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【Objective】 To prepare and preliminarily identify the specific monoclonal antibody (McAb) against-AB blood group antigen. 【Methods】 The human AB red blood cells were used to immunize BALB/c mice for producing monoclonal antibodies against human red cell AB antigens. The cell lines that can secrete AB monoclonal antibodies were obtained by cell fusion and screening using hybridoma cell technology.The specificity and recognized epitopes of anti-AB McAb were preliminarily identified by blood group serological assay. The antibody was also used to detect the clinical samples. 【Results】 One hybridoma cell line secreting IgM monoclonal antibody against human AB antigens was obtained. This anti-AB McAb could agglutinate with human red blood cells of type A, B and AB, but not agglutinate with type O red blood cells. The red blood cell absorption and elution test confirmed that the antibody recognized the common epitope of A and B. The anti-AB McAb obtained and the commercial anti-A and anti-B reagents were used to detect 567 clinical samples in parallel, with the concordance rate at 100%. 【Conclusion】 A hybridoma cell line secreting anti-AB McAb can be successfully prepared.
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This research aims to determine the cytotoxicity and antiproliferation activities of Sida rhombifolia leavesextract against cancer cells MCA-B1, A549, and normal Vero cells. Sida rhombifolia leaves were extractedwith ethanol using ultrasonication method and fractionated using n-hexane, ethyl acetate, and water. The testedsamples were ethanol extract and n-hexane fraction based on the results of cytotoxicity using the Brine ShrimpLethality Test. The antiproliferation activity test by using Trypan Blue Dye method and the cells harvested afterconfluence on the third or fourth day and the total cells were calculated by using the Neubauer Hemocytometer.The result showed that the inhibitory activity of ethanol extract at a concentration of 500 ppm is 69.44% withIC50 202.556 ppm on MCA-B1 cancer cells and 69.44% with IC50 276.836 ppm on A549 cancer cells, whilethe n-hexane fraction at a concentration of 1,000 ppm was 64.13% with IC50 425.969 ppm in MCA-B1 cancercells and 57.18% with IC50 786.617 ppm on A549 cancer cells. After being tested on normal Vero cells, theinhibition of normal Vero cells proliferation is not more than 1%. This indicates that ethanol extracts andn-hexane fraction are safe for normal cells and analysis by using LC-MS/MS showed a benzazepine compoundin the ethanol extract of S. rhombifolia is known for its role as antiproliferation. These results indicate thatS. rhombifolia leaves extract has the potential to be developed as anticancer compounds..
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Objective To investigate the therapeutic effects of anti-B7-H3 blocking monoclonal antibody(McAb) in a murine model of neutrophilic asthma. Methods Twenty-four female BALB/c mice were randomly divided into four groups: PBS control group (group A), neutrophilic asthma group (group B),anti-B7-H3 McAb group (group C) and IgG isotype control group (group D). Those in group A were sensitized by injection of PBS and challenged with PBS through inhalation,while the other mice were sensi-tized by injection of ovalbumin (OVA) plus airway instillation of lipopolysaccharide(LPS),and then chal-lenged with OVA. Moreover,mice in group C and group D were respectively injected with anti-B7-H3 McAb and IgG isotype control in the induction period. Each mouse′s performance was observed. Samples of bron-choalveolar lavage fluid (BALF) and lung tissues were collected. Total and differential cell counts in BALF were determined by using microscope. Levels of cytokines including IFN-γ,IL-4,IL-6,IL-17,tumor nec-rosis factor-α (TNF-α) and granulocyte colony stimulating factor (G-CSF) in BALF were measured by ELISA. Lung sections were stained with hematoxylin and eosin (HE) and periodic acid-Schiff (PAS) to identify tissue inflammation and mucus production,respectively. Immunohistochemistry was used to measure the expression of B7-H3 in frozen mouse lung sections. Results Mice in group B and group D showed asth-matic symptoms such as breathlessness,dysphoria and incontinence after nebulization,while these symptoms in group C were alleviated due to anti-B7-H3 McAb treatment. No abnormalities were observed in group A. Compared with group A,the other three groups showed increased total cell count in BALF and higher per-centages of neutrophil and eosinophil(P<0.05). These three indicators in group C were lower than those in group B and group D (P<0.05). With regard to lung infiltration by Th1,Th2 and Th17 cells,the levels of IFN-γ,IL-4,IL-6,IL-17,TNF-α and G-CSF in BALF were increased in group B,group C and group D as compared with those in group A. Compared with group C,group B and group D showed higher levels of IL-6, TNF-α,IL-17 and G-CSF(P<0.05),but lower level of IL-4(P<0.05). No statistical difference in the level of IFN-γ was observed among group B, group C and group D. Histological staining of lung sections showed that no obvious inflammatory cells and mucus secretion was observed in group A. Massive infiltration of inflammatory cells and neutrophils and mucus hypersecretion were detected in group B and group D. Treatment with anti-B7-H3 McAb inhibited the accumulation of neutrophils and mucus hypersecretion in lung tissues of group C. Compared with group A, levels of B7-H3-positive cells were significantly increased in group B and group D. Anti-B7-H3-treated mice showed reduced levels of B7-H3-positive cells in lung tissues as compared with those in group B and group D(P<0.05). Conclusion Treatment with anti-B7-H3 bloc-king McAb in an early stage can relieve the asthmatic syndrome, reduce airway inflammatory cells, inhibit mucus production and down-regulate Th17 cell-related cytokine secretion,which helps to alleviate airway and systematic inflammation in mice with NA,and partially inhibit the development of NA.
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The rapid spread and outbreak of Middle East respiratory syndrome coronavirus ( MERS-CoV) in the regions of Middle East in 2012 have been a great concern for researchers worldwide. Thus, efficient preventative and therapeutic countermeasures are urgently needed. Clinical studies have con-firmed that high titers of neutralizing antibodies ( Abs) against MERS-CoV in patients during convalescence have protective potency, which indicates that neutralizing Abs are safe and effective for the treatment of MERS-CoV infection. Spike ( S) protein is a key structural protein that mediates MERS-CoV infection and currently a critical protein for studying MERS-CoV neutralizing monoclonal antibodies. This review summari-zes recent advances in identifying neutralizing McAbs against MERS-CoV through describing the structural characteristics of MERS-CoV S protein, different kinds of MERS-CoV vaccines and methods for mAbs screening. Furthermore, we propose the prospects for future research on MERS-CoV neutralizing McAbs ac-cording to the current research progress.
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The rapid spread and outbreak of Middle East respiratory syndrome coronavirus ( MERS-CoV) in the regions of Middle East in 2012 have been a great concern for researchers worldwide. Thus, efficient preventative and therapeutic countermeasures are urgently needed. Clinical studies have con-firmed that high titers of neutralizing antibodies ( Abs) against MERS-CoV in patients during convalescence have protective potency, which indicates that neutralizing Abs are safe and effective for the treatment of MERS-CoV infection. Spike ( S) protein is a key structural protein that mediates MERS-CoV infection and currently a critical protein for studying MERS-CoV neutralizing monoclonal antibodies. This review summari-zes recent advances in identifying neutralizing McAbs against MERS-CoV through describing the structural characteristics of MERS-CoV S protein, different kinds of MERS-CoV vaccines and methods for mAbs screening. Furthermore, we propose the prospects for future research on MERS-CoV neutralizing McAbs ac-cording to the current research progress.
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OBJECTIVE: To investigate the effects of anti CD3 monoclonal antibody (Ant-CD3 McAb) on immune function in cynomolgus monkeys, using the traditional immunotoxicity assessment methods and T cell dependent antibody response (TDAR) test, microarray, etc, and further explore the mechanism of Ant-CD3 McAb. METHODS: Eighteen Cynomolgus monkeys were individually dosed with saline, 0.5 and 2.5 mg·kg-1Ant-CD3 McAb for 7 d. The clinical observation, body weight, hematological test, antibody detection, TDAR test, lymphocyte subsets, bone marrow, histopathological examination and whole blood gene expression profile analysis were evaluated. RESULTS: Ant-CD3 McAb significantly inhibited the immune response to keyhole limpet hemocyanin (KLH) in cynomolgus monkey, and caused the decrease of peripheral white blood cell counts and CD3+ cells, the decrease of lymphocytes in thymus cortex. Microarray test showed that the gene function of differently expressed genes mainly involved in cell proliferation, immune response, cytokine secretion, apoptosis etc. CONCLUSION: Ant-CD3 McAb could induce obvious immunosuppression effects in cynomolgus monkeys. Detection of gene expression profile of whole peripheral blood is feasible in immunotoxicity evaluation. The apoptosis induction and proliferation inhibition of T lymphocytes might be the main cause of the decreased T lymphocytes. IL-8 might play an important role in the mechanism of Ant-CD3 McAb.
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Objective To research and evaluate cell counting random error for cell sheet of T lymphocyte subsets in the practical McAb‐A‐E direct method ,and old method of McAb‐A‐E direct method compared .Methods Randomly selected 20 specimens ,cell sheet of T lymphocyte subsets on counting 200 cell at least 2 area from 2/6~4/6 area ,counting area non‐repeated ,CD4+ and CD8+continuous counter 4 times ,CD3+ continuous counter 2 times .Results In the practical McAb‐A‐E direct method ,random error mean of cell counting was separately CD3+ :7 .86 ,CD4+ :9 .99 ,CD8+ :8 .55 ,CD4+ /CD8+ :0 .33 ,lower than old method of McAb‐A‐E direct method ,that was CD3+ :15 .00 ,CD4+ :13 .80 ,CD8+ :10 .70 ,CD4+ /CD8+ :0 .69 .Conclusion Cell counting random error in practical McAb‐A‐E direct method for cell sheet of T lymphocyte subsets is less than old method of McAb‐A‐E direct method .
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Objective To study the effect of two-step pretargeting radioimmunotherapy of CD45 McAb and 188Re-Avidin on lymphoma Raji cell line.Methods The CD45 McAb and Avidin were directly labeled with 188Re,and the labeling efficiency and radiochemical purity were measured by the paper chromatography.The specific binding test and competition binding test between 188 Re-CD45 McAb and Raji cells in vitro were also performed.CCK-8 assay was used to determine the inhibition effect on Raji cell proliferation in the pretargeted group,188Re-CD45 McAb,188Re-Avidin and 188ReO4 groups,then the cell survival and proliferation inhibition rate were calculated.Results The specific cell binding rate of 188Re-CD45 McAb with Raji cells was (70.92 ± 1.91) ％,in the competition group,the binging rate of 188Re-CD45 McAb with Raji cells was only (7.96 ± 0.87)％.The Raji cells proliferation was inhibited in all groups with 188Re radiolabel,and the inhibition rate was positively correlated with the radioactivity dose (r=0.907-0.992,P ＜0.05).However,at the same dose,the inhibition effect in the group of two-step pretargeting at each time point were all stronger than those of 188Re-CD45 McAb,188Re-Avidin and 188 ReO4-alone (t =124.76-607.98,P ＜ 0.05).But there was no significantly statistical difference in the inhibitory effect between the groups of 188Re-Avidin and 188ReO4-(P ＞ 0.05).Conclusions It is confirmed that 188Re-CD45 McAb could be specifically bound to Raji cells,and the two-step pretargeting of CD45 McAb and 188Re-Avidin has obvious inhibitory effect on the Raji cell proliferation.
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Objective To evaluate the effect of TLR2McAb and TLR4McAb on intestinal flora of DSS-induced colitis in mice. Methods Fifty healthy male BALB/c mice (SPF level), were randomly assigned into five groups: the control group( group A), the UC model group( group B), TLR2McAb intervention group( group C), TLR4McAb intervention group( group D) and TLR2McAb + TLR4McAb intervention group(group E). Clinical symptoms were evaluated by the disease activity index(DAI), while tissue sam ples were evaluated by histological scoring(HS). The quantities of mRNA for IFN-γ, IL-4 and IL-17 were determined by real-time PCR. Meanwhile, fecal samples were obtained directly from the cecum for microbiological studies. Results After the treatment with TLR2McAb and TLR4McAb, DAI and HS were decreased significantly. Compared with group A, inflammatory cytokines such as IFN-γ, IL-4 and IL-17 in group B were higher. Compared with group B, expression of these three cytokines in group C to E was all markedly decreased. Group A showed a considerable predominance of Lactobacillus spp and Bifidobacterium spp,while the UC model group showed a conspicuous increase of Escherichia coli and decreases of Lactobacillus spp and Bifidobacterium spp. After treatment with TLR2McAb or/and TLR4McAb, Lactobacillus spp and Bifidobacterium spp increased to the normal level. But counts of E. Coli in the three intervention groups were not changed. Conclusion TLR2McAb and TLR4McAb suppressed the development of DSS-induced colitis and increase cecum counts of Lactobacilli and Bifidobacteria.
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Objective:To prepare and characterize specific and discrepant mouse hybridoma antibodies on membrane of HL60 and HL60/ADR cell lines.Methods:BALB/c mice were immunized by subtractive immunization induced Cp(Cyclophosphamide).McAbs were prepared by hybridoma technique,screened and detected by FACS and LSCM.Results:51 candidates and discrepant antibodies were found,and one of them (5F6) was purified and identified.Conclusion:Combination of SI with discrepant screening method should facilitate the preparing and identifying discrepant McAbs for identifying antibodies that can distinguish the differences in proteins expressed in HL60 and HL60/ADR,which is a significative and potential method in the research and target therapy associated drug-resistance.
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Objective To investigate the effect of T lymphocyte proliferation,Th1 and Th2 cytokines on T cells from systemic lupus erythematosus (SLE)patients by blocking 4-1BB/4-1BBL.Methods The proliferation of T cells from 30 SLE patients and 20 normal controls was detected by MTT and the levels of patients stimulated with anti-CD3 McAb were significantly higher than those of T cells without stimulation(P＜IFN-γand IL-4 in SLE were significantly higher than those of normal controls(P＜0.01).There were more ex-pression of IFN-γ and IL-4 in supernatant of T cells from SLE after stimulated with anti-CD3 McAb(P＜0.01).However,the production of IFN-γ and IL-4 was inhibited by anti-4-1BB McAb(P＜O.05),and especially the level of IL-4 was markedly decreased.Conclusion Blocking 4-1BB/4-IBBL can significantlv inhibit the abnormal activation of T cells and the secretion of Thl and Th2 cytokines of SLE.
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Objective:To prepare a colloid gold kit for the qualitative detection of the dopes met-amphetamine and morphine by one-step chromatography immunoassay in human samples based on the principle of the highly specific immunochemical reactions between the antigens and their antibodies which were used for the analysis of specific compounds in human urine samples at ideal cut-off concentrations.Methods:The monoclonal antibodies(McAb) of either mouse anti-morphine or mouse met-amphetamine were prepared by hybridomas and the limiting dilution respectively. The titers of McAb were tested by ELISA.The kit was assembled and the specificity,sensitivity,and stability were investigated.In addition,these characters were compared with those of issued Pan Probe single test strip.Results:①The titer of the first McAb against morphine was 1∶3.2?103, as for another, 1∶1.6?103. ②The colloid gold kit was found with following cut-off for the dope concentrations,morphine at 300 ng/ml and met-amphetamine at 1 000 ng/ml. ③The colloid gold kit showed fine specificity without cross-reaction with routine medicines for cold treatment or with the solf-drinks of cokocola or with tobacco for the individuals who did not take the dopes the 7 days.④The colloid gold kit was even more sensitive than the issued strip,because urine sample of the junkies were still positive 4-5 days post-intake.Conclusion:The colloid gold kit for indentifying the drug-users is a rapid,specific, sensitive immunoassay suitable for the simultaneous, qualitative detection of drug abuse.
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Objective:To explore the action of the second signal system in the activatied and proliferating T cells and the induction of apoptosis of the hepatoma cells.Methods:The T cells were costimulated by anti CD28 and anti CD80(B7.1)McAb and acted on the hepatoma cells(BEL 7402),then testing the concentration of cAMP?cGMP and Ca 2+ in the T cells and the apoptosis of the hepatoma cells.Results:The concentration of cAMP was increased temporarily at first,then decreased rapidly,and increased 1 2 times again when acted on the hepatoma cells.The concentration of cGMP was increased 6 8 times fast and the concentration of Ca 2+ obviously increased 2 3 times too.The peak of them was at the fourth day and positive related to apoptosis of the hepatoma cells.Conclusion:The level of the second signal system of cAMP?cGMP and Ca 2+ were significant correlated with the T cells activated and porliferating and the cytotoxic effect.
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Objective To prevent the after cataract induced by lens epithelial cells proliferation from postoperative cataract, monoclonal antibody (McAb) specific for rabbit lens epithelial cell is made, it will be the carrier further to be conjugated with cytotoxin. The conjugations will inhibit lens epithelial cells growth and not damage the other tissues of eye. Thereby McAb is the experimental bases of preventing after cataract.Methods BALB/c mice were immunized by mixture of rabbit lens epithelial cells and Freund's adjuant. The immunized mouse spleen cells were fused with parental mouse myeloma cells (BALB/c SP2/0) using polyethylene glycol (PEG-4000). The fused cells were selectively cultured by hypoxanthine, aminopterin and thymidine (HAT) culture medium. The specificities of the supernatant from hybridomas were tested by indirect immunofluorescence and immunohistochemistry SP (Streptavidin peroxdase conjugated method). The positive hybridomas were further cloned three times by methylcellulose culture medium to ensure monoclonality. At last, the consensual reaction of McAb was tested on human eye tissues.Results Hybridomas were produced by fusion of spleen cells of immunized mice and mouse myoloma cells (SP2/0) with PEG-4000, and grown selectively in medium containing HAT after 16 days. Antibodies of the supernatant from hybridomas were tested on frozen sections of rabbit lens epithelial cell by indirect immunofluorescence. Only a positive clone secreted McAb against antigen of rabbit lens epithelial cell. The specificity of McAb was tested on paraffin sections of whole rabbit eye and whole human eye by immunohistochemistry SP. The results indicated that McAb was only positive to rabbit lens epithelial cell membrane and it was negative to the other tissues of rabbit or whole human eye tissues.Conclusions McAb specific for rabbit lens epithelial cell was manufactured successfully. The specificity of McAb was strong. There was no consensual reaction on human eye tissues. It might be the experimental bases of further targeting chemotherapy on after cataract.
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Objective:To amplify and sequence of the variable region genes of anti CD3 McAb.Methods:The V H?V L genes were amplified by RT PCR from total RNA that were extracted from WuT3 hybridoma.Recombinant cloning vector was constructed and sequenced after the enzyme digestion.Results:It showed that V H gene consisted of 363 bp encoding amine acid residues,belongs to mouse heavy chain subgroup IIB;V L gene consisted of 330 bp encoding amine acid residues,belongs to mouse ? light chain subgroup III.Comparing with Kabat database,the V H?V L genes were in agreement with the characterization of DNA sequences present in the mouse Ig V H?V L regions respectively.Conclusion:The success of cloning of the V H?V L genes of WuT3 McAb lay a good foundation for the construction and expression of chimeric antibody.
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objective : To prepare mouse anti-human CD40 antigen functional monoclonal antibody and to further study it's biological effects by triggering the Cd40 molecules on B cells and dendritic cells(DCs) Methods: Using cell fusion, McAb screening immunofluorescence analysis immunoblotting and competition test obtain mouse anti-human CD40 McAb;by the proliferation assay of B cells and DCsand the analysis of expression of differentiation antigens on DCs. Results: On the basis of phenotype analysis, Western blotting and competition test, it is verified that 5Cll recognizes human CD40 antigens specially; 5Cll can augement the proliferation of tonsil B cells in the LCD32cells and IL-4 culturing system; 5Cll can medicate DCs to get proliferation and maturatio. Conclusion: 5Cll+LCD32 +IL4 can make tonsil resting B cells survive and long-term proliferate in vitro, the setup of CD40 System furnish necessary tool for the study of B cells; McAb5Cll also triggered the generation, proliferation and maturation of dendritic cells from peripheral blood monocytes. Thus 5Cll is a McAb withspecial function and important application value.
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In order to reduce its immunogenicity and increase its therapeutic usefullness, the murine anti-gastric cancer McAb 3H11 was genetically engineered into a human-mouse chimeric antibody. The 3H11 light and heavy chain variable genes were inserted into chimeric antibody expression vectors respectively. The chimeric light chain expression vector was first transfected into murine Sp2/0 myeloma cells using lipofectin. Transfectants that stably expressed human-mouse chimeric light chain were isolated by mycophenolic acid selection. Then the chimeric heavy chain expression vector was transfected into the transfectomas. Histidinol resistent cells were obtained and among these, clones stably secreting human-mouse chimeric antibodies were selected. The expression of human-mouse chimeric light and heavy chain mRNAs were proved by RT-PCR and the expressed chimeric antibody was demonstrated capable of binding to human gastric cancer cell MGC803
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A total of twenty eight HSV isolates from the patients were typed by using HSV typespecific monoclonal antibodies (McAb). Comparison of typing results with McAb in three Sero-lminunological methods with molecular hybridization indicated 100% concordance in the results. But the typing rates were quite different among the various methods (ELISA 64%, IFA and EIA 82%, Hybridization 100%). The results demonstrate that the molecular hybrydization with HSV type-specific probes was highly sensitive and specific, and the method of EIA with HSV type-specific McAb was accurate, cheap, rapid and practical.
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Two monoclonal antibodies of anti-mouse pancreatic cancer, 2B_6and 2B~(10), were purified by the use of salt fractionation-exchange chromatography(DEAE-52) or affinity chromatography from culture supernatants and mice ascites of the hybridomas. After assayed by the use of SDS-PAGE and labeled with FITC, they were used to analyse related antigen site of 35 tissue samples of several human tumor by means of direct-fluorescence method. There was an intense positive reaction between 2B_(10)and 4 samples of gastric cancer. 2B_6or 2B_(10)and 3 tissur samples of ovary cancer and 1 clonal cell of ovary cancer also had intense positive reaction. These results showed that there exist common tumor antigen in both human and mice.
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The BALB/c mice were immunized with human O blood cells. Their spleen cells were fused with murine myeloma cells Ns-1 with PEG. The monoclonal antibodies were identified with human panelled cells. Two clones secreting monoclonal anti-M(2Al and 3F6)and another two clones secreting monoclonal anti-N(4E3 and 7G5)were identified. All these clones could consistently secrete IgM antibodies. In typing 300 different RBC samples,the specificity of antibodies were identical to those of commercial humen anti-M and anti-N typing sere reagent. Significantly, anti-N reagent 4E3 differed from anti-N reagent 7G5, which the former one couldn't be absorbed by M erythrocytes at all,while the latter one absorbed by M erythroeytes.