重组人促红细胞生成素抑制内皮细胞凋亡的实验研究

目的：探讨重组人促红细胞生成素（Recombinant human eryfhropoietin,rHuEPO)对脂多糖（Lipopolysaccharide,LPS)诱导的人脐静脉内皮细胞（Human umbilical vein endotheilial cells,HUVECs)凋亡的影响。方法：实验分为四组;第一组仅用LPS处理;第二组预先用rHuEPO孵育后再用LPS处理;第三组只用rHuEPO处理;对照组用无血清的M199培养。用流式细胞仪DNA分析检测细胞凋亡。结果：与对照组相比,LPS处理后的细胞DNA亚二倍体含量显著增加（P＜0．05）,预先用rHuEPO处理后则可显著降低LPS的这种作用（P＜0．05）。结论：rHuEPO抑制了LPS诱导的内皮细胞凋亡,这种保护作用可能是rHuEPO诱导内皮细胞生长和血管生成的一个重要因素。     

重组人红细胞生成素单克隆抗体的制备

本文应用常规淋巴细胞杂交瘤技术制备了４株能稳定分泌抗人重组红细胞生成素（ｒＨｕＥＰＯ）单克隆抗体（ＭｃＡｂ）的小鼠杂交瘤细胞系ＢⅡ１Ｂ５、ＤⅡ６Ｂ９、ＭⅡ１Ｈ４和ＧＩ３Ｅ７。用鼠单克隆抗体分型试剂盒鉴定，其分泌的ＭｃＡｂ的类分别是ＩｇＭ、ＩｇＭ、ＩｇＧ１和ＩｇＧ２ａ。间接ＥＬＩＳＡ法测定细胞上清的效价为１×１０－２～１．２５×１０－４，腹水效价为１×１０－２～１×１０－８。培养上清经ＥＬＩＳＡ鉴定，与ＩＬ－２、ＧＭ－ＣＳＦ、ＩＦＮ－α等细胞因子均无交叉反应，只与ｒＨｕＥＰＯ特异性结合。     

单克隆抗体亲和层析一步纯化重组人生长激素

单克隆抗体亲和层析技术可用来从体液、组织抽提液、细胞培养液中,特别是从基因工程菌体破碎液等复杂组成物中分离出微量蛋白质和多肽.由于其特异性高,操作简便而迅速,在高价值蛋白质和治疗用多肽的下游处理过程中,作为一种良好的分离纯化工具,有较广泛的适用性.本文运用该技术从人生长激素(hGH)基因工程菌发酵破碎液中一步分离制备高纯度并具有生物活性的重组hGH.     

重组人促红细胞生成素治疗胃肠道恶性肿瘤化疗相关性贫血的疗效观察

目的 观察使用重组人促红细胞生成素治疗胃肠道恶性肿瘤化疗相关性贫血的临床效果。方法 选择我院2017年8月~2018年8月收治的80例胃肠道恶性肿瘤化疗相关性贫血患者作为研究对象,随机分为对照组和观察组,各40例。对照组患者进行常规治疗,观察组在此基础上采用重组人促红细胞生成素治疗,比较两组的临床疗效、并发症发生率,记录两组治疗前后血红蛋白、血细胞比容等指标和患者生活质量评分情况。结果 观察组治疗总有效率高于对照组,差异有统计学意义（P＜0.05）;观察组血红蛋白、血细胞比容、血细胞计数和血小板计数水平均高于对照组,差异具有统计学意义（P＜0.05）;观察组患者生活质量评分高于对照组,差异具有统计学意义（P＜0.05）;两组患者的不良反应发生率比较,差异无统计学意义（P＞0.05）。结论 重组人促红细胞生成素治疗胃肠道恶性肿瘤化疗相关性贫血的效果较好,可显著提高患者的临床疗效,改善患者贫血状况,提高其生活质量。     

重组人促红细胞生成素治疗预防早产儿支气管肺发育不良的效果分析

目的:研究重组人促红细胞生成素(Recombinant human erythropoietin,rhEPO)对早产儿支气管肺发育不良(Bronchopulmonary dysplasia,BPD)的预防效果.方法:选取我院新生儿科2018年1月至2020年1月收治的早产儿80例,随机分为实验组和对照组(n=40).对照组患儿口服复方硫酸亚铁颗粒(5mg?kg-1)纠正贫血,实验组在此基础上于出生72 h后皮下注射rhEPO(375 U?kg-1),2次?w-1.治疗4 w后,比较两组患儿治疗前后支气管肺发育不良的发生率、住院时间及呼吸机治疗时间的差异.结果:实验组患儿治疗4 w后支气管肺发育不良的发生率、住院时间和呼吸机治疗时间均明显低于对照组(P<0.05).结论:早期给予重组人促红细胞生成素能够预防早产儿支气管肺发育不良的发生,缩短呼吸机治疗时间及住院时间.     

重组人促红细胞生成素对小鼠胚胎干细胞向心肌细胞分化的影响

目的初步观察外源性细胞因子重组人促红细胞生成素（NEPO）对小鼠胚胎干细胞（ESC）向心肌细胞分化的影响。方法将NEPO加到细胞培养基中配成终浓度分别为0．5、1．O、5．0U／ml的诱导分化液，分别在分化第8、12天观察ESC自发及在rhEPO诱导情况下心肌细胞分化率；在分化第7天应用RT-PCR检测自发分化组及NEPO诱导组心肌早期转录因子GATA-4、Nkx2．5mRNA的相对表达。结果在分化第8、12天rhEPO诱导（1．0U/ml）心肌细胞分化率分别显著高于对应分化天数的自发心肌细胞分化率（0．11±0．02对0．05±0．01，P〈0．05；0．86±0．02对0．65±0．05，P〈0．05）；与自发分化组比较．NEPO干预（1．0U／ml）上调GATA-4、Nkx2．5mRNA的相对表达。结论外源性rhEPO早期干预能够促进ESC向心肌细胞分化。     

重组人红细胞生成素治疗时产生的高血压症

高血压是重组人红细胞生成素（ｒｈＥｐｏ）治疗尿毒症贫血过程中的主要并发症，其发生率与ｒｈＥｐｏ的给药方式及剂量有关。导致高血压的主要机制是外周血管阻力增加。其预防措施有加强监测、合理选择给药剂量及方式、控制Ｈｂ及红细胞比容（ＨＣＴ）的升高速率及目标值以及药物预防，其治疗方法为充分透析达干体重状态、抗高血压药物治疗、减少ｒｈＥｐｏ用量及停药。     

人肺癌相关单克隆抗体的制备及其抗原的纯化

目的：研制抗人肺癌细胞（A549）单克隆抗体，并对其所识别的抗原进行免疫亲和层析纯化。方法：以人肺癌细胞系A549免疫BALB／c小鼠，常规融合，以间接ELISA筛选，免疫组化研究单克隆抗体的特性，通过免疫亲和层析纯化所识别相关的抗原。结果：成功获得了一株能够稳定分泌抗人肺癌相关抗原单克隆抗体的杂交瘤细胞株289，并提取了其识别的相关抗原。结论：2139单克隆抗体及其所识别抗原有可能进一步用于肺癌的实验室诊断。     

亲和层析纯化重组人源性抗HBsAg Fab抗体

目的：纯化酵母发酵产生的重组人源性抗HBsAg Fab抗体，建立稳定、适于生产应用的纯化工艺。方法：以原核表达的重组人源性抗HBs scFv免疫BALB／c小鼠，经杂交瘤技术制备大量分泌mAb的杂交瘤细胞株14F7。将该细胞株注入小鼠腹腔制备mAb腹水，经辛酸沉淀纯化后，制备亲和层析柱。纯化酵母发酵产生的重组人源性抗HBsAg Fab抗体。结果：用抗scFv mAb亲和层析柱纯化的重组人源性抗HBsAg Fab的纯度可达95％以上，回收率可达70％～85％。结论：人源性抗HBs scFv制备mAb作为亲和层析的介质，可有效地从酵母发酵上清中纯化重组人源性抗HBsAg Fab，适用于大规模生产重组人源性抗HBsAg Fab。     

阳离子交换一步层析法纯化抗gp130单克隆抗体

目的:建立从小鼠腹水中纯化抗gp130单克隆抗体(mAb)B-S12的一步层析方法。方法:小鼠mAb腹水样品经离心后,进行阳离子交换层析柱纯化。检查了上样缓冲液的pH值和洗脱液的离子强度梯度对mAb纯度的影响。纯化后mAb的生物学活性用MTT比色法检测。结果:在pH4.0、20mmol/LHEPES缓冲液条件下上样,用0～1.0mol/L的NaCl梯度洗脱,可获得纯度超过90%的mAbB-S12,回收率为52%。纯化后的mAb对XG-2细胞有明显的促增殖作用。结论:建立的一步纯化方法操作简便,所得mAb的纯度高及生物学活性好。     

Purification of biologically active rubella virus antigens by immunoaffinity chromatography

A general procedure for isolating biologically active rubella virus antigens (VPI, Mr = 61,000; VP2, Mr = 45,000; VP3, Mr = 36,000) by monoclonal antibody affinity chromatography is described. Complexes formed between monoclonal antibodies and rubella virus antigens were found to be stable either at low pH or in Tris buffer containing detergent and high salt, but were efficiently dissociated by 5% diethanolamine, pH 11.5, or 50 mM lithium diiodosalicylate buffer, pH 8.0. Chromatographically purified rubella viral antigens retained their antigenicity as determined by enzyme-linked immunosorbent assays. Biological studies showed that rubella structural proteins VP2 and VP3 had no hemagglutinin function while the mixture of VP1 and VP2 and VP3 directly demonstrated hemagglutination activity. These results indicate that VP1 is at least in part responsible for the hemagglutinin function of rubella virus.     

重组人源细胞珠蛋白单抗制备及检测方法建立

目的制备重组人源细胞珠蛋白（recombinanthumanCytoglobin,rhCygb）单克隆抗体,并建立检测该蛋白双抗体夹心ELISA法,为下一步研究rhCygb药代动力学做准备。方法用纯化的rhCygb免疫BALB/c小鼠,采用甲基纤维素半固体培养基法获得抗rhCygb的单克隆抗体杂交瘤细胞,间接ELISA法筛选制备单克隆抗体,建立双抗体夹心ELISA法。结果筛选获取了稳定分泌单克隆抗体的杂交瘤细胞株,通过抗原表位相加法实验获得5株表位不同的细胞株,Western-blotting验证能与rhCygb特异性结合,间接ELISA法验证其不与本实验室制备的其它PET28a-BL21蛋白及BL21裂解液发生交叉反应。本方法灵敏度为1.25ng/ml,在浓度为10～1250ng/ml时,线性关系良好,相关性达0.9931,实验内和实验间平均变异系数分别为6.2%和10.92%。结论成功建立了灵敏度好、特异性高的双抗体夹心法,为下一步研究rhCygb药代动力学奠定了基础。     

Development and characterisation of an immunoaffinity column for the selective extraction of methandrostenolone from food and feed samples

Methandrostenolone (MA) is used as veterinary medicine for promoting food-producing animal growth. Accumulation of MA in a human body would cause multiple side effects. In this study, an anti-MA monoclonal antibody (mAb)-based immunoaffinity column (IAC) was prepared and its application to the selective extraction of MA from actual samples was evaluated. The employed antibody exhibited good sensitivity and specificity towards MA with an IC₅₀ value of 7.89 ng/mL and less than 1% cross-reactivity towards structurally related compounds. By coupling CNBr-activated Sepharose with the antibody, an IAC was prepared. Two percent methanol and 70% methanol were selected, respectively, as loading and eluting solution by optimisation. The maximum binding capacity of the column for MA reached 4760 ng/mL gel. The average recovery of 50, 250 and 500 ng MA standards from IACs was 88.6% with a relative standard deviation (RSD) among columns of 8.5%. After three times usage, the binding capacity and recovery still remained 93.4% and 81.1%, respectively. To further verify the effect of matrices on the extraction efficiency, the IACs were challenged with MA-fortified animal tissue and feed samples and the recovery were found to be in the range of 78.7%–95.1%.     

Development and application of an immunoaffinity column clean-up for enrofloxacin determination in food samples

Enrofloxacin (ENR) is a synthetic fluoroquinolone antibiotic used for the treatment of infection in animals. The use of ENR has been strictly regulated because of its harmful effect on consumers of animal products. In this study the generation of polyclonal antibody (pAb) against ENR, the preparation of an immunoaffinity chromatography column and its potential application to the selective extraction of ENR residues from food samples were described. The produced pAb exhibited good sensitivity to ENR with an IC₅₀ value of 5.4?ng/mL. The cross-reactivity values of the antibody with ENR structurally related compounds were lower than 0.5%. An immunoaffinity column was constructed by coupling antibody covalently with chitosan microspheres. 0.8% NaOH/0.01?mol/L phosphate-buffered saline (PBS) (pH 7.4) and 85% methanol were selected as loading and eluting solution by careful optimization. Further characterization indicated that the binding capacity of the column for ENR was approximately 3780?ng/mL gel. The immunoaffinity columns were then challenged with ENR-fortified pork, and the recoveries of ENR were found to be in the range of 79.8–94.9%, demonstrating the feasibility of the prepared immunoaffinity column for sample clean-up in ENR residue determination.     

结肠癌相关抗原的制备及其临床意义

目的从培养的结肠癌细胞LOVO中纯化结肠癌相关抗原,探讨其在结肠癌患者血清中的表达。方法裂解结肠癌细胞LOVO,以抗结肠癌相关抗原单克隆抗体(mAb)4D10作为配基进行亲和层析纯化结肠癌相关抗原,SDS-PAGE电泳和Western blot进行鉴定,并用双抗夹心ELISA法检测该相关抗原在结肠癌患者及正常人血清中的表达。结果纯化所获与4D10特异结合的结肠癌相关抗原,其相对分子质量(Mr)约为65000,该抗原由Mr约为30000和35000两种亚基组成。人血清检测实验表明,该抗原在结肠癌患者血清中有较高的表达,与正常人血清相比存在显著的统计学差异(P<0.01)。结论利用mAb4D10进行免疫亲和层析,从结肠癌细胞LOVO中获得纯化的肿瘤相关抗原,该抗原有可能在临床上为结肠癌的诊断提供一定的参考价值。     

Rapid and efficient purification of mouse monoclonal antibodies from ascites fluid using high performance liquid chromatography

Techniques for the rapid and efficient purification of mouse monoclonal antibodies from murine ascites are described that utilize anion exchange and gel permeation chromatography using high performance liquid chromatography (HPLC). Anion exchange chromatography was performed at neutral pH using a hydrophilic resin conjugated with a substituted amine (Mono Q column, Pharmacia Fine Chemicals). Various subclasses of mouse IgG monoclonals were assessed for their binding to this matrix, with all of the antibodies tested eluting at relatively low concentrations of sodium chloride. In some instances, a protein tentatively identified as transferrin was co-purified using this anion exchange procedure. However, this protein was easily removed from the IgG using gel permeation chromatography (Bio-Sil TSK-250, Bio-Rad), also performed at neutral pH.     

One-step purification of mouse monoclonal antibodies from ascites fluid by hydroxylapatite chromatography

A single-step method for purification of mouse monoclonal antibodies directly from ascitic fluids using hydroxylapatite column chromatography is described. The procedure yields highly purified IgG or IgM antibodies. The purified immunoglobulin is essentially free of contaminating mouse albumin, transferrin, and other ascites proteins, as determined by SDS-polyacrylamide gel electrophoresis. Hydroxylapatite chromatography can also separate monoclonal IgG antibodies from contaminating IgG antibodies found in ascites fluid of animals that have been immunosuppressed prior to ascites induction. Furthermore, the evidence presented here suggests that some hybridomas of SP2/0 origin synthesize an extraneous light chain resulting in the secretion of hybrid antibody molecules.