Preparation and characterization of anti-hBLyS monoclonal antibody by DNA immunization

AIM:To establish the monoclonal antibody against human B lymphocyte stimulator (hBLyS) by DNA immunization and analyse its characterization. METHODS:The 858 bp DNA fragment of hBLyS was cloned into pcDNA3 plasmids. The cloned insert was identified by both sequence analysis and double digestion of the recombinant plasmid with restriction enzymesXho Iand EcoR I. After the splenocytes from BALB/c mice immunized with the recombinant plasmid of pcDNA3/hBLyS were fused with myeloma cells SP2/0,the hybridoma which can produce monoclonal antibodies against hBLyS were obtained. The specificity of anti-BLyS monoclonal antibody from hybridoma was verified by ELISA, Western blot and flow cytometry. RESULTS:The recombinant mammalian cell expression vector of pcDNA3/hBLyS was constructed,the sequence of the insert gene was identified to be the sequence encoding hBLy S antigen. The culture supernatants of hybridoma 9c10 were tested to be the monoclonal antibody with specificity against hBLyS on human peripheral blood CD3⁺T cell activated by hIFN-γ by ELISA,Western blot and flow cytometry.CONCLUSION:The monoclonal antibodies against hBLyS with high activity and specificity have been established successfully, and will be an useful tool in the studies of relationship between hBLyS and human autoimmunity diseases.     

Production and identification of monoclonal anti-idiotypic antibody against uterine cervical cancer

AIM: To produce monoclonal anti-idiotypic antibody against uterine cervical cancer and identify its antigenic mimicry properties. METHODS and RESULTS: Mouse splenocytes were immunized in serum-free medium containing immunoactive reagents by using monoclonal antibody AU14-1 (Ab1) recognizing common epitopes shared with both mouse and human uterine cervical cancer-associated antigen molecule as immunogen. The in vitro immunized lymphocytes were fused with murine myeloma SP2/0. Hybridoma cell line secreting monoclonal anti-idiotypic antibody (Ab2) was established by screening and cloning. The identification of antigenic mimicry properties of Ab2 with ELISA, combinding and competing inhibition assays, and immunohistochemistry staining showed that this monoclonal anti-idiotypic antibody was Ab2β, which carried an “internal image” of cervical cancer cell membrane surface antigen. CONCLUSION: A monoclonal anti-idiotypic antibody with an “internal image” of cervical cancer antigen was obtained.     

Effects of αvβ6 integrin-mediated cell adhesion on 5-fluorouracil induced apoptosis in human colon carcinoma cell lines

AIM: To investigate the effects of αvβ6 integrin-mediated cell adhesion on 5-fluorouracil (5-FU) induced apoptosis in colon carcinoma cell lines.METHODS: The expression of the αvβ6 integrin in colon carcinoma cell lines HT-29 and WiDr cells was analyzed by flow cytometry. The apoptosis induced by 5-FU and the effects of αvβ6 integrin-mediated cell adhesion on 5-FU induced cell apoptosis were measured by enzyme-linked immunosorbent assay (ELISA) method and acridine orange-ethidium bromide (AO-EB) double fluorescent dye staining.RESULTS: Both the colon carcinoma cell lines HT-29 and WiDr cells expressed the αvβ6 integrin. The percentages of HT-29 and WiDr cells expression were 80.82% and 82.96%. 5-FU induced the apoptosis of colon carcinoma cell lines HT-29 and WiDr. The result of ELISA method displayed that enrichment factor (EF) of HT-29 and WiDr cells planted on fibronectin (FN)-ligand of αvβ6 integrin was lower significantly than the EF of HT-29 and WiDr cells planted on non-integrin ligand polylisin (1.11±0.04 vs 3.68±0.03, 1.09±0.02 vs 3.72±0.02, P<0.01) after cultured in medium containing 20 mg/L 5-FU for 48 h. When HT-29 and WiDr cells preincubated with αvβ6 integrin blocking antibody were planted on FN again, the EF of HT-29 and WiDr cells was higher significantly than those directly planted on FN without being blocked by αvβ6 integrin antibody (2.12±0.04 vs 1.11±0.04, 2.14±0.03 vs 1.09±0.02, P<0.01). The AO-EB double fluorescent dye staining displayed that the apoptosis percents of HT-29 and WiDr cells planting on FN were (5.6±1.1)% and (5.3±0.7)%, which were lower significantly than those planting on polylisin (37.0±1.4)%, (38.5±0.9)%, P<0.01. When HT-29 and WiDr cells preincubated with αvβ6 integrin blocking antibody were planted on FN again the percents of HT-29 and WiDr cells apoptosis were (19.5±1.2)% and (20.0±0.7)%, which increased significantly compared with those directly planting on FN without being blocked by αvβ6 integrin antibody (P<0.01). CONCLUSION: Colon carcinoma cell lines HT-29 and WiDr cells expressed αvβ6 integrin. The cell adhesion with FN mediated by αvβ6 integrin inhibits 5-FU-induced colon carcinoma cell apoptosis. The results suggest that cell adhesion may enhance drug resistance in colon carcinoma cell lines through inhibiting the cell apoptosis.     

Cloning of hCD154 gene from human activated peripheral blood mononuclear cell and expression of hCD154-GST fusion protein in prokaryote

AIM:To express recombinant hCD154-GST fusion protein, to prepare anti-hCD154 monoclonal antibody, and to investigate the effect of anti-hCD154 monoclonal antibody on graft rejection. METHODS AND RESULTS: Total RNA was prepared from human peripheral blood mononuclear cell (PBMC) activated with 10ng/mL PMA and 1 μg/mL PHA for 8h, the total RNA was reversetranscribed to cDNA. The entire coding region and a part of the 3'non-coding regions were amplified by PCR using a pair of primers designed and synthesized according to the sequence of human CD154 gene from gene bank. The amplified product, a 820bp DNA fragment was cloned into pGEX-4T-1 plasmid expressing glutathione S-transferase(GST). The cloned insert was identified by double digestion of the cloned pGEX-4T-1 plasmid with retriction enzymes BamHⅠand EcoRⅠ.The fusion protein expression plasmid of PGEX-4T-1/hCD154 was constructed, then transformed to E coli BL21. The human CD154-GST fusion protein expression was induced by IPTG in BL21. The expression of recombinant 26kD GST and 55kD human CD154-GST fusion protein were confirmed by SDS-PAGE. CONCLUSION: We have express the recombinant human CD154-GST fusion protein. The expressed hCD154-GST fusion protein will be used to prepare anti-hCD154 monoclonal antibody, to investigate the role of anti-CD154 monoclonal antibody on graft rejection.     

Expression of the novel serine protease SNC19 in different kinds of cells lines and its effects on cellular biological behaviors

AIM: To investigate the physiological function of the novel serine protease SNC19 protein and its possible role in cancer invasion and metastasis. METHODS: Monoclonal antibodies directly against SNC19 extracellular domain was prepared. The protein and SNC19 mRNA expression were determined in different kinds of cell lines respectively by Western blot and Northern blot analysis. Cellular migration and adhesion abilities were assayed by monoclonal antibody blocking method. RESULTS: Western blot analysis showed there were two bands of SNC19 protein in BCAP37, COLO205, SW480 cells at about 120 kD and 60 kD while only one band in SW620 cells at 60 kD; Northern blots showed a approximate 3.4-kilobase fragment appearing in most epithelial-derived cell lines with this only form and high levels but no detection was obtained in OV, TCA8113, KB and SGC7901 cells. In antibody blocking experiments, the migration of SW480 cells was significantly inhibited compared with the control and the abilities of SW480/SW480, SW480/NIH3T3 adhesion increased at the beginning of the experiments, but the difference reduced along with the time passed.CONCLUSION: SNC19 protein is closely related with cellular homogeneous and heterogeneous adhesion as well as cellular motility. As a novel serine protease, it may participate both in physiological and pathological processes, such as cell migration, tissue remodeling and cancer invasion and metastasis.     

Generation and characterization of a series of monoclonal antibodiesagainst recombinant human augmenter of liver regeneration

AIM:To generate monoclonal antibodies against human augmenter of liver regeneration (rhALR). METHODS:After BALB/C mice were immunized by the purified rhALR, the cells of spleen were fused with the cells of SP2/0; The titer and speciality were respectively fathomed from ascites or foster fluid by ELISA and Western-blot test. RESULTS:2 hybridoma cell lines were successfully obtained. The McAbs titer from ascites and foster fluid are respectively about 10^-3-10^-5 and 10^-2-10^-3. It is evident that the two McAbs were directed at different epitopes. CONCLUSIONS:The McAbs have higher speciality. It is significantly useful of the value that how hALR distribute in tissue organs, how the hALR signals the metabolism in the body and the control distribution of the hALR on cell growth on the translational level and so on is researched.     

Effect of epidermal growth factor receptor antibody on human colon carcinoma

AIM: To observe the effect of monoclonal antibody of epidermal growth factor receptor(EGFR) on human colon carcinoma cell lines. METHODS: Cell counting, growth curve measurement and MTT method were applied in this study to examine the proliferation of cultured cells in vitro when different dosage of EGFR McAb is used to treat LST174 colon carcinoma cell lines. RESULT: The proliferation of cultured human colon carcinoma cells could be significantly inhibited in a dose dependent manner by EGFR antibody Compared with the control group, the cell number was decreased by 61.3% and 33.8% respectively when treated with 0.625 mL/L or 2.5 mL/L of EGFR McAb CONCLUSION: EGFR McAb can inhibit cell growth of human colon carcinoma LST174.     

Screening,identifying and sequencing of human single-chain variable fragment specific to hepatitis B virus core protein

AIM:To Screen and identify human single-chain variable fragment (ScFv) specific to hepatitis B virus core protein and determine its gene sequence.METHODS:The recombinant phages were panned by HBcAg coated in a 96-pore plate and 48 clones were identified specific to HBc after three rounds of panning. The specificity of ScFv from the positive clone was determined by ELISA. Then, the soluble ScFv was expressed in E.coli.HB2151 and secreted in the supernatant. Subsequently, SDS-PAGE and dot blot were performed to identify the ScFv in the supernatant and cell lysate. The gene of ScFv specific to hepatitis B virus core protein was sequenced.RESULTS:The ScFv screened from phage antibodies has a specific combination character with hepatitis B virus core antigen. Soluble ScFv was confirmed to express in E.coli.HB2151 and secrete in the supernatant. The sequence of ScFv gene conformed to that of heavy chain and kappa chain of human immunoglubulin. CONCLUSION:Human ScFv specific to hepatitis B virus core protein has been identified by means of the phage display technology, and its gene sequence has been determined.     

Expression of IgG in bladder carcinoma and the effect of the antibody against human IgG on tumor cell apoptosis

AIM: To investigate the expression of immunoglobulin G (IgG) in bladder transitional carcinoma cells (BTCC) and the effect of the antibody against human IgG on tumor cell apoptosis.METHODS: The expression of IgG of BTCC was detected by immunohistochemistry, Western blotting, enzyme linked immunosorbent assay (ELISA) and flow cytometry (FCM). The expression of IgG mRNA was tested by hybridization in situ method and RT-PCR in vitro. Antiproliferation effects of the antibody of antihuman IgG on T24 and BIU-87 cell lines were measured by MTT assay. Cell apoptosis was assessed by FCM.RESULTS: The expression of IgG was 91.1％ (51/56) in clinical cases, and 45.4％ (5/11) in normal controls. Immunohistochemistry and Western blotting showed that the IgG was significantly expressed in T24 and BIU-87. FCM indicated the IgG was mainly expressed in cytoplasma. No IgG was detected by ELISA in supernatant of cell culture medium. RT-PCR and hybridization in situ demonstrated IgG mRNA was significantly expressed in two cell lines. Under the treatment of 25 mg/L of goat nonspecific IgG and the antibody of antihuman IgG, the inhibition ratio of cell growth in T24 and BIU-87 were (4.73±3.73)% vs (24.98±3.81)% and (5.36±1.53)% vs (22.70±3.72)%, respectively (P<0.05). The percentages of apoptotic cells in T24 and BIU-87 were 2.3% vs 20.7% and 1.3% vs 15.3%, respectively.CONCLUSION: IgG is significantly expressed in bladder transitional carcinoma cell. The antibody of antihuman IgG inhibits cancer cell growth and induces tumor cell apoptosis.     

The anti VEGF monoclonal antibody conjugated with nanometer particle of 5-FU

AIM:The characteristics of nanometer particles, which were prepared by the conjugation of anti-VEGF monoclonal antibodies and 5-fluorouracil-loaded polylactic acid nanometer particles (5-FU-NPs), were investigated for improving the anticancer activity of 5-FU. METHODS:The method of couple linkage of chemical bonds was used to prepare the 5-FU-NPs with VEGF antibody, then the appearance, distribution of particle diameter, releasing in vitro and the immunological activity were detected. RESULTS:The 5-FU-Ab-NPs appeared as regular globular, the average particle diameter was (202±23)nm. The 5-FU-Ab-NPs possessed the similar delayed release character of 5-FUs. More than 80% of the immmunogical activity were detected in conjugates-retained antibody by the immunological methods and electron microscopy. CONCLUSION:The 5-FU-Ab-NPs possess the similar delayed released activity to 5-FU-NPs and have double activity of immune targeting and delayed releasing, which may increase the local concentration of 5-FU.     

The preparation,purification and biological activity of anti-cobra immunoglobulin yolk

AIM:In order to substitute antivenom from horse, the biologic activity and preparing methods were investigated in immunoglobulin yolk (IgY) of anti-cobra. METHODS:Leghorn hens were immunized with cobra venom. The methods of three stages，water dilution- ammonium sulfate salting out- anion exchange chromatogram, were used to purify IgY. SDS- PAGE was used to measure the purity of antibody. The titer variety of IgY was observed by indirect ELISA and double immunodiffusion. The method of pre-incubating the venom and IgY in vitro was used to test the protective effect of IgY. RESULTS:The total protein recovery rate of IgY fraction through water dilution method（IgY-WD）followed by 60% ammonium sulphate deposited (IgY-AS) was 40.36%. When applied to DEAE chromatogram further, the total protein recovery rate reduced to 14.86%, but the titer increased by 24 times. When venom at dose of 1.24 mg/kg (2×LD50) was injected into mice intraperitoneally, all the animals died within 24 h, but when pre-incubated with 2.28 mg/kg of IgY before venom treatment, all animals survived. CONCLUSION:Taking the original cobra venom as antigen, and using the water dilution-salting out- DEAE chromatogram method for purification, high purity IgY can be withdrew from the yolk.     

Preparation and characterization of the antibodies against human myofibrillogenesis regulator 1 and application in neonatal rat cardiomyocytes

AIM: To prepare and purify the polyclonal antibodies against human myofibrillogenesis regulator 1 (hMR-1), then to characterize the purity, titer, specificity and the availability.METHODS: Two polypeptides named peptide 1 and 2 were synthesized based on the bioinformatics analysis of the sequence of hMR-1 by using software TMHMM and DNAStar, then coupled with keyhole limpet hemocyanin (KLH) for immunization. These peptides for immunization were mixed and injected into New Zealand rabbits to prepare antibodies specifically against hMR-1. ELISA assay was used to detect the titers of the antibodies. After purification by immunoaffinity chromatography, antibodies were identified by Western blotting and immunocytofluorescent assays. Applications of the antibodies on neonatal rat cardiomyocytes were also employed.RESULTS: (1)The titers of antibodies were 1∶105. In WB assay, a specific 17kD band was detected, corresponding to the predicted molecular weight of hMR-1; the positive fluorescent signals were distinct. (2)On the neonatal rat cardiomyocytes model, we observed a peri-nucleus location. The fluorescent signal of hMR-1 overexpression group was much stronger than that in vector control and normal control groups.CONCLUSION: All these results indicate that the antibodies obtained from poly peptides mixture immunization have either human original or rat original antigens. The antibody is available for using in Western blotting or immunofluorescent assays.     

Establishment of human lung infection model in vitro and mechanisms of NTHi-induced inflammatory responses

AIM: To investigate the key signal pathways of inflammatory responses in lung tissues induced by the infection of nontypeable Haemophilus influenzae(NTHi). METHODS: Human lung tissues were co-incubated with NTHi (10¹⁰ CFU/L) for 4 h and 24 h, respectively. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) was detected by Western blotting. The nuclear translocation of nuclear factor (NF)-κB was examined by electrophoretic mobility shift assay (EMSA). The expression of Toll-like receptor (TLR) 2 was measured by real-time quantitative RT-PCR, and the level of interleukin (IL)-8 was detected by enzyme-linked immunosorbent assay (ELISA). Furthermore, lung tissues were incubated with anti-TLR2 monoclonal antibody (5 mg/L), p38 MAPK inhibitor SB203580 (20 μmol/L), or NF-κB inhibitor PDTC (25 μmol/L) for 2 h, then stimulated with NTHi (10¹⁰ CFU/L) for another 24 h. The supernatants were collected for IL-8 detection. RESULTS: The TLR2-p38 MAPK-NF-κB signaling pathway in lung tissues was rapidly activated 4 h after NTHi stimulation. IL-8 secreted from lung tissues infected with NTHi was significantly increased compared with uninfected lungs (P<0.05). The pre-incubation with anti-TLR2 antibody, p38 MAPK inhibitor or NF-κB inhibitor markedly decreased IL-8 production induced by NTHi. CONCLUSION: NTHi induces inflammatory responses in lung tissues by activation of TLR2-p38 MAPK-NF-κB signaling pathway. Human lung infection model provides a new research tool for the study of interaction between pathogens and hosts.     

Activation of vascular endothelial cells improves the homing of hematopoietic stem cells

AIM: To study the effect of endothelial cell activation on the homing of hematopoietic stem cells (HUHSC) during transplantation. METHODS: Human umbilical vein endothelial cells (HUVEC) were cultured to single layer and activated by vascular endothelial growth factor (EVGF), granulocyte colony stimulating factor (G-CSF) and lipopolysaccharide (LPS), respectively. The HUHSC, enriched by eliminating red blood cells, granulocytes, monocytes and lymphocytes from cord blood, were cocultured with activated HUVEC to make adhesion. The adhesive ability of activated HUVEC to C-Kit⁺ HUHSC was assayed by ELISA. Anti-VCAM-1 monoclonal antibody was used to detect the effect of activation on HUVEC and HUHSC interactions. RESULTS: Resting HUVEC had a little adhesive ability to HUHSC. A great enhancement of adhesive ability was showed when HUVEC was activated by VEGF, G-CSF and LPS. In the presence of anti-VCAM-1, the adhesive ability of activated HUVEC was decreased remarkablely. CONCLUSION: HUHSC homing may be related to the activation of endothelial cells and adhesion molecules.     

Effect of monocyte-secreted VEGF induced by electrical burn serum on monocyte-endothelial cell adhesion

AIM: To observe the level of vascular endothelial growth factor (VEGF) secreted by monocytes cultured with electrical burn serum, and to explore the effect of VEGF on monocyte-endothelial cell adhesion.METHODS: The electrical burn serum of the rat was prepared. The normal serum from the rats without treating electric current was also collected for control. The contents of VEGF and its soluble receptor sFlt-1 in electrical burn group were determined by double-antibody sandwich ELISA. THP-1 cells were randomly divided into normal serum group and electrical burn serum group. The contents of VEGF and sFlt-1 in the culture supernatants were measured by double-antibody sandwich ELISA. THP-1 cells were also randomly divided into another 4 groups:normal serum group, electrical burn serum group, normal serum+inhibitor group and electrical burn serum+inhibitor group. THP-1 cells, which were incubated with the serum for 3 h and 6 h, were labeled with calcein-AM and then were added into the well with monolayer of endothelial cell line EA.hy926 to detect monocyte-endothelial cell adhesion.RESULTS: The levels of serum VEGF of the rats with electrical burns were significantly increased, the levels of serum sFlt-1 were significantly decreased as compared with the controls. The levels of VEGF secreted by THP-1 cells cultured with electrical burn serum were significantly increased, the levels of sFlt-1 were decreased correspondingly. Electrical burn serum enhanced monocyte-endothelial cell adhesion, sFlt-1 inhibited the adhesion between monocytes and endothelial cells.CONCLUSION: The monocytes exposed to the electrical burn serum secrete VEGF, which enhance the adhesion between monocytes and endothelial cells. Blockage of VEGF activity may effectively inhibit monocyte-endothelial cell adhesion.     

Construction of atherosclerosis full-length fully human antibody libraries by mammalian cell surface display technique

AIM: To construct atherosclerosis (As) full-length fully human antibody libraries by mammalian cell surface display technique for looking for the atherosclerotic therapeutic antibody. METHODS: The vector pcDNA-DHL was constructed by remounding the vector pcDNA5/FRT. The human total RNA from human peripheral blood mononuclear cells was isolated, and then reverse transcripation of RNA to cDNA was performed. The genes encoding the heavy chain variable regions and kappa light chains of the antibodies were amplified by PCR, and the product of PCR was connected to the vector pcDNA-DHL and the vector pDHL-As. The library DNAs were transfected into Flp-In^TM-CHO (FCHO) cells, the expression of the full-length human antibodies on the surface of cells was analyzed by flow cytometry. The cells that were successfully transfected with pDHL-As were screened with hygromycin, and then the full-length antibody library of atherosclerosis was obtained. RESULTS: The vector pcDNA-DHL were constructed successfully. The heavy chain gene library constructed showed a diversity of 1.79×10⁵, and the kappa light chain gene library had a diversity of 1.80×10⁵. Theoretically, the diversity of antibody library is 2.32×10¹⁰. The antibody library of atherosclerosis pDHL-As expressed on the surface of Flp-In^TM-CHO cells. CONCLUSION: The full-length human antibody cell library of atherosclerosis was constructed successfully, and 45 FCHO cells that expressing anti-oxidized low density lipoprotein antibodies were selected.     

Effect of nerve growth factor on proliferation and survival of breast cancer cell line MDA-MB-231 and MCF-7

AIM: To study the effect of nerve growth factor (NGF) on the proliferation and survival of breast cancer cell lines MDA-MB-231 and MCF-7. METHODS: Two breast cancer cell lines MDA-MB-231 and MCF-7 were used in the experiment. The expression of NGF and its receptor tyrosine kinase A (TrkA) was determined by the method of immunofluorescence. The NGF autocrine was detected by the method of enzyme linked immunosorbent assay (ELISA). The expression of TrkA was measured by Western blotting. The effect of NGF blocking agent Ro 08-2750 on the proliferation of the cells was evaluated by MTT assay (mono-nuclear cell direct cytotoxicity assay). The cell apoptosis and the change of the cell cycle after exposed to Ro 08-2750 were observed by flow cytometry. RESULTS: Both cell lines expressed NGF and TrkA. Ro 08-2750 inhibited the proliferation of both cell lines in a dose-dependent manner. According to the results of flow cytometry, the S-phase cells in both cell lines increased but the G₂/M-phase cells decreased after treated with Ro 08-2750. An apoptotic peak occurred in MDA-MB-231 cells. CONCLUSION: The results suggest that proliferation of MDA-MB-231 and MCF-7 cell lines is dependent on NGF. NGF promotes the survival of MDA-MB-231 cells.     

Effect of conditioned medium of human multiple myeloma RPMI 8226 cells on osteoclastogenesis of RAW264.7 cells

AIM: To investigate the osteoclastogenic effect of conditioned medium of human multiple myeloma RPMI 8226 cells on preosteoclast RAW264.7 cells. METHODS: The protein expression of soluble receptor activator of NF-κB ligand (sRANKL) was detected by Western blotting. The morphological changes of RAW264.7 cells were observed after tartrate-resistant acid phosphatase (TRAP) staining. The mRNA expression of TRAP and cathepsin K was evaluated by RT-PCR. RESULTS: The result of Western blotting showed that conditioned medium of RPMI 8226 cells contained sRANKL. RPMI 8226 cell conditioned medium induced RAW264.7 cells to differentiate into TRAP-positive multinuclear osteoclasts. Human neutralized RANKL monoclonal antibody suppressed the differentiation of preosteoclasts in a dose-dependent manner, which was induced by 30% RPMI 8226 cell conditioned medium. RPMI 8226 cell conditioned medium increased the mRNA expression of TRAP and cathepsin K in RAW264.7 cells. CONCLUSION: The sRANKL in conditioned medium of human multiple myeloma RPMI 8226 cells has the bioactivity to induce preosteoclast RAW264.7 cells to differentiate into TRAP-positive multinuclear osteoclasts. Human neutralized RANKL monoclonal antibody suppresses the differentiation of preosteoclasts induced by sRANKL in a dose-dependent manner.