Cell Fusion Technology Research Articles

Silver amplified immunosensor via effective fluorogenic Ag+-imidazole aggregation for detection of AFB1

BackgroundCereals are susceptible to aflatoxin contamination during storage and transportation, which is highly carcinogenic and teratogenic, and seriously threaten human health. The accurate and rapid detection of total aflatoxin (including aflatoxin B1, B2, G1, and G2) is of great importance for food safety. Conventional fluorescence immunoassays have the advantage of being sensitive and fast; however, these methods can be affected by strong background and matrix interference. Therefore, the development of ultrasensitive, cost-effective, and interference rejection sensors for detecting aflatoxins in moldy grains is vital for food safety and human health. ResultsIn this paper, a broad-spectrum aflatoxin monoclonal antibody was prepared by using hybridoma cell fusion technology. An aggregation-induced emission (AIE) based immunosensor via silver amplification coupled with a fluorogenic Ag+ probe was established for AFB1 analysis. Silver nanoparticles are decomposed into numerous Ag+ by H2O2, and then Ag+ further specifically binds with imidazole-modified AIE molecules, improving the sensitivity and anti-interference ability of the method. The IC50 and IC15 of AIE-based immunosensor for AFB1 were 0.019 and 0.0014 μg/L, respectively, 2.3-fold and 5.8-fold higher than those of icELISA. The AIE-based immunosensor was also used to analyze AFB1 from actual cereal samples, with spiked recoveries ranging from 72.91 to 115.92 %. In addition, the method was used to detect total aflatoxins in moldy grains. SignificanceBased on the advantages of broad-spectrum aflatoxin monoclonal antibody, high-efficiency metal signal amplification, and functional AIE molecule, a sensitive, accurate, cost-effective, and time-saving method was developed for the analysis of total aflatoxins in cereals. Moreover, the proposed signal amplification strategy shows great potential for analyzing other trace-level small molecular pollutants.

Modern Technologies Provide New Opportunities for Somatic Hybridization in the Breeding of Woody Plants.

Advances in cell fusion technology have propelled breeding into the realm of somatic hybridization, enabling the transfer of genetic material independent of sexual reproduction. This has facilitated genome recombination both within and between species. Despite its use in plant breeding for over fifty years, somatic hybridization has been limited by cumbersome procedures, such as protoplast isolation, hybridized-cell selection and cultivation, and regeneration, particularly in woody perennial species that are difficult to regenerate. This review summarizes the development of somatic hybridization, explores the challenges and solutions associated with cell fusion technology in woody perennials, and outlines the process of protoplast regeneration. Recent advancements in genome editing and plant cell regeneration present new opportunities for applying somatic hybridization in breeding. We offer a perspective on integrating these emerging technologies to enhance somatic hybridization in woody perennial plants.

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.

Consideration on the Research and Development of Anti-tumor Bispecific Antibody Drugs

以程序性死亡受体1及其配体1免疫检查点抑制剂为代表的药物上市，开启了抗肿瘤免疫治疗的新时代，该类药物在某些肿瘤中表现出较为突出的持续应答，如霍奇金淋巴瘤，但仍在部分肿瘤中应答率低。近年来，双特异性抗体是通过细胞融合、重组DNA、蛋白质工程等技术制备的人工抗体，可同时或先后特异性结合两个抗原或抗原表位，在肿瘤治疗中发挥协同作用，能够有效抑制肿瘤免疫逃逸，提高抗肿瘤治疗效果，成为肿瘤研发热点。本文就双特异抗体研究进展和临床研发关注要点进行综述，以期为药物研发的相关人员提供参考。

Development of sensitive and portable immunosensors based on signal amplification probes for monitoring the mercury(II) ions

Mercury ion (Hg2+) as a major environmental pollutant threatens human health even at very low concentrations, so it is essential to monitor mercury residues in food. In this study, Hg2+ was conjugated with protein carrier using 1-(4-Isothiocyanobenzyl) ethylenediamine N, N, N′, N′-tetraacetic acid (ITCBE) as a bifunctional chelator. 7A1 monoclonal antibody (mAb) against Hg2+-ITCBE with high affinity (7.3 × 109 L/moL) and good specificity was obtained by cell fusion technology and performed to establish immunosensors. Immunochromatographic test strip using colloidal gold nanoparticles (AuNP with an average diameter of 18 nm) as signal reporter showed low sensitivity. Signal amplification probes including larger multi-branched gold nanoflowers (AuNF) and latex microspheres (LM) were employed to enhance the sensitivity of immunosensors. The visible limit of detection (vLOD) of the AuNF- and LM-based strip were determined to be 50 ng/mL and 25 ng/mL respectively, showing more sensitive than that of AuNP-based strip (200 ng/mL). Quantitative analysis showed that AuNF-based strip exhibited lower quantitative limit of detection (qLOD) (0.44 ng/mL) which was 20-fold lower than that of AuNP-based strip (8.92 ng/mL) for determination of Hg2+, and LM-based strip (0.49 ng/mL) was 18 times as sensitive as AuNP-based strip. In summary, the developed immunosensors using AuNF and LM as signal amplification probes exhibited excellent sensitivity and provided portable, on-site detection for Hg2+.

Development of a highly sensitive and selective electrochemical immunosensor for controlling of rhodamine B abuse in food samples

Here, a highly sensitive and selective electrochemical immunosensor was established for the rapid screening of Rhodamine B (Rh B) in paprika and capsicol. Rh B complete antigen was prepared and used as immunogen to obtain anti-Rh B monoclonal antibody (anti-Rh B-mAb) through cell fusion technology. A layer of cobalt hydroxide and gold nanoparticles composite material (Co(OH)2–Au NPs) was modified on the surface of screen-printed electrode (SPE) by one-step electrodeposition. Subsequently, Rh B antigen was coated on the modified SPE as coating antigen, then Rh B and anti-Rh B-mAb labeled with cerium oxide (anti-Rh [email protected]2) were overlay simultaneously. Rh B can compete with the Rh B coating antigen to combine the anti-Rh B-mAb which causing electrical signal changes. After optimization of the assembled immunosensor, the assay showed a linear range when the concentration of Rh B ranged from 0.01 ng/mL to 10000 ng/mL. The average recovery rate of spiked samples was 85.7%∼106.8%, which was in good accordance with the standard HPLC determination. The immunosensor behaves high sensitivity and selectivity, and can be used in the rapid screening of Rh B in paprika and capsicol.

An improved iliac lymph node method for production of monoclonal antibodies.

Monoclonal antibodies have been applied in a wide range of biological and medical studies since the advent of cell fusion technology. Although cell fusion techniques have been improved by using myelomas and reagents, researchers still find it difficult to produce monoclonal antibodies because of the long protocols, high costs, and low efficiency of obtaining hybridomas. To solve these problems, we first developed an iliac lymph node method in 1995 using rats. In this method, an antigen emulsion is injected intramuscularly into the tail base, and then B lymphocytes are isolated from the enlarged iliac lymph nodes. This method is approximately 10 times more productive than the conventional spleen method. Here, we present further improvements to the iliac lymph node method to render it easily applicable in both mice and rats. We found that the frequency of hybridomas secreting specific antibodies was over five times higher using the electro cell fusion method than using the polyethylene glycol (PEG) fusion method. This frequency using the iliac lymph node method with electro cell fusion is at least 50 times higher than that using the traditional spleen method, thereby leading to the reduction in the number of mice or rats to be sacrificed. In addition, only a single injection for immunization is necessary for the iliac lymph node method, opposed to three for the spleen method. Therefore, this method is rapid, inexpensive, and ethical for producing monoclonal antibodies.

In vitro immunization approach to generate specific murine monoclonal IgG antibodies

Generating a monoclonal antibody to date is a time intense process that requires immunization of laboratory animals. The transfer of the humoral immune response into in vitro settings enables a shortening of this process and circumvents the necessity of in vivo immunization. However, to orchestrate the complex interplay of dendritic cells, T and B lymphocytes in vitro is very challenging. We therefore aimed for a simplified approach focusing on the protagonist of antibody production: the B lymphocyte. We activated purified murine B lymphocytes alone in vitro by using combinations of antigen and stimuli. We were able to induce a specific antibody response within ten days of culture against a viral coat protein as model antigen. Antibodies were of both IgM and IgG subclass. The stimulated B lymphocytes were transformed into permanently antibody-producing hybridomas by cell fusion technology. We furthermore used this method to induce a specific antibody response against L. pneumophilain vitro. We thus established a useful and effective in vitro protocol to generate monoclonal antibodies. By overcoming the necessity of in vivo immunization this protocol may be the first step towards a universal strategy to generate antibodies from various species.

Lactoferrin-containing immunocomplex mediates antitumor effects by resetting tumor-associated macrophages to M1 phenotype

BackgroundTumor-associated macrophages (TAMs) resemble M2-polarized cells with potent immunosuppressive activity and play a pivotal role in tumor growth and progression. Converting TAMs to proinflammatory M1-like phenotype is thus an attractive...

Production and characterization of monoclonal antibodies against Toxoplasma gondii ROP18 with strain-specific reactivity.

The rhoptry kinase 18 of Toxoplasma gondii (TgROP18) has been identified as a key virulence factor that allows the parasite to escape from host immune defences and promotes its proliferation in host cells. Although much research is focused on the interaction between host cells and TgROP18, the development of monoclonal antibodies (mAbs) against TgROP18 has not been reported till date. To produce mAbs targeting TgROP18, two hybridomas secreting mAbs against TgROP18, designated as A1 and T2, were generated using cell fusion technology. The subtypes of the A1 and T2 mAbs were identified as IgG3 λ and IgM κ, and peptide scanning revealed that the core sequences of the antigenic epitopes were 180LRAQRRRSELVFE192 and 351NYFLLMMRAEADM363, respectively. The T2 mAb specifically reacted with both T. gondii type I and Chinese I, but not with T. gondii type II, Plasmodium falciparum or Schistosoma japonicum. Finally, the sequences of heavy chain and light chain complementarity-determining regions of T2 were amplified, cloned and characterized, making the modification of the mAb feasible in the future. The development of mAbs against TgROP18 would aid the investigation of the molecular mechanisms underlying the modulation of host cellular functions by TgROP18, and in the development of strategies to diagnose and treat Toxoplasmosis.

Ultrasensitive anti-melamine monoclonal antibody and its use in the development of an immunochromatographic strip

ABSTRACT The hapten 1 which was formed by the reaction of 2-chloro-4,6-diamine-1,3,5-triazine (CAAT) with 6-aminocaproic acid, conjugated with keyhole limpet haemocyanin to prepare the immunogen. And coating antigen was prepared by the coupling of ovalbumin and hapten 2 which was synthesized by the reaction of CAAT and 3-mercapto propionic acid. Based on immunological and cell fusion technology, the anti-melamine monoclonal antibody was prepared and characterized. The half inhibitory concentration of the antibody for melamine was 1.8 ng/mL, and the linear range was 0.61–5.56 ng/mL. An immunochromatographic (ICT) strip, based on the antibody, was developed for the rapid detection of melamine residues in milk samples. The ICT strip had a visual cut-off value of 10 ng/mL for milk samples. Therefore, the colloidal gold immunoassay with the advantage of high affinity and sensitivity can be used for the ultrasensitive detection of melamine residues in milk samples.

Generation, characterization and application of monoclonal antibodies against matrix protein of hirame novirhabdovirus (HIRRV) in flounder.

Hirame novirhabdovirus (HIRRV) causes severe disease in fish cultures, resulting in great economic loss in Asia and Europe. In this study, the matrix protein (M) of HIRRV was recombinantly expressed as the immunogen to produce monoclonal antibodies (MAbs) using hybridoma cell fusion technology, and 3 MAbs were produced and characterized by indirect ELISA, Western blotting and immunofluorescence assay (IFA). Western blotting and mass spectrometric analysis showed that the MAbs could specifically react with the nature M protein of HIRRV. The MAbs were employed to detect virions in HIRRV-infected epithelioma papulosum cyprini (EPC) cells and flounder Paralichthys olivaceus by IFA and immunohistochemistry (IHC). In the virus-infected EPC cells, the virions were mainly located in the cytoplasm, whereas in flounder, HIRRV was present in all 10 tested tissues, and the positive signals in spleen, head-kidney and heart were higher than in other tissues, consistent with the results obtained by RT-PCR. Moreover, strong positive signals were observed in the endothelial cells of blood vessels, but only the leukocytes were infected by HIRRV in the whole blood cells. These results indicate that the high susceptibility to HIRRV of leukocytes and endothelial cells may facilitate the spread of HIRRV and finally cause systemic infection in flounder. This study provides a foundation for further studies on rapid diagnosis of HIRRV and its infection mechanisms.

Automatic cell fusion via optically-induced dielectrophoresis and optically-induced locally-enhanced electric field on a microfluidic chip.

Cell fusion technology has been exploited in a wide variety of biomedical applications, and physical, chemical, and biological approaches can all be used to fuse two different types of cells; however, no current technique is adept at inducing both cell pairing and fusion at high efficiencies and yields. Hence, we developed a new method featuring the use of optically induced dielectrophoresis (ODEP) in conjunction with an optically induced, locally enhanced electric field for accurate and automatic cell pairing and fusion on a microfluidic device. After pairing cells via ODEP, a locally enhanced electric field generated by "virtual electrodes" by projecting light patterns was enacted to induce a proper transmembrane potential at the cell contact area such that cell fusion could be triggered by white light exposure. As a fusion yield of 9.67% was achieved between Pan1 and A549 cells, we believe that this may be a promising technique for automatically fusing different cell types.

Ultrasensitive immunochromatographic strip for detection of cyproheptadine

ABSTRACT Using immunological and cell fusion technology, group-specific monoclonal antibodies were produced against cyproheptadine. The 50% inhibitory concentration of the antibody for cyproheptadine was 0.37 ng/mL, the linear range was 0.16–0.85 ng/mL, and recoveries of antibody in spiked negative pig urine were 80–120%. An ultrasensitive immunochromatographic strip, based on the antibody, was developed for the rapid detection of cyproheptadine in pig urine. The test strip had a cut-off value of 5 ng/mL.

Epigenetic mechanism of FMR1 inactivation in Fragile X syndrome.

Fragile X syndrome is the most frequent cause of inherited intellectual disability. The primary molecular defect in this disease is the expansion of a CGG repeat in the 5' region of the fragile X mental retardation1 (FMR1) gene, leading to de novo methylation of the promoter and inactivation of this otherwise normal gene, but little is known about how these epigenetic changes occur during development. In order to gain insight into the nature of this process, we have used cell fusion technology to recapitulate the events that occur during early embryogenesis. These experiments suggest that the naturally occurring Fragile XFMR1 5' region undergoes inactivation post implantation in a Dicer/Ago-dependent targeted process which involves local SUV39H-mediated tri-methylation of histone H3K9. It thus appears that Fragile X syndrome may come about through inadvertent siRNA-mediated heterochromatinization.

A Novel Murine Anti-Lactoferrin Monoclonal Antibody Activates Human Polymorphonuclear Leukocytes through Membrane-Bound Lactoferrin and TLR4.

Soluble lactoferrin (LTF) is a versatile molecule that not only regulates the iron homeostasis, but also harbors direct microbicidal and immunomodulating abilities in mammalian body fluids. In contrast, little is known about the function of membrane-bound LTF (mbLTF), although its expression on human polymorphonuclear leukocytes (huPMNs) has been reported for decades. Given that LTF/anti-LTF antibodies represent a potential diagnostic/prognostic biomarker and a therapeutic target in patients with immune disorders, we wished, in the present study, to generate a novel human LTF- (huLTF-) specific mAb suitable for detailed analyses on the expression and function of mbLTF as well as for deciphering the underlying mechanisms. By using the traditional hybridoma cell fusion technology, we obtained a murine IgG1 (kappa) mAb, M-860, against huLTF. M-860 recognizes a conformational epitope of huLTF as it binds to natural, but not denatured, huLTF in ELISA. Moreover, M-860 detects mbLTF by FACS and captures endogenous huLTF in total cell lysates of huPMNs. Functionally, M-860 induces the activation of huPMNs partially through TLR4 but independently of phagocytosis. M-860 is thus a powerful tool to analyze the expression and function of human mbLTF, which will further our understanding of the roles of LTF in health and disease.

Preparation and validation of monoclonal antibody-based indirect competitive ELISA for detecting testosterone levels

This paper presents the generation of monoclonal antibodies (mAbs) against testosterone (TES) through cell fusion technology, and the development of a mAb-based indirect competitive enzyme-linked immunosorbent assay (icELISA) method to detect TES residue in bovine edible tissues. Under optimal conditions, this assay exhibited a working range of 0.04–19 ng/mL, with half-maximum inhibition (IC50) and limit of detection values of 0.27 and 0.02 ng/mL, respectively. After three sample pretreatment procedures were checked, a simple dilution method was adopted for further use. The stabilisation studies demonstrated that the icELISA kits can be stored for at least 180 to 240 days, at 4°C and −20°C, respectively. When applied to bovine samples, the data from icELISA and gas chromatography coupled to mass spectrometry showed excellent correlation (r2=0.9923 in muscle, 0.9884 in liver and 0.9957 in kidney). Therefore, this assay has the potential to be incorporated into a quantitative monitoring programme for the rapid screening of TES residue in foods.

Development of Monoclonal Antibody Based Heterologous Immunoassay for Ractopamine Residue

This study aimed to develop a monoclonal antibody based icELISA method for Ractopamine (Rac) residue. For this purpose, mixed anhydride method was employed to synthesize the immunogen of Rac-BSA and 1, 4-butanediol diglycidyl ether was used to prepare the coating antigen of Rac-OVA, thus pursue the heterologous sensitivity. Through cell fusion technology, four Hybridoma named R1-B5, R2-B3, R2-C6, and R4-C8 were screened out, and the Kas of all mAbs were between 2.7 and 4.8×109 L/mol. Based on the R1-B5 mAb, a heterologous icELISA standard curve was developed. The working range was from 0.013 to 33.7 ng/mL, with LOD and IC50 value of 0.007 ng/mL and 0.67 ng/mL, respectively. Therefore, this icELISA can be used for detecting Rac residue in animal products.

Preparation and Immunological Traits of Monoclonal Antibody against Sarafloxacin

Through cell fusion technology, five hybridoma lines of sarafloxacin (SAR), named S1-B2, S2-C6, S2-E7, S3-C5, and S3-E5, were identified and their corresponding mAbs were of the IgG1 isotype with a k light chain. The Kaffs of all mAbs were between 2.8 and 4.6×109 L/mol. The titers and IC50 values of purified ascite fluids were in the range of 0.512–2.56×106 and 0.32–0.48 ng/mL, respectively. The performances of S1-B2 and S2-C6 were more consistent in the stability experiments. Based on the S1-B2 hybridoma, an icELISA method was developed. The dynamic range was from 0.004 to 18 ng/mL, with a detection limit for the assay and IC50 values of 0.002 and 0.32 ng/mL, respectively. Therefore, the establishment of these hybridomas may provide an alternative method for the detection of SAR residues in food-original animals.

A Monoclonal Antibody Against Human FXYD6

Previous research has shown that FXYD6 (FXYD domain-containing ion transport regulator 6) is highly increased in bile duct tumor. However, the biological function of FXYD6 is unclear. We aim to prepare and identify a monoclonal antibody against FXYD6, which will be used in diagnostics and as a tool in understanding the role of FXYD6 in pathogenesis of hepatobiliary cancer. In this study, hybridoma cell fusion technology is used for production of FXYD6 monoclonal antibody. BALB/c mice are immunized with FXYD6 synthetic peptide fragment. Hybridoma clones are screened using indirect enzyme-linked immunosorbent assay (ELISA). FXYD6 monoclonal antibody is produced by ascites revulsion. Protein A affinity chromatography is used for the purification of FXYD6 monoclonal antibody. Titer and specificity of monoclonal antibody are assessed by ELISA. Expression of FXYD6 in pancreatic cancer is detected by immunohistochemistry. As a result, one stable hybridoma cell clone producing FXYD6 monoclonal antibody has been established. 2.86 mg monoclonal antibody against FXYD6 with high specificity is prepared with titer of 1:5400. Immunohistochemistry shows that FXYD6 positive staining occurs in the cell membrane of pancreatic cancer, which results in an advantage in investigating the tissue distribution and biological function of FXYD6.