ANALYTICA CHIMICA ACTA
·
2022
·
IF 6.911
Bing Rui,Yangrui Feng,Yanan Wang,Jiawu Deng,Mingqiang Wang,Yi Lyu,Lan Luo
In this work, a novel isophorone-derived fluorescent probe , ISOP-SFT , was developed for the detection of sulfite , and, for the first time, it was applied into monitoring the state of hybridoma cells. ISOP-SFT could exhibit a red-emission response to sulfite with the excitation at 455 nm. It also indicated practical advantages such as wide pH tolerance within 6.0–9.0 and high storage steadiness within at least one week. The linear range was relatively long (0–20 Eq) and reliable (Pearson's correlation coefficient : 0.9989), while Limit of Detection was determined as 0.0095 Eq (95 nM). For the selectivity , ISOP-SFT could distinguish sulfite from other analytes, and the response of ISOP-SFT towards sulfite was not seriously interfered in co-existence system. With low cytotoxicity, ISOP-SFT achieved monitoring both the exogenous and endogenous sulfite in living MCF-7 cells. As a novel attempt, ISOP-SFT was applied into monitoring the state of hybridoma cells, and associated with the quality control of producing antibodies. The result preliminarily inferred the positive correlation between the production efficiency of antibodies in hybridoma cells and the intracellular sulfite level, including the conditions of serum starvation, oxidative stress , severe hypoxia and endogenous induction. We hope that the information in this work could lead to new applications of fluorescent probes in biological industry in future.
研究领域:
生物化学
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分子生物学
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细胞生物学
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免疫学
✓ 已验证
DOI
PubMed
高影响因子
CYTOTECHNOLOGY
·
2011
·Vol. 64
·期 2
·157-163
·
IF 1.277
Yi Yu,Wang Zhuhuan,Li Min,Zhu Keyin,Ying Guoqing
Monoclonal antibodies (McAbs) against chloramphenicol (CAP) were produced to detect CAP residues, which could be toxic and possesses a potential threat to human health. The CAP-BSA conjugate was obtained by bovine serum albumin (BSA) coupled with CAP, and used to immunize the mice. The splenocytes from the immunized mice were fused with mouse myeloma cells SP2/0 to form hybridoma, which may secrete McAbs against CAP. Hybridomas 1D 1 and 3G 12 secreting McAbs against CAP were obtained by screening. Ascites containing McAbs were prepared by injecting 1 x 10 6 cells of hybridoma 1D 1 and 3G 12 into the abdomen of mice. Protein A affinity chromatography was used to purify McAbs against CAP in a single chromatographic step with recovery yield above 80% and purity above 95% and full recovery of antibody activity. Experiments showed that McAb 3G 12 was highly specific for CAP and had no cross-reactivity with analogues which have a structure similar to CAP. The IC 50 value was 50.8 ng/mL.
研究领域:
免疫学
·
药理学
·
毒理学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2018
·
IF 6.042
Xiuqing Li,Hongfen Bian,Siming Yu,Wei Xiao,Jianying Shen,Caifeng Lan,Kenan Zhou,Caihong Huang,Lei Wang,Dan Du,Yuehe Lin,Yong Tang
Using an enzyme-linked immunosorbent assay (ELISA) and limited dilution methods to screen and clone antigen-specific hybridoma cells is extremely time-consuming and labor-intensive. This work features a simple and rapid cell surface fluorescence immunosorbent assay (CSFIA), designed for the detection and isolation of antigen-specific hybridoma clones. In this assay, antigens are first anchored to the hybridoma cell surface through a dual-functioning molecular Oleyl-PEG4000-NHS. Specific antibodies secreted from hybridoma cells are then captured by the antigens on the cell surface. Positive hybridoma cells are stained using a fluorescently labeled anti-mouse IgG-Fc antibody. After the addition of a methylcellulose semisolid medium, positive clones are easily picked using a pipet. These positive cell clones can be used to produce monoclonal antibodies after direct expansion. Using this method, positive hybridoma clones against both malachite green and porcine epidemic diarrhea virus are selected with high efficiency. Compared to the ELISA-based method, the CSFIA-based method achieved the capability of isolating >2-fold more hybridoma clones in <25% of the corresponding processing time. In brief, the CSFIA-based method is highly efficient and inexpensive with a simple and direct operation, which is an excellent candidate method for antigen-specific positive clone isolation in a monoclonal antibody preparation.
研究领域:
免疫学
·
分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
FOOD CHEMISTRY
·
2022
·
IF 9.231
Peng Dai,Yan Zhang,Yanping Hong,Jianhua Xiong,Huaying Du,Luying Duan,Dan Wang,Yubo Wang,Weijie Deng,Bruce D. Hammock,Wuying Yang
To determine gentamicin residues in animal tissues, a monoclonal antibody (Mab) was produced and a sensitive indirect competitive chemiluminescent enzyme immunoassay (icCLEIA) was developed. At first, gentamicin was conjugated with bovine serum albumin as immunogens which were used to immunize BALB/c mice. Then, an anti-gentamicin Mab was prepared by hybridoma technology. Finally, a sensitive icCLEIA was developed with an 50% inhibition concentration (IC 50 ) of 0.067 ng/mL for gentamicin. The limit of detection of the icCLEIA was 0.002 ng/mL. The cross reactivity of the Mab with structural analogues were<0.01%. The recoveries of gentamicin ranged from 80 to 101% and coefficient of variation was <6.4% in pork and fish samples. Samples were detected by UPLC-MS/MS for evaluating reliability of the icCLEIA. The results suggested that the prepared anti-gentamicin Mab can be used for rapid and convenient immunoassays to detect gentamicin residues in animal tissues.
研究领域:
兽医学
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免疫学
·
分析化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2024
·
IF 6.7
Hang Ao,Wencheng Xiao,Wenrui Hu,Jie Wu,Huangxian Ju
This work designed a DNA conformation-regulated hemin switch for rapid chemiluminescent (CL) detection of a monoclonal antibodies. This switch was performed with an affinity probe and an inhibition probe, which were conveniently prepared by hybridizing hemin-labeled DNA1 with KHL peptide-labeled DNA2 and binding biotin-labeled DNA3 to streptavidin, respectively. In the absence of the target antibody, streptavidin-DNA3 could hybridize with hemin-DNA1/KHL-DNA2 to release KHL-DNA2, which led to the loss of hemin activity due to the affinity hindrance of streptavidin-DNA3. After the KHL peptide was recognized by the target antibody, the strand replacement hybridization could be inhibited by the bound antibody, which retained the high catalytic activity of hemin overhung on the antibody-bound affinity probe for a CL reaction, leading to a “signal-on” process for CL antibody detection. Using a KHL-specific antibody, anti-proprotein convertase subtilisin/kexin type 9 antibody (PCSK9-Ab), as a target model and common L012–1,2,4-triazole–H2O2 CL system, the designed switch showed a detection range of 10 ng mL–1 to 1 μg mL–1 with a detection limit of 4.16 ng mL–1 (56.2 pM) and a short analytical time of 6.5 min. The proposed quick method could simply be used for lab-on-chip CL detection of PCSK9-Ab in situ-secreted from PCSK9-6E3 hybridoma cells, which showed an accuracy of 90.2% compared with the statistical results from general fluorescence imaging, providing a potential technique for screening specific hybridoma cells.
研究领域:
生物化学
·
分析化学
·
生物技术
·
分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
TALANTA
·
2024
·
IF 5.6
Chengchen Pang,Bei Yuan,Keyun Ren,Haitao Xu,Kunying Nie,Chunlei Yu,Zhanli Liu,Yanyan Zhang,Sibel A. Ozkan,Qingqing Yang
In preparing monoclonal antibodies by hybridoma cell technology, the quality of B lymphocytes used for cell fusion directly affects the sensitivity of monoclonal antibodies. To obtain B-lymphocytes producing high-quality specific antibodies for cell fusion during the immunization phase of the antigen, we prepared a TH2-Cell stimulatory delivery system as a novel adjuvant. Astragalus polysaccharide has a good ability to enhance antigenic immune response, and it was encapsulated in biocompatible materials PLGA as an immunostimulatory factor to form the delivery system (APS-PLGA). The preparation conditions of APSP were optimized using RSM to attain the highest utilization of APS. Immunization against ZEN-BSA antigen using APSP as an adjuvant to obtain B lymphocytes producing ZEN-specific antibodies for cell fusion. As results present, APSP could induce a stronger TH2 immune response through differentiating CD4 T cells and promoting IL-4 and IL-6 cytokines. Moreover, it could slow down the release efficiency of ZEN-BSA and enhance the targeting of ZEN-BSA to lymph nodes in vivo experiments. Ultimately, the sensitivity of mouse serum ZEN-specific antibodies was enhanced upon completion of immunization, indicating a significant upregulation of high-quality B lymphocyte expression. In the preparation of monoclonal antibodies, the proportion of positive wells for the first screening was 60%, and the inhibition rates of the antibodies were all similar (>50%). Then we obtained the ZEN monoclonal antibody with IC50 of 0.049 ng/mL, which was more sensitive than most antibodies prepared under conventional adjuvants. Finally, a TRFIAS strip assay was preliminarily established with a LOD value of 0.246 ng/mL.
研究领域:
免疫学
·
生物技术
·
纳米技术
·
药学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2016
·
IF 2.857
Feng Wang,Hong Wang,Yu-Dong Shen,Yong-Jun Li,Jie-Xian Dong,Zhen-Lin Xu,Jin-Yi Yang,Yuan-Ming Sun,Zhi-Li Xiao
A new multianalyte immunoassay was designed to screen furaltadone metabolite 5-morpholinomethyl-3-amino-2-oxazolidone (AMOZ), malachite green (MG), and leucomalachite green (LMG) in aquatic products using a bispecific monoclonal antibody (BsMAb). Gradient drug mutagenesis methods were separately used to prepare an anti-3-nitrobenzaldehyde-derivatized AMOZ (3-NPAMOZ) hybridoma cell line that was hypoxanthine-guanine-phosphoribosyltransferase (HGRPT) deficient and an anti-LMG hybridoma cell line that was thymidine kinase (TK) deficient. BsMAb recognizing 3-NPAMOZ and LMG was generated using hybrid–hybridomas of HGRPT and TK deficient cell lines. For AMOZ and LMG, respectively, the BsMAb-based indirect competitive ELSIA (ic-ELISA) values of 1.7 ng/mL and 45.3 ng/mL and detection limits of 0.2 ng/mL and 4.8 ng/mL. To establish the ic-ELISA, 3-NPAMOZ derivatized from AMOZ with 3-nitrobenzaldehyde and LMG reduced from MG by potassium borohydride was recognized by BsMAb. Recoveries of AMOZ, MG, and LMG in aquatic products were satisfactory and correlated with HPLC analysis. Thus, the multianalyte ic-ELISA is suitable for rapid quantification of AMOZ, MG, and LMG in aquatic products.
研究领域:
分析化学
·
食品科学
·
免疫学
·
水产养殖
✓ 已验证
产品关联
DOI
PubMed
高影响因子
FOOD AND AGRICULTURAL IMMUNOLOGY
·
2021
·
IF 3.101
Xinzhu Chen,Huaying Du,Yanping Hong,Yubo Wang,Mei Shu,Dan Wang,Peng Dai,Weijie Deng,Jianhua Xiong,Wuying Yang
To determine furaltadone metabolites 5-methylmorpholino-3-amino-2-oxazolidinone (AMOZ) residual levels in ctenopharyngodon idellus, direct competitive enzyme-linked immunosorbent assay (dcELISA) was developed based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein. Firstly, VH and VL gene sequences were cloned from hybridoma cell lines secreting monoclonal antibody against AMOZ derivative, and then the productive VH and VL genes were assembled to a scFv gene and engineered to produce scFv-AP fusion protein. The fusion protein was further identified as a bifunctional reagent for immunoassay, then a sensitive one-step dcELISA against AMOZ derivative was developed with a half-maximal inhibitory concentration (IC50) and limit of detection (LOD) of 10.62 and 4.83 ng/mL, respectively. The recovery values of AMOZ from spiked fish samples determined by dcELISA were in 71.31%∼82.88%. The results suggested that the prepared scFv-AP fusion protein in this work can be used for rapid and convenient immunoassays to detect AMOZ residues in fish samples.
研究领域:
免疫学
·
生物化学
·
分析化学
·
水产养殖
·
食品安全
✓ 已验证
产品关联
DOI
高影响因子
JOURNAL OF APPLIED ELECTROCHEMISTRY
·
2023
·
IF 2.9
Zhan Ke,Du Yongkun,Liu Qingqing,Cui Chenxu,Xing Guangxu,Li Shanshan,Ren Hongtao,Wang Fan,Xu Chao,Wang Na
Wheat gliadin is thought to be the main cause of wheat allergy leading to celiac disease. In this study, we reported an electrochemical immunosensor based on AuNPs/Zn/Ni-ZIF-8-800@graphene for simple, efficient, and sensitive detection of gliadin. A monoclonal antibody against gliadin were prepared by hybridoma technique. Zn/Ni-ZIF-8-800 with high stability was prepared by using zinc–nickel bimetallic organic framework MOFs, and graphene and gold nanoparticles with large surface area and excellent conductivity were introduced. Chitosan was used as a binder to fixed the composite on the surface of glassy carbon electrode. The fabricated materials and the corresponding sensors were comprehensively characterized by X-ray diffractometer, scanning electron microscope, transmission electron microscopy, Fourier transform infrared spectroscopy, Brunauer–Emmett–Teller, and cyclic voltammetry analyses. Under the optimal conditions, the linear range of the electrochemical immunosensor was 0.1–100 μg mL −1 , and the detection limit was 0.950 μg mL −1 . In addition, the fabricated sensor exhibited high selectivity and long-term stability. The recovery test showed that the prepared sensor was suitable for the detection of gliadin in real food and feed, exhibiting great promising of the developed sensor in practical application. Graphical abstract
研究领域:
分析化学
·
生物化学
·
材料科学
·
食品科学
·
免疫学
✓ 已验证
DOI
高影响因子
危险品化学品经营许可证（带存储）