OBJECTIVETo screen and characterize the variable region gene about prostate specific membrane antigen (PSMA) of the Chinese Fab fragment, and to establish a new approach to researches on PSMA and prostate gene therapy.

METHODSWe used purified PSMA protein as antigen, stuck it on the ELISA plate and scanned the phage Fab fragment antibody library by phage display technology. After five cycles of "absorbing-elution-amplification", we got the Fab fragment phage antibody of PSMA with high antigen binding ability and specificity, and tested it with immunodetection and sequencing.

RESULTSThe sequence of Fd fragment was 696 base pairs encoding 232 amino-acid residues, with 98% homological similarity to the human immunoglobulin gamma chain, while the light chain was constructed by 630 base pairs encoding 210 amino-acid residues, with 93% homological similarity to kappa chain.

CONCLUSIONUsing phage display technology, we obtained the gene sequence of Fab antibody fragment specific to PSMA, and the antibody gene has the classic structural features of immunoglobulin light chain and heavy chain. The coding output of the antibody gene has the specificity and immunological competence to PSMA.

Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Genetic Code ; Humans ; Immunoglobulin Fab Fragments ; genetics ; Immunoglobulin Variable Region ; genetics ; Male ; Peptide Library ; Prostate-Specific Antigen ; immunology

AIMTo construct recombinant retroviruses expressing anti-CD20 scFv/CD80/CD28/zeta gene and detect its expression in Jurkat cells.

METHODCD28-zetacDNA were amplified from plasmids pBULLET and inserted into pLNCX vector that contained anti-CD20 scFv/CD80 gene. The recombinant plasmids were transfected into PA 317 cells. Retroviruses were harvested from culture medium of PA 317 cells. Then NIH 3T3 were transfected with retroviruses. Objective gene expression was determined by PCR and FACS. Jurkat cells were transfected with high titer of retroviruses and resistant clones were obtained by G418 selection. Objective mRNA was determined by RT- PCR.

RESULTSThe recombinant eukaryotic vector was constructed successfully by PCR and enzyme digestion analysis and objective gene was amplified from NIH 3T3 cells transfected with retroviruses by PCR; FACS showed that objective protein could be expressed in NIH 3T3 cells. Objective gene was amplified from Jurkat cells transfected with retroviruses by RT-PCR.

CONCLUSIONRecombinant retrovirus expressing anti-CD20 scFv/CD80/CD28/zeta gene was successfully constructed and objective protein could be expressed in Jurkat cells.

Antigens, CD20 ; genetics ; metabolism ; B7-1 Antigen ; genetics ; metabolism ; CD28 Antigens ; genetics ; metabolism ; Genetic Vectors ; Humans ; Immunoglobulin Fab Fragments ; genetics ; metabolism ; Immunoglobulin Variable Region ; genetics ; metabolism ; Jurkat Cells ; Recombinant Fusion Proteins ; genetics ; metabolism ; Retroviridae ; genetics ; T-Lymphocytes ; metabolism ; Transfection

OBJECTIVETo screen anti-c-Met Fab from a phage antibody library and identify its binding activity.

METHODSThe expression of c-Met of HCC lines was identified by Western blot and immunofluorescence. Antibodies against c-Met were screened with immobilized antigen. After five rounds of panning, 30 randomly selected clones were identified by phage ELISA to select specific clones with high affinity. The positive clones were selected for Fab soluble expression in TOP10F and the binding activities were analysed in HCC lines.

RESULTSc-Met expressed in HCC membrane was confirmed by Western blot and immunofluorescence. A Fab fragment named AM2-26 with fine activity to c-Met was selected. AM2-26 binding specificity was confirmed by IP, FACS and immunofluorescence.

CONCLUSIONThe anti-c-Met Fab binding to c-Met in HCC provides a promising candidate for the biotherapy of hepatoma.

Antibodies ; immunology ; isolation & purification ; Cell Line, Tumor ; Cloning, Molecular ; Gene Library ; Humans ; Immunoglobulin Fab Fragments ; immunology ; Immunoglobulin Variable Region ; immunology ; Peptide Library ; Proto-Oncogene Proteins c-met ; immunology ; Recombinant Fusion Proteins ; immunology

OBJECTIVETo investigate the efficacy of anti-idiotype antibody 3F6 and its single-chain variable fragment (3F6 ScFv) to induce humoral and cellular immune responses against small-cell-lung cancer (SCLC).

METHODS3F6 and 3F6 ScFv (Ab2) were used to immunize BALB/c mice. The reaction of antibodies (Ab3) with specific antigen on NCI-H128 cells was tested by ELISA and Western blot, and the antibody binding inhibition assays were performed by competitive Western blot. Cellular immunity against SCLC induced by Ab2 was detected by a delayed-type hypersensitivity response and mouse lymphocyte proliferation assay.

RESULTSThe sera immunized with Ab2 showed significant reaction (P < 0.001, as compared to control sera) with SCLC-specific antigen on NCI-H128 cells and specifically competed the binding of 2F7 (Ab1) to the specific antigen. DTH responses challenged with NCI-H128 cells were significantly (P < 0.001) stronger in mice immunized with Ab2 as compared to mice immunized with normal mouse IgG. T cell proliferation was significantly higher in Ab2-immunized mice (P < 0.05) than in control mice.

CONCLUSIONThe two kinds of anti-idiotypic antibodies successfully mimic the SCLC-specific antigen on NCI-H128 cell and induce strong humoral and cellular immune responses to SCLC-specific antigen in syngeneic mice. They may become novel vaccines against human small-cell-lung cancer and worthy of further investigation.

Animals ; Antibodies, Anti-Idiotypic ; immunology ; Antibodies, Neoplasm ; immunology ; Antigens, Neoplasm ; immunology ; Cancer Vaccines ; immunology ; Carcinoma, Small Cell ; immunology ; Cytotoxicity, Immunologic ; drug effects ; Immunoglobulin Fab Fragments ; immunology ; Immunoglobulin Fragments ; immunology ; Immunoglobulin Variable Region ; immunology ; Lung Neoplasms ; immunology ; Mice ; Mice, Inbred BALB C

OBJECTIVESequences analysis of Ig variable regions from the peripheral blood mononuclear cells (PBMC) of patients with chronic B lymphocytic leukemia.

METHODSTotal RNA was isolated from PBMC of patients with chronic B lymphocytic leukemia, oligo-dT-primed cDNA was synthesized from RNA. The cDNA was amplified by Taq DNA polymerase with a set of specific 5' primers corresponding to Ig FR1 and 3' primers corresponding to CH1 (C micro /C) or CL (Ckappa/Clambda), the PCR products of variable regions of Ig heavy (IgH) and light (IgL) chains were sequenced by ABI PRISM Dye terminator cycle sequencing ready reaction kit and ABI PRISM 310 Genetic Analyzer. The gene homology of variable regions of IgH and IgL chains was compared by using DNA tools 5.1 system and "the international immunogenetics database".

RESULTSFour light chains and 3 heavy chains were amplified from 4 and 3 patients respectively. Homology analysis of the sequences of 4 light chains and 3 heavy chains were performed by DNA tools system. The sequences of light chains are high homologous. And the sequences of heavy chains are quite different. The homologous analysis of the sequences of variable region by using "the international immunogenetics database" showed that the sequences were higher homologous to idiotype gene of some B lymphocytic leukemia. Four VL genes belong to human Ig Vkappa subgroup I, 2 of 3 VH genes belong to VH3 family and 1 belongs to VH5 family.

CONCLUSIONIg genes have idiotype and same disease may have same idiotype.

Base Sequence ; Cloning, Molecular ; DNA, Complementary ; chemistry ; Gene Rearrangement ; Genes, Immunoglobulin ; Humans ; Immunoglobulin Fab Fragments ; genetics ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Leukemia, Lymphocytic, Chronic, B-Cell ; genetics ; immunology ; Molecular Sequence Data ; Polymerase Chain Reaction

The characterization of the human T-cell receptor (TCR) repertoire has made remarkable progress, with most of the work focusing on the TCRβ chains. Here, we analyzed the diversity and complexity of both the TCRα and TCRβ repertoires of three healthy donors. We found that the diversity of the TCRα repertoire is higher than that of the TCRβ repertoire, whereas the usages of the V and J genes tended to be preferential with similar TRAV and TRAJ patterns in all three donors. The V-J pairings, like the V and J gene usages, were slightly preferential. We also found that the TRDV1 gene rearranges with the majority of TRAJ genes, suggesting that TRDV1 is a shared TRAV/DV gene (TRAV42/DV1). Moreover, we uncovered the presence of tandem TRBD (TRB D gene) usage in ~2% of the productive human TCRβ CDR3 sequences.
Complementarity Determining Regions ; genetics ; DNA Primers ; chemistry ; genetics ; Female ; Gene Rearrangement, beta-Chain T-Cell Antigen Receptor ; genetics ; Gene Rearrangement, delta-Chain T-Cell Antigen Receptor ; genetics ; Genes, T-Cell Receptor beta ; genetics ; Genetic Variation ; High-Throughput Nucleotide Sequencing ; Humans ; Immunoglobulin Joining Region ; genetics ; Immunoglobulin Variable Region ; genetics ; Male ; Receptors, Antigen, T-Cell, alpha-beta ; genetics

OBJECTIVETo establish a three-step purification method of preparative-scale antiCD20 (Fab')2 using AKTA prime.

METHODSAntiCD20 (Fab')2 was extracted by hyperosmotic solution and then purified by CM sepharose FF, phenyl sepharose FF, and protein G sepharose FF.

RESULTSAround 8 mg anti-CD20 (Fab')2, whose purification was 96.678%, was purified. The antigen-binding activity of antiCD20 (Fab')2 was similar to that of antiCD20 (Fab')2 purified by protein G sepharose FF and S-100.

CONCLUSIONThe three-step purification method can obtain high-purity preparative-scale antiCD20 (Fab')2 in a simple way.

Antibodies ; immunology ; isolation & purification ; Antigens, CD20 ; immunology ; Chromatography ; methods ; Humans ; Immunoglobulin Fab Fragments ; immunology ; isolation & purification

Platelets play an important role in the development of acute coronary syndrome. Platelet-inhibiting drugs, such as glycoprotein IIb/IIIa inhibitors, can be beneficial when they are administered at the time of primary percutaneous coronary intervention for acute coronary syndrome. Although an increased risk for bleeding complications is well recognized, the risk associated with diffuse alveolar hemorrhage is much less reported. We report a case of diffuse alveolar hemorrhage after using abciximab.
Acute Coronary Syndrome ; Antibodies, Monoclonal ; Blood Platelets ; Glycoproteins ; Hemoptysis ; Hemorrhage ; Immunoglobulin Fab Fragments ; Percutaneous Coronary Intervention

The V(H) and V(L) gene fragments of anti-CD28 mAb were combined to form anti-CD28 ScFv gene by using TP-PCR method. Sequence analysis showed that 6 x His tag was added to it for the ease of purification and the V(H), and V(L) gene fragments were connected by a linker containing 15 amino acids which are biased by the baculovirus promoter, ph. Then ScFv gene fragment was inserted into baculovirus transfer vector pBacPAK8. The recombinant transfer vector, pBacPAK8/CD28-ScFv was constructed successfully. The pBacPAK8/CD28-ScFv and the linear Bm-BacPAK6 were co-transfected into the cell line of Bombyx mori (BmN) with the help of Lipofectin,then the product was purified by plaque assay and identified by PCR method. The recombinant virus, Bm-BacPAK6 CD28-ScFv, was obtained successfully. The BmN cells and the larvae of Bombyx mori were infected by the recombinant baculovirus and harvested every 24h postinfection. SDS-PAGE and Western Blotting analysis confirmed the expression of ScFv with the molecular weight of about 28 kD. The expression in BmN cells was detected 24h post infection and it peaked at 72 h, while in the larvae of Bombyx mori, the expression was detected 48 h post infection and it peaked at 120 h.
Animals ; Antibodies, Anti-Idiotypic ; biosynthesis ; genetics ; Antibodies, Monoclonal ; immunology ; Baculoviridae ; genetics ; metabolism ; Bombyx ; cytology ; genetics ; metabolism ; CD28 Antigens ; genetics ; immunology ; Cell Line ; Humans ; Immunoglobulin Fab Fragments ; genetics ; immunology ; Immunoglobulin Variable Region ; genetics ; immunology ; Larva ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection

Glycoprotein IIb/IIIa antagonists are well established for their effectiveness in improving clinical outcomes in acute coronary syndrome patients undergoing percutaneous coronary intervention. Acute profound thrombocytopenia is a rare complication of abciximab. We present a case which was managed successfully for the rare complication of acute profound thrombocytopenia after using abciximab and an intra-aortic balloon pump for the treatment of a no-reflow phenomenon and consecutive cardiogenic shock during primary percutaneous coronary intervention.
Acute Coronary Syndrome ; Antibodies, Monoclonal ; Humans ; Immunoglobulin Fab Fragments ; Myocardial Infarction ; No-Reflow Phenomenon ; Percutaneous Coronary Intervention ; Shock, Cardiogenic ; Thrombocytopenia