and in those patients with structural abnormalities of the urinary tract. In addition to the production of hemolysin and urease, fimbriae-mediated adherence to uroepithelial cells and kidney epithelium may be essential for virulence of P. mirabilis. A single P. mirabilis strain is capable of expressing several morphologically distinct fimbrial species, which can each be favoured by specific in vitro growth conditions. The fimbrial species reported to date include mannose-resistant/Proteus-like fimbriae, ambient temperature fimbriae, P. mirabilis fimbriae, and nonagglutinating fimbriae (NAF). Here, using intact bacteria or purified NAF as immunogens, we have generated the first reported NAF-specific monoclonal antibodies (mAbs). Bacteria expressing NAF as their only fimbrial species adhered strongly to a number of cell lines in vitro, including uroepithelial cell lines. Binding of P. mirabilis was markedly reduced following preincubation with NAF-specific mAbs and Fab fragments. The presence of NAF with highly conserved N-terminal sequences on all P. mirabilis strains so far examined, combined with the ability of both anti-NAF mAbs and purified NAF molecules to inhibit P. mirabilis adherence in vitro, suggests that NAF may contribute to the pathogenesis of P. mirabilis.Key words: fimbriae, adherence, monoclonal antibodies, Proteus mirabilis, receptors.

Production of species-specific monoclonal antibodies that react with surface components on zoospores and cysts of Phytophthora nicotianae

1998 ◽  
Vol 44 (12) ◽  
pp. 1161-1170 ◽  
Author(s):  
A V Robold ◽  
A R Hardham
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Lines ◽  
Phytophthora Cinnamomi ◽  
Phytophthora Nicotianae ◽  
The Other ◽  
Hybridoma Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Antibodies ◽  
Hybridoma Cell Lines ◽  
Species Specific

Monoclonal antibodies were generated against components on the surface of zoospores and cysts of the Oomycete, Phytophthora nicotianae, with the aim of obtaining antibodies diagnostic for this species of plant pathogen. A dipstick version of an enzyme-linked immunosorbent assay was used to screen hybridoma cell lines produced by following a coimmunization protocol in which a mouse was immunized with Phytophthora nicotianae cysts mixed with murine antisera raised against cysts of Phytophthora cinnamomi and Phytophthora cryptogea. Of the nine hybridoma cells lines which remained positive, five produced antibodies that reacted with species-specific epitopes on the surface of the spores. Immunofluorescence, immunogold, and immunoblot labelling showed that three of the five species-specific antibodies reacted with a polypeptide of relative molecular mass greater than 205 kDa which was distributed over the entire zoospore surface, including that of the two flagella. These antibodies also labelled the surface of cysts to varying degrees. The other two species-specific antibodies bound to the shaft of tubular mastigonemes that form two rows on the anterior flagellum. In immunoblots, one of these antibodies recognised a 40-kDa glycoprotein. Antibodies produced by the other four hybridoma cell lines reacted with all Phytophthora and Pythium species tested. The results (i) showed that the coimmunization technique effectively produced antibodies directed towards components specific for Phytophthora nicotianae in the presence of antigens common to many Phytophthora species, and (ii) revealed for the first time the biochemical nature of molecular constituents of flagellar mastigonemes in the Oomycetes.Key words: cell surface, flagella, immunodiagnostics, mastigonemes, monoclonal antibodies.

Connecting gene expression subtypes of colorectal cancer (CRC) with cell lines and drug resistance.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e14544-e14544
Author(s):  
Eva Budinska ◽  
Jenny Wilding ◽  
Vlad Calin Popovici ◽  
Edoardo Missiaglia ◽  
Arnaud Roth ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Clinical Significance ◽  
Cell Lines ◽  
Drug Response ◽  
Drug Sensitivity ◽  
Expression Profiles ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis

e14544 Background: We identified CRC gene expression subtypes (ASCO 2012, #3511), which associate with established parameters of outcome as well as relevant biological motifs. We now substantiate their biological and potentially clinical significance by linking them with cell line data and drug sensitivity, primarily attempting to identify models for the poor prognosis subtypes Mesenchymal and CIMP-H like (characterized by EMT/stroma and immune-associated gene modules, respectively). Methods: We analyzed gene expression profiles of 35 publicly available cell lines with sensitivity data for 82 drug compounds, and our 94 cell lines with data on sensitivity for 7 compounds and colony morphology. As in vitro, stromal and immune-associated genes loose their relevance, we trained a new classifier based on genes expressed in both systems, which identifies the subtypes in both tissue and cell cultures. Cell line subtypes were validated by comparing their enrichment for molecular markers with that of our CRC subtypes. Drug sensitivity was assessed by linking original subtypes with 92 drug response signatures (MsigDB) via gene set enrichment analysis, and by screening drug sensitivity of cell line panels against our subtypes (Kruskal-Wallis test). Results: Of the cell lines 70% could be assigned to a subtype with a probability as high as 0.95. The cell line subtypes were significantly associated with their KRAS, BRAF and MSI status and corresponded to our CRC subtypes. Interestingly, the cell lines which in matrigel created a network of undifferentiated cells were assigned to the Mesenchymal subtype. Drug response studies revealed potential sensitivity of subtypes to multiple compounds, in addition to what could be predicted based on their mutational profile (e.g. sensitivity of the CIMP-H subtype to Dasatinib, p<0.01). Conclusions: Our data support the biological and potentially clinical significance of the CRC subtypes in their association with cell line models, including results of drug sensitivity analysis. Our subtypes might not only have prognostic value but might also be predictive for response to drugs. Subtyping cell lines further substantiates their significance as relevant model for functional studies.

scholarly journals Obtaining and Characterization of the Monoclonal Antibodies Against G-Protein of the Respiratory Syncytial Virus

2020 ◽  
pp. 7-14
Author(s):  
N. A. Demidova ◽  
R. R. Klimova ◽  
A. A. Kushch ◽  
E. I. Lesnova ◽  
O. V. Masalova ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Respiratory Syncytial Virus ◽  
G Protein ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rsv Infection ◽  
Hybridoma Technology ◽  
Infected Cells ◽  
Syncytial Virus

The aim of this study was to obtain hybridomas producing monoclonal antibodies (Mabs) to the G-protein of the respiratory syncytial virus (RSV), and to evaluate their immunological characteristics and virus-neutralizing activity.Material and methods. Mouse Mabs were obtained using hybridoma technology. The properties of Mabs were studied by enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining (IF) of infected cells, as well as by biological neutralization test in vitro (NT). To identify epitopes recognized by the Mabs on G protein ELISA additivity test was used.Results. Hybridization of splenocytes with Sp2/0 myeloma cells and primary screening showed that 75 hybridomas produce antibodies interacting with purified virus, 17 of them also react with the recombinant G-protein in ELISA. In NT 4, hybridomas suppressed in vitro RSV infection by more than 50%. Cloning of these hybridomas revealed 4 monoclones producing the most active Mabs. Mab 1C11 was IgG2a, 3 others (5D4, 5G11 and 6H4) were IgM. Three IgM Mabs actively reacted with both RSV A2 and Long, and with G-protein; Mab 1C11 was less reactive with all antigens tested. All Mabs suppressed RSV infection, while Mab 5D4 supressed it almost completely (98%). IF analysis showed that all Mabs detected RSV G-protein in the cell cytoplasm, the largest number of infected cells was detected using Mab 5D4 (80%). It was shown that the isolated Mabs were directed to two non-overlapping epitopes on the RSV G-protein.Conclusion. The isolated Mabs can be used to detect RSV in clinical samples by ELISA and IF. The isolated Mabs can be used for humanized recombinant antibodies construction and for the treatment of RSV infection in future.

scholarly journals Adapter Chimeric Antigen Receptor (AdCAR)-Engineered NK-92 Cells for the Multiplex Targeting of Bone Metastases

Cancers ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 1124
Author(s):  
Stefan Grote ◽  
Frank Traub ◽  
Joerg Mittelstaet ◽  
Christian Seitz ◽  
Andrew Kaiser ◽  
...  
Keyword(s):  
Bone Metastases ◽  
Cell Lines ◽  
Tumor Antigens ◽  
Fatal Outcome ◽  
In Vivo Models ◽  
Established Cell ◽  
Metastatic Spreading ◽  
Pro Inflammatory Cytokines

Background: Since metastatic spreading of solid tumor cells often leads to a fatal outcome for most cancer patients, new approaches for patient-individualized, targeted immunotherapy are urgently needed. Methods: Here, we established cell lines from four bone metastases of different tumor entities. We assessed AdCAR NK-92-mediated cytotoxicity in vitro in standard cytotoxicity assays as well as 3D spheroid models Results: AdCAR-engineered NK-92 cells successfully demonstrated distinct and specific cytotoxic potential targeting different tumor antigens expressed on cell lines established from bone metastases of mammary, renal cell and colorectal carcinoma as well as melanomas. In that process AdCAR NK-92 cells produced a multitude of NK effector molecules as well as pro inflammatory cytokines. Furthermore, AdCAR NK-92 showed increased cytotoxicity in 3D spheroid models which can recapitulate in vivo architecture, thereby bridging the gap between in vitro and in vivo models. Conclusions: AdCAR NK-92 cells may provide an interesting and promising “off-the-shelf” cellular product for the targeted therapy of cancers metastasizing to the bone, while utilization of clinically approved, therapeutic antibodies, as exchangeable adapter molecules can facilitate quick clinical translation.

scholarly journals Multiplexed Engineered, Off-the-Shelf T Cells Carrying Three Tumor-Associated Antigen-Targeting Modalities: CAR + Pan-Tumor Targeting TCR + CD16 Fc Receptor

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-32
Author(s):  
Cokey Nguyen ◽  
Eigen Peralta ◽  
Chia-Wei Chang ◽  
Wen-I Yeh ◽  
Yijia Pan ◽  
...  
Keyword(s):  
T Cells ◽  
Monoclonal Antibodies ◽  
T Cell ◽  
Tumor Heterogeneity ◽  
Tumor Antigens ◽  
Fc Receptor ◽  
Car T Cells ◽  
Tumor Associated Antigen ◽  
Car T

Autologous chimeric antigen receptor (CAR)-T cell therapy has shown great promise in various hematologic malignancies. However, the complexities associated with immune cell evasion are prevalent causes of disease relapse in many cancers. With the advent of pluripotent stem cell (iPSC)-derived CAR-T cells, many factors that hamper therapeutic efficacy of CAR-T cells can be addressed through multiplexed engineering at the clonal level. This includes enhanced potency, increased capacity for multi-antigen targeting, and the consistency of a clonally derived engineered cellular product for off-the-shelf patient administration. In particular, strategies to mitigate antigen escape and address tumor heterogeneity may help promote durable responses. To combine the potent targeted therapy of the CAR with universal targeting of secondary and tertiary antigens, we expressed an MR1 clonal T cell receptor (TCR) and a high-affinity, non-cleavable CD16 Fc receptor (hnCD16) in our iPSC-derived CAR19 T cells (CAR19-iT cells) directed to leukemia and lymphoma and CAR-MICA/B T cells directed to solid tumors. The MR1-TCR allows highly specific recognition of tumor associated antigen presented by the MR1 protein. The non-polymorphic MHC class I-related protein MR1 is widely expressed with minimal variability among patients and enables the unique prospect to be a universal cancer immunotherapy by using the cognate MR1-TCR. The hnCD16 Fc receptor has been shown to improve antibody-dependent cellular cytotoxicity (ADCC) leveraging the broad range of available therapeutic monoclonal antibodies to target clinically validated tumor antigens. A preliminary assessment demonstrated that MR1-TCR overexpressed in T cells allowed for enhanced recognition of multiple hematological and solid tumor cell lines. Notably, prominent target specific killing was seen in A549 lung carcinoma cells (&gt;75% reduction in total viable cells) with the directed cytotoxicity specifically inhibited by an MR1 blocking antibody. Next, in vitro functional testing was performed on the engineered CAR19-iT cells in co-culture assays where we measured killing of tumor cells via MR1-TCR engagement and via hnCD16 mediated ADCC. Specifically, we show that CAR19-iT cells expressing hnCD16 can be efficiently directed to lyse CD20+ Raji cells in the presence of rituximab or HER2+ SKOV3 cells in the presence of Herceptin, demonstrating the potential to target both hematological malignancies and solid tumors with one target modality in combination with various monoclonal antibodies. Moreover, CAR19-iT cells expressing either MR1-TCR or hnCD16 show the ability to control growth of CD19 KO lymphoma cells in the co-culture assays, further highlighting the unique ability to elicit multiple ways to target antigen escape. Further in vitro and in vivo combinatorial targeting studies focused on antigen escape and tumor heterogeneity are ongoing and will be discussed. In summary, the advances presented here demonstrate that both the MR1-TCR and hnCD16 modalities synergize with CAR-iT cells as an off-the-shelf therapeutic that can provide durable responses and enable broad applicability for targeting of additional tumor antigens where single-agent therapeutics fail to provide clinical benefit for patients. Disclosures Nguyen: Fate Therapeutics, Inc.: Current Employment. Peralta:Fate Therapeutics, Inc.: Current Employment. Lu:Fate Therapeutics, Inc.: Current Employment. Sung:Fate Therapeutics, Inc.: Current Employment. Lee:Fate Therapeutics, Inc.: Current Employment.

Mini-review: The market growth of diagnostic and therapeutic monoclonal antibodies; SARS CoV-2 an example

2021 ◽  
pp. 1-10
Author(s):  
Yasmine El Abd ◽  
Ashraf Tabll ◽  
Robert Smolic ◽  
Martina Smolic
Keyword(s):  
Monoclonal Antibodies ◽  
Main Body ◽  
Global Public Health ◽  
Emerging Viruses ◽  
Human Form ◽  
Market Growth ◽  
Biopharmaceutical Company ◽  
Hybridoma Technology ◽  
Antiviral Medications ◽  
Therapeutic Monoclonal Antibodies

BACKGROUND: The emergence of novel viruses poses severe challenges to global public health highlighting the crucial necessity for new antivirals. MAIN BODY: Monoclonal antibodies (mAbs) are immunoglobulins that bind with a single epitope. Mouse mAbs are generated by classic hybridoma technology and are mainly used for immunodiagnostics. For immunotherapy, it is critical to use monoclonal antibodies in the human form to minimize adverse reactions. They have been successfully used to treat numerous illnesses, accordingly, an increasing number of mAbs, with high potency against emerging viruses is the target of every biopharmaceutical company. The diagnostic and therapeutic mAbs market grows rapidly into a multi-billion-dollar business. Biopharmaceuticals are innovative resolutions which revolutionized the treatment of significant chronic diseases and malignancies. Currently, a variety of therapeutic options that include antiviral medications, monoclonal antibodies, and immunomodulatory agents are available in the management of COVID-19. SHORT CONCLUSION: The invasion of mAbs in new medical sectors will increase the market magnitude as it is expected to generate revenue of about 300 billion $ by 2025. In the current mini-review, the applications of monoclonal antibodies in immune-diagnosis and immunotherapy will be demonstrated, particularly for COVID-19 infection and will focus mainly on monoclonal antibodies in the market.

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