抗H7N9流感病毒中和抗体快速检测方法的建立及应用

目的：对建立的抗H7N9流感病毒中和抗体快速检测方法进行方法学验证及初步应用。方法：分别采用不同代次细胞对高、中、低不同滴度的阳性血清进行多次平行检测,考察细胞代次对检验结果的影响;采用NIBSC提供的参考品对方法学的特异性进行验证;同时应用抗H7N9的阳性血清检测进一步评估方法的准确性和精密性;采用ELISA-MNT法和血凝抑制（hemagglutination inhibition,HI）试验分别接种H7N9灭活流感疫苗免疫的小鼠血清样本20份,分析两种方法检测结果的相关性。结果：采用ELISA-MNT中和法,使用不同代次MDCK细胞（25、30和35代）检测相同的血清样本的中和抗体滴度结果相同;该方法只对羊抗H7N9的血清具有较高保护力,对其他血清基本没有交叉反应;该方法准确性良好;该方法组间、组内精密性的平均变异系数分别为4%和11%。该方法测定H7N9型流感疫苗免疫后的小鼠血清抗体效价,其结果与HI抗体的相关系数为0.61,表明两种方法的检测结果之间呈良好的正相关性。结论：建立的微量病毒中和法能够满足H7N9流感病毒中和抗体效价检测的要求,可用于H7N9新型大流行流感疫苗的免疫评价。     

快速荧光灶抑制试验测定的人抗狂犬病免疫球蛋白对狂犬病主动免疫的抑制作用

以往报道快速荧光灶抑制试验(RFFIT)可代替小鼠中和试验(MNT)检测狂犬病免疫血清(RIS)和人狂犬病免疫球蛋白(HRIG),两法结果是可比的。近来文献相继报道RFFIT检测结果要比MNT法低2～10倍。如用RFF-IT测抗体,那么实际使用时HRIG用量将要比常规推荐量大2～10倍,会干扰接种疫苗者的抗体生成。最近,由于全欧药典委员     

小鼠中和试验与快速荧光灶抑制试验检测狂犬病抗体结果的比较

作者用标准的小鼠中和试验(MNT)和快速荧光灶抑制试验(RFFIT)测定了16批狂犬病免疫球蛋白(RIG)和6批狂犬病免疫马血清(RIS)中的抗体. 按Atanasiu法作MNT,市售RIG岔量为150IU/ml,用两倍稀释法以1∶500稀释至1∶16,000.市售RIS含量为200IU/ml,由1∶750稀释至1∶24,000.狂犬病标准马血清WS1从0.2IU/ml稀释至0.0125IU/ml.给10只NMRI小鼠脑内接种各个稀释度的病毒-抗体混合物,每只0.03ml,同时接种等量的CVS狂犬病病毒.     

狂犬病病毒中和单抗表位鉴定和中和广谱性评价研究进展

目的：对狂犬病病毒（RABV）中和单抗的研发进展及其表位和中和广谱性研究评价进行综述，为行业提供参考。方法：通过文献检索，对国内外开展的狂犬病病毒中和单抗研究进行梳理和汇总。结果与结论：中和抗体在狂犬病暴露后预防中发挥着重要的作用，目前国内外已有多个RABV单抗品种上市并有多个品种处于临床评价阶段。RABV具有较高的突变频率且感染发病后致死率极高，为了最大程度地中和自然流行的RABV病毒株，以防止感染后狂犬病的发生，在单抗候选药物研发时应对其表位和广谱性进行充分的研究评价。     

献浆员中新型冠状病毒SARS-CoV-2中和抗体检测分析

目的： 静注人免疫球蛋白（IVIG）和新冠肺炎恢复者的血浆已被推荐用于重症新冠肺炎患者的治疗。因此,血浆中新型冠状病毒（SARS-CoV-2）中和抗体水平需要得以及时评价。本研究利用不需要在生物安全三级实验室操作的SARS-CoV-2假病毒中和抗体检测方法,建立了快速检测中和抗体的策略。方法： 采用新型冠状病毒SARS-CoV-2假病毒,对1080份健康献血员血浆及26批次IVIG的SARSCoV-2中和抗体进行检测。初筛采用1：30倍稀释检测;对初筛阳性（抑制率≥ 50%为阳性）的样本进行复试,复试采用1：30、1：90和1：270三个梯度;对于复试阳性的样本进行中和特异性分析,进一步检测针对水疱性口炎病毒（VSV）、严重急性呼吸道综合征冠状病毒（SARS-CoV）和中东呼吸综合征冠状病毒（MERS-CoV）的中和活性。结果与结论： 在1：30倍稀释时,26批次IVIG的SARS-CoV-2中和抗体均为阴性,1080份献血员血浆中SARS-CoV-2中和抗体阳性率为0.6%（6/1180）;对6份阳性血浆的特异性分析显示均为对VSV、SRAS-CoV或MESR-CoV假病毒的非特异中和作用。本研究表明该假病毒中和抗体检测方法和快速检测策略可以用于IVIG、健康献血员和感染者恢复期血浆中的中和抗体检测,发现IVIG针对SARS-CoV-2无特异的中和抗体活性,IVIG在临床重症治疗中发挥的是非病毒特异的辅助治疗作用。     

联合酶联免疫的微量病毒中和法检测大流行流感疫苗血清中和抗体的可行性研究

目的:建立联合酶联免疫的微量病毒中和法(ELISA-MVN法),用于大流行流感疫苗流感病毒中和抗体效价的检测,并进行方法学验证。方法:采用4种羊抗流感病毒血清参考品,与人感染禽流感H5N1病毒疫苗株(A/Vietnam/1194/2004-NIBRG-14)病毒液,分别于37℃孵育18 h后,固定细胞,ELISA检测细胞内流感病毒核蛋白表达,确定其中和抗体效价,考察该方法的特异性;通过对高中低不同抗体滴度的血清样本的多次平行检测来评价该方法的重复性;通过ELISAMVN法和血凝抑制试验(HI试验)分别检测大流行流感疫苗接种者血清样本,比较2种方法的灵敏性和相关性。结果:ELISA-MVN结果显示羊抗H5N1的血清中和抗体检测滴度为5 120,其他3种血清的中和抗体滴度小于10,该方法特异性良好;采用ELISA-MVN法对3种不同滴度的血清进行重复检测,组内重复性试验的6次平行试验结果的RSD为3.5%。组间重复性5次结果,RSD为4.8%~8.6%。2种方法测定疫苗接种者血清样本抗体效价的结果具有显著相关性(P<0.001),相关系数r=0.932。ELISA-MVN法检测结果的几何平均滴度高于HI试验检测结果,该方法较HI试验在检测大流行流感疫苗血清样本灵敏度高。结论:ELISA-MVN可满足大流行流感疫苗血清学检测的要求,并可用于评价大流行流感疫苗的免疫效果。     

用双抗夹心ELISA法快速检测灭活乙型脑炎疫苗的效价

目的：用单克隆抗体制备的双抗夹心ELISA法快速检测灭活乙型脑炎疫苗中的乙型脑炎病毒包膜蛋白（E）的含量,并探讨其替代蚀斑减少中和试验法的可行性。方法：将疫苗样品用蚀斑减少中和试验法测定,同时选定一批疫苗为参考疫苗,用双抗夹心ELISA法检测。结果：用双抗夹心ELISA法测得的中和指数（TE）为1.595-1.655。用蚀斑减少中和试验法测得的中和指数（TP）为1.367-1.932。经F检测,两种检测方法的灵敏度有极显著差异（F=56.25,P〈0.01）,用t检验法对这两种检测方法进行统计分析,表明两组测定值的差异无统计学意义（t=0.079 3,P〉0.05）。结论：双抗夹心ELISA法与蚀斑减少中和试验法测得的中和指数（T）呈正相关性;与蚀斑减少中和试验法比,双抗夹心ELISA法具有重复性好、成本低、快速等优点。用双抗夹心ELISA法取代蚀斑减少中和试验法测灭活乙型脑炎疫苗效价是可行的。     

肠道病毒71型假病毒荧光定量检测方法在疫苗中和抗体检测中的应用

目的假病毒荧光定量方法（PVLA）应用于EV71疫苗动物样本和人体样本检测的适用性研究。方法分别采用细胞病变法（CPE）和PVLA方法检测82份EV71试验小鼠血清以及27份疫苗临床试验人体血清样本,比较两者检测的一致性。结果应用两种方法检测82份疫苗免疫小鼠血清,EV71中和抗体滴度的相关系数为0．842,Bland—Altman分析显示高度一致性;检测27份EV71临床试验人体血清样本的中和抗体,两种方法具有高度一致性。结论PVLA与CPE方法具有高度一致性,适用于疫苗免疫动物样本和临床评价人体样本EV71中和抗体的检测。     

RFFIT法血清系列参考品的建立

目的建立血清参考品,用于RFFIT法检测抗狂犬病毒中和抗体。方法狂犬病疫苗免疫后人血清,采用小鼠脑内中和试验确定抗狂犬病毒中和抗体滴度后,混匀,分装,以第2批人免疫球蛋白国际标准品为参考品用RFFIT方法进行标定,并考察所建参考品的稳定性。结果检测用标准血清效价为22.14IU.mL-1,弱阳性、强阳性参考品分别1.11IU.mL-1,9.20IU.mL-1,阴性参考品低于0.1IU.mL-1。结论建立的系列参考品适用于RFFIT法检测人血清中抗狂犬病中和抗体水平。     

抗柯萨奇病毒A组10型中和抗体检测用毒株的应用研究

目的 研究抗柯萨奇病毒A组10型（Coxsackievirus A10,CV-A10）中和抗体检测用毒株的应用。方法 对CV-A10毒株进行扩增培养,建立3级种子批并进行相关检定。采用3人3次独立测定病毒滴度,对工作种子批进行滴度标定,通过分别与肠道病毒71型（enterovirus A71,EV-A71）、CV-A16、CV-A6免疫血清进行交叉中和反应,评价该毒株的专属性。对CV-A10自然感染人血清和CV-A10小鼠免疫血清样品进行中和抗体效价检测,对其应用进行评价研究。结果 获得一株CV-A10中和抗体检测用毒株,3级种子批的无菌检查、支原体检查等项目均符合中国药典2020年版三部相关要求;经标定,平均病毒滴度为7.903 lg半数细胞培养物感染量（50% cell culture infectious dose,CCID₅₀）/ml（95%置信区间：7.868~7.937 lgCCID₅₀/ml）,变异系数为1.10%;与EV-A71、CV-A16、CV-A6免疫血清无交叉反应,专属性良好;检测CV-A10自然感染人血清和CV-A10小鼠免疫血清,中和抗体的最大值与最小值倍数差均小于4,中和抗体检测变异系数分别为6.38%和3.64%。结论 抗CV-A10中和抗体检测用毒株具有较好的专属性可很好的应用于后续抗CV-A10中和抗体的相关检测。     

Neutralization of heparin activity

Heparin is the mainstay in the treatment and prevention of thrombosis in such diverse clinical settings as venous thromboembolism, acute coronary syndrome, cardiopulmonary bypass, and hemodialysis. However, the major complication of heparin - like that of all anticoagulants - is bleeding. Heparin may need to be reversed in the following settings: clinically significant bleeding; prior to an invasive procedure; at the conclusion of a procedure involving extracorporeal circulation (e.g., cardiopulmonary bypass, dialysis). This chapter discusses protamine sulfate, as well as several other agents that are able to neutralize heparin, including their pharmacological properties, indications, dosing, and efficacy.     

Neutralization of alpha-bungarotoxin by monoclonal antibodies

We have produced monoclonal antibodies against alpha-bungarotoxin from Bungarus multicinctus, and controls were performed for their specificity and monoclonal character. The antibodies protected mice against the toxic effect of alpha-bungarotoxin for a few hours to six days, depending upon the chosen antibody (in comparison to a survival time of 79 min for the standard toxin dose). Antibodies can be grouped in several sets according to their duration of protection.     

Neutralization of LSD by active immunization

Mice immunized with a lysergic acid derivatized protein showed subsequent resistance to the effects of intravenously administered LSD as measured by the poke and rearing tests. Mice immunized with a tryptophyl conjugated protein displayed similar refractoriness relative to normal control mice. Thus, significant immunological cross reactivity as expressed by neutralization, was evident due to the common indole moiety of the two haptenic groups studied.Supported by a grant from the Pharmaceutical Manufacturers Association Foundation, Inc.     

中和滴定法测定牛磺酸含量

采用酸碱中和滴定法测定了牛磺酸含量。结果表明 ,该法准确、可靠 ,所用仪器简单、方便易行。     

Neutralization of saxitoxin by anti-saxitoxin rabbit serum

This study examined the ability of anti-saxitoxin rabbit serum to neutralize saxitoxin, both in vitro and in vivo. In vitro, two rabbit antisera decreased [3H]-saxitoxin binding to specific sites in rat brain membranes. The more potent of these sera, antiserum A, when combined with saxitoxin in vitro, decreased saxitoxin's lethal potency based on mouse bioassay. Antiserum A also neutralized saxitoxin in vivo, as illustrated by the fact that mice injected i.p. with antiserum A (1:4) survived a s.c. injection 1 hr later of 16.7 micrograms saxitoxin/kg (1 LD99). Finally, antiserum A prevented death when injected i.v. immediately after s.c. injection of 16.7 micrograms saxitoxin/kg, however, antiserum injected by the i.p. and i.m. routes caused no significant increase in survival. This study indicates that antiserum can neutralize saxitoxin both in vitro and in vivo.     

中和法在微生物限度检查中的应用

目的为有抑菌性的药物提供中和剂,建立有效的微生物限度检验方法。方法通过葡萄糖酸氯己定软膏,盐酸多塞平乳膏和盐酸二甲双胍控释片三种药物为例,说明如何采用中和法进行微生物限度方法学验证。结果中和剂的合理运用,可更好满足微生物限度试验的要求。结论中和法在微生物限度检查中的广泛应用,可明显降低抑菌性药物的抑菌能力,提高菌的回收率,简化试验操作。     

Neutralization epitopes on HIV pseudotyped with HTLV-I

Summary During the last decade, research efforts have been aimed at development of an HIV vaccine. Until now it has not been possible to identify conserved epitopes on the envelope structure of HIV, which might be targets for neutralizing antibodies. However, it is clear that the conformation of gp120 is important for the development of broadly neutralizing antibodies, and that glycan structures of gp120 are of importance in this context. HIV-infected patients are often co-infected with other viruses, and one mechanism for expanding the cellular tropism of HIV in vitro is through formation of phenotypically mixed particles (pseudotypes) with HTLV-I. Previously, pseudotypes were found to allow penetration of HIV particles into CD4-negative cells, which are otherwise nonsusceptible to HIV infection. In the search for epitopes that might be the target for an HIV vaccine, the pseudotype phenomenon should be taken into account. An HIV-neutralizing antibody, induced by a vaccine, should also be able to neutralize potential pseudotypes. The in vitro infection of CD4-negative cells with pseudotypes could be blocked with anti-HTLV-I serum, but failed to be significantly inhibited with antibodies directed at HIV. These findings suggest that pseudotypes may represent a way to escape neutralization by the immune system in vivo. Previous reports have indicated that carbohydrate structures may be conserved neutralization epitopes on retroviruses. Lectins and monoclonal antibodies directed to carbohydrate structures were found to block HIV/HTLV-I pseudotype infection in CD4-negative cells. Therefore, although viral cofactors might expand the tropism of HIV in vivo, HIV and HTLV-I seem to induce common carbohydrate neutralization epitopes. Taken together, these results indicate that carbohydrate structures might induce protective antibodies, in spite of HIV envelope protein variability and the possibility of pseudotype production, which is why the results may be used in the search of an HIV vaccine or immune-stimulating agents.