Hepatitis B virus(HBV)reactivation poses a significant clinical challenge,espe-cially in patients undergoing immunosuppressive therapies,including mono-clonal antibody treatments.This manuscript briefly explores the c...Hepatitis B virus(HBV)reactivation poses a significant clinical challenge,espe-cially in patients undergoing immunosuppressive therapies,including mono-clonal antibody treatments.This manuscript briefly explores the complex rela-tionship between monoclonal antibody therapy and HBV reactivation,drawing upon current literature and clinical case studies.It delves into the mechanisms underlying this phenomenon,highlighting the importance of risk assessment,monitoring,and prophylactic measures for patients at risk.The manuscript aims to enhance the understanding of HBV reactivation in the context of monoclonal antibody therapy,ultimately facilitating informed clinical decision-making and improved patient care.This paper will also briefly review the definition of HBV activation,assess the risks of reactivation,especially in patients treated with monoclonal antibodies,and consider management for patients with regard to screening,prophylaxis,and treatment.A better understanding of patients at risk can help clinicians provide optimum management to ensure successful patient outcomes and prevent morbidity.展开更多
Aphid-borne Zucchini yellow mosaic virus (ZYMV) is one of the most economically important viruses of cucurbitaceous plants. To survey and control this virus, it is necessary to develop an efficient detection techniq...Aphid-borne Zucchini yellow mosaic virus (ZYMV) is one of the most economically important viruses of cucurbitaceous plants. To survey and control this virus, it is necessary to develop an efficient detection technique. Using purified ZYMV virion and the conventional hybridoma technology, three hybridoma cell lines (16A11, 5A7 and 3B8) secreting monoclonal antibodies (MAbs) against ZYMV Zhejiang isolate were obtained. The working titers of the ascitic fluids secreted by the three hybridoma cell lines were up to 10^-7 by indirect enzyme-linked immunosorbent assay (ELISA). All MAbs were isotyped as IgG1, kappa light chain. Western blot analysis indicated that the MAb 3B8 could specifically react with the coat protein of ZYMV while MAbs 5A7 and 16A11 reacted strongly with a protein of approximately 51 kDa from the ZYMV-infected leaf tissues. According to this molecular weight, we consider this reactive protein As likely to be the HC-Pro protein. Using these three MAbs, we have now developed five detection assays, i.e., antigen-coated-plate ELISA (ACP-ELISA), dot-ELISA, tissue blot-ELISA, double-antibody sandwich ELISA (DAS-ELISA), and immunocapture-RT-PCR (IC-RT-PCR), for the sensitive, specific, and easy detection of ZYMV. The sensitivity test revealed that ZYMV could be readily detected respectively by ACP-ELISA, dot-ELISA, DAS-ELISA and IC-RT-PCR in 1:163840, 1:2560, 1:327680 and 1:1 310720 (w/v, g mL-1) diluted crude extracts from the ZYMV-infected plants. We demonstrated in this study that the dot-ELISA could also be used to detect ZYMV in individual viruliferous aphids. A total of 275 cucurbitaceous plant samples collected from the Zhejiang, Jiangsu, Shandong and Hainan provinces, China, were screened for the presence of ZYMV with the described assays. Our results showed that 163 of the 275 samples (59%) were infected with ZYMV. This finding indicates that ZYMV As now widely present in cucurbitaceous crops in China. RT-PCR followed by DNA sequencing and sequence analyses confirmed the accuracy of the five assays. We consider that these detection assays can significantly benefit the control of ZYMV in China.展开更多
To purify Methamidophos (Met) monoclonal antibodies with two methods and compare immune activity of purified antibodies. Method Caprylic acid ammonium sulphate precipition (CAASP) method and Sepharose protein-A (S...To purify Methamidophos (Met) monoclonal antibodies with two methods and compare immune activity of purified antibodies. Method Caprylic acid ammonium sulphate precipition (CAASP) method and Sepharose protein-A (SPA) affinity chromatography method were used to purify Met monoclonal antibodies, UV spectrum scanning was used to determine protein content and recovery of purified antibodies, sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the purity of purified antibodies, and enzyme-linked immunosorbent assay (ELISA) was used to determine immune activity of purified antibodies. Results Antibody protein content and recovery rate with CAASP method were 7.62 mg/mL and 8.05% respectively, antibody protein content and recovery rate with SPA method were 6.45 mg/mL and 5.52% respectively. Purity of antibodies purified by SPA method was higher than that by CAASP method. The half-maximal inhibition concentration (IC50) of antibodies purified by SPA to Met was 181.26 靏/mL, and the linear working range and the limit of quantification (LOD) were 2.43-3896.01 靏/mL and 1.03 靏/mL, respectively. The IC50 of antibodies purified by CAASP to Met was 352.82 靏/mL, and the linear working range and LOD were 10.91-11412.29 靏/mL and 3.42 靏/mL, respectively. Conclusion Antibodies purified by SPA method are better than those by CAASP method, and Met monoclonal antibodies purified by SPA method can be used to prepare gold-labelled testing paper for analyzing Met residue in vegetable and drink water.展开更多
Cardiac troponin I(cTnI) was separated and purified from human left ventricular tissue by affinity chromatographic method and used to immunize Balb/c mice by intraperitoneal injection and four hybridoma cell lines, wh...Cardiac troponin I(cTnI) was separated and purified from human left ventricular tissue by affinity chromatographic method and used to immunize Balb/c mice by intraperitoneal injection and four hybridoma cell lines, which secreted monoclonal antibody(mAb) against human cTnI, were obtained by cell fusion, identification and cloning twice. Three mAbs(9F5, 2F11, 8C12) were produced from the ascites of Balb/c mice injected intraperitoneally the hybridoma cells and characterized by means of a surface plasmon resonance(SPR) biosensor. An optimal and specific sensing membrane for troponin I was prepared with staphylococcal protein A(SPA) as the intermediate layer and mAb against human cTnI as the capture antibody. On the basis of the sensing membrane, two modes of operation of the SPR biosensor were developed, i.e ., a direct detection of antigen antibody affinity and a sandwich assay. In the sandwich assay detection mode, the mAbs competition was measured by monitoring whether the secondary antibody had been attached to the cTnI already captured by the first antibody on the sensor surface. The SPR biosensor was shown to be able to directly detect the antigen antibody affinity and the order of the affinity was found to be 9F5>2F11>8C12. In the sandwich detection mode, it was found that the different epitopes on the cTnI molecules were recognized by the three mAbs respectively, but the asymmetrical competition was shown between 2F11 and 8C12 and no competition was found between 9F5 and 2F11 or 8c12. Based on these results, a double monoclonal sandwich immunoassay for cTnI was developed by using the optimal antibody pair of 9F5 and 2F11 and the SPR biosensor with SPA substrate membrane, which showed an excellent sensitivity of 0.8 μg/L for both the buffer and the serum samples compared with the direct detection of cTnI for the buffer with the lowest detection limit of 4 μg/L and conventional ELISA with the sensitivity of 1.9 μg/L.展开更多
Tomato yellow leaf curl virus(TYLCV)is a species of the family Geminiviridae,causing serious yield losses in tomato production.The coat protein(CP)gene of TYLCV isolate SH2 was expressed in Escherichia coli BL21(...Tomato yellow leaf curl virus(TYLCV)is a species of the family Geminiviridae,causing serious yield losses in tomato production.The coat protein(CP)gene of TYLCV isolate SH2 was expressed in Escherichia coli BL21(DE3)using pET-32a as the expression vector.The recombinant protein was purified through Ni+-NTA affinity column and used to immunize BALB/c mice.Three hybridoma cell lines(2B2,2E3 and 3E10)secreting monoclonal antibodies(MAbs)against TYLCV CP were obtained by fusing mouse myeloma cells(Sp 2/0)with spleen cells from the immunized BALB/c mouse.The titers of ascitic fluids of three MAbs ranged from 10-6 to 10-7 in indirect-ELISA.Isotypes and subclasses of all the MAbs belonged to IgG1,κ light chain.Triple antibody sandwich enzyme-linked immunosorbent assay(TAS-ELISA)showed that the MAb 3E10 could react with five begomoviruses infecting tomato,while the other two(2B2 and 2E3)mainly reacted with TYLCV.TAS-ELISA was set up using the MAb 3E10,and the established method could successfully detect virus in plant sap at 1:2 560(w/v,g mL-1).Detection of field samples showed that begomoviruses were common in tomato crops in Zhejiang Province,China.展开更多
To produce high-affinity monoclonal antibodies against pesticide carbofuran, and the develop immunochemical assays for people's health and environmental protection, the hapten 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofu...To produce high-affinity monoclonal antibodies against pesticide carbofuran, and the develop immunochemical assays for people's health and environmental protection, the hapten 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy) carbonyl]- amino]-butanoic acid (BFNB) of carbofuran was synthesized and Balb/c mice were immunized by the hapten-carrier (BFNB-bovine serum albumin, BFNB-BSA) conjugates. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by the indirect enzyme-linked immunoabsorbent assay (ELISA), based on BFNB-ovoalbumin conjugates (BFNB-OVA). Purified monoclonal antibody (McAb) was obtained from fluids of ascites, deposited by octanoic acid and ammonium sulfate. The affinity and the specificity of McAb were characterized by ELISA or indirect competitive ELISA. A hybridoma cell line (5D3) secreting anti-carbofuran McAb had been established. The titer of culture medium and ascites was up to 1:2.048 × 10^3 and 1:1.024 × 10^6, respectively, and the subtype of the McAb was IgG1. The affinity constant of the McAb was about 2.54 × 10×9 L mol^-1, with an IC50 value of 1.18 ng mL^-1 and a detection limit of 0.01 ng mL^-1. Cross-reactivity studies showed that the McAb was quiet specific for carbofuran, as among the four analogous compounds, they were all hardly recognized (4.59 × 10^-4% for 2,3-dihydro-2,2- dimethyl-7-benzofuranol and less than 3.0 × 10^4% for others). The prepared McAb had a very high affinity and specificity, and it could be used to develop ELISA for rapid determination of carbofuran.展开更多
With the size of the biopharmaceutical market exponentially increasing,there is an aligned growth in the importance of data-rich analyses,not only to assess drug product safety but also to assist drug development driv...With the size of the biopharmaceutical market exponentially increasing,there is an aligned growth in the importance of data-rich analyses,not only to assess drug product safety but also to assist drug development driven by the deeper understanding of structure/function relationships.In monoclonal antibodies,many functions are regulated by N-glycans present in the constant region of the heavy chains and their mechanisms of action are not completely known.The importance of their function focuses analytical research efforts on the development of robust,accurate and fast methods to support drug development and quality control.Released N-glycan analysis is considered as the gold standard for glycosylation characterisation;however,it is not the only method for quantitative analysis of glycoform heterogeneity.In this study,ten different analytical workflows for N-glycan analysis were compared using four monoclonal antibodies.While observing good comparability between the quantitative results generated,it was possible to appreciate the advantages and disadvantages of each technique and to summarise all the observations to guide the choice of the most appropriate analytical workflow according to application and the desired depth of data generated.展开更多
Objective To produce anti‐19‐Nortestosterone (NT) monoclonal antibodies and identify their immunological characteristics. Methods Hybridomas were prepared by fusing NS0 mouse myeloma cells with splenocytes isolate...Objective To produce anti‐19‐Nortestosterone (NT) monoclonal antibodies and identify their immunological characteristics. Methods Hybridomas were prepared by fusing NS0 mouse myeloma cells with splenocytes isolated from immunized BALB/c mice. Noncompetitive and competitive indirect ELISA were employed to screen positive cell clones. A caprylic acid ammonium sulphate (CAAP) method was used to purify NT mAb, and the Batty saturation method was used to determine the affinity constant (Kaff). Results Five hybridoma cell lines, named NT‐1, NT‐2, NT‐3, NT‐4, and NT‐5, were identified and their corresponding mAbs were of the IgG 1 isotype with a k light chain. The Kaffs of all mAbs were between 2.6 and 4.7×10 9 L/mol. The titers and IC 50 values of purified ascite fluids were in the range of (0.64–2.56)×10 5 and (0.55–1.0) ng/mL, respectively. Of all the cross‐reacting steroids, α ‐NT was the most reactive with the mAbs at 62% with NT‐1 mAb and 64% with NT‐2 mAb. Negligible cross‐reactivity (0.01%) with other steroids was observed. Conclusion The establishment of these hybridomas allows the potential development of a rapid test kit, and may provide an alternative method for the detection of NT residues in food producing animals.展开更多
Monoclonal antibodies(mAbs) are widely used in virus research and disease diagnosis. The nucleoprotein(NP) of influenza A virus(IAV) plays important roles in multiple stages of the virus life cycle. Therefore, generat...Monoclonal antibodies(mAbs) are widely used in virus research and disease diagnosis. The nucleoprotein(NP) of influenza A virus(IAV) plays important roles in multiple stages of the virus life cycle. Therefore, generating conserved mAbs against NP and characterizing their properties will provide useful tools for IAV research. In this study, two mAbs against the NP protein, 10 E9 and 3 F3, were generated with recombinant truncated NP proteins(NP-1 and NP-2) as immunogens. The heavy-chain subclass of both 10 E9 and 3 F3 was determined to be IgG2α, and the light-chain type was κ. Truncation and site-specific mutation analyses showed that the epitopes of mAbs 10 E9 and 3 F3 were located in the N terminal 84–89 amino acids and the C terminal 320–324 amino acids of the NP protein, respectively. We found that mAbs 10 E9 and 3 F3 reacted well with the NP protein of H1–H15 subtypes of IAV. Both 10 E9 and 3 F3 can be used in immunoprecipitation assay, and 10 E9 was also successfully applied in confocal microscopy. Furthermore, we found that the 10 E9-recognized _(84) SAGKDP_(89) epitope and 3 F3-recognized 320 ENPAH324 epitope were highly conserved in NP among all avian and human IAVs. Thus, the two mAbs we developed could be used as powerful tools in the development of diagnostic methods of IAV, and also surely promote the basic research in understanding the replication mechanisms of IAV.展开更多
Therapeutic monoclonal antibodies have become one of the central components of the healthcare system and continuous efforts are made to bring innovative antibody therapeutics to patients in need.It is equally critical...Therapeutic monoclonal antibodies have become one of the central components of the healthcare system and continuous efforts are made to bring innovative antibody therapeutics to patients in need.It is equally critical to acquire sufficient knowledge of their molecular structure and biological functions to ensure the efficacy and safety by incorporating new detection approaches since new challenges like individual differences and resistance are presented.Conventional techniques for determining antibody disposition including plasma drug concentration measurements using LC-MS or ELISA,and tissue distribution using immunohistochemistry and immunofluorescence are now complemented with molecular imaging modalities like positron emission tomography and near-infrared fluorescence imaging to obtain more dynamic information,while methods for characterization of antibody’s interaction with the target antigen as well as visualization of its cellular and intercellular behavior are still under development.Recent progress in detecting therapeutic antibodies,in particular,the development of methods suitable for illustrating the molecular dynamics,is described here.展开更多
The neutralizing activities of eight monoclonal antibodies (MAbs) against white spot syndrome virus (WSSV) (2D2, 2B2,1D2, 1D5, 1C2, 4A1, 6A4 and 6B4) were analyzed by in vivo experiments. Gills from WSSV-infecte...The neutralizing activities of eight monoclonal antibodies (MAbs) against white spot syndrome virus (WSSV) (2D2, 2B2,1D2, 1D5, 1C2, 4A1, 6A4 and 6B4) were analyzed by in vivo experiments. Gills from WSSV-infected shrimp were homogenized and ten-fold serially diluted by PBS, and then incubated with MAbs (hybridoma culture supernatant), respectively. The mixture of WSSV and MAbs were injected into crayfish (Procambarus clarkii). After challenge, the death rates of crayfish were counted to determine the neutralizing activities of MAbs. At the same time, the mixture of myeloma culture supernatant and WSSV or PBS was served as positive or negative control, respectively. The results showed that, at each virus dilution, the mean time to death of the crayfish injected with MAb-treated virus was significantly longer than that in the positive control, though they all showed 100% mortality within 25 d, and meanwhile, few crayfish died in the negative control. Among the eight MAbs, 2D2, 2B2, 1D2 and 1D5, especially the former two, delayed the mortality significantly, and 1 C2, 4A1 and 6A4 delayed the mortality as well but not so efficiently, while MAb 6IM was efficient only when the virus concentration increased. The results indicated that the anti-WSSV MAbs can neutralize WSSV in different virus dilutions.展开更多
Objective:Rabies is invariably a fatal encephalomyelitis that is considered to be a serious public health problem.It is necessary to develop standard rabies virus diagnostic tools,especially for diagnosing the strain...Objective:Rabies is invariably a fatal encephalomyelitis that is considered to be a serious public health problem.It is necessary to develop standard rabies virus diagnostic tools,especially for diagnosing the strains prevalent in China.Methods:Monoclonal antibodies(MAbs)specific to rabies virus were produced and characterized by enzyme linked immunosorbent assay(ELISA),isotyping,affinity assay,immunofluorescence assay(IFA),and immunocytochemistry.The MAb,whose affinity was higher for antigen,was used to establish an antigen captureELISA(AC-ELISA)detection system and test the efficiency by using clinical samples.Results:The heavy chain subclasses of two MAbs were all determined to be IgG2a.The 3C7 MAb showed stronger reactivity with rabies virus protein than the 2C5 MAb in an ELISA analysis,whereas the 3C7 MAb showed the highest affinity for antigen.IFA and immunocytochemistry results also indicated that the two MAbs could recognize rabies virus protein in its native form in cell samples.Data obtained using clinical samples showed that rabies virus could be detected by AC-ELISA detection system using the 3C7 MAb.Conclusion:It was potentially useful for the further development of highly sensitive,easily handled,and relatively rapid detection kits/tools for rabies surveillance in those areas where rabies is endemic,especially in China.展开更多
To prepare the PrP specific monoclonal antibodies (mAbs) that can be used for the detection of mammalian prions and study of pathogenesis of prion diseases. Methods Several BALB/c mice were immunized with recombinan...To prepare the PrP specific monoclonal antibodies (mAbs) that can be used for the detection of mammalian prions and study of pathogenesis of prion diseases. Methods Several BALB/c mice were immunized with recombinant hamster prion protein (HaPrP). Three hybridoma cell lines designated as B7, B9, and B10, secreting monoclonal antibodies against HaPrP, were established by hybridoma technique. The mAbs reactivities were evaluated with ELISA, Western blot, and immunohistochemistry. Results The mAbs produced by these cell lines reacted well with different recombinant hamster PrP proteins. Western blot analyses showed that mAbs B7 and B9 reacted with PrP^Sc from the scrapie-infected animals after proteinase K digestion with three glycosylated forms. The mAbs exhibited cross-reactivity with various PrPc from several other mammalian species, including humans and cattles, lmmunohistochemistry assays confirmed that mAbs B7 and B9 could recognize not only extracellular but also intracellular PrPso. Conclusion The mAbs of prion protein are successfully generated by hybridoma technique and can be applied for the diagnosis of prion associated diseases.展开更多
Murine monoclonal antibody (MoAb) BB4.3, raised against the human gastric cancer cell line BGC823, was puriffied with Protein A-Sepharose CL-4B affinity chromatography and identified as IgG2a. It was then conjugated w...Murine monoclonal antibody (MoAb) BB4.3, raised against the human gastric cancer cell line BGC823, was puriffied with Protein A-Sepharose CL-4B affinity chromatography and identified as IgG2a. It was then conjugated with a hematoporphyrin derivative (HPD) by using carbodiimide. The qualitative analysis of this conjugate showed that the amount of free HPD was negligible and there were no IgG aggregates among the conjugates. The conjugate retained both the antibody and photochemical activity of HPD.In vitro, the phototoxic effect of this HPD-BB4.3 conjugate on target cells was about 15 times higher than that of free HPD. The quality of selective photocytotoxicity was proven by the greater cytotoxi-city this conjugate showed than that of corresponding normal mouse IgG (NIgG) conjugated with HPD. It showed less cytotoxicity to colon cancer cell line B-80 (negative reaction to MoAb BB4.3) than to BGC825. Moreover, its cytotoxicity to BGC823 cells could be blocked specifically by excess BB4.3 antibody, but not by another MoAb 3G9, which combines with BGC823 at different binding sites from MoAb BB4.3.Nude mice inoculated with 2 × 10- BGC823 cells were given HPD-BB4.3, HPD, HPD-NIgG, HPD plus BB4.3 and PBS, respectively then exposed to light. Four out of six animals treated with the HPD-BB4.3 conjugate remained tumor-free for a long period. Although two developed tumors, there was a significant difference between the HPD-BB4.3-treated group and all the control groups in tumor induction time, tumor growth rate, and survival time (p<0.001). The HPD-BB4.3 conjugate inhibited the growth of established tumors by more than 40% in comparison with control groups (p<0.05).展开更多
Spleen cells from Balb/c mice immunized with five human gastric cancer cell lines in sequence were fused with murine myeloma cell line SP2/0, and hybridomas 3F4, 3G9 and 3H11, secreting monoclonal antibodiees (MoAbs) ...Spleen cells from Balb/c mice immunized with five human gastric cancer cell lines in sequence were fused with murine myeloma cell line SP2/0, and hybridomas 3F4, 3G9 and 3H11, secreting monoclonal antibodiees (MoAbs) against gastric cancer, were obtained through selective culture and screening. These MoAbs have both good selectivity and a high positive rate of reaction for gastric cancer, reaching 5/5 and 84.8% to 93.5% for gastric cancer cells and tissues respectively. The reaction of MoAbs with normal cells and tissues was neglible.The corresponding antigens of the MoAbs were sensitive to digestion by trypsin and pronase and resistant to treatment with sodium periodate, indicating their nature as proteins. The antigen 3G9 could be visualized with Western blotting as two bands with molecular weights of 100KD and 70KD, however no band was found for antigens 3F4 and 3H11. There was a high expression of antigens in the majority of gastric cancer cells and tissues independent of his-topathological type of gastric cancer. A low expression of antigens was seen with other tumors and fetal gastrointestinal tissues. These could be considered as gastric cancer-associated antigens or on-cofetal antigens with a quite extensive distribution.展开更多
The histological specificities of 4 monoclonal antibodies against cytokeratins (HK2, HK5, K174 and K27) were investigated in various kinds of human and rat tissues with ABC immunohistochemical technique. K174 was show...The histological specificities of 4 monoclonal antibodies against cytokeratins (HK2, HK5, K174 and K27) were investigated in various kinds of human and rat tissues with ABC immunohistochemical technique. K174 was shown to have the same recognition spectrum with a polyclonal antibody (RAK1) to epidermal keratin. HK2 and HK5 were similar except the difference in reaction with stratified squqmous epithelium. K27 could only label the suprabasal layers of the squamous epithelium, and could be used as a marker of cornified or cornifying epithelium. This study provided a sound basis for the use of this group of antibodies in the subtyping of different epithelial tissues and the tumors originating from them.展开更多
In the present study, an indirect assay was employed to investigate 5 anti-gastric cancer monoclonal antibodies for their cytotoxic potential as ricin A chain-containing immunotoxins. The tumor cell, were treated with...In the present study, an indirect assay was employed to investigate 5 anti-gastric cancer monoclonal antibodies for their cytotoxic potential as ricin A chain-containing immunotoxins. The tumor cell, were treated with dilutions of tested antibody followed by ricin A chain coupled to goat anti-mouse immunoglobulin. The cytotoxic effect was determined with tetrazolium colorimetric assay. The results showed that among the 5 antibodies chosen, MGb2 and MG7 could be well used for preparation of effective A chain immunotoxins.展开更多
Objective In 2017,China launched a new round of medical reform(NMR)to address the inaccessibility of high-priced drugs for patients with serious diseases.This study explored the impact of the NMR on the accessibility ...Objective In 2017,China launched a new round of medical reform(NMR)to address the inaccessibility of high-priced drugs for patients with serious diseases.This study explored the impact of the NMR on the accessibility and affordability of high-priced monoclonal antibodies(mAbs),and the effective promotion policies after the NMR.Methods We used a standard method developed by the World Health Organization to conduct two surveys on the availability of mAbs and their prices before and after the NMR in the public hospitals in Hubei province,China.By interviewing hospital pharmacy experts,we identified the potential value of the current NMR in improving the access to therapeutic mAbs.Results The average availability of 13 mAbs increased by 8.1%in the surveyed hospitals of Hubei province after the NMR.The median unit price of 10 mAbs dropped by 34.3%.The average affordability of a treatment cycle of 10 mAbs dropped from 680 days to 298 days of the disposable daily income for a middle-income resident(56.2%reduction).The drug price negotiation of medical insurance inclusion and the promotion of consistent evaluation of generic and original drugs could effectively promote the accessibility of mAbs.However,the zero markup of drug pricing and the limit on the proportion of drug revenues in public hospitals showed certain negative effects on the availability of mAbs.Conclusion Not all current NMR policies play a positive role in promoting the accessibility of mAbs.To further improve the accessibility of mAbs in the future in China,it is therefore critical to increase the investment in independent research and development of high-quality mAbs,establish localized guidelines for the rational use of mAbs in clinical practice,and have a cost-sharing mechanism for high-priced drugs with multiple stakeholders.展开更多
Nervous necrosis virus(NNV)can infect more than 120 fish species worldwide and has caused high mortality and sig-nificant economic losses to the aquaculture industry.Among different genotypes of NNV,the red-grouper ne...Nervous necrosis virus(NNV)can infect more than 120 fish species worldwide and has caused high mortality and sig-nificant economic losses to the aquaculture industry.Among different genotypes of NNV,the red-grouper nervous necrosis virus(RGNNV)is the most widely distributed one with the highest number of susceptible fish species.In this study,the capsid protein(Cp)gene of RGNNV was recombined and expressed in Escherichia coli strain BL21(DE3)and the recombinant Cp(rCp)was used as an immunogen to produce monoclonal antibodies(MAbs)through hybridoma cell fusion technology.Three MAbs were produced and characterized by indirect enzyme-linked immunosorbent assay(ELISA),western blotting,and immunofluorescence assay(IFA).Wes-tern blotting result showed that the MAbs could specifically react with the capsid protein of RGNNV.The result of IFA showed that the MAbs could recognize virions in RGNNV-infected grouper spleen(GS)cells.These results indicate that the MAbs can specifi-cally recognize RGNNV virions and can be used to produce a rapid detection method.This study provides a foundation for further studies on the rapid diagnosis of RGNNV and its infection mechanisms.展开更多
Thirty-five hybridoma cell lines against cytoke-ratins, isolated from Hela cells and human cellus respectively, were generated by fusion of immunized spleen cells of BALB/C mice with P3×63-Ag8.653, a mouse myelom...Thirty-five hybridoma cell lines against cytoke-ratins, isolated from Hela cells and human cellus respectively, were generated by fusion of immunized spleen cells of BALB/C mice with P3×63-Ag8.653, a mouse myeloma cell line. Two of them (HI and C53) were characterized by indirect immu-nofluorescence, ABC immunostaining and immuno-blotting. The results of immunofluorescence and ABC immunostaining suggested that both monoclonal antibodies were specific for keratin-type intermediate filaments. However, the two monoclonal antibodies showed different specificities in normal tissues and neoplasms as observed on both frozen and deparaf-finized sections. In normal tissues, H1 stained transitional epithelium and all types of simple epithelium except endothelium and mesothelium but did not stain stratified squamous epithelium. In contrast, C35 recognized only stratified squamous epithelium, but failing to react with simple epithelium. Both monoclonal antibodies did not react with nonepithelia cells and tissues. In neoplasms, H1 stained varieties of adenocaroinomas and C35 recognized merely squamous cell carcinomas. Therefore, all the epithelial tissues can nearly be recognized by combination of the two monoclonal antibodies. All the results indicated that H1 and C35 can be used in cell biology and histology studies, and can be used in differential diagnosis of adenocarcinoma and squamous cell carcinoma in pathology.展开更多
文摘Hepatitis B virus(HBV)reactivation poses a significant clinical challenge,espe-cially in patients undergoing immunosuppressive therapies,including mono-clonal antibody treatments.This manuscript briefly explores the complex rela-tionship between monoclonal antibody therapy and HBV reactivation,drawing upon current literature and clinical case studies.It delves into the mechanisms underlying this phenomenon,highlighting the importance of risk assessment,monitoring,and prophylactic measures for patients at risk.The manuscript aims to enhance the understanding of HBV reactivation in the context of monoclonal antibody therapy,ultimately facilitating informed clinical decision-making and improved patient care.This paper will also briefly review the definition of HBV activation,assess the risks of reactivation,especially in patients treated with monoclonal antibodies,and consider management for patients with regard to screening,prophylaxis,and treatment.A better understanding of patients at risk can help clinicians provide optimum management to ensure successful patient outcomes and prevent morbidity.
基金supported by the National Natural Science Foundation of China(31272015)the National Basic Research Program(973)of China(2014CB138400)the Special Fund for Agro-scientific Research in the Public Interest,China(201303021,201303028)
文摘Aphid-borne Zucchini yellow mosaic virus (ZYMV) is one of the most economically important viruses of cucurbitaceous plants. To survey and control this virus, it is necessary to develop an efficient detection technique. Using purified ZYMV virion and the conventional hybridoma technology, three hybridoma cell lines (16A11, 5A7 and 3B8) secreting monoclonal antibodies (MAbs) against ZYMV Zhejiang isolate were obtained. The working titers of the ascitic fluids secreted by the three hybridoma cell lines were up to 10^-7 by indirect enzyme-linked immunosorbent assay (ELISA). All MAbs were isotyped as IgG1, kappa light chain. Western blot analysis indicated that the MAb 3B8 could specifically react with the coat protein of ZYMV while MAbs 5A7 and 16A11 reacted strongly with a protein of approximately 51 kDa from the ZYMV-infected leaf tissues. According to this molecular weight, we consider this reactive protein As likely to be the HC-Pro protein. Using these three MAbs, we have now developed five detection assays, i.e., antigen-coated-plate ELISA (ACP-ELISA), dot-ELISA, tissue blot-ELISA, double-antibody sandwich ELISA (DAS-ELISA), and immunocapture-RT-PCR (IC-RT-PCR), for the sensitive, specific, and easy detection of ZYMV. The sensitivity test revealed that ZYMV could be readily detected respectively by ACP-ELISA, dot-ELISA, DAS-ELISA and IC-RT-PCR in 1:163840, 1:2560, 1:327680 and 1:1 310720 (w/v, g mL-1) diluted crude extracts from the ZYMV-infected plants. We demonstrated in this study that the dot-ELISA could also be used to detect ZYMV in individual viruliferous aphids. A total of 275 cucurbitaceous plant samples collected from the Zhejiang, Jiangsu, Shandong and Hainan provinces, China, were screened for the presence of ZYMV with the described assays. Our results showed that 163 of the 275 samples (59%) were infected with ZYMV. This finding indicates that ZYMV As now widely present in cucurbitaceous crops in China. RT-PCR followed by DNA sequencing and sequence analyses confirmed the accuracy of the five assays. We consider that these detection assays can significantly benefit the control of ZYMV in China.
基金National Natural Science Foundation of China (39970519) Guangxi Basic Studying Fund (0236053) +1 种基金 Guangdong Province Natural Science Foundation (990683) Core Teacher Project of Ministry of Education and Chinese Postdoctoral Fu
文摘To purify Methamidophos (Met) monoclonal antibodies with two methods and compare immune activity of purified antibodies. Method Caprylic acid ammonium sulphate precipition (CAASP) method and Sepharose protein-A (SPA) affinity chromatography method were used to purify Met monoclonal antibodies, UV spectrum scanning was used to determine protein content and recovery of purified antibodies, sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the purity of purified antibodies, and enzyme-linked immunosorbent assay (ELISA) was used to determine immune activity of purified antibodies. Results Antibody protein content and recovery rate with CAASP method were 7.62 mg/mL and 8.05% respectively, antibody protein content and recovery rate with SPA method were 6.45 mg/mL and 5.52% respectively. Purity of antibodies purified by SPA method was higher than that by CAASP method. The half-maximal inhibition concentration (IC50) of antibodies purified by SPA to Met was 181.26 靏/mL, and the linear working range and the limit of quantification (LOD) were 2.43-3896.01 靏/mL and 1.03 靏/mL, respectively. The IC50 of antibodies purified by CAASP to Met was 352.82 靏/mL, and the linear working range and LOD were 10.91-11412.29 靏/mL and 3.42 靏/mL, respectively. Conclusion Antibodies purified by SPA method are better than those by CAASP method, and Met monoclonal antibodies purified by SPA method can be used to prepare gold-labelled testing paper for analyzing Met residue in vegetable and drink water.
基金Supported by National Natural Science Fundation of China(Project No.2 9875 0 10 ) and the Health Department of JilinProvince(Project No.980 4 7) ,China
文摘Cardiac troponin I(cTnI) was separated and purified from human left ventricular tissue by affinity chromatographic method and used to immunize Balb/c mice by intraperitoneal injection and four hybridoma cell lines, which secreted monoclonal antibody(mAb) against human cTnI, were obtained by cell fusion, identification and cloning twice. Three mAbs(9F5, 2F11, 8C12) were produced from the ascites of Balb/c mice injected intraperitoneally the hybridoma cells and characterized by means of a surface plasmon resonance(SPR) biosensor. An optimal and specific sensing membrane for troponin I was prepared with staphylococcal protein A(SPA) as the intermediate layer and mAb against human cTnI as the capture antibody. On the basis of the sensing membrane, two modes of operation of the SPR biosensor were developed, i.e ., a direct detection of antigen antibody affinity and a sandwich assay. In the sandwich assay detection mode, the mAbs competition was measured by monitoring whether the secondary antibody had been attached to the cTnI already captured by the first antibody on the sensor surface. The SPR biosensor was shown to be able to directly detect the antigen antibody affinity and the order of the affinity was found to be 9F5>2F11>8C12. In the sandwich detection mode, it was found that the different epitopes on the cTnI molecules were recognized by the three mAbs respectively, but the asymmetrical competition was shown between 2F11 and 8C12 and no competition was found between 9F5 and 2F11 or 8c12. Based on these results, a double monoclonal sandwich immunoassay for cTnI was developed by using the optimal antibody pair of 9F5 and 2F11 and the SPR biosensor with SPA substrate membrane, which showed an excellent sensitivity of 0.8 μg/L for both the buffer and the serum samples compared with the direct detection of cTnI for the buffer with the lowest detection limit of 4 μg/L and conventional ELISA with the sensitivity of 1.9 μg/L.
基金supported by the Special Fund for Agro-Scientific Research in the Public Interest from the Ministry of Agriculture,China(201003065)
文摘Tomato yellow leaf curl virus(TYLCV)is a species of the family Geminiviridae,causing serious yield losses in tomato production.The coat protein(CP)gene of TYLCV isolate SH2 was expressed in Escherichia coli BL21(DE3)using pET-32a as the expression vector.The recombinant protein was purified through Ni+-NTA affinity column and used to immunize BALB/c mice.Three hybridoma cell lines(2B2,2E3 and 3E10)secreting monoclonal antibodies(MAbs)against TYLCV CP were obtained by fusing mouse myeloma cells(Sp 2/0)with spleen cells from the immunized BALB/c mouse.The titers of ascitic fluids of three MAbs ranged from 10-6 to 10-7 in indirect-ELISA.Isotypes and subclasses of all the MAbs belonged to IgG1,κ light chain.Triple antibody sandwich enzyme-linked immunosorbent assay(TAS-ELISA)showed that the MAb 3E10 could react with five begomoviruses infecting tomato,while the other two(2B2 and 2E3)mainly reacted with TYLCV.TAS-ELISA was set up using the MAb 3E10,and the established method could successfully detect virus in plant sap at 1:2 560(w/v,g mL-1).Detection of field samples showed that begomoviruses were common in tomato crops in Zhejiang Province,China.
基金This study was supported by the Key Project of Natural Science Foundation of Guangdong Province, China (036842) Key Project of Key Technologies R&D Program of Guangzhou City, China (2004Z2-E0031 ,2004Z1-E0061)+2 种基金 Natural Science Foundation of Guangdong Province, China (06300421) National HighTech Research and Development Program of China (863 Program) (2006AA10Z447) and Key Technologies R&D Program during the 10th Five-Year Plan Period, China (2002BA518A06-04).
文摘To produce high-affinity monoclonal antibodies against pesticide carbofuran, and the develop immunochemical assays for people's health and environmental protection, the hapten 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy) carbonyl]- amino]-butanoic acid (BFNB) of carbofuran was synthesized and Balb/c mice were immunized by the hapten-carrier (BFNB-bovine serum albumin, BFNB-BSA) conjugates. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by the indirect enzyme-linked immunoabsorbent assay (ELISA), based on BFNB-ovoalbumin conjugates (BFNB-OVA). Purified monoclonal antibody (McAb) was obtained from fluids of ascites, deposited by octanoic acid and ammonium sulfate. The affinity and the specificity of McAb were characterized by ELISA or indirect competitive ELISA. A hybridoma cell line (5D3) secreting anti-carbofuran McAb had been established. The titer of culture medium and ascites was up to 1:2.048 × 10^3 and 1:1.024 × 10^6, respectively, and the subtype of the McAb was IgG1. The affinity constant of the McAb was about 2.54 × 10×9 L mol^-1, with an IC50 value of 1.18 ng mL^-1 and a detection limit of 0.01 ng mL^-1. Cross-reactivity studies showed that the McAb was quiet specific for carbofuran, as among the four analogous compounds, they were all hardly recognized (4.59 × 10^-4% for 2,3-dihydro-2,2- dimethyl-7-benzofuranol and less than 3.0 × 10^4% for others). The prepared McAb had a very high affinity and specificity, and it could be used to develop ELISA for rapid determination of carbofuran.
文摘With the size of the biopharmaceutical market exponentially increasing,there is an aligned growth in the importance of data-rich analyses,not only to assess drug product safety but also to assist drug development driven by the deeper understanding of structure/function relationships.In monoclonal antibodies,many functions are regulated by N-glycans present in the constant region of the heavy chains and their mechanisms of action are not completely known.The importance of their function focuses analytical research efforts on the development of robust,accurate and fast methods to support drug development and quality control.Released N-glycan analysis is considered as the gold standard for glycosylation characterisation;however,it is not the only method for quantitative analysis of glycoform heterogeneity.In this study,ten different analytical workflows for N-glycan analysis were compared using four monoclonal antibodies.While observing good comparability between the quantitative results generated,it was possible to appreciate the advantages and disadvantages of each technique and to summarise all the observations to guide the choice of the most appropriate analytical workflow according to application and the desired depth of data generated.
基金supported by the Henan Innovation Project for University Prominent Research Talents, 2010HASTIT026
文摘Objective To produce anti‐19‐Nortestosterone (NT) monoclonal antibodies and identify their immunological characteristics. Methods Hybridomas were prepared by fusing NS0 mouse myeloma cells with splenocytes isolated from immunized BALB/c mice. Noncompetitive and competitive indirect ELISA were employed to screen positive cell clones. A caprylic acid ammonium sulphate (CAAP) method was used to purify NT mAb, and the Batty saturation method was used to determine the affinity constant (Kaff). Results Five hybridoma cell lines, named NT‐1, NT‐2, NT‐3, NT‐4, and NT‐5, were identified and their corresponding mAbs were of the IgG 1 isotype with a k light chain. The Kaffs of all mAbs were between 2.6 and 4.7×10 9 L/mol. The titers and IC 50 values of purified ascite fluids were in the range of (0.64–2.56)×10 5 and (0.55–1.0) ng/mL, respectively. Of all the cross‐reacting steroids, α ‐NT was the most reactive with the mAbs at 62% with NT‐1 mAb and 64% with NT‐2 mAb. Negligible cross‐reactivity (0.01%) with other steroids was observed. Conclusion The establishment of these hybridomas allows the potential development of a rapid test kit, and may provide an alternative method for the detection of NT residues in food producing animals.
基金supported by the Natural Science Foundation of Heilongjiang Province,China(JQ2019C005)the National Natural Science Foundation of China(31702265 and 32172847)。
文摘Monoclonal antibodies(mAbs) are widely used in virus research and disease diagnosis. The nucleoprotein(NP) of influenza A virus(IAV) plays important roles in multiple stages of the virus life cycle. Therefore, generating conserved mAbs against NP and characterizing their properties will provide useful tools for IAV research. In this study, two mAbs against the NP protein, 10 E9 and 3 F3, were generated with recombinant truncated NP proteins(NP-1 and NP-2) as immunogens. The heavy-chain subclass of both 10 E9 and 3 F3 was determined to be IgG2α, and the light-chain type was κ. Truncation and site-specific mutation analyses showed that the epitopes of mAbs 10 E9 and 3 F3 were located in the N terminal 84–89 amino acids and the C terminal 320–324 amino acids of the NP protein, respectively. We found that mAbs 10 E9 and 3 F3 reacted well with the NP protein of H1–H15 subtypes of IAV. Both 10 E9 and 3 F3 can be used in immunoprecipitation assay, and 10 E9 was also successfully applied in confocal microscopy. Furthermore, we found that the 10 E9-recognized _(84) SAGKDP_(89) epitope and 3 F3-recognized 320 ENPAH324 epitope were highly conserved in NP among all avian and human IAVs. Thus, the two mAbs we developed could be used as powerful tools in the development of diagnostic methods of IAV, and also surely promote the basic research in understanding the replication mechanisms of IAV.
基金We acknowledge financial support from the National Natural Science Foundation of China(81903574)the Fundamental Research Funds for the Central Universities(2019QNA7046,2020QNA7001).
文摘Therapeutic monoclonal antibodies have become one of the central components of the healthcare system and continuous efforts are made to bring innovative antibody therapeutics to patients in need.It is equally critical to acquire sufficient knowledge of their molecular structure and biological functions to ensure the efficacy and safety by incorporating new detection approaches since new challenges like individual differences and resistance are presented.Conventional techniques for determining antibody disposition including plasma drug concentration measurements using LC-MS or ELISA,and tissue distribution using immunohistochemistry and immunofluorescence are now complemented with molecular imaging modalities like positron emission tomography and near-infrared fluorescence imaging to obtain more dynamic information,while methods for characterization of antibody’s interaction with the target antigen as well as visualization of its cellular and intercellular behavior are still under development.Recent progress in detecting therapeutic antibodies,in particular,the development of methods suitable for illustrating the molecular dynamics,is described here.
基金The National Basic Research Program of China under contract No 2006CB101806the National"863"Project of China under con-tract No 2006AA100312
文摘The neutralizing activities of eight monoclonal antibodies (MAbs) against white spot syndrome virus (WSSV) (2D2, 2B2,1D2, 1D5, 1C2, 4A1, 6A4 and 6B4) were analyzed by in vivo experiments. Gills from WSSV-infected shrimp were homogenized and ten-fold serially diluted by PBS, and then incubated with MAbs (hybridoma culture supernatant), respectively. The mixture of WSSV and MAbs were injected into crayfish (Procambarus clarkii). After challenge, the death rates of crayfish were counted to determine the neutralizing activities of MAbs. At the same time, the mixture of myeloma culture supernatant and WSSV or PBS was served as positive or negative control, respectively. The results showed that, at each virus dilution, the mean time to death of the crayfish injected with MAb-treated virus was significantly longer than that in the positive control, though they all showed 100% mortality within 25 d, and meanwhile, few crayfish died in the negative control. Among the eight MAbs, 2D2, 2B2, 1D2 and 1D5, especially the former two, delayed the mortality significantly, and 1 C2, 4A1 and 6A4 delayed the mortality as well but not so efficiently, while MAb 6IM was efficient only when the virus concentration increased. The results indicated that the anti-WSSV MAbs can neutralize WSSV in different virus dilutions.
基金supported by the National High Technology Research and Development Program of China(863 Program,No.2007AA02Z418)
文摘Objective:Rabies is invariably a fatal encephalomyelitis that is considered to be a serious public health problem.It is necessary to develop standard rabies virus diagnostic tools,especially for diagnosing the strains prevalent in China.Methods:Monoclonal antibodies(MAbs)specific to rabies virus were produced and characterized by enzyme linked immunosorbent assay(ELISA),isotyping,affinity assay,immunofluorescence assay(IFA),and immunocytochemistry.The MAb,whose affinity was higher for antigen,was used to establish an antigen captureELISA(AC-ELISA)detection system and test the efficiency by using clinical samples.Results:The heavy chain subclasses of two MAbs were all determined to be IgG2a.The 3C7 MAb showed stronger reactivity with rabies virus protein than the 2C5 MAb in an ELISA analysis,whereas the 3C7 MAb showed the highest affinity for antigen.IFA and immunocytochemistry results also indicated that the two MAbs could recognize rabies virus protein in its native form in cell samples.Data obtained using clinical samples showed that rabies virus could be detected by AC-ELISA detection system using the 3C7 MAb.Conclusion:It was potentially useful for the further development of highly sensitive,easily handled,and relatively rapid detection kits/tools for rabies surveillance in those areas where rabies is endemic,especially in China.
文摘To prepare the PrP specific monoclonal antibodies (mAbs) that can be used for the detection of mammalian prions and study of pathogenesis of prion diseases. Methods Several BALB/c mice were immunized with recombinant hamster prion protein (HaPrP). Three hybridoma cell lines designated as B7, B9, and B10, secreting monoclonal antibodies against HaPrP, were established by hybridoma technique. The mAbs reactivities were evaluated with ELISA, Western blot, and immunohistochemistry. Results The mAbs produced by these cell lines reacted well with different recombinant hamster PrP proteins. Western blot analyses showed that mAbs B7 and B9 reacted with PrP^Sc from the scrapie-infected animals after proteinase K digestion with three glycosylated forms. The mAbs exhibited cross-reactivity with various PrPc from several other mammalian species, including humans and cattles, lmmunohistochemistry assays confirmed that mAbs B7 and B9 could recognize not only extracellular but also intracellular PrPso. Conclusion The mAbs of prion protein are successfully generated by hybridoma technique and can be applied for the diagnosis of prion associated diseases.
文摘Murine monoclonal antibody (MoAb) BB4.3, raised against the human gastric cancer cell line BGC823, was puriffied with Protein A-Sepharose CL-4B affinity chromatography and identified as IgG2a. It was then conjugated with a hematoporphyrin derivative (HPD) by using carbodiimide. The qualitative analysis of this conjugate showed that the amount of free HPD was negligible and there were no IgG aggregates among the conjugates. The conjugate retained both the antibody and photochemical activity of HPD.In vitro, the phototoxic effect of this HPD-BB4.3 conjugate on target cells was about 15 times higher than that of free HPD. The quality of selective photocytotoxicity was proven by the greater cytotoxi-city this conjugate showed than that of corresponding normal mouse IgG (NIgG) conjugated with HPD. It showed less cytotoxicity to colon cancer cell line B-80 (negative reaction to MoAb BB4.3) than to BGC825. Moreover, its cytotoxicity to BGC823 cells could be blocked specifically by excess BB4.3 antibody, but not by another MoAb 3G9, which combines with BGC823 at different binding sites from MoAb BB4.3.Nude mice inoculated with 2 × 10- BGC823 cells were given HPD-BB4.3, HPD, HPD-NIgG, HPD plus BB4.3 and PBS, respectively then exposed to light. Four out of six animals treated with the HPD-BB4.3 conjugate remained tumor-free for a long period. Although two developed tumors, there was a significant difference between the HPD-BB4.3-treated group and all the control groups in tumor induction time, tumor growth rate, and survival time (p<0.001). The HPD-BB4.3 conjugate inhibited the growth of established tumors by more than 40% in comparison with control groups (p<0.05).
文摘Spleen cells from Balb/c mice immunized with five human gastric cancer cell lines in sequence were fused with murine myeloma cell line SP2/0, and hybridomas 3F4, 3G9 and 3H11, secreting monoclonal antibodiees (MoAbs) against gastric cancer, were obtained through selective culture and screening. These MoAbs have both good selectivity and a high positive rate of reaction for gastric cancer, reaching 5/5 and 84.8% to 93.5% for gastric cancer cells and tissues respectively. The reaction of MoAbs with normal cells and tissues was neglible.The corresponding antigens of the MoAbs were sensitive to digestion by trypsin and pronase and resistant to treatment with sodium periodate, indicating their nature as proteins. The antigen 3G9 could be visualized with Western blotting as two bands with molecular weights of 100KD and 70KD, however no band was found for antigens 3F4 and 3H11. There was a high expression of antigens in the majority of gastric cancer cells and tissues independent of his-topathological type of gastric cancer. A low expression of antigens was seen with other tumors and fetal gastrointestinal tissues. These could be considered as gastric cancer-associated antigens or on-cofetal antigens with a quite extensive distribution.
文摘The histological specificities of 4 monoclonal antibodies against cytokeratins (HK2, HK5, K174 and K27) were investigated in various kinds of human and rat tissues with ABC immunohistochemical technique. K174 was shown to have the same recognition spectrum with a polyclonal antibody (RAK1) to epidermal keratin. HK2 and HK5 were similar except the difference in reaction with stratified squqmous epithelium. K27 could only label the suprabasal layers of the squamous epithelium, and could be used as a marker of cornified or cornifying epithelium. This study provided a sound basis for the use of this group of antibodies in the subtyping of different epithelial tissues and the tumors originating from them.
文摘In the present study, an indirect assay was employed to investigate 5 anti-gastric cancer monoclonal antibodies for their cytotoxic potential as ricin A chain-containing immunotoxins. The tumor cell, were treated with dilutions of tested antibody followed by ricin A chain coupled to goat anti-mouse immunoglobulin. The cytotoxic effect was determined with tetrazolium colorimetric assay. The results showed that among the 5 antibodies chosen, MGb2 and MG7 could be well used for preparation of effective A chain immunotoxins.
基金supported by the grants from the National Natural Science Foundation of China(No.70903025,No.71373089).
文摘Objective In 2017,China launched a new round of medical reform(NMR)to address the inaccessibility of high-priced drugs for patients with serious diseases.This study explored the impact of the NMR on the accessibility and affordability of high-priced monoclonal antibodies(mAbs),and the effective promotion policies after the NMR.Methods We used a standard method developed by the World Health Organization to conduct two surveys on the availability of mAbs and their prices before and after the NMR in the public hospitals in Hubei province,China.By interviewing hospital pharmacy experts,we identified the potential value of the current NMR in improving the access to therapeutic mAbs.Results The average availability of 13 mAbs increased by 8.1%in the surveyed hospitals of Hubei province after the NMR.The median unit price of 10 mAbs dropped by 34.3%.The average affordability of a treatment cycle of 10 mAbs dropped from 680 days to 298 days of the disposable daily income for a middle-income resident(56.2%reduction).The drug price negotiation of medical insurance inclusion and the promotion of consistent evaluation of generic and original drugs could effectively promote the accessibility of mAbs.However,the zero markup of drug pricing and the limit on the proportion of drug revenues in public hospitals showed certain negative effects on the availability of mAbs.Conclusion Not all current NMR policies play a positive role in promoting the accessibility of mAbs.To further improve the accessibility of mAbs in the future in China,it is therefore critical to increase the investment in independent research and development of high-quality mAbs,establish localized guidelines for the rational use of mAbs in clinical practice,and have a cost-sharing mechanism for high-priced drugs with multiple stakeholders.
基金supported by the National Natural Science Foundation of China (No. 31972834)the National Key Research and Development Program of China (No. 2018YFD0900505)
文摘Nervous necrosis virus(NNV)can infect more than 120 fish species worldwide and has caused high mortality and sig-nificant economic losses to the aquaculture industry.Among different genotypes of NNV,the red-grouper nervous necrosis virus(RGNNV)is the most widely distributed one with the highest number of susceptible fish species.In this study,the capsid protein(Cp)gene of RGNNV was recombined and expressed in Escherichia coli strain BL21(DE3)and the recombinant Cp(rCp)was used as an immunogen to produce monoclonal antibodies(MAbs)through hybridoma cell fusion technology.Three MAbs were produced and characterized by indirect enzyme-linked immunosorbent assay(ELISA),western blotting,and immunofluorescence assay(IFA).Wes-tern blotting result showed that the MAbs could specifically react with the capsid protein of RGNNV.The result of IFA showed that the MAbs could recognize virions in RGNNV-infected grouper spleen(GS)cells.These results indicate that the MAbs can specifi-cally recognize RGNNV virions and can be used to produce a rapid detection method.This study provides a foundation for further studies on the rapid diagnosis of RGNNV and its infection mechanisms.
文摘Thirty-five hybridoma cell lines against cytoke-ratins, isolated from Hela cells and human cellus respectively, were generated by fusion of immunized spleen cells of BALB/C mice with P3×63-Ag8.653, a mouse myeloma cell line. Two of them (HI and C53) were characterized by indirect immu-nofluorescence, ABC immunostaining and immuno-blotting. The results of immunofluorescence and ABC immunostaining suggested that both monoclonal antibodies were specific for keratin-type intermediate filaments. However, the two monoclonal antibodies showed different specificities in normal tissues and neoplasms as observed on both frozen and deparaf-finized sections. In normal tissues, H1 stained transitional epithelium and all types of simple epithelium except endothelium and mesothelium but did not stain stratified squamous epithelium. In contrast, C35 recognized only stratified squamous epithelium, but failing to react with simple epithelium. Both monoclonal antibodies did not react with nonepithelia cells and tissues. In neoplasms, H1 stained varieties of adenocaroinomas and C35 recognized merely squamous cell carcinomas. Therefore, all the epithelial tissues can nearly be recognized by combination of the two monoclonal antibodies. All the results indicated that H1 and C35 can be used in cell biology and histology studies, and can be used in differential diagnosis of adenocarcinoma and squamous cell carcinoma in pathology.