Porcine proliferative enteropathy(PPE),an important infectious disease in pig production caused by an obligate intracellular bacterium Lawsonia intracellularis,is commonly associated with diarrhea and reduced weight g...Porcine proliferative enteropathy(PPE),an important infectious disease in pig production caused by an obligate intracellular bacterium Lawsonia intracellularis,is commonly associated with diarrhea and reduced weight gain in growing pigs widespread.An accurate method for detecting L.intracellularis is particularly important for preventing and controlling PPE.Heat shock protein 60(Hsp60)is an immunodominant bacterial antigen found in all eukaryotic and prokaryotic organisms.Thus,the purpose of the current investigation was to produce a novel L.intracellularis Hsp60 monoclonal antibody(mAb)useful for immunodiagnostics.Three hybridomas secreted anti-Hsp60 termed 3E5,4E2,and 9G6 were generated,and the titers of ascitic fluids of 3E5,4E2,9G6 were 1:1024000,1:2048000 and 1:2048000,respectively.The Western blotting analysis demonstrated that recombinant Hsp60(rHsp60)was recognized by mAbs 3E5,4E2 and 9G6.Subsequently,analyses of specificity showed all the mAbs were highly specific to L.intracellularis while could not significantly react with other enteric bacteria commonly found in the ileum of pigs,such as Escherichia coli,Salmonella Choleraesuis,Salmonella Typhimurium,and Brachyspira hyodysenteriae.Furthermore,the mAbs were useful for detecting L.intracellularis in the infected monolayer cells and histological sections of the ileum from PPE-affected pigs.Our research will provide a foundation for the development of immunological diagnostic tests.展开更多
Morphine is a frequently used analgesic that activates the mu-opioid receptor(MOR),which has prominent side effects of tolerance.Although the inefficiency of morphine in inducing the endocytosis of MOR underlies the d...Morphine is a frequently used analgesic that activates the mu-opioid receptor(MOR),which has prominent side effects of tolerance.Although the inefficiency of morphine in inducing the endocytosis of MOR underlies the development of morphine tolerance,currently,there is no effective therapy to treat morphine tolerance.In the current study,we aimed to develop a monoclonal antibody(mAb)precisely targeting MOR and to determine its therapeutic efficacy on morphine tolerance and the underlying molecular mechanisms.We successfully prepared a mAb targeting MOR,named 3A5C7,by hybridoma technique using a strategy of deoxyribonucleic acid immunization combined with cell immunization,and identified it as an immunoglobulin G mAb with high specificity and affinity for MOR and binding ability to antigens with spatial conformation.Treatment of two cell lines,HEK293T and SH-SY5Y,with 3A5C7 enhanced morphine-induced MOR endocytosis via a G protein-coupled receptor kinase 2(GRK2)/b-arrestin2-dependent mechanism,as demonstrated by immunofluorescence staining,flow cytometry,Western blotting,coimmunoprecipitation,and small interfering ribonucleic acid(siRNA)-based knockdown.This mAb also allowed MOR recycling from cytoplasm to plasma membrane and attenuated morphine-induced phosphorylation of MOR.We established an in vitro morphine tolerance model using differentiated SH-SY5Y cells induced by retinoic acid.Western blot,enzyme-linked immunosorbent assays,and siRNA-based knockdown revealed that 3A5C7 mAb diminished hyperactivation of adenylate cyclase,the in vitro biomarker of morphine tolerance,via the GRK2/b-arrestin2 pathway.Furthermore,in vivo hotplate test demonstrated that chronic intrathecal administration of 3A5C7 significantly alleviated morphine tolerance in mice,and withdrawal jumping test revealed that both chronic and acute 3A5C7 intrathecal administration attenuated morphine dependence.Finally,intrathecal electroporation of silencing short hairpin RNA illustrated that the in vivo anti-tolerance and anti-dependence efficacy of 3A5C7 was mediated by enhanced morphine-induced MOR endocytosis via GRK2/b-arrestin2 pathway.Collectively,our study provided a therapeutic mAb,3A5C7,targeting MOR to treat morphine tolerance,mediated by enhancing morphine-induced MOR endocytosis.The mAb 3A5C7 demonstrates promising translational value to treat clinical morphine tolerance.展开更多
Process analytical technology(PAT) is gaining more interest in the biomanufacturing industry because of its potential to improve operational control and compliance through real-time quality assurance.Currently, biopha...Process analytical technology(PAT) is gaining more interest in the biomanufacturing industry because of its potential to improve operational control and compliance through real-time quality assurance.Currently, biopharmaceutical producers mainly monitor chromatographic processes with ultraviolet/visible(UV/Vis) absorbance. However, this measurement has a very limited correlation with purity and quantity. The current study aims to determine the concentration of monoclonal antibody(mAb) and host cell proteins(HCPs) using a build-in UV/Vis monitoring during Protein A affinity chromatography and to optimize the separation conditions for high purity of mAb and minimizing the HCPs content. The eluate was analyzed through in-line UV/Vis at 280 and 410 nm, representing mAb and HCPs concentration,respectively. Each 0.1 column volume(CV) fraction of UV/Vis chromatogram peak area were calculated,and different separation conditions were then compared. The optimum conditions of mAb separation were found as 12 CV loading, elution at pH 3.5, and starting the collection at 0.5 CV point, resulting in high m Ab recovery of 95.92% and additional removal of 49.98% of HCP comparing with whole elution pool. This study concluded that UV/Vis-based in-line monitoring at 280 and 410 nm showed a high potential to optimize and real-time control Protein A affinity chromatography for mAb purification from HCPs.展开更多
The purpose of this study was to investigate the clinical application of severe acute respiratory distress syndrome coronavirus-2(SARS-CoV-2)specific antibody detection and anti-SARS-CoV-2 specific monoclonal antibodi...The purpose of this study was to investigate the clinical application of severe acute respiratory distress syndrome coronavirus-2(SARS-CoV-2)specific antibody detection and anti-SARS-CoV-2 specific monoclonal antibodies(mAbs)in the treatment of coronavirus infectious disease 2019(COVID-19).The dynamic changes of SARS-CoV-2 specific antibodies during COVID-19 were studied.Immunoglobulin M(IgM)appeared earlier and lasted for a short time,while immunoglobulin G(IgG)appeared later and lasted longer.IgM tests can be used for early diagnosis of COVID-19,and IgG tests can be used for late diagnosis of COVID-19 and identification of asymptomatic infected persons.The combination of antibody testing and nucleic acid testing,which complement each other,can improve the diagnosis rate of COVID-19.Monoclonal anti-SARS-CoV-2 specific antibodies can be used to treat hospitalized severe and critically ill patients and non-hospitalized mild to moderate COVID-19 patients.COVID-19 convalescent plasma,highly concentrated immunoglobulin,and anti-SARS-CoV-2 specific mAbs are examples of anti-SARS-CoV-2 antibody products.Due to the continuous emergence of mutated strains of the novel coronavirus,especially omicron,its immune escape ability and infectivity are enhanced,making the effects of authorized products reduced or invalid.Therefore,the optimal application of anti-SARS-CoV-2 antibody products(especially anti-SARS-CoV-2 specific mAbs)is more effective in the treatment of COVID-19 and more conducive to patient recovery.展开更多
Background: The coronavirus disease 2019 (COVID-19) pandemic is a distinct public health issue that calls for the quick development of novel treatments and viral detection. Due to their high specificity and reliabilit...Background: The coronavirus disease 2019 (COVID-19) pandemic is a distinct public health issue that calls for the quick development of novel treatments and viral detection. Due to their high specificity and reliability, monoclonal antibodies (mAbs) have emerged as useful diagnostic and therapeutic tools for a variety of diseases. As a result, several scientists have jumped right into developing Ab-based assays for the identification of SARS-CoV-2 and Ab drugs for use as COVID-19 therapy agents. Since the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is essential for viral infection and has a known precise structure, it has become a key target for the creation of therapeutic antibodies. The use of Ab cocktails is anticipated to be a key component of an efficient COVID-19 treatment plan since SARS-CoV-2 is an RNA virus with a high mutation rate, particularly when subjected to the selection pressure of aggressively applied preventive vaccinations and neutralizing Abs. Furthermore, SARS-CoV-2 infection could provoke an overzealous immune response, leading to a cytokine storm that accelerates the onset of a severe disease. Abs to counteract cytokine storms are also actively being researched as COVID-19 therapies. Abs are now used in SARS-CoV-2 detection assays, including immunoglobulin and antigen tests, in addition to their use as medicines. In order to stop the spread of COVID-19, such Ab-based detection tests are essential surveillance tools. In this article, we’ll go over several important ideas related to mAb-based COVID-19 pandemic detection tests and treatments. Objective: To understand the role of hybridoma technology in therapeutic implications. 1) To study the basic concepts and options in hybridoma technology;2) To study the applications of hybridoma technology;3) To explore how hybridoma technology is applied in diagnostic histopathology. Method: For this method generally there is use of mouse or mammals are transfect with the Ags to find out the formation of antibody afterwards isolate the antibody which has been formed after injecting the antigens for a number of weeks. Following are the steps for mAbs: Step 1: In this step immunization of mouse is done;Step 2: Spleen is used for the isolation of B cells;Step 3: Cultivation of cancerous cells;Step 4: Merging of B cells with Myeloma cells;Step 5: This step cell lines are separated;Step 6: in the next step screening the suitable cell lines;Step 7: observation of multiplication in vitro as well as in vivo;Step 8: Harvesting. Discussion: Now a day there are many diseases which has been cured easily at the mean time it’s very difficult to diagnose and get the treatment. Due to advancement of monoclonal antibodies are used in the diagnosis and treatments such as COVID-19, SARS and SARS COV-2. Therefore important part of the monoclonal antibodies are its used in the diagnosis as well as in the treatment tools.展开更多
Citrus yellow vein clearing virus (CYVCV) is considered as the causal agent of Citrus yellow vein clearing disease and belongs to the genus Mandarivirus in the family Alphaflexiviridae. Capsid protein (CP) of CYVC...Citrus yellow vein clearing virus (CYVCV) is considered as the causal agent of Citrus yellow vein clearing disease and belongs to the genus Mandarivirus in the family Alphaflexiviridae. Capsid protein (CP) of CYVCV Chongqing isolate (CYVCV- CQ) was produced using a prokaryotic expression system and used as the immunogen for monoclonal antibody (MAb) production. Four highly specific and sensitive murine MAbs and one polyclonal antibody were prepared in this study. Titers of the four MAbs in ascites fluids ranged from 10-6 to 10-7 as determined by indirect enzyme-linked immunosorbent assay (ELISA). Three serological assays, including dot enzyme-linked immunosorbent assay (dot-ELISA), tissue blot-ELISA, and double-antibody sandwich (DAS)-ELISA, were developed for quick and reliable detections of CYVCV in citrus samples. The developed dot-ELISA and DAS-ELISA methods could detect CYVCV in the infected citrus leaf crude extracts diluted at 1:2 560 and 1:10 240 (w/v, g mL^-1), respectively. The detection result of 125 citrus leaf samples collected from citrus groves in Yunnan Province and Chongqing Municipality of China showed that approximately 36% samples were positive for CYVCV. This virus was, however, not'detected in any sample collected from Zhejiang or Jiangxi Province, China.展开更多
Objective To prepare monoclonal antibodies against a newly discovered and conserved linear epitope of Rabies virus nucleoprotein and to use them in a rabies diagnostic test. Methods Synthetic peptide containing the ep...Objective To prepare monoclonal antibodies against a newly discovered and conserved linear epitope of Rabies virus nucleoprotein and to use them in a rabies diagnostic test. Methods Synthetic peptide containing the epitope was used as immunogen to prepare hybridoma cell lines by classical hybridoma technology. Anti-peptide monoclonal antibodies produced in ascites of inoculated Balb/c mice were labeled with fluorescein isothiocyanate (FITC) after purification and used in fluorescent antibody test (FAT). Results Two positive hybridoma cell lines, RVNP-mAbl-CL and RVNP-mAb2-CL, were obtained. RVNP- mAbl-CL produced a higher concentration of monoclonal antibody RVNP-mAbl in Balb/c ascites. FITC-labeled RVNP-mAbl showed correct results on certain Rabies virus-positive canine brain tissue samples and cells of a small subclone of baby hamster kidney 21 cell line (BSR). Conclusion FITC-labeled RVNP-mAbl has potential application for laboratory diagnosis of rabies展开更多
Objective Determination of estrone (E1) levels has a significant meaning in evaluating physiological effect and diagnosing some diseases. In order to detect free E1 in biological fluids, a monoclonal antibody specifi...Objective Determination of estrone (E1) levels has a significant meaning in evaluating physiological effect and diagnosing some diseases. In order to detect free E1 in biological fluids, a monoclonal antibody specific for E1 was prepared after the complete antigen of E1 was synthesized. The purified monoclonal antibody was fully characterized for later immunoassay. Methods 3-O-carboxymethyl ether derivative of E1 was synthesized and in turn coupled to bovine serum albumin (BSA) to form complete antigen E1-BSA. A monoclonal antibody (McAb) specific for E1 was produced both in vitro and in vivo by a hybridoma anti-E1. Anti-E1 was prepared by fusion of SP2/0 murine myeloma cells with spleen cells isolated from immunized BALB/c mouse. The McAb was characterized by enzyme-linked immunosorbent assay (ELISA), SDS-PAGE and Western-blotting. The specificity of the immunoassay was investigated by determining the cross-reactions of E1 analogs when free E1 was detected by competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). Results Analysis revealed that anti-E1 McAb (E1-McAb) was of the IgG1 type, the molecular weight of E1-McAb was 164 000 daltons. The affinity constant of E1-McAb with coated complete antigen was 8.2108L/mol. The linear range for free E1 determined by CI-ELISA was 10pg/mL^10ng/mL. The detection limit was 21.4 pg/mL (defined as twice the standard deviation of the blank). Conclusion The CI-ELISA developed with E1-McAb was both sensitive and specific. The prepared E1-McAb can be used in some immunoassays.展开更多
To prepare monoclonal antibodies (MAb) and antisera specific for Escherichia coli (E.coli) O157, and to develop a sandwich enzyme-linked immunosorbent assay (ELISA) to detect Eocoli O157 in foods. Methods Spleen...To prepare monoclonal antibodies (MAb) and antisera specific for Escherichia coli (E.coli) O157, and to develop a sandwich enzyme-linked immunosorbent assay (ELISA) to detect Eocoli O157 in foods. Methods Spleen cells from BALB/c mice immunized with the somatic antigen of E.coli O157:H7 were fused with routine Sp2/0 myeloma cells. The hybridoma cell line specific for E.coli O157 was established after having been subcloned. Antisera specific for E.coli O157 was prepared by intravenous injection into New Zealand rabbits with a stain of E.coli O157:H7. The sandwich ELISA was developed with the polyclonal antibody as the capture antibody and the MAb 3A5 as the detection antibody. The inoculated ground poultry meat and pasteurized miLk were tested to confirm efficiency of the method. Results MAb 3A5 specific for E.coli O157 and O 113:H21 belonged to subtype IgM. The ascetic titers of the antibody was 1:1× 10^6. No cross-reactivity of the MAb was observed with strains of Salmonella spp, Yersinia enterocolitica, Shigella dysenteriae, etc. The purified polyclonal antibody had a titer of 1: 1× 10^5 with E.coli O 157. The detection limit of this sandwich ELISA was 10^3- 10^4 cfu E.coli O157/mL in pure culture with a high specificity, which was characterized by every non-O157 strain with negative response. With 10h enrichment procedure, E.coli O157:H7 recovered well from inoculated ground poultry meat and pasteurized milk at levels of 0.1 cfu/g and 0.1 cfu/mL. Conclusion MAb 3A5 specific for E.coli O 157 and O 113:H21 can be produced by immunizing BALB/c mice with a strain of E.coli O157:H7. Then a sandwich ELISA can be developed with the polyclonal antibody as the capture antibody and the MAb 3A5 as the detection antibody. The method is proved to be a sensitive and specific technique to detect low number of E.coli O157 in food.展开更多
Recently, serum Golgi protein 73 (GP73) levels have been found to be elevated in patients with hepatocellu- lar carcinoma (HCC), and GP73 has been proposed as a novel marker for HCC. However, GP73 levels in patien...Recently, serum Golgi protein 73 (GP73) levels have been found to be elevated in patients with hepatocellu- lar carcinoma (HCC), and GP73 has been proposed as a novel marker for HCC. However, GP73 levels in patients remain controversial due to the specificity of the anti-GP73 antibody-based enzyme linked immunosorbent as- say (ELISA). Therefore, an anti-GP73 antibody with high specificity was highly demanded. In the present study, by hybridoma screening, we generated an anti-GP73 monoclonal antibody (mAb) designated as 6A2 using recom- binant GP73 protein produced by prokaryotic expression. The specificity of 6A2 was evaluated by Western blot- ting, immunohistochemistry and immunoprecipitation. The results showed that 6A2 recognized GP73 in both native and denatured forms. In addition, we have developed a sandwich ELISA using 6A2 and GP73 polyclonal antibody generated in New Zealand white rabbits according to standard procedures, and measured the serum GP73 level of patients using this assay. Our results showed that serum GP73 levels of HCC patients were significantly higher than those of healthy controls (P = 0.0036). Furthermore, for the first time, GP73 serum level was found to be elevated in patients with breast cancer compared with healthy controls (P = 0.0172).展开更多
To produce the monoclonal antibodies (mAbs) against hygromycin B phosphotransferase (HPT) and to develop immunoassay based on mAbs for biosafety assessment of HPT in genetically modified rice (GM rice). Methods ...To produce the monoclonal antibodies (mAbs) against hygromycin B phosphotransferase (HPT) and to develop immunoassay based on mAbs for biosafety assessment of HPT in genetically modified rice (GM rice). Methods BALB/c mice were immunized with purified recombinant 6His. HPT protein, and the conventional hybridoma technology was used to generate the monoclonal hybridoma cells. ELISA and Western blot were used to analyze the specificity of mAbs recognizing HPT and the cross reaction with other proteins. A double-Ab sandwich ELISA method was established to detect HPT expression level in the sck gene-modified rice plants. Results Four hybridomas, named F1,D4-2, D4-4, and D4-5, producing the mAbs against HPT were successfully obtained with the titer of ascetic mAbs ranging from 10×10^-4 to10×10^-5. Identification of subclass showed that all the produced mAbs belonged to IgG1. Western blot showed specific binding reaction between the mAbs to the HPT proteins expressed in the GM rice. A double sandwich ELISA coated with anti-HPT polyclonal antibody was established with mAbs as sandwich antibody, which showed a sensitivity of 30ng/mL and did not crossreact with other proteins. The expression level of HPT in the leaves of sck-transformed lines was detected (80-150ng/mL). But HPT protein in the grain and seed of GM rice could not be detected using this ELISA assay, Conclusion Anti-HPT mAbs prepared herein have a high specificity and can be used for rapid assay of HPT antigen. The expression level of HPT in the GM rice grain and seed is lower than our ELISA detection limit.展开更多
Objective:The mainstay treatment of esophageal squamous cell carcinoma(ESCC)involves chemotherapy and immunotherapy.However,alternative therapies are required for patients who are refractory or intolerant to existing ...Objective:The mainstay treatment of esophageal squamous cell carcinoma(ESCC)involves chemotherapy and immunotherapy.However,alternative therapies are required for patients who are refractory or intolerant to existing therapies.Methods:In this single-arm,multicenter,open-label phase Ib study,30 patients received an intravenous infusion of SCT200,an antiepidermal growth factor receptor(EGFR)monoclonal antibody,6.0 mg/kg once a week for 6 weeks,followed by 8.0 mg/kg once every 2 weeks until disease progression or intolerable toxicity.The primary endpoint was the objective response rate(ORR).The secondary endpoints were progression-free survival(PFS),overall survival(OS),and safety.Results:Thirty patients were enrolled between July 2018 and May 2019.The ORR was 16.7%(95%CI:5.6%–34.7%).The median PFS and OS were 3.1 months(95%CI:1.5–4.3)and 6.8 months(95%CI:4.7–10.1),respectively.A numerical difference without any statistical significance in ORR was observed in patients with different EGFR expressions(≥50%:25.0%vs.<50%:0%,P=0.140)or TP53 mutation abundance(<10%:23.8%vs.≥10%:0%,P=0.286).Improved median PFS(3.4 vs.1.4 months,P=0.006)and OS(8.0 vs.4.2 months,P=0.027)were associated with TP53 mutation abundance of<10%.The most common treatment-related adverse events of grade 3 or 4(occurring in≥2 patients)were hypomagnesemia[7(23.3%)]and rash[2(6.7%)].No treatmentrelated death occurred.Conclusions:SCT200 monotherapy as the second-or further-line treatment for advanced ESCC showed favorable efficacy,with an acceptable safety profile.TP53 mutation abundance might serve as a potential predictive biomarker.展开更多
Despite the significant resources dedicated to the development of monoclonal antibody(m Ab)therapies for solid tumors,the clinical success,thus far,has been modest.Limited efficacy of m Ab in solid tumors likely relat...Despite the significant resources dedicated to the development of monoclonal antibody(m Ab)therapies for solid tumors,the clinical success,thus far,has been modest.Limited efficacy of m Ab in solid tumors likely relates to unique aspects of tumor physiology.Solid tumors have an aberrant vasculature and a dense extracellular matrix that slow both the convective and diffusive transport of m Abs into and within tumors.For m Abs that are directed against cellular antigens,high antigen expression and rapid antigen turnover can result in perivascular cells binding to and eliminating a significant amount of extravasated m Ab,limiting m Ab distribution to portions of the tumor that are distant from functional vessels.Many preclinical investigations have reported strategies to improve m Ab uptake and distribution;however,to our knowledge,none have translated into the clinic.Here,we provide an overview of several barriers in solid tumors that limit m Ab uptake and distribution and discuss approaches that have been utilized to overcome these barriers in preclinical studies.展开更多
The present study aimed at the preparation of monoclonal antibody against the recombinant PthA-NLS and the isolation of the relative ScFv (single chain variable fragment) genes, providing the possibility to better u...The present study aimed at the preparation of monoclonal antibody against the recombinant PthA-NLS and the isolation of the relative ScFv (single chain variable fragment) genes, providing the possibility to better understand the pathogenesis mechanism via PthA, and developing proper construct for future experimentation to obtain citrus plants resistant to canker disease by transformation and plant antibody techniques. The recombinant polypeptide PthA-NLS was injected into Balb/c mice to produce monoclonal antibody. Total RNA was isolated from the hybridoma cell line 3D10H2 which secreted anti- PthA-NLS McAb, and the variable region genes were amplified with specific primers by RT-PCR and SOE-PCR (splicing by overlap extension), and then the ScFv gene was isolated. The recombinant ScFv gene was cloned into pGEM-T and pET32a(+) vector. The later plasmid was transferred into E. coli BL21 (DE3) and the expression of the recombinant protein was induced. Three cell lines producing monoclonal antibody against PthA-NLS were acquired and named 1C8H1, 2D12B6, and 3D8A10. The recombinant ScFv gene of about 750 bp was constructed. The sequencing results showed that the ScFv gene consists of a 360 bp heavy chain, a 342 bp light chain, and a 45 bp linker region. The recombinant fusion ScFv protein was expressed by IPTG induction, and a 44.5 kDa of recombinant fusion protein was obtained. In conclusion, we obtained three cell lines stably producing monoclonal antibody specifically bound to PthA-NLS, and the relative ScFv gene was constructed and successfully expressed in E. coli. These results may play an important role in further understanding the pathogenesis mechanism and in the development of possible citrus resistant to canker disease by genetic transformation and plant antibiobody.展开更多
Ensuring the removal of host cell proteins(HCPs) during downstream processing of recombinant proteins such as monoclonal antibodies(m Abs) remains a challenge.Since residual HCPs might affect product stability or safe...Ensuring the removal of host cell proteins(HCPs) during downstream processing of recombinant proteins such as monoclonal antibodies(m Abs) remains a challenge.Since residual HCPs might affect product stability or safety,constant monitoring is required to demonstrate their removal to be below the regulatory accepted level of 100 ng/mg.The current standard analytical approach for this procedure is based on ELISA;however,this approach only measures the overall HCP content.Therefore,the use of orthogonal methods,such as liquid chromatography-mass spectrometry(LC-MS),has been established,as it facilitates the quantitation of total HCPs as well as the identification and quantitation of the individual HCPs present.In the present study,a workflow for HCP detection and quantitation using an automated magnetic bead-based sample preparation,in combination with a data-independent acquisition(DIA) LC-MS analysis,was established.Employing the same instrumental setup commonly used for peptide mapping analysis of m Abs allows for its quick and easy implementation into pre-existing workflows,avoiding the need for dedicated instrumentation or personnel.Thereby,quantitation of HCPs over a broad dynamic range was enabled to allow monitoring of problematic HCPs or to track changes upon altered bioprocessing conditions.展开更多
Liquid chromatography tandem mass spectrometry(LC-MS/MS)has gradually become a promising alternative to ligand binding assay for the bioanalysis of biotherapeutic molecules,due to its rapid method development and high...Liquid chromatography tandem mass spectrometry(LC-MS/MS)has gradually become a promising alternative to ligand binding assay for the bioanalysis of biotherapeutic molecules,due to its rapid method development and high accuracy.In this study,we established a new LC-MS/MS method for the determination of the anti-sclerostin monoclonal antibody(SHR-1222)in cynomolgus monkey serum,and compared it to a previous electrochemiluminescence method.The antibody was quantified by detecting the surrogate peptide obtained by trypsin digestion.The surrogate peptide was carefully selected by investigating its uniqueness,stability and MS response.The quantitative range of the proposed method was 2.00-500μg/mL,and this verified method was successfully applied to the toxicokinetic assessment of SHR-1222 in cynomolgus monkey serum.It was found that the concentrations of SHR-1222 in cynomolgus monkeys displayed an excellent agreement between the LC-MS/MS and electrochemiluminescence methods(ratios of drug exposure,0.8-1.0).Notably,two monkeys in the60 mg/kg dose group had abnormal profiles with a low detection value of SHR-1222 in their individual sample.Combining the high-level anti-drug antibodies(ADAs)in these samples and the consistent quantitative results of the two methods,we found that the decreased concentration of SHR-1222 was due to the accelerated clearance mediated by ADAs rather than the interference of ADAs to the detection platform.Taken together,we successfully developed an accurate,efficient and cost-effective LC-MS/MS method for the quantification of SHR-1222 in serum samples,which could serve as a powerful tool to improve the preclinical development of antibody drugs.展开更多
Targeted therapy has been widely demonstrated as an effective strategy to treat cancers,the leading cause of death in the world.This minireview summarizes the technical platforms and methodologies utilized to develop ...Targeted therapy has been widely demonstrated as an effective strategy to treat cancers,the leading cause of death in the world.This minireview summarizes the technical platforms and methodologies utilized to develop and engineer therapeutic monoclonal antibodies and antibody-drug conjugates.First,the USA FDA approved monoclonal antibody(mAb)-based targeted therapies are reviewed.Then the representative innovative chimeric,humanized and fully human anti-cancer antibodies and antibody-drug conjugates are described.Finally,the past and predictive market trend of therapeutic antibodies is discussed.展开更多
Testis-specific protease 50(TSP50) has been identified as a testis-specific protein that is expressed abnormally in most human breast cancer samples,which makes it an attractive molecular marker and a potential targ...Testis-specific protease 50(TSP50) has been identified as a testis-specific protein that is expressed abnormally in most human breast cancer samples,which makes it an attractive molecular marker and a potential target for diagnosis and therapy.In the present study,we prepared a panel of monoclonal antibodies(mAbs) with high specificity and sensitivity against TSP50 by hybridoma method and characterized them by ELISA,Western blot,immunofluroescence and immunohistochemical analyses.The results show that all of the 9 different clones can specifically bind to TSP50.The mAbs against TSP50 we generated could be good tools for both basic and clinical studies.展开更多
Rice ragged stunt virus(RRSV) is a serious rice disease in Asia, causing serious yield losses on rice. The capsid protein(CP) gene of the major outer capsid protein of RRSV was expressed in Escherichia coli BL21(...Rice ragged stunt virus(RRSV) is a serious rice disease in Asia, causing serious yield losses on rice. The capsid protein(CP) gene of the major outer capsid protein of RRSV was expressed in Escherichia coli BL21(DE3) using the pMAL-C2 X expression vector. The recombinant protein was used as the immunogen to immunize BALB/c mice. A hybridoma cell line 8A12 secreting monoclonal antibody(MAb) against RRSV was obtained by fusing mouse myeloma cells(Sp 2/0) with spleen cells from the immunized BALB/c mice. Western blot analysis showed that the MAb 8A12 can specifically react with RRSV CP. Using the MAb, an antigen-coated-plate enzyme-linked immunosorbent assay(ACP-ELISA), a dot enzyme-linked immunosorbent assay(dot-ELISA), and immunocapture-RT-PCR(IC-RT-PCR) assay were developed to detect RRSV. The established ACP-ELISA, dot-blot ELISA and IC-RT-PCR methods could detect RRSV in infected rice tissue crude extracts with dilutions of 1:40 960, 1:1 280 and 1:655 360(w/v, g mL-1), respectively. The ACP-ELISA and dot-blot ELISA methods could detect RRSV in infected insect vector crude extracts with dilutions of 1:12 800 and 1:1 600(an individual planthopper μL-1), respectively. The field survey revealed that Rice ragged stunt disease occurs on rice in Hainan, Yunnan, Guangxi, Sichuan, Guizhou, Fujian, Hunan, Jiangxi and Zhejiang in China.展开更多
Anti-HER2 monoclonal antibody (Sc7301)-paclitaxel (TAX) immunoconjugate was pre- pared and its specific binding to tumor cells was investigated in this study. Sc7301 was conjugated to TAX by the active ester metho...Anti-HER2 monoclonal antibody (Sc7301)-paclitaxel (TAX) immunoconjugate was pre- pared and its specific binding to tumor cells was investigated in this study. Sc7301 was conjugated to TAX by the active ester method and then the TAX-Sc7301 immunoconjugate was obtained. After purification and labeling by Cyano-fluorescein isothiocyanate (FITC), the specific binding of TAX-Sc7301 to HER2-positive tumor cells (SKOV3) and HER2-negative tumor cells (HepG2) was evaluated respectively. TAX-Sc7301 (20 nmol/L) showed distinct specific binding to SKOV3 cells rather than HepG2 cells. And the uptake of the immunoconjugate by SKOV3 cells was increased with the TAX-So7301 concentration (3-48 nmol/L) and the incubation time (P〈0.05). It was concluded that the TAX-Sc7301 immunoconjugate is ootentially applicable as a targeted agent against HER2-10ositive tumor cells.展开更多
基金supported by the National Key Research and Development Program of China(2021YFD1800400)the National Natural Science Foundation of China(31872480)+1 种基金the Jiangsu Agriculture Science and Technology Innovation Fund,China(CX(19)2020)the Priority Academic Program Development of Jiangsu Higher Education Institutions,China(PAPD).
文摘Porcine proliferative enteropathy(PPE),an important infectious disease in pig production caused by an obligate intracellular bacterium Lawsonia intracellularis,is commonly associated with diarrhea and reduced weight gain in growing pigs widespread.An accurate method for detecting L.intracellularis is particularly important for preventing and controlling PPE.Heat shock protein 60(Hsp60)is an immunodominant bacterial antigen found in all eukaryotic and prokaryotic organisms.Thus,the purpose of the current investigation was to produce a novel L.intracellularis Hsp60 monoclonal antibody(mAb)useful for immunodiagnostics.Three hybridomas secreted anti-Hsp60 termed 3E5,4E2,and 9G6 were generated,and the titers of ascitic fluids of 3E5,4E2,9G6 were 1:1024000,1:2048000 and 1:2048000,respectively.The Western blotting analysis demonstrated that recombinant Hsp60(rHsp60)was recognized by mAbs 3E5,4E2 and 9G6.Subsequently,analyses of specificity showed all the mAbs were highly specific to L.intracellularis while could not significantly react with other enteric bacteria commonly found in the ileum of pigs,such as Escherichia coli,Salmonella Choleraesuis,Salmonella Typhimurium,and Brachyspira hyodysenteriae.Furthermore,the mAbs were useful for detecting L.intracellularis in the infected monolayer cells and histological sections of the ileum from PPE-affected pigs.Our research will provide a foundation for the development of immunological diagnostic tests.
基金supported by the National Basic Research Program of China(Grant No.:2015CB553701)the National Science and Technology Major Project,China(Grant No.:2019ZX09732001).
文摘Morphine is a frequently used analgesic that activates the mu-opioid receptor(MOR),which has prominent side effects of tolerance.Although the inefficiency of morphine in inducing the endocytosis of MOR underlies the development of morphine tolerance,currently,there is no effective therapy to treat morphine tolerance.In the current study,we aimed to develop a monoclonal antibody(mAb)precisely targeting MOR and to determine its therapeutic efficacy on morphine tolerance and the underlying molecular mechanisms.We successfully prepared a mAb targeting MOR,named 3A5C7,by hybridoma technique using a strategy of deoxyribonucleic acid immunization combined with cell immunization,and identified it as an immunoglobulin G mAb with high specificity and affinity for MOR and binding ability to antigens with spatial conformation.Treatment of two cell lines,HEK293T and SH-SY5Y,with 3A5C7 enhanced morphine-induced MOR endocytosis via a G protein-coupled receptor kinase 2(GRK2)/b-arrestin2-dependent mechanism,as demonstrated by immunofluorescence staining,flow cytometry,Western blotting,coimmunoprecipitation,and small interfering ribonucleic acid(siRNA)-based knockdown.This mAb also allowed MOR recycling from cytoplasm to plasma membrane and attenuated morphine-induced phosphorylation of MOR.We established an in vitro morphine tolerance model using differentiated SH-SY5Y cells induced by retinoic acid.Western blot,enzyme-linked immunosorbent assays,and siRNA-based knockdown revealed that 3A5C7 mAb diminished hyperactivation of adenylate cyclase,the in vitro biomarker of morphine tolerance,via the GRK2/b-arrestin2 pathway.Furthermore,in vivo hotplate test demonstrated that chronic intrathecal administration of 3A5C7 significantly alleviated morphine tolerance in mice,and withdrawal jumping test revealed that both chronic and acute 3A5C7 intrathecal administration attenuated morphine dependence.Finally,intrathecal electroporation of silencing short hairpin RNA illustrated that the in vivo anti-tolerance and anti-dependence efficacy of 3A5C7 was mediated by enhanced morphine-induced MOR endocytosis via GRK2/b-arrestin2 pathway.Collectively,our study provided a therapeutic mAb,3A5C7,targeting MOR to treat morphine tolerance,mediated by enhancing morphine-induced MOR endocytosis.The mAb 3A5C7 demonstrates promising translational value to treat clinical morphine tolerance.
基金supported by the National Key Research & Development Program of China (2021YFE0113300)the National Natural Science Foundation of China (22078286 and 21878263)+1 种基金Zhejiang Universitythe Talent-Introduction Program of China for the Postdoctoral Researcher for the financial support。
文摘Process analytical technology(PAT) is gaining more interest in the biomanufacturing industry because of its potential to improve operational control and compliance through real-time quality assurance.Currently, biopharmaceutical producers mainly monitor chromatographic processes with ultraviolet/visible(UV/Vis) absorbance. However, this measurement has a very limited correlation with purity and quantity. The current study aims to determine the concentration of monoclonal antibody(mAb) and host cell proteins(HCPs) using a build-in UV/Vis monitoring during Protein A affinity chromatography and to optimize the separation conditions for high purity of mAb and minimizing the HCPs content. The eluate was analyzed through in-line UV/Vis at 280 and 410 nm, representing mAb and HCPs concentration,respectively. Each 0.1 column volume(CV) fraction of UV/Vis chromatogram peak area were calculated,and different separation conditions were then compared. The optimum conditions of mAb separation were found as 12 CV loading, elution at pH 3.5, and starting the collection at 0.5 CV point, resulting in high m Ab recovery of 95.92% and additional removal of 49.98% of HCP comparing with whole elution pool. This study concluded that UV/Vis-based in-line monitoring at 280 and 410 nm showed a high potential to optimize and real-time control Protein A affinity chromatography for mAb purification from HCPs.
文摘The purpose of this study was to investigate the clinical application of severe acute respiratory distress syndrome coronavirus-2(SARS-CoV-2)specific antibody detection and anti-SARS-CoV-2 specific monoclonal antibodies(mAbs)in the treatment of coronavirus infectious disease 2019(COVID-19).The dynamic changes of SARS-CoV-2 specific antibodies during COVID-19 were studied.Immunoglobulin M(IgM)appeared earlier and lasted for a short time,while immunoglobulin G(IgG)appeared later and lasted longer.IgM tests can be used for early diagnosis of COVID-19,and IgG tests can be used for late diagnosis of COVID-19 and identification of asymptomatic infected persons.The combination of antibody testing and nucleic acid testing,which complement each other,can improve the diagnosis rate of COVID-19.Monoclonal anti-SARS-CoV-2 specific antibodies can be used to treat hospitalized severe and critically ill patients and non-hospitalized mild to moderate COVID-19 patients.COVID-19 convalescent plasma,highly concentrated immunoglobulin,and anti-SARS-CoV-2 specific mAbs are examples of anti-SARS-CoV-2 antibody products.Due to the continuous emergence of mutated strains of the novel coronavirus,especially omicron,its immune escape ability and infectivity are enhanced,making the effects of authorized products reduced or invalid.Therefore,the optimal application of anti-SARS-CoV-2 antibody products(especially anti-SARS-CoV-2 specific mAbs)is more effective in the treatment of COVID-19 and more conducive to patient recovery.
文摘Background: The coronavirus disease 2019 (COVID-19) pandemic is a distinct public health issue that calls for the quick development of novel treatments and viral detection. Due to their high specificity and reliability, monoclonal antibodies (mAbs) have emerged as useful diagnostic and therapeutic tools for a variety of diseases. As a result, several scientists have jumped right into developing Ab-based assays for the identification of SARS-CoV-2 and Ab drugs for use as COVID-19 therapy agents. Since the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is essential for viral infection and has a known precise structure, it has become a key target for the creation of therapeutic antibodies. The use of Ab cocktails is anticipated to be a key component of an efficient COVID-19 treatment plan since SARS-CoV-2 is an RNA virus with a high mutation rate, particularly when subjected to the selection pressure of aggressively applied preventive vaccinations and neutralizing Abs. Furthermore, SARS-CoV-2 infection could provoke an overzealous immune response, leading to a cytokine storm that accelerates the onset of a severe disease. Abs to counteract cytokine storms are also actively being researched as COVID-19 therapies. Abs are now used in SARS-CoV-2 detection assays, including immunoglobulin and antigen tests, in addition to their use as medicines. In order to stop the spread of COVID-19, such Ab-based detection tests are essential surveillance tools. In this article, we’ll go over several important ideas related to mAb-based COVID-19 pandemic detection tests and treatments. Objective: To understand the role of hybridoma technology in therapeutic implications. 1) To study the basic concepts and options in hybridoma technology;2) To study the applications of hybridoma technology;3) To explore how hybridoma technology is applied in diagnostic histopathology. Method: For this method generally there is use of mouse or mammals are transfect with the Ags to find out the formation of antibody afterwards isolate the antibody which has been formed after injecting the antigens for a number of weeks. Following are the steps for mAbs: Step 1: In this step immunization of mouse is done;Step 2: Spleen is used for the isolation of B cells;Step 3: Cultivation of cancerous cells;Step 4: Merging of B cells with Myeloma cells;Step 5: This step cell lines are separated;Step 6: in the next step screening the suitable cell lines;Step 7: observation of multiplication in vitro as well as in vivo;Step 8: Harvesting. Discussion: Now a day there are many diseases which has been cured easily at the mean time it’s very difficult to diagnose and get the treatment. Due to advancement of monoclonal antibodies are used in the diagnosis and treatments such as COVID-19, SARS and SARS COV-2. Therefore important part of the monoclonal antibodies are its used in the diagnosis as well as in the treatment tools.
基金supported by the Special Fund for Agro-scientific Research in the Public Interest,China(201203076-05)the National Basic Research Program of China(2014CB138400)
文摘Citrus yellow vein clearing virus (CYVCV) is considered as the causal agent of Citrus yellow vein clearing disease and belongs to the genus Mandarivirus in the family Alphaflexiviridae. Capsid protein (CP) of CYVCV Chongqing isolate (CYVCV- CQ) was produced using a prokaryotic expression system and used as the immunogen for monoclonal antibody (MAb) production. Four highly specific and sensitive murine MAbs and one polyclonal antibody were prepared in this study. Titers of the four MAbs in ascites fluids ranged from 10-6 to 10-7 as determined by indirect enzyme-linked immunosorbent assay (ELISA). Three serological assays, including dot enzyme-linked immunosorbent assay (dot-ELISA), tissue blot-ELISA, and double-antibody sandwich (DAS)-ELISA, were developed for quick and reliable detections of CYVCV in citrus samples. The developed dot-ELISA and DAS-ELISA methods could detect CYVCV in the infected citrus leaf crude extracts diluted at 1:2 560 and 1:10 240 (w/v, g mL^-1), respectively. The detection result of 125 citrus leaf samples collected from citrus groves in Yunnan Province and Chongqing Municipality of China showed that approximately 36% samples were positive for CYVCV. This virus was, however, not'detected in any sample collected from Zhejiang or Jiangxi Province, China.
基金supported by research grants from the Diagnosis of Infectious Pathogens and Combination of Diagnostic Technologies (2008ZX10004-002)Prevention and Control of Major Infectious Disease such as AIDS and Viral Hepatitis,State Eleventh Five-Year Plan
文摘Objective To prepare monoclonal antibodies against a newly discovered and conserved linear epitope of Rabies virus nucleoprotein and to use them in a rabies diagnostic test. Methods Synthetic peptide containing the epitope was used as immunogen to prepare hybridoma cell lines by classical hybridoma technology. Anti-peptide monoclonal antibodies produced in ascites of inoculated Balb/c mice were labeled with fluorescein isothiocyanate (FITC) after purification and used in fluorescent antibody test (FAT). Results Two positive hybridoma cell lines, RVNP-mAbl-CL and RVNP-mAb2-CL, were obtained. RVNP- mAbl-CL produced a higher concentration of monoclonal antibody RVNP-mAbl in Balb/c ascites. FITC-labeled RVNP-mAbl showed correct results on certain Rabies virus-positive canine brain tissue samples and cells of a small subclone of baby hamster kidney 21 cell line (BSR). Conclusion FITC-labeled RVNP-mAbl has potential application for laboratory diagnosis of rabies
基金This work was supported by National Science Foundation of China (Grant no.20075001).
文摘Objective Determination of estrone (E1) levels has a significant meaning in evaluating physiological effect and diagnosing some diseases. In order to detect free E1 in biological fluids, a monoclonal antibody specific for E1 was prepared after the complete antigen of E1 was synthesized. The purified monoclonal antibody was fully characterized for later immunoassay. Methods 3-O-carboxymethyl ether derivative of E1 was synthesized and in turn coupled to bovine serum albumin (BSA) to form complete antigen E1-BSA. A monoclonal antibody (McAb) specific for E1 was produced both in vitro and in vivo by a hybridoma anti-E1. Anti-E1 was prepared by fusion of SP2/0 murine myeloma cells with spleen cells isolated from immunized BALB/c mouse. The McAb was characterized by enzyme-linked immunosorbent assay (ELISA), SDS-PAGE and Western-blotting. The specificity of the immunoassay was investigated by determining the cross-reactions of E1 analogs when free E1 was detected by competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). Results Analysis revealed that anti-E1 McAb (E1-McAb) was of the IgG1 type, the molecular weight of E1-McAb was 164 000 daltons. The affinity constant of E1-McAb with coated complete antigen was 8.2108L/mol. The linear range for free E1 determined by CI-ELISA was 10pg/mL^10ng/mL. The detection limit was 21.4 pg/mL (defined as twice the standard deviation of the blank). Conclusion The CI-ELISA developed with E1-McAb was both sensitive and specific. The prepared E1-McAb can be used in some immunoassays.
文摘To prepare monoclonal antibodies (MAb) and antisera specific for Escherichia coli (E.coli) O157, and to develop a sandwich enzyme-linked immunosorbent assay (ELISA) to detect Eocoli O157 in foods. Methods Spleen cells from BALB/c mice immunized with the somatic antigen of E.coli O157:H7 were fused with routine Sp2/0 myeloma cells. The hybridoma cell line specific for E.coli O157 was established after having been subcloned. Antisera specific for E.coli O157 was prepared by intravenous injection into New Zealand rabbits with a stain of E.coli O157:H7. The sandwich ELISA was developed with the polyclonal antibody as the capture antibody and the MAb 3A5 as the detection antibody. The inoculated ground poultry meat and pasteurized miLk were tested to confirm efficiency of the method. Results MAb 3A5 specific for E.coli O157 and O 113:H21 belonged to subtype IgM. The ascetic titers of the antibody was 1:1× 10^6. No cross-reactivity of the MAb was observed with strains of Salmonella spp, Yersinia enterocolitica, Shigella dysenteriae, etc. The purified polyclonal antibody had a titer of 1: 1× 10^5 with E.coli O 157. The detection limit of this sandwich ELISA was 10^3- 10^4 cfu E.coli O157/mL in pure culture with a high specificity, which was characterized by every non-O157 strain with negative response. With 10h enrichment procedure, E.coli O157:H7 recovered well from inoculated ground poultry meat and pasteurized milk at levels of 0.1 cfu/g and 0.1 cfu/mL. Conclusion MAb 3A5 specific for E.coli O 157 and O 113:H21 can be produced by immunizing BALB/c mice with a strain of E.coli O157:H7. Then a sandwich ELISA can be developed with the polyclonal antibody as the capture antibody and the MAb 3A5 as the detection antibody. The method is proved to be a sensitive and specific technique to detect low number of E.coli O157 in food.
基金supported by the Science and Technology Foundation of Nanjing Medical University (No. 07NMUZ005).
文摘Recently, serum Golgi protein 73 (GP73) levels have been found to be elevated in patients with hepatocellu- lar carcinoma (HCC), and GP73 has been proposed as a novel marker for HCC. However, GP73 levels in patients remain controversial due to the specificity of the anti-GP73 antibody-based enzyme linked immunosorbent as- say (ELISA). Therefore, an anti-GP73 antibody with high specificity was highly demanded. In the present study, by hybridoma screening, we generated an anti-GP73 monoclonal antibody (mAb) designated as 6A2 using recom- binant GP73 protein produced by prokaryotic expression. The specificity of 6A2 was evaluated by Western blot- ting, immunohistochemistry and immunoprecipitation. The results showed that 6A2 recognized GP73 in both native and denatured forms. In addition, we have developed a sandwich ELISA using 6A2 and GP73 polyclonal antibody generated in New Zealand white rabbits according to standard procedures, and measured the serum GP73 level of patients using this assay. Our results showed that serum GP73 levels of HCC patients were significantly higher than those of healthy controls (P = 0.0036). Furthermore, for the first time, GP73 serum level was found to be elevated in patients with breast cancer compared with healthy controls (P = 0.0172).
基金This work was supported by 973 National Fundamental Science Program Funds (2001CB109007 and 2001CB10901)and 863 National High-Tech Program Funds (2004AA212221 and 2002AA212041).
文摘To produce the monoclonal antibodies (mAbs) against hygromycin B phosphotransferase (HPT) and to develop immunoassay based on mAbs for biosafety assessment of HPT in genetically modified rice (GM rice). Methods BALB/c mice were immunized with purified recombinant 6His. HPT protein, and the conventional hybridoma technology was used to generate the monoclonal hybridoma cells. ELISA and Western blot were used to analyze the specificity of mAbs recognizing HPT and the cross reaction with other proteins. A double-Ab sandwich ELISA method was established to detect HPT expression level in the sck gene-modified rice plants. Results Four hybridomas, named F1,D4-2, D4-4, and D4-5, producing the mAbs against HPT were successfully obtained with the titer of ascetic mAbs ranging from 10×10^-4 to10×10^-5. Identification of subclass showed that all the produced mAbs belonged to IgG1. Western blot showed specific binding reaction between the mAbs to the HPT proteins expressed in the GM rice. A double sandwich ELISA coated with anti-HPT polyclonal antibody was established with mAbs as sandwich antibody, which showed a sensitivity of 30ng/mL and did not crossreact with other proteins. The expression level of HPT in the leaves of sck-transformed lines was detected (80-150ng/mL). But HPT protein in the grain and seed of GM rice could not be detected using this ELISA assay, Conclusion Anti-HPT mAbs prepared herein have a high specificity and can be used for rapid assay of HPT antigen. The expression level of HPT in the GM rice grain and seed is lower than our ELISA detection limit.
基金supported by a grant from the Science&Technology Development Fund of the Tianjin Education Commission for Higher Education(Grant No.2018KJ046).
文摘Objective:The mainstay treatment of esophageal squamous cell carcinoma(ESCC)involves chemotherapy and immunotherapy.However,alternative therapies are required for patients who are refractory or intolerant to existing therapies.Methods:In this single-arm,multicenter,open-label phase Ib study,30 patients received an intravenous infusion of SCT200,an antiepidermal growth factor receptor(EGFR)monoclonal antibody,6.0 mg/kg once a week for 6 weeks,followed by 8.0 mg/kg once every 2 weeks until disease progression or intolerable toxicity.The primary endpoint was the objective response rate(ORR).The secondary endpoints were progression-free survival(PFS),overall survival(OS),and safety.Results:Thirty patients were enrolled between July 2018 and May 2019.The ORR was 16.7%(95%CI:5.6%–34.7%).The median PFS and OS were 3.1 months(95%CI:1.5–4.3)and 6.8 months(95%CI:4.7–10.1),respectively.A numerical difference without any statistical significance in ORR was observed in patients with different EGFR expressions(≥50%:25.0%vs.<50%:0%,P=0.140)or TP53 mutation abundance(<10%:23.8%vs.≥10%:0%,P=0.286).Improved median PFS(3.4 vs.1.4 months,P=0.006)and OS(8.0 vs.4.2 months,P=0.027)were associated with TP53 mutation abundance of<10%.The most common treatment-related adverse events of grade 3 or 4(occurring in≥2 patients)were hypomagnesemia[7(23.3%)]and rash[2(6.7%)].No treatmentrelated death occurred.Conclusions:SCT200 monotherapy as the second-or further-line treatment for advanced ESCC showed favorable efficacy,with an acceptable safety profile.TP53 mutation abundance might serve as a potential predictive biomarker.
基金funded by the National Institutes of Health/National Cancer Institute(Grant Nos.CA204192 and CA246785)。
文摘Despite the significant resources dedicated to the development of monoclonal antibody(m Ab)therapies for solid tumors,the clinical success,thus far,has been modest.Limited efficacy of m Ab in solid tumors likely relates to unique aspects of tumor physiology.Solid tumors have an aberrant vasculature and a dense extracellular matrix that slow both the convective and diffusive transport of m Abs into and within tumors.For m Abs that are directed against cellular antigens,high antigen expression and rapid antigen turnover can result in perivascular cells binding to and eliminating a significant amount of extravasated m Ab,limiting m Ab distribution to portions of the tumor that are distant from functional vessels.Many preclinical investigations have reported strategies to improve m Ab uptake and distribution;however,to our knowledge,none have translated into the clinic.Here,we provide an overview of several barriers in solid tumors that limit m Ab uptake and distribution and discuss approaches that have been utilized to overcome these barriers in preclinical studies.
基金supported by the Scientific and Technical Project of Hunan Province,China(04NK1005)
文摘The present study aimed at the preparation of monoclonal antibody against the recombinant PthA-NLS and the isolation of the relative ScFv (single chain variable fragment) genes, providing the possibility to better understand the pathogenesis mechanism via PthA, and developing proper construct for future experimentation to obtain citrus plants resistant to canker disease by transformation and plant antibody techniques. The recombinant polypeptide PthA-NLS was injected into Balb/c mice to produce monoclonal antibody. Total RNA was isolated from the hybridoma cell line 3D10H2 which secreted anti- PthA-NLS McAb, and the variable region genes were amplified with specific primers by RT-PCR and SOE-PCR (splicing by overlap extension), and then the ScFv gene was isolated. The recombinant ScFv gene was cloned into pGEM-T and pET32a(+) vector. The later plasmid was transferred into E. coli BL21 (DE3) and the expression of the recombinant protein was induced. Three cell lines producing monoclonal antibody against PthA-NLS were acquired and named 1C8H1, 2D12B6, and 3D8A10. The recombinant ScFv gene of about 750 bp was constructed. The sequencing results showed that the ScFv gene consists of a 360 bp heavy chain, a 342 bp light chain, and a 45 bp linker region. The recombinant fusion ScFv protein was expressed by IPTG induction, and a 44.5 kDa of recombinant fusion protein was obtained. In conclusion, we obtained three cell lines stably producing monoclonal antibody specifically bound to PthA-NLS, and the relative ScFv gene was constructed and successfully expressed in E. coli. These results may play an important role in further understanding the pathogenesis mechanism and in the development of possible citrus resistant to canker disease by genetic transformation and plant antibiobody.
基金funding from Thermo Fisher Scientific as part of a funded collaborative agreement with NIBR。
文摘Ensuring the removal of host cell proteins(HCPs) during downstream processing of recombinant proteins such as monoclonal antibodies(m Abs) remains a challenge.Since residual HCPs might affect product stability or safety,constant monitoring is required to demonstrate their removal to be below the regulatory accepted level of 100 ng/mg.The current standard analytical approach for this procedure is based on ELISA;however,this approach only measures the overall HCP content.Therefore,the use of orthogonal methods,such as liquid chromatography-mass spectrometry(LC-MS),has been established,as it facilitates the quantitation of total HCPs as well as the identification and quantitation of the individual HCPs present.In the present study,a workflow for HCP detection and quantitation using an automated magnetic bead-based sample preparation,in combination with a data-independent acquisition(DIA) LC-MS analysis,was established.Employing the same instrumental setup commonly used for peptide mapping analysis of m Abs allows for its quick and easy implementation into pre-existing workflows,avoiding the need for dedicated instrumentation or personnel.Thereby,quantitation of HCPs over a broad dynamic range was enabled to allow monitoring of problematic HCPs or to track changes upon altered bioprocessing conditions.
基金supported by the National Natural Science Foundation of China(Grant No.81521005)the National Key Research Project of the Chinese Academy of Sciences(Grant No.XDA12050306)。
文摘Liquid chromatography tandem mass spectrometry(LC-MS/MS)has gradually become a promising alternative to ligand binding assay for the bioanalysis of biotherapeutic molecules,due to its rapid method development and high accuracy.In this study,we established a new LC-MS/MS method for the determination of the anti-sclerostin monoclonal antibody(SHR-1222)in cynomolgus monkey serum,and compared it to a previous electrochemiluminescence method.The antibody was quantified by detecting the surrogate peptide obtained by trypsin digestion.The surrogate peptide was carefully selected by investigating its uniqueness,stability and MS response.The quantitative range of the proposed method was 2.00-500μg/mL,and this verified method was successfully applied to the toxicokinetic assessment of SHR-1222 in cynomolgus monkey serum.It was found that the concentrations of SHR-1222 in cynomolgus monkeys displayed an excellent agreement between the LC-MS/MS and electrochemiluminescence methods(ratios of drug exposure,0.8-1.0).Notably,two monkeys in the60 mg/kg dose group had abnormal profiles with a low detection value of SHR-1222 in their individual sample.Combining the high-level anti-drug antibodies(ADAs)in these samples and the consistent quantitative results of the two methods,we found that the decreased concentration of SHR-1222 was due to the accelerated clearance mediated by ADAs rather than the interference of ADAs to the detection platform.Taken together,we successfully developed an accurate,efficient and cost-effective LC-MS/MS method for the quantification of SHR-1222 in serum samples,which could serve as a powerful tool to improve the preclinical development of antibody drugs.
基金supported by National Institutes of Health R21CA226491-01A1 (X.M.L.),1R01CA238273-01A1 (X.M.L.) and 1R01CA242917-01A1(X.M.L.)
文摘Targeted therapy has been widely demonstrated as an effective strategy to treat cancers,the leading cause of death in the world.This minireview summarizes the technical platforms and methodologies utilized to develop and engineer therapeutic monoclonal antibodies and antibody-drug conjugates.First,the USA FDA approved monoclonal antibody(mAb)-based targeted therapies are reviewed.Then the representative innovative chimeric,humanized and fully human anti-cancer antibodies and antibody-drug conjugates are described.Finally,the past and predictive market trend of therapeutic antibodies is discussed.
基金Supported by the National Natural Science Foundation of China(No.30672068)the Distinguished Young Scholars Fund of Jilin Province,China(No.20050114)+1 种基金the Key Grant of Jilin Province Science & Technology Committee,China(Nos.20060923-01,200805131)the Key Grant of Changchun City Science & Technology Committee,China(No.06GG147)
文摘Testis-specific protease 50(TSP50) has been identified as a testis-specific protein that is expressed abnormally in most human breast cancer samples,which makes it an attractive molecular marker and a potential target for diagnosis and therapy.In the present study,we prepared a panel of monoclonal antibodies(mAbs) with high specificity and sensitivity against TSP50 by hybridoma method and characterized them by ELISA,Western blot,immunofluroescence and immunohistochemical analyses.The results show that all of the 9 different clones can specifically bind to TSP50.The mAbs against TSP50 we generated could be good tools for both basic and clinical studies.
基金supported by the National Basic Research Program of China(2010CB126203)the special fund for Agro-scientific Research in the Public Interest,China(201003031)+1 种基金Earmarked Funds for Modern Agro-industry Technology Research SystemZhejiang Provincial Natural Science Foundation of China(Z3090039)
文摘Rice ragged stunt virus(RRSV) is a serious rice disease in Asia, causing serious yield losses on rice. The capsid protein(CP) gene of the major outer capsid protein of RRSV was expressed in Escherichia coli BL21(DE3) using the pMAL-C2 X expression vector. The recombinant protein was used as the immunogen to immunize BALB/c mice. A hybridoma cell line 8A12 secreting monoclonal antibody(MAb) against RRSV was obtained by fusing mouse myeloma cells(Sp 2/0) with spleen cells from the immunized BALB/c mice. Western blot analysis showed that the MAb 8A12 can specifically react with RRSV CP. Using the MAb, an antigen-coated-plate enzyme-linked immunosorbent assay(ACP-ELISA), a dot enzyme-linked immunosorbent assay(dot-ELISA), and immunocapture-RT-PCR(IC-RT-PCR) assay were developed to detect RRSV. The established ACP-ELISA, dot-blot ELISA and IC-RT-PCR methods could detect RRSV in infected rice tissue crude extracts with dilutions of 1:40 960, 1:1 280 and 1:655 360(w/v, g mL-1), respectively. The ACP-ELISA and dot-blot ELISA methods could detect RRSV in infected insect vector crude extracts with dilutions of 1:12 800 and 1:1 600(an individual planthopper μL-1), respectively. The field survey revealed that Rice ragged stunt disease occurs on rice in Hainan, Yunnan, Guangxi, Sichuan, Guizhou, Fujian, Hunan, Jiangxi and Zhejiang in China.
基金supported by a grant from the National Natural Science Foundation of China(No.30300429)
文摘Anti-HER2 monoclonal antibody (Sc7301)-paclitaxel (TAX) immunoconjugate was pre- pared and its specific binding to tumor cells was investigated in this study. Sc7301 was conjugated to TAX by the active ester method and then the TAX-Sc7301 immunoconjugate was obtained. After purification and labeling by Cyano-fluorescein isothiocyanate (FITC), the specific binding of TAX-Sc7301 to HER2-positive tumor cells (SKOV3) and HER2-negative tumor cells (HepG2) was evaluated respectively. TAX-Sc7301 (20 nmol/L) showed distinct specific binding to SKOV3 cells rather than HepG2 cells. And the uptake of the immunoconjugate by SKOV3 cells was increased with the TAX-So7301 concentration (3-48 nmol/L) and the incubation time (P〈0.05). It was concluded that the TAX-Sc7301 immunoconjugate is ootentially applicable as a targeted agent against HER2-10ositive tumor cells.