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

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

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

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

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

OBJECTIVETo express and purify HBoV VP2 protein, and the monoclonal antibody against HBoV VP2 protein was prepared with hybridoma technique.

METHODSThe HBoV VP2 cloned into vector pET-30a was expressed in E. coil. After purified by immobilized metal affinity chromatography, the BALB/c mouse was immunized with purified protein as antigen. The positive hybridoma cells were screened with hybridoma technique and ELISA assay. Isotype and titer of the monoclonal antibody were detected.

RESULTSThe recombinant HBoV VP2 protein was expressed and purified, and then the monoclonal antibody was obtained with hybridoma technique. The titer of the IgG monoclonal antibody was up to 1:4 x 10(5).

CONCLUSIONMonoclonal antibody against recombinant HBoV VP2 protein was prepared and the antibody titer was high. This work may provide a new method in rapid diagnosis and study of HBoV.

Animals ; Antibodies, Monoclonal ; immunology ; Capsid Proteins ; genetics ; immunology ; Human bocavirus ; immunology ; Hybridomas ; Mice ; Mice, Inbred BALB C ; Plasmids ; Recombinant Proteins ; biosynthesis ; immunology ; isolation & purification

UNLABELLEDTo reduce the huge labor-cost in the screening in traditional monoclonal antibody generation, We established a new system for monoclonal antibody generation integrating with protein array. BALB/c mice were immunized by eight recombinant proteins respectively, and the positive hybridoma cells were obtained by cell fusion and ELISA screening. All the eight kinds of positive hybridoma cells were mixed, cloned, screened by protein array, and definite dilution cloned.

RESULTS175 single cell clones were obtained by complex cloning, and 119 of those were positive clones. Then 8 positive cell lines were generated by the following 2 rounds definite dilution cloning. By comparing with the traditional method, we got 8 monoclonal antibodies using the combined protein array screening and multiplex cloning method in 1 cycle, and fewer amounts of antigens were used. As a result, the combined protein array and multiplex cloning method could be used as an economical, rapid and simple tool applying in high throughput monoclonal antibody generation.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; isolation & purification ; Female ; Humans ; Hybridomas ; metabolism ; Mice ; Mice, Inbred BALB C ; Protein Array Analysis

OBJECTIVETo prepare the monoclonal antibody (mAb) against tissue inhibitor of metalloproteinases I (TIMP-I) fusion protein.

METHODSTIMP-I gene was amplified from fibrotic human liver tissue by RT-PCR, then ligated with pQE31 to form recombinant plasmid pQE-TIMP-I and transformed into E. coli BL21. The protein induced by IPTG was purified by 6 x His-tag and used to immunize the BALB/c mice. The specific monoclonal antibodies (mAbs) were prepared by the cell fusion technique. Western Blot were used to detect specificity of mAbs.

RESULTSThe prokaryotic plasmid expressing the recombinant protein was constructed, and the TIMP-I recombinant protein was expressed and purified. Four hybridoma cell lines that secreted anti-TIMP-I mAbs were obtained. 3 of 4 mAbs were the IgG1 subtype. Western Blot indicated the mAbs showed specific combination with TIMP-I protein.

CONCLUSIONThe TIMP-I recombinant protein is highly purified and has strong antigenicity. The anti- TIMP-I mAbs were prepared successfully.

Animals ; Antibodies, Monoclonal ; immunology ; Cloning, Molecular ; Humans ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; immunology ; isolation & purification ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; immunology

Diphtheria toxin A fragment (DTA) is an essential catalytic domain of diphtheria toxin (DT)-based immunotoxin. DTA protein and its antibodies play an important role in the studies on toxicology, purification and identification of DT-based immunotoxins. In this paper, DTA was expressed and purified from E. coli. After Q-Sepharose FF chromatography and (Ni+)-Sepharose affinity chromatography, 6 x His-DTA fusion protein with 90% purity was achieved. Using the purified DTA as antigen to immunize BalB/c mice, 2 hybridoma cell lines (designated as 3B6 and 3B9, respectively) secreting monoclonal antibodies (McAbs) against DTA were established. Investigations showed that both McAbs were characterized as IgG1 with titers of 1: 10(6). The binding of the McAbs to DTA was competitively inhibited by horse sera against DT. The fact that anti-DTA McAbs could be used in western blot analysis and affinity chromatography purification of DT-based immunotoxins implied that they will be useful agents in the studies on DT-based immunotoxins.
Animals ; Antibodies, Bacterial ; genetics ; Antibodies, Monoclonal ; biosynthesis ; genetics ; Chromatography, Affinity ; Diphtheria Toxin ; immunology ; Escherichia coli ; genetics ; Female ; Immunotoxins ; isolation & purification ; Mice ; Mice, Inbred BALB C ; Peptide Fragments ; immunology ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; isolation & purification

Total RNA was isolated from the hybridoma cell line (LC-1), which secretes anti-lung adenocarcinoma monoclonal antibody, and was transferred into cDNA. Based on the FR1 (framework region 1) and FR4 conserved regions of LC-1 gene, the variable regions of heavy chain (Vh) and light chain (Vl) were amplified, and the Vh and modified Vl were connected to single chain Fv (ScFv) by SOE-PCR (splice overlap extension PCR). The modified ScFv was fused with green fluorescent protein (GFP) and introduced into E. coli JM109. The fusion protein induced by IPTG (Isopropylthiogalactoside) was about 57000 on a 10% SDS-PAGE gel (10% Sds Polyacrylamide Gel Electrophoresis), and primarily manifested as inclusion bodies. The renatured protein purified by Ni-NTA Superflow resins showed ability to bind to antigen on SPC-A-1 lung adenocarcinoma. In addition, the induced host cells fluoresced bright green under 395 nm wavelength, which indicated that the expected protein with dual activity was expressed in the prokaryotic system. The ScFv with GFP tag used in this research can be applied as a new reagent to detect immunological dye, and provide a feasible way to detect adenocarcinoma in a clinical setting.
Adenocarcinoma ; immunology ; Antibodies, Monoclonal ; biosynthesis ; genetics ; immunology ; isolation & purification ; Cell Line ; Cell Line, Tumor ; Cloning, Molecular ; methods ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; Hybridomas ; metabolism ; Immunoglobulin Fragments ; biosynthesis ; genetics ; immunology ; isolation & purification ; Lung Neoplasms ; immunology ; Protein Engineering ; methods ; Recombinant Fusion Proteins ; biosynthesis ; immunology ; isolation & purification