茯苓多糖PCP-Ⅰ和PCP-Ⅱ作为疫苗佐剂的免疫原性

目的研究茯苓多糖(PCP)中PCP-Ⅰ和PCP-Ⅱ作为疫苗佐剂的免疫原性。方法 1采用钥孔戚血蓝蛋白(KLH)和牛血清白蛋白(BSA)为载体蛋白分别与PCP-Ⅰ或PCP-Ⅱ连接制备免疫抗原KLHPCP-Ⅰ和KLH-PCP-Ⅱ及筛选抗原BSA-PCP-Ⅰ和BSA-PCP-Ⅱ。KLH-PCP-Ⅰ和KLH-PCP-Ⅱ分别与弗氏佐剂联用id免疫家兔2次,ELISA检测家兔血清中抗多糖抗体。2PCP-Ⅰ或PCP-Ⅱ单独im免疫小鼠2次,ELISA检测小鼠血清中抗多糖抗体。3PCP-Ⅰ或PCP-Ⅱ为佐剂分别配伍重组乙肝病毒表面抗原(HBs Ag)和猪繁殖与呼吸综合征病毒灭活疫苗(PRRSV)im或sc免疫小鼠2次,ELISA检测小鼠血清中抗多糖抗体。结果1KLH-PCP-Ⅰ或KLH-PCP-Ⅱ与弗氏佐剂联用免疫2次,可产生抗KLH和抗多糖抗体。2PCP-Ⅰ或PCP-Ⅱ单独im免疫小鼠2次,检测出低水平Ig M抗体,未检测出IgG抗体。3HBs Ag或PRRSV抗原联用PCP-Ⅰ或PCP-Ⅱ免疫小鼠2次,未检出抗多糖IgG抗体。结论 PCP-Ⅰ和PCP-Ⅱ本身免疫原性较弱,作为疫苗佐剂可能具有良好的安全性。     

TDAR在药物临床前毒理学研究中的应用

目的 TDAR试验被认为是检测药物潜在免疫毒性预测性较好的功能性试验,本文综述总结TDAR检测方法在药物临床前毒理学研究中应用的一般原则,以便同行参考。方法通过综合分析、对比,总结国内外文献资料中关于TDAR的实验方法和评价,得出在药物临床前毒理学研究中应用的一般原则。结果 T细胞依赖性抗体反应（TDAR）是检测免疫功能的主要试验。TDAR通过人为引入外来抗原,反映药物或化学物质对免疫系统整体的影响,目前在药物免疫毒理学研究中逐渐得到广泛应用。结论 TDAR试验是检测药物潜在免疫毒性预测性较好的功能性试验,该试验可以用来在临床前的反复给药毒性试验或者临床试验中检测免疫功能的改变,可以对候选药物的免疫调节和免疫毒性特点进行早期预测。     

小鼠树突状细胞过继转移免疫作用的研究

目的 探讨树突状细胞（DCs)在过继免疫中的作用。以及递呈不同性质抗原的特点,为应用DCs诱导免疫耐受或治疗肿瘤奠定基础。方法 分别用绵羊红细胞（SRBC）和人白蛋白单次免疫和再次免疫小鼠,分离脾DCs和腹腔巨噬细胞（Mφ）,并分别转移至各组小鼠体内,检测抗SRBC和抗人白蛋白抗体效价。结果 实验组中不论是DCs组,还是Mφ组产生的抗体效价均较高于对照组。过继转移白蛋白时,DCs组产生抗体水平较Mφ组低;过继转移SRBC时,DCs组产生的抗体水平较Mφ组稍高,但无统计学意义,而血清中的抗体水平与抗体形成细胞（AFC）水平发生分离。结论 DCs既能处理递呈可溶性抗原,又具有较强的处理,递呈颗粒性抗原通过过继转移给受体,使受体T、B细胞活化。     

单纯疱疹病毒感染患者抗原和抗体检测结果分析

目的 探讨单纯疱疹病毒(HSV-Ⅱ)感染患者抗原、抗体的相互关系.方法 对89例皮肤性病门诊的疑似女性病人的疱液或分泌物和血清用酶联免疫法(ELISA)进行抗原抗体检测.结果 抗原阳性的患者,其抗体不一定阳性,抗原阴性的患者,其抗体检测也不一定阴性,对于性病门诊患者,最好同时检测抗原、抗体,以便临床确诊.结论 ELISA法检测疱疹病毒抗原抗体具有简便、快速、特异的特点.     

柯萨奇病毒A组10型抗原双抗体夹心ELISA定量方法的建立及应用

目的 建立柯萨奇病毒A组10型（coxsackievirus A10,CV-A10）抗原双抗体夹心ELISA定量检测方法,用于CV-A10生产过程样品和四价手足口病疫苗的质量控制。方法  以纯化的CV-A10抗原分别免疫绵羊和小鼠获得羊免疫血清和杂交瘤单克隆抗体(单抗)细胞株,以杂交瘤细胞株免疫小鼠制备腹水,羊血清和小鼠腹水分别经亲和层析后获得纯化羊抗CV-A10多克隆抗体（多抗）和小鼠抗CV-A10单抗。采用微量细胞病变法测定纯化多抗和单抗中和抗体效价。分别以纯化羊多抗作为包被抗体、小鼠单抗作为检测抗体,进行配对筛选研究,建立CV-A10抗原定量双抗体夹心ELISA;对方法的线性、专属性、准确度、精密度、范围进行验证,并对CV-A10抗原生产过程中的样品和成品进行检测。结果 纯化抗CV-A10单抗和多抗均具有中和抗体活性。以多抗作为包被抗体、7株单抗作为检测抗体进行配对,其中5株单抗配对成功,选择CY166-14R作为检测抗体用于CV-A10抗原定量检测ELISA的建立;对检测方法进行优化,确定以羊多抗作为包被抗体的使用稀释比例为1：5 000〜1：10 000、小鼠单抗检测抗体稀释比例1 ：2 000〜1：4 000。建立的CV-A10抗原定量检测方法范围为0.42〜10. 00 U/ml,线性决定系数≥0. 99;该方法仅能特异性检测CV-A10抗原,与其他抗原或物质（肠道病毒71型、CV-A6、CV-A16、M199、DMEM、Vero细胞蛋白、牛血清）无交叉反应;对高、中、低浓度样品进行测定,测定值/理论值在95%〜110%之间,相对标准偏差在15%以内。结论 建立了 CV-A10抗原定量双抗体夹心ELISA,该方法在一定范围内的线性、专属性、准确度、精密度均较好,可用于CV-A10抗原生产过程样品和成品的质量控制,为CV-A10抗原的体外效力评价提供方法学基础。     

单价和多价KLH结合疫苗免疫后抗体水平比较

神经节苷脂 GD3、中性糖脂 L e Y和粘蛋白 MUC1、MUC2等抗原在多种肿瘤细胞表面过量表达。现已证实上述抗原与匙孔血蓝蛋白 ( KLH)的结合及免疫佐剂 QS- 2 1的应用是诱生高滴度 Ig M和 Ig G抗体的最适方法。这些抗体能与肿瘤细胞表面的天然抗原结合 ,并介导补体依赖的细胞毒性和 /或抗体依赖细胞介导的细胞毒性。研究证明单价KL H结合疫苗安全 ,并具有较好的免疫原性 ,目前正在制备用于临床的多价 KLH结合疫苗 ,并评估多价疫苗各组分的免疫原性是否与单价疫苗相同及疫苗配方和接种途径是否对免疫原性产生影响。　　 GD3- KLH…     

酶联免疫白喉抗体定量试剂的研制及应用

目的 研制白喉抗体定量试剂,用于白喉疫苗免疫后抗体水平监测.  方法 采用双抗原夹心法原理,用白喉抗体国家参考品为定量依据,建立ELISA定量检测系统.  结果 试剂经系统优化后,检测抗体浓度在10～120 IU/L之间,呈良好的线性关系(r=0.996).精密度(CV)≤4.6%.实际应用验证,用ELISA试剂对不同年龄组的检测结果显示,白喉抗体水平的变化趋势与国家计划免疫程序密切相关.在白喉疫苗效力检定中,用ELISA试剂直接测定免疫后小鼠的抗体水平与Vero细胞法测定结果之间具有良好的相关性(r=0.974).  结论 该试剂可用于白喉抗体的定量检测.     

鼠和兔中流行性感冒病毒抗体的制备及测定

报道了流行性感冒病毒抗体的制备及其抗体价的检测方法。利用鼠、兔对流行性感冒(IFV)抗原的免疫反应制备抗体；酶联免疫法(ELISA)测定抗体价。结果显示：Freund佐剂与抗原的混合乳液，具有流行性感冒病毒的抗原特异性，且能够增强抗原的免疫原性，并改变宿主的免疫反应原性。     

应用T细胞依赖性抗体反应模型评价参麦注射液对大鼠免疫功能的影响

目的建立T细胞依赖性抗体反应(TDAR)大鼠模型,研究参麦注射液(SMIJ)对大鼠免疫功能的影响。方法 SD大鼠按分组分别ig给予环孢素A(CSA)0.02 g·kg-1,iv给予SMIJ溶剂或SMIJ 0.6,1.2和2.4 g·kg-1,每天1次,连续28 d。于给药第15天和第22天经左后足趾sc给予钥孔其虫戚血蓝蛋白(KLH)2.4 mg·kg-1进行免疫刺激。实验期间观察大鼠的一般状态,每周测体质量和摄食量。在第20天和第29天用ELISA法分别测定大鼠血清KLH-Ig M和KLH-Ig G抗体浓度,第29天活杀大鼠,应用全自动血液分析仪测定血液红细胞和白细胞数及白细胞分类,用天平称取肺、肝、脾和胸腺质量并计算脏器系数,采用HE染色法观察肺、肝、脾、胸腺和淋巴结组织病理变化。结果实验期间未见大鼠死亡,各给药组体质量增长和摄食量无异常。与正常对照组相比,模型组大鼠血清KLH-Ig M和KLH-Ig G抗体浓度均明显增高(P<0.01),提示KLH引起机体免疫应答;与模型组相比,血清KLH-Ig M和KLH-Ig G抗体浓度在CSA组均明显降低(P<0.01),而在SMIJ溶剂组和给药组则无明显变化。给药后第29天,与正常对照组相比,模型组大鼠各项血液学指标和脏器系数无差异,各组织脏器亦无明显病变;与模型组相比,CSA组白细胞、嗜酸性粒细胞和淋巴细胞数、脾和胸腺脏器系数均明显降低(P<0.05,P<0.01),脾出现白髓范围和脾小体淋巴细胞数目减少及淋巴小结界限不清等免疫抑制现象,而SMIJ溶剂和给药组各项血液学和脏器系数均无明显改变,各组织脏器亦无明显病变。结论建立了SD大鼠TDAR研究模型,连续尾静脉给予SMIJ 28 d对SD大鼠TDAR无明显影响。     

甘草酸人工抗原的合成鉴定及免疫原性分析

目的制备中药甘草的活性成分甘草酸(GA)的人工抗原及抗血清,为制备GA的单克隆抗体、并建立快速检测GA的酶联免疫吸附测定(ELISA)提供技术基础。方法将GA与载体蛋白牛血清白蛋白(BSA)偶联起来制得完全抗原后,经基质辅助激光解吸飞行时间质谱鉴定其相对分子质量,用此抗原免疫BALB/c小鼠,制备抗血清,并通过间接ELISA法和竞争ELISA法检测其抗体效价和特异性。结果合成的人工抗原GA-BSA中GA与BSA的结合比约为7∶1;免疫小鼠得到特异针对GA的多抗血清,GA抗体的效价为1∶8000。结论成功地合成了GA的人工抗原,且该抗原有较好的免疫原性,可应用于建立GA的免疫分析方法。     

Retrospective evaluation of the impact of functional immunotoxicity testing on pesticide hazard identification and risk assessment

Conduct of a T-cell-dependent antibody response (TDAR) assay in rodents according to Environmental Protection Agency (EPA) Test Guideline OPPTS 870.7800 is now required for chemical pesticide active ingredients registered in the United States. To assess potential regulatory impact, a retrospective analysis was developed using TDAR tests conducted on 78 pesticide chemicals from 46 separate chemical classes. The objective of the retrospective analysis was to examine the frequency of positive responses and determine the potential for the TDAR to yield lower endpoints than those utilized to calculate reference doses (RfDs). A reduction in the TDAR response was observed at only the high-dose level in five studies, while it was unaltered in the remaining studies. Importantly, for all 78 pesticide chemicals, the TDAR no-observed-adverse-effect levels (TDAR NOAELs) were greater than the NOAELS currently in use as risk assessment endpoints. The TDAR NOAELs were higher than the current EPA-selected endpoints for the chronic RfD, short-term, intermediate and long-term exposure scenarios by 3–27,000, 3–1,688, 3–1,688 and 4.9–1,688 times, respectively. Based on this analysis, conduct of the TDAR assay had minimal impact on hazard identification and did not impact human health risk assessments for the pesticides included in this evaluation. These data strongly support employment of alternative approaches including initial weight-of-evidence analysis for immunotoxic potential prior to conducting functional immunotoxicity testing for pesticide active ingredients.     

Specific antibody responses of primary cells from different cell sources are able to predict immunotoxicity in vitro

Alternative methods for the prediction of immunotoxicity are highly desirable. However, until now no in vitro test for this purpose has been fully validated or accepted by regulatory authorities. MD cultures are in vitro equivalent to the widely used ex vivo primary T cell dependent antibody responses (TDAR), which has been identified in a regulatory context as a main functional test for immunotoxicological investigations. The purpose of the present study was to use MD cultures of spleen and blood cells to compare data from three different chemicals using SRBC as antigen in two different species. Using this approach we were able to show that cell sources from both rats and mice were able to correctly predict all tested compounds and to clearly distinguish immunosuppressants from control substances. Furthermore, animal studies can be refined by using MD cultures of PBMC. During a 28d benzo(a)pyrene treatment of rats we were able to follow the kinetic of an immune response by in vitro analyses. Additionally evaluation of in vitro antibody responses of spleen cells and PBMC from rats treated with cyclophosphamide revealed similar results compared to the conventional ex vivo plaque forming cell assay (PFCA).In conclusion, investigation of in vitro antibody responses is a sensitive and reliable approach for detection of a compound induced specific effect on the immune system. MD cultures may not only replace the ex vivo TDAR in the future, but their implementation in routine toxicology also enables refinement of existing in vivo studies by reducing the numbers of animals.     

Immune function tests for hazard identification: a paradigm shift in drug development

Routine immune function testing in preclinical drug development was established as a regulatory requirement in June of 2000 under the Committee of Proprietary Medicinal Products (CPMP) Note for Guidance on Repeated Dose Toxicity (CPMP/SWP/1042/99). The purpose of the more stringent approach to immunotoxicology testing was to better identify unintended immunosuppression; however, the requirement was met with much discussion and debate. At the center of the discussion was an attempt to reconcile opposing regulatory directives from agencies outside of Europe that adhere to a more selective, weight-of-evidence approach to functional evaluations. Uncertainty over the predictive value of the recommended immune function tests relative to conventional toxicology parameters prompted an investigation by the International Committee on Harmonization (ICH). The results of a preliminary, industry-wide survey indicated that only a low percentage of pharmaceuticals adversely affect immune function without alterations to standard toxicology parameters. Expected ICH guidelines will ultimately determine to what extent and for what purpose immune function tests will be conducted. In the meantime, optimization of the recommended immune function tests is ongoing. The T-cell dependent antibody response (TDAR) by either conventional Sheep Red Blood Cell (SRBC) plaque assay or by the modified ELISA method using either SRBC or keyhole limpet hemocyanin (KLH) as antigen is being extensively evaluated to determine best practices and procedures for preclinical immunotoxicity evaluations. This review addresses some aspects of the debate concerning the appropriateness of immune function tests for hazard identification, along with recommendations for optimizing TDAR methodology to ensure adequate sensitivity and predictability in risk assessments for immunotoxicity.     

Developmental immunotoxicity in male rats after juvenile exposure to di-n-octyltin dichloride (DOTC)

To determine relevant endpoints for evaluating developmental immunotoxicity due to juvenile exposure and optimal age of the animals at assessment, a wide range of immunological parameters were assessed in a juvenile toxicity study. Rats were exposed to di-n-octyltin dichloride (DOTC) by gavage from postnatal day (PND) 10 through PND 21 and via the diet after weaning using a benchmark dose (BMD) approach. Immune assessments were performed in male rats on PNDs 21, 42, and 70 and a subset of animals was used to evaluate the T-cell dependent antibody response (TDAR) to Keyhole limpet hemocyanin. Immune effects were more pronounced on PND 21 and 42 and observed at lower doses than developmental effects. The most sensitive immune parameters affected included TDAR parameters and thymocyte subpopulations with lower confidence limits of the benchmark doses (BMDLs) below the overall no-observed-adverse-effect-level (NOAEL) for DOTC reported so far in literature. These findings illustrate the relative sensitivity of the developing immune system for DOTC, the additional value of assessing functional immune parameters, and underscore the relevance of juvenile immunotoxicity testing in view of the risk assessment of chemicals.     

Prenatal administration of indomethacin modulates Th2 cytokines in juvenile rats

Indomethacin (IND) suppresses the T-dependent antibody response (TDAR) in juvenile males when it is administered to pregnant rats during late gestation. In this study, the effect of IND on cytokine production in juvenile rats was examined to investigate the mechanism behind the suppression of antibody production. IND was orally administered to pregnant SD rats on days 18–21 of gestation. After parturition, the spleen cells isolated from 3-week-old pups were incubated with concanavalin A (Con A) or lipopolysaccharide (LPS). The level of cytokines in the culture supernatant was measured. IL-10 decreased significantly in the males, and IL-6 and TNF-α tended to decrease in both sexes.     

抗人死亡受体5单链抗体的构建表达纯化及活性分析

目的构建、表达、纯化抗人死亡受体5(DR5)单链抗体,并检测其诱导肿瘤细胞凋亡的活性。方法采用RT-PCR获取鼠源性抗人DR5单克隆抗体重链和轻链可变区基因序列,以一柔性连接肽连接二者,转入表达载体,以大肠杆菌表达融合蛋白,亲和色谱纯化后用MTT实验和凋亡检测试剂盒检测其凋亡诱导活性。结果获得的序列经比对为抗体重链和轻链可变区基因,表达纯化后的重组蛋白具有接近完整抗体的肿瘤细胞凋亡诱导活性。结论抗人DR5 scFv可作为诱导肿瘤细胞凋亡的候选药物,为肿瘤免疫学研究提供材料。     

An Enzyme-Linked Immunosorbent Assay (ELISA) for Quantitation of Adducts of Granulocyte–Macrophage Colony Stimulating Factor (GM-CSF) and Human Serum Albumin (HSA) in Stressed Solution Mixtures

HPLC analyses of GM-CSF in solution mixtures containing both GM-CSF and HSA showed losses of GM-CSF which could not be accounted for using conventional electrophoretic and/or RP-HPLC techniques. Further investigation of these mixtures by immunoblotting and by immunoaffinity chromatography demonstrated the presence of high molecular weight (>67,000) GM-CSF related species. No such species was detectable in solutions of GM-CSF alone. This experiment pointed to the formation of an adduct between GM-CSF and HSA in the solution mixtures. To probe further the hypothesis of a GM-CSF/HSA adduct, an immunologically based test was conceived which could react only with this type of hybrid molecule. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed using two antibodies, anti-GM-CSF (capture antibody) and anti-HSA (detection antibody), as part of the quantitation of GM-CSF/HSA adducts. After confirming its existence by ELISA, a GM-CSF/HSA adduct was isolated from the solution mixture containing both GM-CSF and HSA. This isolate served as a primary reference standard in the ELISA assay. The immunoassay has a subnanogram sensitivity and is highly specific for GM-CSF/HSA adducts in the presence of either free GM-CSF or free HSA. As a verification, conjugates of GM-CSF/HSA were synthesized using a cross-linking reagent. These covalent conjugates reacted positively in the ELISA and are employed as a convenient alternative reference standard.