1.Characterization of specific monoclonal antibodies to Aspergillus conidia by flow cytometry.
Nan YU ; Xiao-peng YUAN ; Jing JIN ; Wei HAO ; Yan-fang WANG ; Xiao-yan CHE
Journal of Southern Medical University 2011;31(3):487-489
OBJECTIVETo characterize the specific monoclonal antibodies to Aspergillus conidia.
METHODSFlow cytometry was used to examine the reactivity of the specific monoclonal antibodies to Aspergillus conidia.
RESULTSBoth the monoclonal antibodies MA3 and Con2 showed specific reactivity to Aspergillus conidia suspensions. MA3 was capable of binding to the conidia of A.fumigatus, A.flavus, A.niger and A.terreus, while Con2 was reactive only to the conidia of A.fumigatus.
CONCLUSIONTwo specific monoclonal antibodies (MA3 and Con2) to Aspergillus conidia have been obtained.
Antibodies, Fungal ; immunology ; isolation & purification ; Antibodies, Monoclonal ; immunology ; isolation & purification ; Antibody Specificity ; Aspergillus ; immunology ; Flow Cytometry ; Spores, Fungal ; immunology
2.Preparation and identification of monoclonal antibody against PNAS-2 protein.
Jia LIU ; Fang-Yuan CHEN ; Hai-Rong WANG ; Ji-Hua ZHONG ; Hua ZHONG ; Jie-Ying HAN ; Ren-Rong OUYANG
Journal of Experimental Hematology 2009;17(5):1269-1272
This study was purposed to prepare and primarily identify the specific monoclonal antibodies (McAbs) against the apoptosis related protein PNAS-2 so as to provide the essential tool for study of PNAS-2 function. The McAbs against PNAS-2 were prepared via the immunization of mice, cell fusion and cloning using synthetic peptide of PNAS-2 as immunogen; the specificity, titer and subtype of McAb were detected by Western blot, ELISA and immunofluorescence. The results showed that the stable hybridoma cell line S-31-7 producing McAbs against PNAS-2 protein was successfully obtained. The immunoglobulin of the McAb was identified to be IGg1lambda. The titer of ascetic fluid fled McAb were 1:8,000. A single specific band with 28 kD was shown in Western blot test, and the antigen recognized was present in cell cytoplasm by immunofluorescence. In conclusion, the obtained McAb against PNAS-2 displays strong specificity and high titer, which may be applied to the advanced research on PNAS-2 protein.
Animals
;
Antibodies, Monoclonal
;
immunology
;
isolation & purification
;
Antibody Specificity
;
immunology
;
Apoptosis Regulatory Proteins
;
immunology
;
Female
;
Mice
;
Mice, Inbred BALB C
3.Characterization and functional studies of vWF A3 domain monoclonal antibodies that inhibit binding of vWF to collagen.
Yi-Ming ZHAO ; Ning-Zheng DONG ; Fei SHEN ; Li-Qian XIE ; Chang-Geng RUAN
Chinese Journal of Hematology 2008;29(3):171-174
OBJECTIVETo prepare anti-von Willebrand factor A3 (vWF-A3) domain monoclonal antibodies(mAbs) which block vWF-A3 binding to collagen, and characterize their biochemical properties and functions.
METHODSBALB/c mice were immunized with purified recombinant vWF-A3 protein (rvWF-A3). Murine anti-human vWF-A3 mAbs were developed by standard hybridoma technology and identified with ELISA. The recognition of the mAbs with rvWF -A3 and reduced human vWF was identified by Western-blot. The effect of mAbs on binding of purified human vWF to human placenta or calf skin collagen III was studied with collagen binding inhibition test.
RESULTSA group of 30 murine anti-human vWF-A3 mAbs was obtained, from which 2 clones were identified as inhibitory ones and designated as SZ-123 and SZ-125. SZ-123 and SZ-125 could react specifically with human vWF and rvWF-A3 respectively, while neither of them reacted with rvWF-A1 and rvWF-A2. Western-blot showed that SZ-123 and SZ-125 could recognize a 27 x 10(3) band of rvWF-A3 and 2 reduced human vWF bands at 250 x 10(3) and 170 x 10(3). SZ-123 and SZ-125 not only inhibited the binding of purified human vWF (1.5 and 3.0 microg/ml) to human type III collagen and to calf skin collagen III in a dose dependent manner, but also inhibited the binding of plasma vWF from human, rhesus monkeys or Beagle dogs to the two collagens.
CONCLUSIONSZ-123 and SZ-125 are neutralizing mAbs against vWF-A3 domain and may have therapeutic potential as an antithrombotic agent.
Animals ; Antibodies, Monoclonal ; immunology ; isolation & purification ; Collagen ; immunology ; Mice ; Mice, Inbred BALB C ; von Willebrand Factor ; immunology
4.Preparation of immunoaffinity column of zearalenone.
Ying ZHANG ; Zhi-bing HUANG ; Shun-zhou DENG ; Yang XU
Chinese Journal of Preventive Medicine 2007;41(2):110-113
OBJECTIVETo prepare immunoaffinity column of zearalenone.
METHODSThe zearalenone immunoaffinity column (IAC) was prepared by coupling CNBr-activated Sepharose 4 Fast Flow (4FF) with the anti-zearalenone monoclonal antibody which was purified by caprylic acid-ammonium sulfate method. The coupling reaction was identified by UV-absorbance measurements, and the IAC prepared was evaluated by indirect-competition ELISA and HPLC.
RESULTSThe column capacity was determined to be 0.40 microg when using 0.5 ml of CNBr activated Sepharose 4FF and 350 microg of purified anti-zearalenone monoclonal antibody. The mean true recoveries were in the range 76.33% - 90.10% and RSD was 6.68% - 10.93% at levels of 60 microg/kg - 300 microg/kg. 30 samples of wheat and maize were detected by the anti-ZEN IAC produced by the laboratory, 17 samples were observed to be contaminated in a comparable range from 31.33 microg/kg - 377.84 microg/kg. Detection limit based on a signal-to-noise ratio 3:1 was 10. 00 microg/kg for ZEN in wheat and maize.
CONCLUSIONIAC, a simple separating method which is used in ZEN extraction from cereals, is able to purify and condense ZEN in one step. The cost of detection can be lowered down because the IAC developed is hopefully to substitute the imported IAC.
Antibodies, Monoclonal ; isolation & purification ; Chromatography, Affinity ; methods ; Chromatography, High Pressure Liquid ; Zearalenone ; antagonists & inhibitors ; immunology
5.Purification and characterization of recombinant human anti-HAV monoclonal antibody.
Jing-Shuang WEI ; Ran TAO ; Wei-Wei SUN ; Qia JIA ; Chuan LI ; Mi-Fang LIANG
Chinese Journal of Biotechnology 2004;20(2):257-261
In order to obviate the drawbacks of plasma immunoglobulins, the whole molecular recombinant human anti-HAV (hepatitis A virus) monoclonal antibody (anti-HAV IgG) produced and secreted by rCHO cells was purified and its physicochemical properties were extensively characterized. The rCHO cells were cultured in serum-free medium and the supernatants were collected. The recombinant human IgG molecules were sequentially purified by ultrafiltration, rProtein A Sepharose Fast Flow affinity chromatography, ion exchange chromatography and diafiltration. In affinity chromatography, prior to the target protein elution, an intermediate high salt wash step was inserted, different pH and salt concentrations were evaluated for the capacity of removing host cell DNA. The yield of the downstream purification process was approximately 40%. The purity of anti-HAV IgG thus generated was assayed with SEC-HPLC method, integration result showed that the monomeric IgG content was more than 99%. Western-blot was carried out with AP-antiHuman IgG (Fab specific) and AP-antiHuman IgG (Fc specific) respectively, the blot result demonstrated that the anti-HAV IgG is human antibody with Fab and Fc structure. The specific anti-HAV activity determined by ELISA was 100 IU/mg, with anti-HAV immunoglobulin as the working standard reference. Ligand leakage in the eluate of the affinity column was approximately 32 ng/mg IgG, while after further purification steps, it was decreased to less than 2 ng/mg IgG. Residual host cell DNA was monitored with solid dot blot assay, DNA can be removed effectively with intermediate high salt wash step in the affinity chromatography. Free sulfhydryl content of anti-HAV IgG was assayed with fluorescent spectrophotometer, the low molecular weight bands appeared in non-reducing SDS-PAGE may be caused by the presence of free sulfhydryl. The endotoxin content was less than 1EU/ mg examined by standard LAL test procedures. Anti-HAV IgG prepared with this process is able to fulfill the regulatory requirements of State Food and Drug Administration for recombinant products.
Antibodies, Monoclonal
;
biosynthesis
;
immunology
;
isolation & purification
;
Chromatography, Affinity
;
methods
;
Hepatitis A Antibodies
;
biosynthesis
;
immunology
;
isolation & purification
;
Hepatitis A virus
;
immunology
;
Humans
;
Immunoglobulin G
;
biosynthesis
;
immunology
;
isolation & purification
;
Recombinant Proteins
;
biosynthesis
;
immunology
;
isolation & purification
6.Preparation and characterization of monoclonal antibodies against SARS-associated coronavirus nucleocapsid protein.
Yan-bin WANG ; Zhao-rui CHANG ; Jian-wei WANG ; Rong JI ; Chun-hui HAN ; Li ZHAO ; Li-li REN ; Yan-gong CHAO ; Jian-guo QU ; Cheng-yi QU ; Tao HUNG
Chinese Journal of Experimental and Clinical Virology 2004;18(4):316-320
OBJECTIVETo obtain monoclonal antibodies (McAbs) against severe acute respiratory syndrome (SARS) associated coronavirus (SARS-CoV) nucleocapsid (N) protein to develop diagnostic test for SARS and study the pathogenesis of the disease.
METHODSBALB/c mice were immunized with purified N protein of SARS-CoV. Hybridoma cell lines secreting monoclonal antibodies against SARS-associated coronavirus nucleocapsid were established after cell fusion with mouse splenic cells and SP2/0 cells. The specificity of the McAbs obtained was examined by Western blot and indirect fluorescence assay. Epitopes reacted with the McAbs were preliminarily located through Western blot by expressing truncated N proteins.
RESULTSAfter cell fusion and three rounds of cell cloning, six hybridoma cell lines secreting monoclonal antibodies specifically against SARS-CoV nucleocapsid were obtained. Western blot and indirect fluorescence assay showed that the McAbs reacted specifically with nucleocapsid protein and SARS-CoV. Among the six McAbs, three recognize the epitopes located in the N-terminus of the protein, whereas the others reacted with those located in the C-terminus.
CONCLUSIONThe anti-SARS-CoV nucleocapsid McAbs were developed and these McAbs may be useful in the development of diagnosis assays and basic research of SARS.
Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibodies, Viral ; biosynthesis ; immunology ; Antibody Specificity ; Female ; Hybridomas ; secretion ; Mice ; Mice, Inbred BALB C ; Nucleocapsid Proteins ; immunology ; isolation & purification ; SARS Virus ; chemistry ; immunology
7.Purification of recombinant lysostaphin by monoclonal antibody affinity chromatography.
Jinjiang HUANG ; Hongyu WU ; Jien ZHANG ; Qingshan HUANG
Chinese Journal of Biotechnology 2009;25(1):147-151
Lysostaphin, a specific endopeptidase enzyme derived from Staphylococcus aureus, is a bactericidal agent against Staphylococcus and difficult to be drug-resistant. This study established the monoclonal antibody affinity chromatography to obtain lysostaphin of high purity for drug-use standard. The purified Lysostaphin was of > 95% purity and its recovery rate more than 90%. Moreover, the affinity column kept its efficiency of purification invariable after more than 30 times repeat. Also, the dye release assay validated that the purified lysostaphin had significant bactericidal activity. This method was simple and of high efficacy for the lysostaphin purification and showed its potency in commercial production.
Antibodies, Monoclonal
;
immunology
;
Chromatography, Affinity
;
methods
;
Lysostaphin
;
biosynthesis
;
isolation & purification
;
Recombinant Proteins
;
biosynthesis
;
isolation & purification
;
Staphylococcus aureus
;
enzymology
8.Preparation and activity detection of monoclonal antibody against anti-CD3 ScFv.
Xiao-Feng SHAO ; Ying-Dai GAO ; Juan-Ni LIU ; Jin-Hong WANG ; Yuan-Fu XU ; Dong-Mei FAN ; Chun-Zheng YANG ; Dong-Sheng XIONG
Acta Academiae Medicinae Sinicae 2008;30(3):354-359
OBJECTIVETo prepare monoclonal antibody (McAb) against anti-CD3 ScFv for purifying and detecting serum anti-CD3 antibody concentration.
METHODSMcAb against anti-CD3 ScFv was prepared by hybridoma technique and used to prepare affinity chromatography column, which was used to purify anti-CD3 ScFv and Diabody [CD3 x Pgp] without E-tag. The binding activities of anti-CD3 ScFv, Diabody [CD3 x Pgp] without E-tag, and Diabody [CD3 x Pgp] purified by anti-CD3 affinity chromatography column or anti-E-tag affinity chromatography column against K562/A02 cell and Jurket cells were detected by fluorescence activated cell sorting (FACS) method. ELISA was used to identify the specificity of the McAb.
RESULTSMcAb against anti-CD3 ScFv specifically detected serum anti-CD3 ScFv without reacting with sera. The anti-CD3 ScFv purified by anti-CD3 affinity chromatography column and purified by anti-E-tag affinity chromatography column had the same specific binding activity with Jurkat cells. The positive binding rates of Diabody [CD3 x Pgp] without E-tag to K562/A02 and Jurkat cells were 89.87% and 83.95%, respectively. In the competitive binding experiments with K562/A02 and Jurkat cells, the binding rates of Diabody [CD3 x Pgp] without E-tag decreased to 56.30% and 43.78%, respectively.
CONCLUSIONThe McAb against anti-CD3 ScFv prepared in our lab can be used to purify and detect serum anti-CD3 antibody concentration.
Antibodies, Monoclonal ; biosynthesis ; immunology ; isolation & purification ; CD3 Complex ; immunology ; Cell Line ; Chromatography, Affinity ; Humans ; Hybridomas ; metabolism ; Jurkat Cells ; K562 Cells
9.Application of McAbs against rabies nucleocapsid in diagnosis of rabies street virus.
Ge-lin XU ; Jia-xin YAN ; F LARROUS ; Yu-tao ZHU ; P COZETTE ; Hong-gang XUE ; Qiao-ling HU ; H BOURHY
Chinese Journal of Epidemiology 2005;26(2):113-115
OBJECTIVEMcAbs against rabies nucleocapsid were used to detect rabies street viruses in animal brain specimens with indirect immunofluorescent assay to evaluate the sensitivity and specificity of this assay.
METHODS62 specimen from rabid animal brains including genotype 1 to 7 and 271 specimens from different normal animal brains collected in Pasteur Institute in 2003 were tested and compared, using indirect immunofluorescent assay. All these specimens were identified and compared using rapid rabies enzyme immunodiagnosis, fluorescent antibody test and rabies virus isolation assay in neuroblastoma cell culture which were all provided by Pasteur Institute.
RESULTSBoth sensitivity and the specificity of the indirect immunofluorescent assay were 100%.
CONCLUSIONThe results showed a positive of rabies virus detection with these methods.
Animals ; Antibodies, Monoclonal ; immunology ; Antibodies, Viral ; immunology ; Brain ; virology ; Dogs ; Fluorescent Antibody Technique ; methods ; Genotype ; Nucleocapsid ; immunology ; Rabies virus ; isolation & purification ; Sensitivity and Specificity
10.Production of polyclonal and monoclonal antibodies against citrus tristeza virus and their efficiency for the detection of the virus.
Cai-Xia WANG ; Guo-Ping WANG ; Ni HONG ; Bo JIANG ; Hui LIU ; Kang-Wei WU
Chinese Journal of Biotechnology 2006;22(4):629-634
Citrus tristeza virus (CTV) was purified from a citrus sample by a modified protocol, and the yield was about 1 mg from 100 g citrus tissues. Polyclonal antibody was prepared by immunizing rabbits with the purified CTV preparation with a titer 1:25600 in indirect ELISA test. Eighteen hybridoma-cell lines secreting monoclonal antibodies (MAbs) against CTV were screened after the fusion of mouse myeloma cells (SP2/0) with spleen cells from BALB/c immunized with the virus preparation. Four hybridoma-cell lines were selected randomly for later analysis. The results indicated that the titers of ascetic fluids against these hybridoma cell lines ranged from 1:51200 to 1:204800 in indirect ELISA, and their isotypes and subclasses were IgG2a for 2G and 3H and IgG2b for IE and 4H. These four Mabs were used to detect CTV in citrus samples in different sources. Results showed that TAS-ELISA with polyclonal antibody as trapping antibody and monoclonal antibody as testing antibody had a higher specificity and sensitivity than PAS-ELISA. Four Mabs showed different intensities of serological reaction with different CTV isolates. However, much work remains for realizing the characteristics and the serological relationships among these isolates.
Animals
;
Antibodies, Monoclonal
;
biosynthesis
;
immunology
;
Antibodies, Viral
;
biosynthesis
;
immunology
;
Blotting, Western
;
Citrus
;
virology
;
Closterovirus
;
immunology
;
isolation & purification
;
Enzyme-Linked Immunosorbent Assay
;
Mice
;
Mice, Inbred BALB C
;
Rabbits