Screening of human anti-TNF-alpha scFv from large phage library

AIM: To clone human anti-TNF-alpha scFv from large phage antibody library. METHODS: Panning of large phage library against TNF-alpha was conducted to select specific antibodies. The antigen binding activity and DNA sequence were determined and analyzed by ELISA, restriction enzyme map. RESULTS: After 4 rounds of panning, 62 positive clones were obtained and 27 clones had specific binding ability to TNF-alpha. DNA fingerprinting of the 27 clones showed 7 different stripes indicated 7 different positive clones. 5 of the 7 clones expressed soluble anti-TNF-alpha activity. DNA sequence analysis showed that the variable regions of these scFvs belonged to different subgroups. CONCLUSION: Human TNF-alpha scFv have been successfully obtained from large phage antibody library.     

奥美拉唑对家兔肾脏内髓集合管细胞H+/K+交换功能的影响

目的探讨奥美拉唑对家兔肾脏IMCD细胞H+/K+交换的影响,以及经洗涤处理是否解除这种影响.方法原代培养家兔肾脏IMCD细胞单层,在100 μmol/L奥美拉唑缓冲液中孵育25 min, 洗涤组则在奥美拉唑缓冲液孵育后,用不含奥美拉唑的缓冲液洗涤IMCD细胞;对照组在不含奥美拉唑的缓冲液孵育25 min;CECF/AM荧光探针法测定各组IMCD细胞H+/K+交换.结果奥美拉唑组的H+/K+交换为(0.016±0.006) dpHi/min(n=8);与对照组的(0.053±0.008) dpHi/min(n=8)相比,相差非常显著(P<0.001);洗涤组的H+/K+交换为(0.016±0.006) dpHi/min(n=6),与对照组(n=6)的(0.052±0.009)dpHi/min相比,相差非常显著(P<0.001).结论 100 mol/L的奥美拉唑对家兔IMCD细胞的H+/K+交换有显著影响,而且洗涤处理不能解除这种抑制.因而,肺心病合并上消化道出血患者使用奥美拉唑时,应综合考虑其利弊.     

Identification of a new HLA-B*56 variant, B*5614

In this report, the novel allele B*5614 is presented. The allele was identified in a Chinese individual by sequence-based typing. HLA-B*5614 differs from B*5608 by a single nucleotide at position 277G-->C in exon 2. This results in an amino acid change from Gly to Arg at codon 93.     

Application of Serum-Free Culture Medium for Preparation of A-NK Cells

To compare the differences between proliferation and cytotoxicity of adherent natural killer （A-NK） cells cultured with serum-free medium AIMV and standard serum-containing medium in vitro, and also observe the assisting effect of IL-12 on the activation and the morphology character of IL-2-treated A-NK cells, cellular proliferation was evaluated by MTT method in vitro. The morphology of the target cells killed by A-NK cells was observed through electroscope. All of the A-NK cells cultured in serum-free medium AIMV could rapidly proliferate and keep high cytotoxicity compared with that in standard serum-containing medium. A-NK cells activated by both moderate-dose IL-2 and IL-12 were superior to the high-dose IL-2-treated A-NK cells. These data indicated that serum-free medium AIMV could replace standard serum-containing medium for culturing A-NK cells, and moderate-dose IL-2 and IL-12 could reduce side effects caused by high-dose IL-2. The study provided a new experimental basis for experimental and clinical preparation of A-NK cells. Cellular ＆ Molecular Immunology.     

Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus

There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103 mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.