Although African swine fever(ASF) has been prevalent for more than a century, it remains the number one swine disease that seriously endangers the global pig industry, and there is no effective means of prevention and...Although African swine fever(ASF) has been prevalent for more than a century, it remains the number one swine disease that seriously endangers the global pig industry, and there is no effective means of prevention and treatment(Wang et al. 2023). Due to its enormous economic and social impact, it is listed as a notifiable animal disease by the World Organization for Animal Health(Costard et al. 2013). Although ASF has been present in Sub-Saharan Africa since its first discovery in Kenya.展开更多
Encephalomyocarditis virus(EMCV),a potential zoonotic pathogen,poses significant socioeconomic and public health challenges across various host species.Although EMCV rarely triggers severe clinical symptoms in humans,...Encephalomyocarditis virus(EMCV),a potential zoonotic pathogen,poses significant socioeconomic and public health challenges across various host species.Although EMCV rarely triggers severe clinical symptoms in humans,its widespread prevalence and unique biological characteristics underscore the need for continuous surveillance and the development of effective therapeutics and prophylactics.In this study,we evaluated the neutralizing effects of a monoclonal antibody derived from the spleens of mice immunized with EMCV virus-like particles(VLPs),both in vitro and in vivo.Using recombinant DNA technology,we engineered a baculovirus system to express EMCVs P12A and 3C,facilitating the production of VLPs in Sf9 cells.These VLPs serve as antigens to immunize mice,leading to the isolation of the monoclonal antibody 45G3.This antibody exhibited high specificity for EMCV confor-mational epitopes,excluding linear epitopes,and demonstrated potent in vitro neutralizing activity,with an IC50 of 0.01873μg/mL.Immunoelectron microscopy(IEM)revealed a strong direct interaction between the 45G3 antibody and EMCV particles.Virus adsorption inhibition assays demonstrated that 45G3 effectively blocked viral attachment,thereby preventing further infection of host cells.These findings further support the notion of a robust interaction between the virus and the antibody.Moreover,in vivo assessments revealed that 45G3 significantly reduced viral loads in treated mice and improved survival outcomes following EMCV exposure.Additionally,posttreatment analysis revealed reduced tissue damage and a markedly decreased inflammatory response in the brain,indicating that the 45G3 antibody effectively blocked viral infection,thereby mitigating tissue damage and enhancing survival.These findings position 45G3 as a promising candidate for EMCV management and provide a strong foundation for the future development of antiviral drugs targeting this widespread virus.展开更多
The monoclonal antibodies consist of an innovative form of immunotherapy,capable of defeating several diseases,such as cancer.It is an emergent and important theme,that advances evaluation,challenges,and future perspe...The monoclonal antibodies consist of an innovative form of immunotherapy,capable of defeating several diseases,such as cancer.It is an emergent and important theme,that advances evaluation,challenges,and future perspectives with high relevance to identify gaps in recent studies and to consolidate this general theme in only one research.Its action in Chronic and Acute Lymphoid Leukemia has been evaluated in several clinical trials,which were selected between 2022 and 2023,in order to understand better the monoclonal antibodies that were most studied.The biopharmaceutical compounds Ibrutinib,Obinutuzumab,Rituximab,Venetoclax,and Inotuzumab Ozogamicin were the ones that most appeared in the most recent publications,indicating the importance of amplifying the studies.The action mechanisms that are used imply that their combined use has more success in the disease remission,showing a lower recurrence,adverse effects,and toxicity.Besides the adverse effects and overwhelming prices of the treatment,these immunotherapies results are promising,amplifying the survival rates,improving the patient’s life quality,and resulting in a precision medicine,aiming a custom treatment.The future perspectives on this therapy consist of its application in the public health system,with patients being able to be submitted to this treatment without any costs and receive a better life quality.展开更多
Dear Editor,Group B coxsackieviruses(CVBs),belonging to the genus Enterovirus(EV)of the family Picornaviridae,comprise six serotypes and share a typical picornaviral structure(Alhazmi et al.,2023).While most CVB infec...Dear Editor,Group B coxsackieviruses(CVBs),belonging to the genus Enterovirus(EV)of the family Picornaviridae,comprise six serotypes and share a typical picornaviral structure(Alhazmi et al.,2023).While most CVB infections are mild and self-limiting,they can cause severe or fatal illness,especially in children(Tracy and Gauntt,2008).展开更多
Lumpy skin disease(LSD)is a highly contagious disease caused by lumpy skin disease virus(LSDV)in bovines.Rapid and accurate diagnosis is very important to controll it.However,current commercial detection kits need to ...Lumpy skin disease(LSD)is a highly contagious disease caused by lumpy skin disease virus(LSDV)in bovines.Rapid and accurate diagnosis is very important to controll it.However,current commercial detection kits need to be improved in terms of sensitivity or specificity.This study aimed to develop a novel diagnostic competitive enzyme-linked immunosorbent assay(cELISA)based on the newly identified antigen gene LSDV034.The rLSDV034 protein was identified as a potential diagnostic antigen,and it was expressed,purified,and used to immunize BALB/c mice.Using laboratory-prepared indirect ELISA(iELISA),the positive cell lines were screened,and their blocking activity was further verified by competitive ELISA(cELISA).The cell line,1H7,was chosen to produce mouse ascites,which were purified for a monoclonal antibody(mAb,5.395 mg/mL).The heavy chain type of the 1H7 mAb was identified as IgG1a,and its light chain subtype was identified as κ.Furthermore,cELISA was developed to detect bovine serum antibodies,with rLSDV034(4μg/mL)as the coating antigen and HRP-1H7 mAb(1:300)as the competitive antibody.The cutoff value of cELISA was 55%,based on 32 negative bovine serum samples.The analytical sensitivity was 1:8,and no cross-reaction was detected with bovine viral diarrhea virus(BVDV),infectious bovine rhinotracheitis virus(IBRV),Pasteurella multocida(P.multocida),or Mycoplasma bovis(M.bovis)from the serum samples.The diagnostic sensitivity and specificity of cELISA were 98.46%(95%confidence interval,CI:91.7–100)and 100%(95%CI:89.1–100),respectively,based on the analysis of 30 LSDV-infected bovine serum samples,35 GTPV-vaccinated samples,and 32 negative samples.The overall coincidence of the cELISA with the virus neutralization test(VNT)reached 98.97%(95%CI:94.4–100).Furthermore,we used cELISA to analyze 230 clinical bovine serum samples(including 59 infected and 171 vaccinated samples)and found that the serum positivity rates of the immunized samples(on d 60 postimmunization)and infected samples were 77.78%(95%CI:70.8–83.8%)and 71.19%(95%CI:57.9–82.2),respectively.These results indicate that the developed cELISA is promising for detecting serum antibodies in naturally infected or vaccinated cattle.展开更多
Porcine deltacoronavirus(PDCoV)is an emerging swine enteropathogenic coronavirus that can cause acute diarrhea and vomiting in newborn piglets and poses a potential risk for cross-species transmission.It is necessary ...Porcine deltacoronavirus(PDCoV)is an emerging swine enteropathogenic coronavirus that can cause acute diarrhea and vomiting in newborn piglets and poses a potential risk for cross-species transmission.It is necessary to develop an effective serological diagnostic tool for the surveillance of PDCoV infection and vaccine immunity effects.In this study,we developed a monoclonal antibody-based competitive ELISA(cELISA)that selected the purified recombinant PDCoV nucleocapsid(N)protein as the coating antigen to detect PDCoV antibodies.To evaluate the diagnostic performance of the cELISA,122 swine serum samples(39 positive and 83 negative)were tested and the results were compared with an indirect immunofluorescence assay(IFA)as the reference method.By receiver operating characteristic(ROC)curve analysis,the optimum cutoff value of percent inhibition(PI)was determined to be 26.8%,which showed excellent diagnostic performance,with an area under the curve(AUC)of 0.9919,a diagnostic sensitivity of 97.44%and a diagnostic specificity of 96.34%.Furthermore,there was good agreement between the cELISA and virus neutralization test(VNT)for the detection of PDCoV antibodies,with a coincidence rate of 92.7%,and theκanalysis showed almost perfect agreement(κ=0.851).Overall,the established cELISA showed good diagnostic performance,including sensitivity,specificity and repeatability,and can be used for diagnostic assistance,evaluating the response to vaccination and assessing swine herd immunity.展开更多
Five monoclonal antibodies（Mabs） to nuclear protein of avain influenza virus（AIV） were developed by syncretizing SP 2/0 and the spleen cells from BALB of mice immuized with H9 subtype AIV. Specificity of these Mab...Five monoclonal antibodies（Mabs） to nuclear protein of avain influenza virus（AIV） were developed by syncretizing SP 2/0 and the spleen cells from BALB of mice immuized with H9 subtype AIV. Specificity of these Mabs were identified by immunofluorescent assay（IFA） and enzyme linked immunosorbent assay （ELISA）. These five Mabs which were named as AIV-NP-2C3, AIV-NP-6A5, AIV-NP-3 H9, AIV-NP-7B4, AIV-NP-2H4 could react with all viruses of AIV-H9 strains in tests. The result of Western blotting showed that only the 60 ku protein antigen of AIV-H9 could be recognized by the Mabs but never recognized by New castle disease virus, REV and infectious bursa disease virus. The result of preliminary application showed that avian influenza viruses could be deetected bv Mabs in IFA and ELISA. All these Mabs will probably play important roles in preventing and monitoring avian influenza viruses.展开更多
Using the purified VP1 protein of Asia 1 type foot-and-mouth disease virus as the antigen, the purified monoclonal antibody was labeled by the sodium periodate method and the monoclonal antibody competitive ELISA was ...Using the purified VP1 protein of Asia 1 type foot-and-mouth disease virus as the antigen, the purified monoclonal antibody was labeled by the sodium periodate method and the monoclonal antibody competitive ELISA was established in this study. Ten positive porcine foot-and-mouth disease serums and more than two hundreds negative serum were tested, and the results were the same as the background of samples. The sensitivity test and replicate test indicated that this method was stable and sensitive, which was suitable for monitoring Asia 1 type porcine foot-and-mouth disease virus antibody.展开更多
The production and characterization of a monoclonal antibody (MAb AB10) against GA 3 glucoside as well as GA 3 is described. MAb AB10 was derived from an immunogen in which human serum albumin (HSA) was linked to G...The production and characterization of a monoclonal antibody (MAb AB10) against GA 3 glucoside as well as GA 3 is described. MAb AB10 was derived from an immunogen in which human serum albumin (HSA) was linked to GA 3 at carbon 3. This antibody showed high affinity for GA 3 glucoside as well as for 13 hydroxy gibberellins (GA 1, GA 3, GA 5, etc). The affinity of MAb AB10 for 13 hydroxy GAs was significantly reduced by methylation of the 7 oic acid but not by glycosylation of 3 hydroxyl group. Based on this antibody, both of competitive enzyme linked immunosorbent assays (ELISAs) for GA 3 glucoside and for GA 3 were developed. These two ELISAs displayed linear detection ranges from 0 2 pmol to 20 pmol. Using these assays, the fluctuation of GA 3 like and GA 3 glucoside like substances in the leaves of Rumex japonicus was investigated. The results indicated that the glycosylation of free GAs was connected with leaf senescence and that the function of 6 benzyl amino purine in retarding the leaf senescence was probably related to delaying the process of glycosylation of free GAs.展开更多
[Objective] The aim of the present study was to develop a direct immunofluorescence method for the diagnosis of canine distemper （CD） with FITC-conjugated monoclonal antibodies （FITC-McAb）.[ Metbod] The McAb again...[Objective] The aim of the present study was to develop a direct immunofluorescence method for the diagnosis of canine distemper （CD） with FITC-conjugated monoclonal antibodies （FITC-McAb）.[ Metbod] The McAb against CDV, designated as CE3, was purified with protein G and labeled with FITC through agitation method. After purification and identification, the optimal working concentration of FITC-labeled CE3 was determined. Then 61 clinical samples of suspected canine distemper were detected by direct immunofluorescence assay. [ Result] The absorption test, blocking test and specificity test showed that the labeled antibody had high specificity and sensitivity, but didn＇t have cross reaction with canine parvovirus （CPV）, canine parainfluenza virus （CPIV）, canine adenovirus （CAV） and rabies virus （RV）. The optimal working concentration was 1：80. The positive rate of clinical suspected samples was 48%. [ Conclusion] The direct immunofluorescence assay developed in this study was rapid, specific and convenient, and had great significance for the early diagnosis of canine distemper.展开更多
BALB/c mice were immunized using synthetic tandem polypeptide of Cap protein epitope of porcine circovirus type 2 （PCV2） as the antigen. By using lym-phocyte hybridoma technique, a hybridoma cellline stably secretin...BALB/c mice were immunized using synthetic tandem polypeptide of Cap protein epitope of porcine circovirus type 2 （PCV2） as the antigen. By using lym-phocyte hybridoma technique, a hybridoma cellline stably secreting monoclonal an-tibody against PCV2-rCap protein was successful y obtained and named as 670#. The ascites titer of the obtained monoclonal antibody was 1∶100 000. Western blot results showed that the monoclonal antibody could react with prokaryotical y ex-pressed PET32a-ORF2 recombinant protein, eukaryotical y expressed ORF1-ORF2 tandem protein and PCV2 whole virus celllysate. Indirect EILSA demonstrated that the monoclonal antibody could bind with ORF1-ORF2 tandem protein. Indirect im-munofluorescence assay （IFA） indicated that the monoclonal antibody could identify native PCV2 virus. The preparation of this monoclonal antibody provided technical tools for epitope analysis and molecular diagnosis of PCV2 virus.展开更多
Salicylic acid (SA) is widely distributed in many monocots and dicots and has many physiological effects. It can induce heat production in the thermogenic inflorescences of Arum lily([1]), block the biosynthesis of et...Salicylic acid (SA) is widely distributed in many monocots and dicots and has many physiological effects. It can induce heat production in the thermogenic inflorescences of Arum lily([1]), block the biosynthesis of ethylene, and more attractively, it seems to be an important natural signal molecule in the induction of systemic acquired resistance (SAR) in tobacco, cucumber and other plants([2,3]). Studies in recent years showed that SA was also intimately related to the resistance of plants to aboitic stress, for example, SA increased chilling resistance of maize seedlings. Hence, SA has been accepted as a kind of new plant hormones. Up to date, the quantification of SA usually has been performed by HPLC[4,5], which often needs a large quantity of sample and a verbose pretreatment. Compared to HPLC, immunoassays, including radio-immunoassays (RIA) and enzyme-immunoassays (EIA), are easy to perform and have been widely used in the quantification of other plant hormones, such as IAA([6]), ABA([7,8]), GAs([9]), cytokinins et al([10]), and jasmonic acid (JA)([11]), and other low-molecular-weight, none-immunogenic compounds in plants([12]). Till now, only an indirect enzyme-linked immunosorbent assay (ELISA) for SA based on polyclonal antibodies (PAbs) has been developed by our group([13]), although Bennett et al([14]) had prepared SA PAbs using 4-aminosalicylic acid linked to KLH as immunogen in goat. However, the sensitivity of the ELISA we established formerly was relatively low, and also relatively larger quantity of sample is needed than other ELISAs for plant hormones. In this paper, an ELISA for SA based on monoclonal antibody raised against SA-NH-CH2-NH-KLH was introduced, and the fluctuation of SA content in cucumber leaves after inoculated with Pseudomonas syringae pv. syringae was determined.展开更多
[Objective] This study was conducted to find out an approach for determining trimethoprim residues in water. [Method] Trimethoprim antigen was prepared through a series of reactions from trimethoprim hapten which was ...[Objective] This study was conducted to find out an approach for determining trimethoprim residues in water. [Method] Trimethoprim antigen was prepared through a series of reactions from trimethoprim hapten which was generated through the reaction between trimethoprim and maleic anhydride. And trimethoprim monoclonal antibodies were prepared by animal immune, and used to prepare ELISA kit to detect trimethoprim residues in water. Finally, the limit of detection （LED） of the ELISA kit was determined. [Result] The standard curve covered a concentration range of 0-80 μg/L. The LeD of trimethoprim in water using the ELISA kit was 2.34 μg/kg; the IC50 （half maximal inhibitory concentration） was 4.8 μg/L; the recovery rate of added trimethoprim standard ranged from 60.5% to 79.7%; within-and among-batches RSD was less than 10%. The trimethoprim monoclonal antibody was specific, as the cross-reactivity rate of trimethoprim antibody and diaveridine was less than 1%. The stability tests revealed that the ELISA kit was stable after being stored at 4 ℃ for 12 months. [Conclusion] The results will provide references for controlling the abuse of trimethoprim.展开更多
P65 protein, the major immunodominant protein of Mycoplasma hyopneu-moniae （Mhp） exhibiting no cross-reaction with other mycoplasmas, is general y used as a target protein for Mhp detection. In this study, BALB/c mi...P65 protein, the major immunodominant protein of Mycoplasma hyopneu-moniae （Mhp） exhibiting no cross-reaction with other mycoplasmas, is general y used as a target protein for Mhp detection. In this study, BALB/c mice were immunized with prokaryotical y expressed P65 recombinant protein to prepare monoclonal anti-body. After screening with Mhp whole-cel protein and P65 protein, a specific hy-bridoma cel line, 3G12, was obtained by ELISA. Identification results indicated that the antibody secreted by 3G12 hybridoma cel s could react with P65 protein and Mhp whole-cel protein. According to indirect ELISA assay, 3G12 cel culture super-natant possessed a titer of 1∶12 800 against P65 protein and 1∶3 200 against Mhp whole-cel protein; 3G12 ascites possessed a titer of above 1∶4 000 000 against P65 protein and above 1∶20 000 against Mhp 168 whole-cel protein. After long-term in vitro culture and continuous freezing-thawing, 3G12 cel line could stably secrete antibodies. A monoclonal antibody against P65 protein and Mhp whole-cel protein was successful y obtained in the present study, which provided basis for further in-vestigating the pathogenic mechanism of Mhp and establishing diagnostic methods of Mycoplasmal pneumonia of swine （MPS）.展开更多
Two monoclonal antibodies (McAbs) against Aα chain's C terminus offibrinogen (Fg) have been prepared and designated SZ-78 and SZ-79. Both theantigens in binding assay and immunoblot analysis showed that the two M...Two monoclonal antibodies (McAbs) against Aα chain's C terminus offibrinogen (Fg) have been prepared and designated SZ-78 and SZ-79. Both theantigens in binding assay and immunoblot analysis showed that the two McAbs recognized the epitopes located in residues 549-560 of the Aαchain. The two McAbs couldaccelerate rate of fibrin polymer assembly both in the purified system and in the humanplasma. From the pictures of transmission electronmicroscope, the average diametersof the fibers increase significantly to an average diameters of 375 nm after incubationwith the McAbs, while it was only 75nm without addition of the McAbs. There were al-so more branchings of fibers with addition of McAbs. These observations demonstratethat the amino acid sequences ofα 549-560 in the COOH terminus of the Aα chain mayplay an important role in the assembly of a fibrin clot, presumably being involved in lat-eral aggregation of protofibrils. The preparation of the McAbs supplies a usuful probe for the investigation of the展开更多
AIM To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')2 fragment of mAb HAbl8 against human hepatocellular carcinoma (HCC), and identify its activity in ...AIM To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')2 fragment of mAb HAbl8 against human hepatocellular carcinoma (HCC), and identify its activity in order to use SAg in the targeting therapy of HCC.METHODS MAb HAbl8 was extracted from the abdominal dropsy of Balb/ c mice, and was purified through chromatography column SP-40HR with Fast protein liquid chromatography (FPLC) system. The F(ab')2 fragment of mAb HAb18 was prepared by papainic digestion method. The conjugate of mAb HAb18 F(ab')2fragment and SEA was prepared with chemical conjugating reagent N-succinimidyl-3-( 2-pyridyldithio) propionate (SPDP) and purified through chromatography column Superose 12with FPLC system. The molecular mass and purity of each collected peak were identified with SDS-PAGE assay. The protein content was assayed by Lowry's method. The antibody activity of HAb18 F (ab')2 against HCC in the conjugate was identified by indirect immunocytochemical ABC method, and the activity of SEA in the conjugate to activate peripheral blood mononuclear cells (PBMC) was identified with MTT assay.RESULTS The lgG mAb HAb18 was extracted,and purified successfully. Immunocytochemical staining demonstrated that it reacted with most of HHCC cells of human HCC cell line. There were two peaks in the process of purification of the prepared HAb18 F(ab)2-SEA conjugate. SDS-PAGE assay demonstrated that the molecular mass of the first peak was about 130 ku, and the second peak was the mixture of about 45 ku and a little 100 ku proteins. The immunocytochemical staining was similar in HAb18 F (ab ')2-SEAconjugate and HAb18 F (ab ')2, i.e., thecytoplasm and/or cell membranes of most HHCC cells were positively stained. The MTT assay showed that the optical absorbance (A) value at 490 nm of HAb18 F (ab')2-SEA conjugate was 0.182 ± 0.012, that of negative control was 0.033± 0.009, and there was significant difference between them ( P < 0.05).CONCLUSION SPDP is a good proteinconjugating reagent and can be used in preparing protein conjugate. The conjugate of mAb HAb18F(ab')2 fragment and SEA protein was preparedsuccessfully in present study and can be used in the experimental study of HCC targeting therapy with the conjugate of SAg and anti-HCC mAbs or their fragments.展开更多
AIM To directly radiolabel an anti-hepatomamAb fragment HAb18 F（ab’）<sub>2</sub> with <sup>99m</sup>Tc bystannous-reduced method,and assess thestability,biodistribution and radioimmun-oimag...AIM To directly radiolabel an anti-hepatomamAb fragment HAb18 F（ab’）<sub>2</sub> with <sup>99m</sup>Tc bystannous-reduced method,and assess thestability,biodistribution and radioimmun-oimaging（RⅡ）.METHODS Immunoreactive fraction wasdetermined according to Lindmo’s method.Ellman’s reagent was used to determine thenumber of thiols in the reduced F（ab’）<sub>2</sub>.Labelingefficiency and homogeneity were measured bypaper chromatography,sodium dodecylsulphatepolyacrylamide gel electrophoresis（SDS-PAGE）and autoradiography.Challenge assay involvedthe incubation of aliquots of labeled antibody inethylenediaminetetraacetate（ EDTA ）and L-cysteine（L-cys）solutions with different molarratio at 37℃ for 1h,respectively.Investigationsin vivo utilized nude mice bearing humanhepatocellular carcinoma（HHCC）xenograftswith gamma camera imaging and tissuebiodistribution studies at regular intervals.RESULTS The labeling procedure was finishedwithin 1.5 h compared with the'pretinning'method which would take at least 21h.In vitrostudies demonstrated that the radiolabeled mAbfragment was homogeneous and retained itsimmunoreactivity.Challenge studies indicatedthat <sup>99m</sup>Tc-labeled HAb18 F（ab’）<sub>2</sub> in EDTA is morestable than in L-cys.Imaging and biodistribution showed a significant tumor uptake at 24 h post-injection of <sup>99m</sup>Tc-labeled HAb18 F（ab’）<sub>2</sub>.Theblood,kidney,liver and tumor uptakes at 24hwere 0.56±0.09,56.45±11.36,1.43±0.27 and6.57±3.01（%ID/g）,respectively.CONCLUSION <sup>99m</sup>Tc-HAb18 F（ab’）<sub>2</sub> conjugateprepared by this direct method appears to be aneffective way to detect hepatoma in nude micemodel.展开更多
A protein conjugate of streptomycin （streptomycin-bovine serum albumin （BSA） conjugate） was prepared and used as immunogen to produce monoclonal antibodies （MAb）. One hybridoma secreting anti-streptomycin MAb wa...A protein conjugate of streptomycin （streptomycin-bovine serum albumin （BSA） conjugate） was prepared and used as immunogen to produce monoclonal antibodies （MAb）. One hybridoma secreting anti-streptomycin MAb was obtained and then used to produce MAb. The MAb named 13H5 showed the 50% maximal inhibitory concentra- tion （IC50） value of 4.65 ng/ml and the IC20value of 0.21 ng/ml in phosphate buffered saline （PBS）. At optimum con- ditions, an indirect competitive enzyme-linked immunosorbent assay （ELISA） and a colloidal gold-based immuno- chromatographic assay （CGIA） were developed and applied to detect streptomycin residues in milk and swine urine samples. The developed ELISA showed that the minimum detection limit was 2.0 and 1.9 ng/ml for milk and swine urine samples, respectively, without obvious cross-reactivity to other tested antibiotics except dihydrostreptomycin which gave a 118.32% cross reaction value. Milk and swine urine samples spiked with streptomycin at 10, 50, 100 and 200 ng/rnl were analyzed by the established ELISA. The mean recovery of streptomycin was from 81.9% to 105.5% and from 84.3% to 92.2% for milk and swine urine, respectively. The optimized CGIA showed that the minimum de- tection limit was 20.0 ng/ml for milk and swine urine samples. The results of spiked analysis and specific analysis demonstrate that the CGIA could be applicable for screening milk and swine urine samples for the presence of streptomycin residues on-site. The established ELISA and CGIA allow the rapid, low-cost, and sensitive determination of streptomycin residues in food samples.展开更多
AIM To evaluale the potential role of P-selectinand anti-P-selectin monoclonal antibody(mAb)in apoptosis during hepatic/renal ischemia-reperfusion injury.METHODS Plasma P-selectin level,hepatic/renal P-selectin expres...AIM To evaluale the potential role of P-selectinand anti-P-selectin monoclonal antibody(mAb)in apoptosis during hepatic/renal ischemia-reperfusion injury.METHODS Plasma P-selectin level,hepatic/renal P-selectin expression and cell apoptosiswere detected in rat model of hepatic/ renalischemia-reperfusion injury.ELISA,immunohist-ochemistry and TUNEL were used.Someischemia-reperfusion rats were treated with anti-P-selectin mAb.RESULTS Hepatic/renal function insuffic-iency,up-regulated expression of P-selectin inplasma and hepatic/renal tissue,hepatic/renalhistopathological damages and cell apoptosiswere found in rats with hepatic/renal ischemia-reperfusion injury,while these changes becameless conspicuous in animals treated with anti-P-selectin mAb.CONCLUSION P-selectin might mediateneutrophil infiltration and cell apoptosis andcontribute to hepatic/renal ischemia-reperfusioninjury,anti-P-selectin mAb might be an efficientapproach for the prevention and treatment ofhepatic/renal ischemia-reperfusion injury.展开更多
AIM:To investigate the utility of immunohistochemical(IHC) staining with an antibody to Mycobacterium tuberculosis(M.tuberculosis) for the diagnosis of intestinal tuberculosis(TB).METHODS:We retrospectively identified...AIM:To investigate the utility of immunohistochemical(IHC) staining with an antibody to Mycobacterium tuberculosis(M.tuberculosis) for the diagnosis of intestinal tuberculosis(TB).METHODS:We retrospectively identified 10 patients(4 males and 6 females;mean age = 65.1 ± 13.6 years) with intestinal TB.Clinical characteristics,including age,gender,underlying disease,and symptoms were obtained.Chest radiograph and laboratory tests,including sputum Ziehl-Neelsen(ZN) staining,M.tuberculosis culture,and sputum polymerase chain reaction(PCR) for tubercle bacilli DNA,as well as Tuberculin skin test(TST) and QuantiFERON-TB gold test(QFT),were examined.Colonoscopic records recorded on the basis of Sato's classification were also reviewed,in addition to data from intestinal biopsies examined for histopathological findings,including hematoxylin and eosin staining,and ZN staining,as well as M.tuberculosis culture,and PCR for tubercle bacilli DNA.For the present study,archived formalin-fixed paraffin-embedded(FFPE) intestinal tissue samples were immunohistochemically stained using a commercially available species-specific monoclonal antibody to the 38-kDa antigen of the M.tuberculosis complex.These sections were also stained with the pan-macrophage marker CD68 antibody.RESULTS:From the clinical data,we found that no patients were immunocompromised,and that the main symptoms were diarrhea and weight loss.Three patients displayed active pulmonary TB,six patients(60%) had a positive TST,and 4 patients(40%) had a positive QFT.Colonoscopic findings revealed that all patients had type 1 findings(linear ulcers in a circumferential arrangement or linear ulcers arranged circumferentially with mucosa showing multiple nodules),all of which were located in the right hemicolon and/or terminal ileum.Seven patients(70%) had concomitant healed lesions in the ileocecal area.No acid-fast bacilli were detected with ZN staining of the intestinal tissue samples,and both M.tuberculosis culture and PCR for tubercle bacilli DNA were negative in all samples.The histopathological data revealed that tuberculous granulomas were present in 4 cases(40%).IHC staining in archived FFPE samples with anti-M.tuberculosis monoclonal antibody revealed positive findings in 4 patients(40%);the same patients in which granulomas were detected by hematoxylin and eosin staining.M.tuberculosis antigens were found to be mostly intracellular,granular in pattern,and primarily located in the CD68 + macrophages of the granulomas.CONCLUSION:IHC staining with a monoclonal antibody to M.tuberculosis may be an efficient and simple diagnostic tool in addition to classic examination methods for the diagnosis of intestinal TB.展开更多
基金supported by the National Key Research and Development Program of China (2021YFD1800100)the earmarked fund for China Agriculture Research System (CARS-35)。
文摘Although African swine fever(ASF) has been prevalent for more than a century, it remains the number one swine disease that seriously endangers the global pig industry, and there is no effective means of prevention and treatment(Wang et al. 2023). Due to its enormous economic and social impact, it is listed as a notifiable animal disease by the World Organization for Animal Health(Costard et al. 2013). Although ASF has been present in Sub-Saharan Africa since its first discovery in Kenya.
基金funded by the National Key Research and Development Program of China(grant number:2023YFC2306501)the Hubei Provincial Fund for Supporting High-Quality Development of the Seed Industry"Conservation and Utilization of Agricultural Germplasm Resources"Project(grant number:HBZY2023A001-16)。
文摘Encephalomyocarditis virus(EMCV),a potential zoonotic pathogen,poses significant socioeconomic and public health challenges across various host species.Although EMCV rarely triggers severe clinical symptoms in humans,its widespread prevalence and unique biological characteristics underscore the need for continuous surveillance and the development of effective therapeutics and prophylactics.In this study,we evaluated the neutralizing effects of a monoclonal antibody derived from the spleens of mice immunized with EMCV virus-like particles(VLPs),both in vitro and in vivo.Using recombinant DNA technology,we engineered a baculovirus system to express EMCVs P12A and 3C,facilitating the production of VLPs in Sf9 cells.These VLPs serve as antigens to immunize mice,leading to the isolation of the monoclonal antibody 45G3.This antibody exhibited high specificity for EMCV confor-mational epitopes,excluding linear epitopes,and demonstrated potent in vitro neutralizing activity,with an IC50 of 0.01873μg/mL.Immunoelectron microscopy(IEM)revealed a strong direct interaction between the 45G3 antibody and EMCV particles.Virus adsorption inhibition assays demonstrated that 45G3 effectively blocked viral attachment,thereby preventing further infection of host cells.These findings further support the notion of a robust interaction between the virus and the antibody.Moreover,in vivo assessments revealed that 45G3 significantly reduced viral loads in treated mice and improved survival outcomes following EMCV exposure.Additionally,posttreatment analysis revealed reduced tissue damage and a markedly decreased inflammatory response in the brain,indicating that the 45G3 antibody effectively blocked viral infection,thereby mitigating tissue damage and enhancing survival.These findings position 45G3 as a promising candidate for EMCV management and provide a strong foundation for the future development of antiviral drugs targeting this widespread virus.
文摘The monoclonal antibodies consist of an innovative form of immunotherapy,capable of defeating several diseases,such as cancer.It is an emergent and important theme,that advances evaluation,challenges,and future perspectives with high relevance to identify gaps in recent studies and to consolidate this general theme in only one research.Its action in Chronic and Acute Lymphoid Leukemia has been evaluated in several clinical trials,which were selected between 2022 and 2023,in order to understand better the monoclonal antibodies that were most studied.The biopharmaceutical compounds Ibrutinib,Obinutuzumab,Rituximab,Venetoclax,and Inotuzumab Ozogamicin were the ones that most appeared in the most recent publications,indicating the importance of amplifying the studies.The action mechanisms that are used imply that their combined use has more success in the disease remission,showing a lower recurrence,adverse effects,and toxicity.Besides the adverse effects and overwhelming prices of the treatment,these immunotherapies results are promising,amplifying the survival rates,improving the patient’s life quality,and resulting in a precision medicine,aiming a custom treatment.The future perspectives on this therapy consist of its application in the public health system,with patients being able to be submitted to this treatment without any costs and receive a better life quality.
基金supported by grants from the National Natural Science Foundation of China(82172248,82472253,82272310 and 32470996)the Xiamen Science and Technology Program(2022CXY0102)+1 种基金the Fundamental Research Funds for the Central Universities(20720250004)the Independent Research Project of the State Key Laboratory of Vaccine for Infectious Diseases(2024SKLVDzy03).
文摘Dear Editor,Group B coxsackieviruses(CVBs),belonging to the genus Enterovirus(EV)of the family Picornaviridae,comprise six serotypes and share a typical picornaviral structure(Alhazmi et al.,2023).While most CVB infections are mild and self-limiting,they can cause severe or fatal illness,especially in children(Tracy and Gauntt,2008).
基金financially supported by the National Key Research and Development Program of China(#2022YFD1800701)the Key Research and Development Program of the Ningxia Hui Autonomous Region(#2021BEF02028)the Chinese Agricultural Research System of MOF and MARA(#CARS-37).
文摘Lumpy skin disease(LSD)is a highly contagious disease caused by lumpy skin disease virus(LSDV)in bovines.Rapid and accurate diagnosis is very important to controll it.However,current commercial detection kits need to be improved in terms of sensitivity or specificity.This study aimed to develop a novel diagnostic competitive enzyme-linked immunosorbent assay(cELISA)based on the newly identified antigen gene LSDV034.The rLSDV034 protein was identified as a potential diagnostic antigen,and it was expressed,purified,and used to immunize BALB/c mice.Using laboratory-prepared indirect ELISA(iELISA),the positive cell lines were screened,and their blocking activity was further verified by competitive ELISA(cELISA).The cell line,1H7,was chosen to produce mouse ascites,which were purified for a monoclonal antibody(mAb,5.395 mg/mL).The heavy chain type of the 1H7 mAb was identified as IgG1a,and its light chain subtype was identified as κ.Furthermore,cELISA was developed to detect bovine serum antibodies,with rLSDV034(4μg/mL)as the coating antigen and HRP-1H7 mAb(1:300)as the competitive antibody.The cutoff value of cELISA was 55%,based on 32 negative bovine serum samples.The analytical sensitivity was 1:8,and no cross-reaction was detected with bovine viral diarrhea virus(BVDV),infectious bovine rhinotracheitis virus(IBRV),Pasteurella multocida(P.multocida),or Mycoplasma bovis(M.bovis)from the serum samples.The diagnostic sensitivity and specificity of cELISA were 98.46%(95%confidence interval,CI:91.7–100)and 100%(95%CI:89.1–100),respectively,based on the analysis of 30 LSDV-infected bovine serum samples,35 GTPV-vaccinated samples,and 32 negative samples.The overall coincidence of the cELISA with the virus neutralization test(VNT)reached 98.97%(95%CI:94.4–100).Furthermore,we used cELISA to analyze 230 clinical bovine serum samples(including 59 infected and 171 vaccinated samples)and found that the serum positivity rates of the immunized samples(on d 60 postimmunization)and infected samples were 77.78%(95%CI:70.8–83.8%)and 71.19%(95%CI:57.9–82.2),respectively.These results indicate that the developed cELISA is promising for detecting serum antibodies in naturally infected or vaccinated cattle.
基金supported by the National Key Research and Development Program(2023YFD1800501)the National Natural Science Foundation of China(32373030,32202787)+5 种基金the S&T Program of Hebei(21322401D)the Jiangsu Province Natural Sciences Foundation(BK20221432,BK20210158)the Jiangsu Agricultural Science and Technology Innovation Fund(CX(22)3028)the Special Project of Northern Jiangsu(SZ-LYG202109)the Open Fund of Shaoxing Academy of Biomedicine of Zhejiang Sci-Tech University(SXAB202215)the Open Fund of Key Laboratory for Prevention and Control of Avian Influenza and Other Major Poultry Diseases,Ministry of Agriculture and Rural Affairs(YDWS202213).
文摘Porcine deltacoronavirus(PDCoV)is an emerging swine enteropathogenic coronavirus that can cause acute diarrhea and vomiting in newborn piglets and poses a potential risk for cross-species transmission.It is necessary to develop an effective serological diagnostic tool for the surveillance of PDCoV infection and vaccine immunity effects.In this study,we developed a monoclonal antibody-based competitive ELISA(cELISA)that selected the purified recombinant PDCoV nucleocapsid(N)protein as the coating antigen to detect PDCoV antibodies.To evaluate the diagnostic performance of the cELISA,122 swine serum samples(39 positive and 83 negative)were tested and the results were compared with an indirect immunofluorescence assay(IFA)as the reference method.By receiver operating characteristic(ROC)curve analysis,the optimum cutoff value of percent inhibition(PI)was determined to be 26.8%,which showed excellent diagnostic performance,with an area under the curve(AUC)of 0.9919,a diagnostic sensitivity of 97.44%and a diagnostic specificity of 96.34%.Furthermore,there was good agreement between the cELISA and virus neutralization test(VNT)for the detection of PDCoV antibodies,with a coincidence rate of 92.7%,and theκanalysis showed almost perfect agreement(κ=0.851).Overall,the established cELISA showed good diagnostic performance,including sensitivity,specificity and repeatability,and can be used for diagnostic assistance,evaluating the response to vaccination and assessing swine herd immunity.
基金Supported by the National Key Technologies Research and Develop-ment Program of China during the 10th Five-Year Plan Period(2004BA519A05)Technologies Research and Development Program of China during the 10th Five-Year Plan Period in Jiangsu Province(BE2002346).~~
文摘Five monoclonal antibodies（Mabs） to nuclear protein of avain influenza virus（AIV） were developed by syncretizing SP 2/0 and the spleen cells from BALB of mice immuized with H9 subtype AIV. Specificity of these Mabs were identified by immunofluorescent assay（IFA） and enzyme linked immunosorbent assay （ELISA）. These five Mabs which were named as AIV-NP-2C3, AIV-NP-6A5, AIV-NP-3 H9, AIV-NP-7B4, AIV-NP-2H4 could react with all viruses of AIV-H9 strains in tests. The result of Western blotting showed that only the 60 ku protein antigen of AIV-H9 could be recognized by the Mabs but never recognized by New castle disease virus, REV and infectious bursa disease virus. The result of preliminary application showed that avian influenza viruses could be deetected bv Mabs in IFA and ELISA. All these Mabs will probably play important roles in preventing and monitoring avian influenza viruses.
基金Supported by National High-tech R&D Program (863) Subsidized Project(2006AA10A204)Special Fund for Basic Scientific Research-related Subsidy of State-level and Public-welfare Scientific Research Institutes~~
文摘Using the purified VP1 protein of Asia 1 type foot-and-mouth disease virus as the antigen, the purified monoclonal antibody was labeled by the sodium periodate method and the monoclonal antibody competitive ELISA was established in this study. Ten positive porcine foot-and-mouth disease serums and more than two hundreds negative serum were tested, and the results were the same as the background of samples. The sensitivity test and replicate test indicated that this method was stable and sensitive, which was suitable for monitoring Asia 1 type porcine foot-and-mouth disease virus antibody.
文摘The production and characterization of a monoclonal antibody (MAb AB10) against GA 3 glucoside as well as GA 3 is described. MAb AB10 was derived from an immunogen in which human serum albumin (HSA) was linked to GA 3 at carbon 3. This antibody showed high affinity for GA 3 glucoside as well as for 13 hydroxy gibberellins (GA 1, GA 3, GA 5, etc). The affinity of MAb AB10 for 13 hydroxy GAs was significantly reduced by methylation of the 7 oic acid but not by glycosylation of 3 hydroxyl group. Based on this antibody, both of competitive enzyme linked immunosorbent assays (ELISAs) for GA 3 glucoside and for GA 3 were developed. These two ELISAs displayed linear detection ranges from 0 2 pmol to 20 pmol. Using these assays, the fluctuation of GA 3 like and GA 3 glucoside like substances in the leaves of Rumex japonicus was investigated. The results indicated that the glycosylation of free GAs was connected with leaf senescence and that the function of 6 benzyl amino purine in retarding the leaf senescence was probably related to delaying the process of glycosylation of free GAs.
基金Supported by Science and Technology Foundation of PLA General Lo-gistics Department(06G138)~~
文摘[Objective] The aim of the present study was to develop a direct immunofluorescence method for the diagnosis of canine distemper （CD） with FITC-conjugated monoclonal antibodies （FITC-McAb）.[ Metbod] The McAb against CDV, designated as CE3, was purified with protein G and labeled with FITC through agitation method. After purification and identification, the optimal working concentration of FITC-labeled CE3 was determined. Then 61 clinical samples of suspected canine distemper were detected by direct immunofluorescence assay. [ Result] The absorption test, blocking test and specificity test showed that the labeled antibody had high specificity and sensitivity, but didn＇t have cross reaction with canine parvovirus （CPV）, canine parainfluenza virus （CPIV）, canine adenovirus （CAV） and rabies virus （RV）. The optimal working concentration was 1：80. The positive rate of clinical suspected samples was 48%. [ Conclusion] The direct immunofluorescence assay developed in this study was rapid, specific and convenient, and had great significance for the early diagnosis of canine distemper.
基金Supported by National Natural Science Foundation of China(31302071)AgriculturalScience and Technology Independent Innovation Fund of Jiangsu Province(TechnicalInnovation)[CX(13)3065]~~
文摘BALB/c mice were immunized using synthetic tandem polypeptide of Cap protein epitope of porcine circovirus type 2 （PCV2） as the antigen. By using lym-phocyte hybridoma technique, a hybridoma cellline stably secreting monoclonal an-tibody against PCV2-rCap protein was successful y obtained and named as 670#. The ascites titer of the obtained monoclonal antibody was 1∶100 000. Western blot results showed that the monoclonal antibody could react with prokaryotical y ex-pressed PET32a-ORF2 recombinant protein, eukaryotical y expressed ORF1-ORF2 tandem protein and PCV2 whole virus celllysate. Indirect EILSA demonstrated that the monoclonal antibody could bind with ORF1-ORF2 tandem protein. Indirect im-munofluorescence assay （IFA） indicated that the monoclonal antibody could identify native PCV2 virus. The preparation of this monoclonal antibody provided technical tools for epitope analysis and molecular diagnosis of PCV2 virus.
基金SupportedbytheNationalNaturalScienceFoundationofChina (No .39870 473) .
文摘Salicylic acid (SA) is widely distributed in many monocots and dicots and has many physiological effects. It can induce heat production in the thermogenic inflorescences of Arum lily([1]), block the biosynthesis of ethylene, and more attractively, it seems to be an important natural signal molecule in the induction of systemic acquired resistance (SAR) in tobacco, cucumber and other plants([2,3]). Studies in recent years showed that SA was also intimately related to the resistance of plants to aboitic stress, for example, SA increased chilling resistance of maize seedlings. Hence, SA has been accepted as a kind of new plant hormones. Up to date, the quantification of SA usually has been performed by HPLC[4,5], which often needs a large quantity of sample and a verbose pretreatment. Compared to HPLC, immunoassays, including radio-immunoassays (RIA) and enzyme-immunoassays (EIA), are easy to perform and have been widely used in the quantification of other plant hormones, such as IAA([6]), ABA([7,8]), GAs([9]), cytokinins et al([10]), and jasmonic acid (JA)([11]), and other low-molecular-weight, none-immunogenic compounds in plants([12]). Till now, only an indirect enzyme-linked immunosorbent assay (ELISA) for SA based on polyclonal antibodies (PAbs) has been developed by our group([13]), although Bennett et al([14]) had prepared SA PAbs using 4-aminosalicylic acid linked to KLH as immunogen in goat. However, the sensitivity of the ELISA we established formerly was relatively low, and also relatively larger quantity of sample is needed than other ELISAs for plant hormones. In this paper, an ELISA for SA based on monoclonal antibody raised against SA-NH-CH2-NH-KLH was introduced, and the fluctuation of SA content in cucumber leaves after inoculated with Pseudomonas syringae pv. syringae was determined.
基金Supported by Beijing Municipal Science and Technology Project(Z151100002115059)~~
文摘[Objective] This study was conducted to find out an approach for determining trimethoprim residues in water. [Method] Trimethoprim antigen was prepared through a series of reactions from trimethoprim hapten which was generated through the reaction between trimethoprim and maleic anhydride. And trimethoprim monoclonal antibodies were prepared by animal immune, and used to prepare ELISA kit to detect trimethoprim residues in water. Finally, the limit of detection （LED） of the ELISA kit was determined. [Result] The standard curve covered a concentration range of 0-80 μg/L. The LeD of trimethoprim in water using the ELISA kit was 2.34 μg/kg; the IC50 （half maximal inhibitory concentration） was 4.8 μg/L; the recovery rate of added trimethoprim standard ranged from 60.5% to 79.7%; within-and among-batches RSD was less than 10%. The trimethoprim monoclonal antibody was specific, as the cross-reactivity rate of trimethoprim antibody and diaveridine was less than 1%. The stability tests revealed that the ELISA kit was stable after being stored at 4 ℃ for 12 months. [Conclusion] The results will provide references for controlling the abuse of trimethoprim.
基金Supported by National Natural Science Foundation of China(31100136,3111339)Independent Innovation Fund of Agricultural Science and Technology of Jiangsu Province[CX(13)3066]~~
文摘P65 protein, the major immunodominant protein of Mycoplasma hyopneu-moniae （Mhp） exhibiting no cross-reaction with other mycoplasmas, is general y used as a target protein for Mhp detection. In this study, BALB/c mice were immunized with prokaryotical y expressed P65 recombinant protein to prepare monoclonal anti-body. After screening with Mhp whole-cel protein and P65 protein, a specific hy-bridoma cel line, 3G12, was obtained by ELISA. Identification results indicated that the antibody secreted by 3G12 hybridoma cel s could react with P65 protein and Mhp whole-cel protein. According to indirect ELISA assay, 3G12 cel culture super-natant possessed a titer of 1∶12 800 against P65 protein and 1∶3 200 against Mhp whole-cel protein; 3G12 ascites possessed a titer of above 1∶4 000 000 against P65 protein and above 1∶20 000 against Mhp 168 whole-cel protein. After long-term in vitro culture and continuous freezing-thawing, 3G12 cel line could stably secrete antibodies. A monoclonal antibody against P65 protein and Mhp whole-cel protein was successful y obtained in the present study, which provided basis for further in-vestigating the pathogenic mechanism of Mhp and establishing diagnostic methods of Mycoplasmal pneumonia of swine （MPS）.
文摘Two monoclonal antibodies (McAbs) against Aα chain's C terminus offibrinogen (Fg) have been prepared and designated SZ-78 and SZ-79. Both theantigens in binding assay and immunoblot analysis showed that the two McAbs recognized the epitopes located in residues 549-560 of the Aαchain. The two McAbs couldaccelerate rate of fibrin polymer assembly both in the purified system and in the humanplasma. From the pictures of transmission electronmicroscope, the average diametersof the fibers increase significantly to an average diameters of 375 nm after incubationwith the McAbs, while it was only 75nm without addition of the McAbs. There were al-so more branchings of fibers with addition of McAbs. These observations demonstratethat the amino acid sequences ofα 549-560 in the COOH terminus of the Aα chain mayplay an important role in the assembly of a fibrin clot, presumably being involved in lat-eral aggregation of protofibrils. The preparation of the McAbs supplies a usuful probe for the investigation of the
基金Supported by the National 863 Project of China,No.102-09-01-02National Natural Science Foundation of China,No.39770827
文摘AIM To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')2 fragment of mAb HAbl8 against human hepatocellular carcinoma (HCC), and identify its activity in order to use SAg in the targeting therapy of HCC.METHODS MAb HAbl8 was extracted from the abdominal dropsy of Balb/ c mice, and was purified through chromatography column SP-40HR with Fast protein liquid chromatography (FPLC) system. The F(ab')2 fragment of mAb HAb18 was prepared by papainic digestion method. The conjugate of mAb HAb18 F(ab')2fragment and SEA was prepared with chemical conjugating reagent N-succinimidyl-3-( 2-pyridyldithio) propionate (SPDP) and purified through chromatography column Superose 12with FPLC system. The molecular mass and purity of each collected peak were identified with SDS-PAGE assay. The protein content was assayed by Lowry's method. The antibody activity of HAb18 F (ab')2 against HCC in the conjugate was identified by indirect immunocytochemical ABC method, and the activity of SEA in the conjugate to activate peripheral blood mononuclear cells (PBMC) was identified with MTT assay.RESULTS The lgG mAb HAb18 was extracted,and purified successfully. Immunocytochemical staining demonstrated that it reacted with most of HHCC cells of human HCC cell line. There were two peaks in the process of purification of the prepared HAb18 F(ab)2-SEA conjugate. SDS-PAGE assay demonstrated that the molecular mass of the first peak was about 130 ku, and the second peak was the mixture of about 45 ku and a little 100 ku proteins. The immunocytochemical staining was similar in HAb18 F (ab ')2-SEAconjugate and HAb18 F (ab ')2, i.e., thecytoplasm and/or cell membranes of most HHCC cells were positively stained. The MTT assay showed that the optical absorbance (A) value at 490 nm of HAb18 F (ab')2-SEA conjugate was 0.182 ± 0.012, that of negative control was 0.033± 0.009, and there was significant difference between them ( P < 0.05).CONCLUSION SPDP is a good proteinconjugating reagent and can be used in preparing protein conjugate. The conjugate of mAb HAb18F(ab')2 fragment and SEA protein was preparedsuccessfully in present study and can be used in the experimental study of HCC targeting therapy with the conjugate of SAg and anti-HCC mAbs or their fragments.
基金National Natural Science Foundation of China,No.39700175
文摘AIM To directly radiolabel an anti-hepatomamAb fragment HAb18 F（ab’）<sub>2</sub> with <sup>99m</sup>Tc bystannous-reduced method,and assess thestability,biodistribution and radioimmun-oimaging（RⅡ）.METHODS Immunoreactive fraction wasdetermined according to Lindmo’s method.Ellman’s reagent was used to determine thenumber of thiols in the reduced F（ab’）<sub>2</sub>.Labelingefficiency and homogeneity were measured bypaper chromatography,sodium dodecylsulphatepolyacrylamide gel electrophoresis（SDS-PAGE）and autoradiography.Challenge assay involvedthe incubation of aliquots of labeled antibody inethylenediaminetetraacetate（ EDTA ）and L-cysteine（L-cys）solutions with different molarratio at 37℃ for 1h,respectively.Investigationsin vivo utilized nude mice bearing humanhepatocellular carcinoma（HHCC）xenograftswith gamma camera imaging and tissuebiodistribution studies at regular intervals.RESULTS The labeling procedure was finishedwithin 1.5 h compared with the'pretinning'method which would take at least 21h.In vitrostudies demonstrated that the radiolabeled mAbfragment was homogeneous and retained itsimmunoreactivity.Challenge studies indicatedthat <sup>99m</sup>Tc-labeled HAb18 F（ab’）<sub>2</sub> in EDTA is morestable than in L-cys.Imaging and biodistribution showed a significant tumor uptake at 24 h post-injection of <sup>99m</sup>Tc-labeled HAb18 F（ab’）<sub>2</sub>.Theblood,kidney,liver and tumor uptakes at 24hwere 0.56±0.09,56.45±11.36,1.43±0.27 and6.57±3.01（%ID/g）,respectively.CONCLUSION <sup>99m</sup>Tc-HAb18 F（ab’）<sub>2</sub> conjugateprepared by this direct method appears to be aneffective way to detect hepatoma in nude micemodel.
基金Project (No.2007C22047) supported by the Program of Science and Technology of Zhejiang Province,China
文摘A protein conjugate of streptomycin （streptomycin-bovine serum albumin （BSA） conjugate） was prepared and used as immunogen to produce monoclonal antibodies （MAb）. One hybridoma secreting anti-streptomycin MAb was obtained and then used to produce MAb. The MAb named 13H5 showed the 50% maximal inhibitory concentra- tion （IC50） value of 4.65 ng/ml and the IC20value of 0.21 ng/ml in phosphate buffered saline （PBS）. At optimum con- ditions, an indirect competitive enzyme-linked immunosorbent assay （ELISA） and a colloidal gold-based immuno- chromatographic assay （CGIA） were developed and applied to detect streptomycin residues in milk and swine urine samples. The developed ELISA showed that the minimum detection limit was 2.0 and 1.9 ng/ml for milk and swine urine samples, respectively, without obvious cross-reactivity to other tested antibiotics except dihydrostreptomycin which gave a 118.32% cross reaction value. Milk and swine urine samples spiked with streptomycin at 10, 50, 100 and 200 ng/rnl were analyzed by the established ELISA. The mean recovery of streptomycin was from 81.9% to 105.5% and from 84.3% to 92.2% for milk and swine urine, respectively. The optimized CGIA showed that the minimum de- tection limit was 20.0 ng/ml for milk and swine urine samples. The results of spiked analysis and specific analysis demonstrate that the CGIA could be applicable for screening milk and swine urine samples for the presence of streptomycin residues on-site. The established ELISA and CGIA allow the rapid, low-cost, and sensitive determination of streptomycin residues in food samples.
基金the Scientific Foundation of Ministry of Health of China,No.98-2-283Shanghai Natural Science Foundation,No.98ZB14025
文摘AIM To evaluale the potential role of P-selectinand anti-P-selectin monoclonal antibody(mAb)in apoptosis during hepatic/renal ischemia-reperfusion injury.METHODS Plasma P-selectin level,hepatic/renal P-selectin expression and cell apoptosiswere detected in rat model of hepatic/ renalischemia-reperfusion injury.ELISA,immunohist-ochemistry and TUNEL were used.Someischemia-reperfusion rats were treated with anti-P-selectin mAb.RESULTS Hepatic/renal function insuffic-iency,up-regulated expression of P-selectin inplasma and hepatic/renal tissue,hepatic/renalhistopathological damages and cell apoptosiswere found in rats with hepatic/renal ischemia-reperfusion injury,while these changes becameless conspicuous in animals treated with anti-P-selectin mAb.CONCLUSION P-selectin might mediateneutrophil infiltration and cell apoptosis andcontribute to hepatic/renal ischemia-reperfusioninjury,anti-P-selectin mAb might be an efficientapproach for the prevention and treatment ofhepatic/renal ischemia-reperfusion injury.
文摘AIM:To investigate the utility of immunohistochemical(IHC) staining with an antibody to Mycobacterium tuberculosis(M.tuberculosis) for the diagnosis of intestinal tuberculosis(TB).METHODS:We retrospectively identified 10 patients(4 males and 6 females;mean age = 65.1 ± 13.6 years) with intestinal TB.Clinical characteristics,including age,gender,underlying disease,and symptoms were obtained.Chest radiograph and laboratory tests,including sputum Ziehl-Neelsen(ZN) staining,M.tuberculosis culture,and sputum polymerase chain reaction(PCR) for tubercle bacilli DNA,as well as Tuberculin skin test(TST) and QuantiFERON-TB gold test(QFT),were examined.Colonoscopic records recorded on the basis of Sato's classification were also reviewed,in addition to data from intestinal biopsies examined for histopathological findings,including hematoxylin and eosin staining,and ZN staining,as well as M.tuberculosis culture,and PCR for tubercle bacilli DNA.For the present study,archived formalin-fixed paraffin-embedded(FFPE) intestinal tissue samples were immunohistochemically stained using a commercially available species-specific monoclonal antibody to the 38-kDa antigen of the M.tuberculosis complex.These sections were also stained with the pan-macrophage marker CD68 antibody.RESULTS:From the clinical data,we found that no patients were immunocompromised,and that the main symptoms were diarrhea and weight loss.Three patients displayed active pulmonary TB,six patients(60%) had a positive TST,and 4 patients(40%) had a positive QFT.Colonoscopic findings revealed that all patients had type 1 findings(linear ulcers in a circumferential arrangement or linear ulcers arranged circumferentially with mucosa showing multiple nodules),all of which were located in the right hemicolon and/or terminal ileum.Seven patients(70%) had concomitant healed lesions in the ileocecal area.No acid-fast bacilli were detected with ZN staining of the intestinal tissue samples,and both M.tuberculosis culture and PCR for tubercle bacilli DNA were negative in all samples.The histopathological data revealed that tuberculous granulomas were present in 4 cases(40%).IHC staining in archived FFPE samples with anti-M.tuberculosis monoclonal antibody revealed positive findings in 4 patients(40%);the same patients in which granulomas were detected by hematoxylin and eosin staining.M.tuberculosis antigens were found to be mostly intracellular,granular in pattern,and primarily located in the CD68 + macrophages of the granulomas.CONCLUSION:IHC staining with a monoclonal antibody to M.tuberculosis may be an efficient and simple diagnostic tool in addition to classic examination methods for the diagnosis of intestinal TB.
