Preparation and selection of the monoclonal antibody used for kits to detect BNP32 which was applied to clinical research

目的:制备抗脑钠肽(bnP32)单克隆抗体(MAb),并利用双抗体夹心ElISA法建立bnP抗原检测技术,应用于临床心脏患者脑钠肽水平的检测。方法:以基因工程原核重组表达bnP抗原免疫bAlb/C小鼠,利用常规杂交瘤技术制备MAb,MAb经纯化和HrP标记后,利用双抗体夹心ElISA法筛选检测bnP32蛋白的最佳配对MAb,以其建立bnP32抗原检测技术,并与临床bnP检测的标准实验做平行比较。结果:成功筛选到16株稳定分泌抗bnP32MAb的杂交瘤细胞株,16株MAb的亚型分别为Igg1、Igg2A和IgM,并从中筛选出最佳MAb配对组合,该组合对bnP32蛋白的检测灵敏度为20ng/l。建立的双抗体夹心bnP检测ElISA法与临床bnP检测的标准实验平行比较具有很好的一致性(kAPPA值=0.828),两者没有统计学意义(P>0.05)。结论:成功地建立了bnP32抗原的双抗体夹心ElISA法检测技术,并能够很好地运用于临床心衰患者bnP指标的检测。AIM:To prepare the monoclonal antibody(mAb)used for kits to detect the BNP32 antigen by means of double-antibody sandwich ELISA assay.Comparasion differences on detection BNP between double-antibody sandwich ELISA and Roche ECL.METHODS:BALB/c mice were immunized with purified recombinant BNP32 protein and by routine hybridoma technique,Then comparasion differences on detection BNP between double-antibody sandwich ELISA and Roche ECL.RESULTS:16 hybridoma cell lines secreting potent mAb against BNP32 were obtained.The subtype of these 16 mAb were found to be IgG1,IgG2a and IgM,then their cross-blocking properties were analyzed,when they reacted with the BNP32 protein in direct ELISA in order to offer the valuable data for selecting feasible pair of mAb in the detection of the BNP32 antigen.All the mAbs were used for the detection of BNP32 by double-antibody sandwich ELISA.In addition,the mAbs were purified and HRP-labelled in advance.Finally,we had successfully screened a pair of mAbs,which exhibited a sensitivity of 20 ng/L for the detection of BNP32 antigen.The two detection methods have very good consistency(kappa=0.828>0.75)between double-antibody sandwich ELISA and Roche ECL.There was no statistics significance on differences between these two methods(P>0.05).CONCLUSION:It is evident that this pair of mAb shows excellent detection of BNP32.The double-antibody sandwich ELISA can be good apply to detect BNP level in clinical congestive heart failure patients

人乳头瘤病毒假病毒制备方法的优化

人乳头瘤病毒假病毒可广泛应用于中和抗体鉴定和疫苗免疫性评估等研究.本研究对多质粒共转染293FT细胞生产HPV假病毒的方法进行了深入优化,结果显示不同的HPV结构蛋白表达质粒转染比例对于假病毒生产效率有显著的影响,HPV主要结构蛋白L1的表达水平是影响生产效率的主要因素,L2表达质粒的用量过高或过低均不利于假病毒的生产,而报告质粒的用量对假病毒生产效率的影响相对较小.综合比较显示,在该体系中L1和L2表达质粒和报告质粒以合适比例(如1∶1/10∶1/2)进行共转染可获得更为理想的生产效率.本研究同时也在293FT细胞中对磷酸钙转染方法进行了优化,通过磷酸钙与质粒用量的交叉配比试验获得了较优的转染条件.通过优化,建立了更为高效的HPV假病毒大量制备方法,在HPV16、HPV18、HPV6、HPV11假病毒生产中的应用结果显示较原方法可显著提高生产效率.本研究为HPV假病毒中和实验的规模化应用提供了有利条件

Assessment of single-dose regimen for antimicrobial prophylaxis to prevent perioperative infection in urologic surgery

2007年1年間に施行した尿路内視鏡手術114症例, 清潔手術92症例, 準清潔手術20症例に対し, 周術期感染症阻止薬の術前単回投与を行い, 手術部位感染(SSI), 尿路感染(UTI), 遠隔感染(RI), 術後合併症の発生頻度を前方視的に調査した。その結果, 尿路内視鏡手術ではUTI発生率が2.7%で, RIは認めなかった。清潔手術での発生率はSSI:1.1%, RI:2.2%, UTI:0%, 準清潔手術ではSSI:5%, RI:0%, UTI:5%であった。よって, 泌尿器科領域における尿路内視鏡手術, 清潔手術, 準清潔手術に対する周術期感染症予防抗菌薬の効果は, 術前単回投与で十分に期待できると考えられた。A single-dose ofantimicrobial prophylaxis (AMP) was administered parenterally for prevention of perioperative infection in a total of 206 urologic surgeries including 114 endoscopic-instrumental, 92 clean, and 20 clean-contaminated procedures between January and December, 2007, and surgical site infections (SSI), urinary tract infections (UTI), and remote infections (RI) were prospectively surveyed. The definition of a single-dose of AMP allowed the administration of a second dose of an antimicrobial during a surgical procedure that exceeded 3 hours but not parenteral or oral administration at the end of the procedure, in the recovery room, or at a later time over a period of more than 24 hours. UTI was observed in 3 cases (2.7%) after endoscopic-instrumental procedures and in 1 case (5%) after clean-contaminated procedures while no case was associated with UTI in clean procedures. SSI was seen in 1 case each after clean procedures (1.1%) and after clean-contaminated procedures (5%), and RI was seen in 2 cases (2.2%) after clean procedures. A single-dose regimen of AMP was effective for prevention ofperioperative infections including SSI, UTI, and RI in endoscopic-instrumental, clean, and clean-contaminated surgical procedures in urology