Glycosylation is one of the common post-translational modifications of proteins to regulate the ability of tumor invasion, metastasis and tumor heterogeneity by interacting with glycan-binding proteins such as lectins and antibodies. Glycan microarray can be constructed by chemical synthesis, chemical-enzyme synthesis or natural glycan releasing. Glycan microarray is an essential analytical tool to discover the interaction between glycan and its binding proteins. Here we summarize the standard techniques to construct glycan microarray for the application in cancer vaccine, monoclonal antibody and diagnostic markers.
Antibodies, Monoclonal ; Glycosylation ; Lectins/metabolism* ; Microarray Analysis ; Neoplasms ; Polysaccharides

As one of the proteomic technologies, antibody microarrays can provide a powerful tool for the high-throughput parallel analysis of thousands of proteins in a single experiment. Because antibodies are chemically and structurally complex; immobilizing antibody on solid phase surface of substrates has been a key step during the development of antibody microarrays. This review will focus on the current strategies for antibody immobilization and discuss the advantages and disadvantages of these methods.
Adsorption ; Antibodies, Monoclonal ; chemistry ; metabolism ; Protein Array Analysis ; methods ; Protein Binding

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

Adult ; Antibodies, Monoclonal ; analysis ; DNA Nucleotidylexotransferase ; metabolism ; Female ; Follow-Up Studies ; Humans ; Keratins ; immunology ; Male ; Middle Aged ; Prognosis ; Thymoma ; metabolism ; pathology ; surgery ; Thymus Neoplasms ; metabolism ; pathology ; surgery

The environmental pollution by heavy metals such as mercury, cadmium and lead has become a worldwide public health hazard. To rapidly and inexpensively monitor environmental heavy metals is a prerequisite for minimizing human and animal exposure. The development of immunoassays to detect mercury ion residues has been a promising trend with the advantage of rapid and cheap operation. We reported the isolation and characterization of mercury-specific monoclonal antibodies. Because Hg2+ ions are too small to elicit an immune response, the metal was coupled to protein carrier (keyhole limpet, KLH) using a chelator (diethylenetriamine pentaacetic acid, DTPA). After the synthesis of antigen and characterization, monoclonal antibodies against mercury ions were generated by immunizing BALB/c mice with mercury conjugated antigen (Hg-DTPA-KLH). The stable hybridoma cell lines were produced by fusion of murine splenocytes and SP2/0 myeloma cells. The hybridoma cells were subcloned by the limiting dilution and screened by ELISA, two hybridoma cell lines producing stably specific monoclonal antibodies (MAbs) against mercury ions were obtained, named H2H5 and H1H8. The ascites fluid was produced in BABL/c mice by intraperitoneal injection of 1 x 10(7) H2H5 and H1H8 cells, respectively. The titers of ascites were all above 1:51 200. The isotyping of secrete antibodies from two hybridoma cell lines was IgG1, kappa type. These data laid a potency of establishing immunoassays methods of determining Hg2+ ion residues and had the realistic significance for improving the efficiency and quality of risk assessment.
Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Chelating Agents ; chemistry ; Environmental Pollutants ; analysis ; immunology ; Female ; Hybridomas ; metabolism ; Immunoassay ; methods ; Mercury ; analysis ; immunology ; Mice ; Mice, Inbred BALB C

OBJECTIVETo explore the specificity of anti-phosphotyrosine monoclonal antibody PY20 in bcr-abl+ cells and its possible clinical applications.

METHODSBcr-abl cell lines( K562, MEG-01) and bcr-abl- cells lines( Jurkat, MCF-7 )were stained with PY20. Phosphotyrosine protein of K562 and MEG-01 cells was detected by flow cytometry before and after treatment with imatinib. Phosphotyrosine protein in bone marrow cells from 49 patients with chronic myeloid leukemia (CML), Ph+ acute lymphoblastic leukemia(Ph(+) -ALL), Ph- ALL, acute myeloid leukemia (AML-M1, M2, M3, M5, FAB classification), chronic lymphocytic leukemia (CML) and 3 normal donor. Positive cells over 5% of total cells was considered positive cases for phosphotyrosine protein. The level of tyrosine phosphorylation was determined by median fluorescence intensity (MFI).

RESULTSBcr-abl cell lines and marrow cells from 10 CML patients and 8 ALL patients were all PY20-positive, while bcr-abl- cell lines and marrow cells from 18 leukemia patients and 3 normal donor were all PY20-negative. MFI decreased remarkably after blocked by imatinib in K562 cells and MEG-01 cells. The positive cell percent of marrow cells from 10 newly diagnosed CML patients and 9 imatinib-sensitive CML patients was (54.20 +/- 19.82)% and (14.84 +/- 6.17)% (P < 0.05), while that of 2 cases of imatinib-resistant was 64.3% and 57.2%. There was significant difference of MFI between imatinib-resistant patients and imatinib-sensitive patients (99.42 +/- 4.87 vs 46.41 +/- 4.67, P < 0.01).

CONCLUSIONPY20 monoclonal antibody is highly specific for bcr-abl+ cells. It might be useful in rapid detection of bcr-abl+ cells and sensitivity to imatinib of CML patients.

Antibodies, Monoclonal ; analysis ; Bone Marrow Cells ; metabolism ; Flow Cytometry ; Fusion Proteins, bcr-abl ; analysis ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism ; Leukemia, Myeloid, Acute ; metabolism ; Phosphotyrosine ; analysis ; immunology

This study was aimed to investigate the effects of rituximab (RTX), a chimeric human anti-CD20 monoclonal antibody, on lymphoma cell injury induced by X ray irradiation. The human Burkitt EBV-infected and moderate radioresistance lymphoma cells (Namalwa) were used in the this study. Cytotoxicity of rituximab combined with X ray irradiation on Namalwa cells was measured by sulforhodamine B (SRB)-staining; the apoptosis of Namalwa cells was detected by flow cytometry with FITC-Annexin V/PI double staining; the morphologic changes of cells were observed under transmission electron microscope (TEM) and the change of intracellular free calcium level ([Ca(2+)]i) in response to irradiation and rituximab was determined by means of the fluorescent dye fluo-3 and confocal microscopy. The results showed that the growth inhibition in Namalwa cells exposed to irradiation was enhanced by treatment with rituximab. Compared with irradiation alone, rituximab combined with irradiation significantly induced the cell apoptosis and a sustained rise of intracellular free calcium ([Ca(2+)]i) level in Namalwa cells; the serial apoptotic appearances of cells could be observed under TEM. It is concluded that rituximab can enhance the sensitivity of lymphoma cells on X ray irradiation as to induce cell more apoptosis, in this process the intracellular free calcium ([Ca(2+)]i), as an intracellular signaling molecule probably plays an important role.
Antibodies, Monoclonal, Murine-Derived ; immunology ; pharmacology ; Antigens, CD20 ; immunology ; Apoptosis ; Calcium ; analysis ; Cell Line, Tumor ; Humans ; Lymphoma ; drug therapy ; metabolism ; pathology ; Radiation Tolerance ; drug effects ; Rituximab

The study was aimed to generate monoclonal antibodies (mAbs) against homo sapiens UDP-glucose pyrophosphorylase 2 (UGP2). Normal human liver tissues homogenized, and cytosolic proteins isolated by centrifugation were used to immunize BALB/c mice to generate mAbs by hybridoma technique. The mAbs were identified by ELISA, Western blot, and immunohistochemistry assay. The antibody specificity was confirmed by Uni-ZAP expression library screening. The results indicated that one hybridoma BAD062 secreting specific mAb against UGP2 was established. The Ig subclass of this mAb was IgG(2b) (kappa), and it could be used in ELISA, Western blot, immunohistochemistry assay. The antigen recognized by BAD062 mAb was localized in the hepatocyte cytoplasm, with molecular weight of 56 kD in the cytosolic proteins of human liver tissue. The BAD062 mAb was further confirmed by immunoscreening of Uni-ZAP XR liver cDNA expression library. It is concluded that a hybridoma cell line stably secretes specific mAb against UGP2. This mAb reacted with UGP2 in ELISA, Western blot, immunohistochemistry assay, and would be very useful for the UGP2 studies.
Animals ; Antibodies, Monoclonal ; analysis ; biosynthesis ; Antibody Specificity ; Base Sequence ; Humans ; Hybridomas ; secretion ; Liver ; metabolism ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; UTP-Glucose-1-Phosphate Uridylyltransferase ; immunology

This study was aimed to investigate the importance of chemokine SDF-1 in maintaining proliferation ability of acute myelocytic leukemia cell line HL-60 when the effects of SDF-1 on HL-60 cell proliferation were inhibited. Marrow stromal cells were cultured and co-cultured with HL-60 cells, and SDF-1 activity was blocked with anti-CXCR4 McAb. HL-60 cell activity was detected by MTT while cell cycle and the expression of CXCR4 on HL-60 cell membrane were observed by flow cytometry meanwhile. The internal calcium ionic concentration in HL-60 cell was detected as well before and after treated with 12G5. The results showed that 12G5 down-regulated the expression of CXCR4 on HL-60 cell membrane; HL-60 cells at G(0)/G(1) phase increased, but decreased at S phase; survive rate of leukemia cells reduced; the intercellular calcium ionic concentration of HL-60 cell decreased after treated with 12G5. It was concluded that brockage of the SDF-1 activity may inhibit proliferation of leukemia cell at certain level.
Antibodies, Monoclonal ; pharmacology ; Calcium ; metabolism ; Cell Cycle ; Cell Division ; Cell Survival ; Chemokine CXCL12 ; Chemokines, CXC ; antagonists & inhibitors ; physiology ; HL-60 Cells ; cytology ; Humans ; Receptors, CXCR4 ; analysis

Actins ; immunology ; metabolism ; Antibodies, Monoclonal ; analysis ; Antigens, CD34 ; immunology ; metabolism ; Breast Neoplasms ; metabolism ; Female ; Humans ; Immunohistochemistry ; methods ; Lung Neoplasms ; metabolism ; Membrane Proteins ; immunology ; metabolism ; Tumor Suppressor Protein p53 ; immunology ; metabolism