OBJECTIVEScreening and characterizing high affinity completely humanized single-chain antibody (ScFv) against PreS1 of hepatitis B virus.

METHODSA combinatorial library of phage-displayed human ScFv, genes of which were derived from peripheral blood lymphocytes immunized by PreS1 of Hepatitis B Virus in vitro, was constructed. The library contained 7 10(8) clones.

RESULTSAfter 3 rounds panning, a high affinity (K=10(7) to 10(8) mol/L) ScFv specific to PreS1 was obtained. The V(H) belonged to human V(H4) family, and V(1) to V(4) by sequence analysis.

CONCLUSIONThis study suggests that the method of antigen stimulation in vitro is an expeditious way for the source of human immune antibody. And the ScFv may provide a more satisfactory therapy.

Antibodies, Monoclonal ; biosynthesis ; Antibody Affinity ; Hepatitis B Antibodies ; biosynthesis ; Hepatitis B Surface Antigens ; immunology ; Humans ; Peptide Library ; Protein Precursors ; immunology

OBJECTIVETo develop a monoclonal antibody (mAb) directed to FVIII C2 domain and investigate its effect on FVIII activity.

METHODSFVIII C2 protein was expressed in E. coli and purified. A murine antihuman FVIII C2 domain mAb SZ-132 was developed by standard hybridoma technology and characterized. In coagulation assays, different concentrations of SZ-132 were incubated with freshly collected pooled human plasma and the residual activity of FVIII and activated partial thromboplastin time (APTT) were determined. The effects of SZ-132 on rhFVIII binding to purified human vWF, phosphatidylserine (PS) and platelets were assessed by enzyme linked immunosorbent assays (ELISA).

RESULTSSZ-132 could inhibit FVIII procoagulant activity in a dose-dependent manner within the concentrations of 0-25 microg/ml and the FVIII activity was completely inhibited on above 25 microg/ml. It could also prevent rhFVIII from binding to vWF, PS and platelets.

CONCLUSIONSSZ-132 is a neutralizing mAb against FVIII C2 domain and can inhibit FVIII procoagulant activity by preventing FVIII from binding to vWF and PS.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibodies, Neutralizing ; biosynthesis ; immunology ; Factor VIII ; immunology ; metabolism ; Humans ; Male ; Mice ; Mice, Inbred BALB C

OBJECTIVETo establish a novel immunoassay for qualitative detection of ginsenoside Rb1 in rat serum.

METHODSAnti-G-Rb1 monoclonal antibody (mAb) was through a hybridoma approach. Rat serum containing G-Rb1 was deproteinized with methanol to prepare the sample for testing, which was loaded onto polyethersulfone (PES) membrane and developed in the mixture of acetonitrile, water and acetic acid (25:75:1). After treatment with NaIO(4), the membrane was transferred to 1% BSA solution for immobilization of G-Rb1. The membrane was subsequently treated with anti-G-Rb1 mAb solution, followed by addition of peroxidase-labeled goat anti-mouse IgG and color development using 4-chloro-1-naphthol-0.03% H(2)O(2).

RESULTSOn the PES membrane, a clear blue spot representing G-Rb1 occurred where the rat serum for testing and the standard G-Rb1 samples were blotted. The limit of this immunodetection was 0.25 microg.

CONCLUSIONThis immunoassay has greater specificity and reliability than thin-layer chromatography with a sensitivity similar to that of high-performance liquid chromatography, and does not require sophisticated equipment for convenient G-Rb1 detection in rat serum.

Animals ; Antibodies, Monoclonal ; biosynthesis ; Ginsenosides ; analysis ; blood ; Hybridomas ; Immunoassay ; Limit of Detection ; Rats

Monoclonal antibodies (mAbs) contribute a lot to the development of numerous fields in life science as a pivotal tool in modern biological research. Development of the PCR methods and maturation of antibody production have made it possible to generate mAbs from single human B cells by single cell RT-PCR with successional cloning and expression in vitro. Compared to traditional monoclonal antibody technologies, single B cell technologies require relatively fewer cells, which are highly efficient in obtaining specific mAbs in a rapid way with preservation of the natural heavy and light chain pairing. With so many advantages, single B cell technologies have been proved to be an attractive approach for retrieval of naive and antigen-experienced antibody repertoires generated in vivo, design of rationale structure-based vaccine, evaluation and development of basic B cell biology concepts in health and autoimmunity, and prevention of infectious diseases by passive immunization and therapy for disorders. Accordingly, this review introduced recent progresses in the single B cell technologies for generating monoclonal antibodies and applications.
Antibodies, Monoclonal ; biosynthesis ; genetics ; immunology ; Antibody Specificity ; B-Lymphocytes ; cytology ; immunology ; metabolism ; Humans ; Immunologic Techniques

Monoclonal antibody producted by continuous perfusion culture was recovered and purified by expended bed adsorption chromatography. A packed bed column XK16/20 was used for method scouting with Streamline SP adsorbent. Two expended bed columns Streamline-25 and -50 were used for method optimization and pilot scale experiment, respectively. The recovery yield of monoclonal antibody was above 90% in a 5 - 7 fold enhanced purity and 10 fold increased concentration. According to the different concentration of monoclonal antibody in cell culture broth, about 18 - 50L fluid can be treated in a single cycle. MAb purification from lab scale (400mg per cycle) to a small pilot scale (2g per cycle) has been achieved. Compared with packed bed adsorption, the preparation cycle was half shortened, and the cost of production and the complexity of process were decreased markedly. It has been proven that a purification process based on expended bed adsorption technique is simple, efficient and economical.
Adsorption ; Antibodies, Monoclonal ; biosynthesis ; isolation & purification ; Bioreactors ; Cell Culture Techniques ; methods ; Chromatography ; methods ; Hybridomas ; cytology ; metabolism

OBJECTIVETo prepare rabbit monoclonal antibody (RabMab) against guanosine 3', 5'-cyclic monophosphate (cGMP) and to develop a competitive ELISA for the detection of cGMP.

METHODSNew Zealand white rabbits were immunized with synthesized cGMP-keyhole limpet hemoeyanin (cGMP-KLH) to prepared a RabMAb with monoclonal antibody technique of Epitomics. A competitive ELISA kit was produced with cGMP RabMAb. The specificity, the precision and the recoveries of the method were determined.

RESULTSThe RabMAb with high sensitivity towards cGMP were prepared with an antibody timer of 3.1 ng/mL and 50% inhibitive concentration (IC50) of 12.57 ng/mL. The cGMP RabMAb had 33% cross-reactivity to inosine 3', 5'-cyclic monophosphate (cIMP) and little or no cross-reactivity to other compounds. A competitive ELISA was developed for detection of cGMP. The range of detection was 0~120 ng/mL with a minimal limit of 1.95 ng/mL. The recovery of assay was 89%~103%. The inter-assay and intra-assay coefficient variations were below 11.68% and 13.85%, respectively.

CONCLUSIONThe RabMab against cGMP with high affinity and high specificity has been generated successfully, and a competitive ELISA for detection of cGMP has been developed with the prepared cGMP RabMAb.

Animals ; Antibodies, Monoclonal ; biosynthesis ; Antibody Specificity ; Cross Reactions ; Cyclic GMP ; immunology ; Enzyme-Linked Immunosorbent Assay ; Rabbits

OBJECTIVE: To prepare monoclonal antibody (MAb) against carboxy-terminus of E-phA4 and to analyze the immunological characteristics and significance of the antibody. METHODS: Mice were immunized with a chemically synthesized peptide that had been conjugated to KLH (keyhole limpet hemocyamin). A panel of antibodies specifically against EphA4 were obtained by hybridoma technique. The immunological properties and significance of the MAb were analyzed by immunological and immunochemistry techniques. RESULTS: A hybridoma cell line secreting anti-EphA4 MAb was established. The MAb reacted specifically with human, chicken and mouse EphA4, but did not cross-react with EphA5 and EphA7. The antibody could be used for immunochemical techniques such as ELISA, immunoprecipitation, Western blot and immunohistochemistry. CONCLUSION The anti-EphA4 MAb generated by immunizing a synthetic peptide possesses excellent immunological properties. MAb can be applied for immunological and immunohistochemical purpose and will become an important tool in the study of EphA4 and its ligand.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Hybridomas ; Mice ; Mice, Inbred BALB C ; Peptide Fragments ; immunology ; Receptor, EphA4 ; immunology

OBJECTIVETo express and purify the recombinant human bone morphogenetic protein-2 mature peptide (rhBMP-2m) in prokaryotic system and to develop highly-specific monoclonal antibodies.

METHODSAn engineered E. coli strain expressing rhBMP-2m was fermented. The bacterial cells were firstly lysed and then the rhBMP-2m inclusion bodies were isolated by centrifugation. After the inclusion bodies had been solubilized by high-concentration denaturing agents, denatured rhBMP-2m was purified by cation ion-exchange chromatography. Biologically active rhBMP-2m was obtained by refolding of purified denatured rhBMP-2m through direct dilution. The refolded rhBMP-2m was used to immunize Balb/c mice to develop anti-rhBMP-2m monoclonal antibodies using classic hybridoma technique.

RESULTSrhBMP-2m with a purity greater than 95% was obtained on reduced SDS-PAGE. The refolded rhBMP-2m was measured to be bioactive by the induction of alkaline phosphatase activity in MC3T3-E1 cells. Two hybridoma cell lines that stably secreted anti-rhBMP-2m antibody were developed from the immunized mice.

CONCLUSIONBioactive rhBMP-2m protein and its monoclonal antibodies were successfully prepared, which will provides a solid base for future studies on rhBMP-2.

Animals ; Antibodies, Monoclonal ; biosynthesis ; Bone Morphogenetic Proteins ; biosynthesis ; immunology ; Escherichia coli ; metabolism ; Humans ; Mice ; Mice, Inbred BALB C

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 develop human recombinant neutralizing IgG monoclonal antibodies to hepatitis A virus (HAV) by baculovirus expression system.

METHODSThe heavy and light chain genes of two human-derived neutralizing Fab antibodies to HAV were cloned into baculovirus expression vector Pac-kappa-Fc and Pac-L-Fc, and further expressed in insect cells as IgG antibodies. The IgG products were purified and well characterized.

RESULTSThe baculovirus expressed McAb HAFc16 fully retained the specificity of binding to hepatitis A virus and the competition with mouse anti-hepatitis A virus McAb using ELISA. The viral neutralization assay in vitro demonstrated the retention of antibody function after expression of the human antibody in insect cells. The other expressed antibody HAFc78 also has the neutralizing activity but it is directed against different epitopes of HAV when compared with HAFc16.

CONCLUSIONThe recombinant baculovirus/insect cells expressed human neutralizing IgG antibodies to hepatitis A virus retained all biological functions specific for hepatitis A virus. The results provided the possibility of using these antibodies to rapidly protect high risk or early exposure populations from hepatitis A virus infection.

Antibodies, Monoclonal ; biosynthesis ; immunology ; Baculoviridae ; genetics ; Hepatitis A virus ; immunology ; Hepatitis Antibodies ; biosynthesis ; immunology ; Humans ; Immunoglobulin Fab Fragments ; biosynthesis ; immunology ; Immunoglobulin G ; biosynthesis ; immunology ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Recombinant Proteins ; biosynthesis ; immunology