ABSTRACT
Objective To purify the anti-T cell immunoglobulin mucin ( TIM)-3 monoclonal antibody 4E8 and examine its biological function in vitro. Methods The mouse monoclonal antibody against human TIM-3, clone 4E8, was obtained by standard protocol for monoclonal antibody purification. The cell lines expressing human TIM-3 molecule were obtained by cell transfection technique. We ex-amined the ability of 4E8 binding to human TIM-3 by flow cytometry. The ability of 4E8 blocking the binding of fusion protein TIM-3 Ig-huFc with phosphatidylserine( PtdSer) , the apoptotic cell surface TIM-3 ligand, was also analyzed by flow cytometry. Mixed lympho-cyte reaction ( MLR) and ELISA assays were used to determine the effect of TIM-3 monoclonal antibody ( 4E8) on IFN-γsecretion in CD4+ T cells. Results 4E8 specifically bound to human TIM-3 but could not block the binding of TIM-3 to Ptdser. Compared with the negative control (IFN-γ secretion: 958.3±153.2), 4E8 enhanced the ability of CD4+ T cells to secrete IFN-γ in MLR (4E8 of 10μg/mL group:IFN-γ secretion 2563±150.3 and 4E8 of 3.33 μg/mL group:IFN-γ secretion 1981±211.5) with statistically signifi-cant difference ( P<0.05) . In addition, the combined application of 4E8 with the anti-programmed death-1 ( PD-1) monoclonal anti-body nivolumab showed synergistic effects for increasing IFN-γ secretion in MLR assay ( 4E8 of 10 μg/mL group: IFN-γ secretion 3049±80.5 and 4E8 of 0.33μg/mL group:IFN-γsecretion 1957±321.3) as compared with 4E8 alone (10μg /mL group:IFN-γse-cretion 2563±150.3 and 0.33 μg/mL group:IFN-γ secretion 844±76.2) with statistically significant difference (P<0.05). Conclu-sion We successfully obtained a 4E8 clone of monoclonal antibody to human TIM-3 which may enhance the capacity of IFN-γsecre-tion from CD4+ T cells. The effect of enhancing IFN-γ secretion of CD4+T cells by TIM-3 monoclonal antibody was independent from blocking the binding of TIM-3 with Ptdser.
ABSTRACT
Objective To clone the full length staphylococcal protein A(SPA) gene from Staphylococcus aureus (ATCC6538), and subsequently study the gene structure and antibody binding ability.Methods The full length and the functional region of the SPA gene were cloned into pHisSUMO vector respectively, and expressed in E. coli. The full length and the functional fragment of the SPA protein were detected for antibody binding ability and stability. The functional fragment of the SPA protein fused with SUMO was coupled to the CNBr-activated agarose for antibody purification from rabbit serum. Results A variant of the full length SPA gene was cloned, which has been submitted to GenBank (the accession number is EU695225). Two fusion proteins had the same antibody binding ability as the untagged SPA protein. However, the formers was more stable than the latter at the tested conditions. SUMO-SPA conjugated-agarose kept high efficiency for antibody binding. Conclusion To our knowledge, the full length SPA gene of S.aureus(ATCC6538) is a novel variant. The SUMO tag can improve the stability of the functional region of the SPA protein without damaging the antibody binding ability. This fusion protein has been used for antibody purification successfully.
ABSTRACT
Ever from FDA approved the Rituxan in 1997,an Anti-CD-20 chimeric antibody,there have been more than 20 antibodies approved and they have got into more than 56 countries.About half approvied antibodies are curing kinds of cancers and seven of them have got a excellent sale targets to be "Blockbuster Drug".As the cell culture technology improving,the large scale cell expression titer comes from 1g/L to 5g/L.At the same time,the culture scale is coming form 12,000L to 15,000L,even 20000L and it can run several bioreactors at the same time.All these are make the improvement of the antibody purification.The new edition of media,system,chromatography technology and filtration technology can product the protein to several tens kilogram antibody per cycle and tons per year,although the productivity is only several kilograms per cycle many years ago.Also the new combined technology makes a two-step chromatography production protocol come into been.This will highly improve the antibody productivity and save the investment cost.
ABSTRACT
Glechoma hederocea agglutinin (Gleheda) is a novel glycosylated lectin isolated from the leaves of G. hederacea. Like other glycosylated proteins, the detection of Gleheda by immunological methods is often hampered by the cross-reactivity of the polyclonal antibodies with unrelated glycoproteins. Hence a protocol to purify monospecific polyclonal antibodies from a crude antiserum raised against Gleheda was developed. After selective ammonium sulfate precipitation and successive affinity chromatography on columns of Sepharose 4B with immobilized Gleheda and Robinia pseudoacacia agglutinin (RPA), respectively, ion-exchange chromatography on a column of Q Fast Flow was used for further purification. The specificity of the antibody fractions from each step was tested by double immunodiffusion assay and analyzed by Western blot. Results revealed that affinity chromatography of the immunoglobulin fraction on the immobilized Gleheda antigen yielded an antibody preparation that still cross-reacted with many proteins in leaf extracts. Depletion of nonspecific cross-reacting antibodies directed against the glycan part of the glycoprotein by affinity chromatography on immobilized RPA removed most but not all nonspecifically reacting antibodies. Only upon further purification by ion exchange chromatography an IgG fraction of monospecific antibodies that reacted exclusively with Gleheda could be obtained and accordingly was suitable for immunodetection studies. This antibody purification procedure promises simplicity and efficiency. In addition, this method does not require expensive facilities.