犬传染性肝炎病毒在体外细胞质内的发生

通过对犬传染性肝炎病毒（ＩＣＨＶ）在犬肾传代细胞内形态发生及其抗原定位的电镜和免疫胶体金电镜研究,发现ＩＣＨＶ除了在宿主细胞核内发生外,还有一条细胞质内的发生途径。在细胞质内病毒核壳体的装配是以均质致密包涵体和副晶格包涵体为“基地”,这与人们熟知的细胞核内形态发生方式相似。免疫胶体金标记显示,细胞质包涵体中含有大量的ＩＣＨＶ抗原成分,显核壳体在细胞质内装配病毒的结构蛋白来源。此外,在感染的细胞质内还观察到与核内相同的病毒核心样结构。     

Melatonin and schistosomal antigens ameliorate the anti-oxidative and biochemical response to Schistosoma mansoni infection in hamster

The present study was designed to investigate the potential protective effect of melatonin as an antioxidant separately or in combination with antigens (cercarial; CAP or soluble worm; SWAP) against Schistosoma mansoni infection in hamsters. Each hamster was sensitized with an initial immunization of 0.6 ml of the extracted antigen (30 μg protein/mL). After four days,a second injection of 0.4 mL was given (20 μg protein/mL). Then,each hamster was exposed to 260±20 S.mansoni cercariae followed with melatonin...     

Microwave oven-based technique for immunofluorescent staining of paraffin-embedded tissues

Immunohistochemical analysis of formalin-fixed paraffin-embedded tissues can be challenging due to potential modifications of protein structure by exposure to formalin. Heat-induced antigen retrieval techniques can reverse reactions between formalin and proteins that block antibody recognition. Interactions between antibodies and antigens are further enhanced by microwave irradiation, which has simplified immunohistochemical staining protocols. In this report, we modify a technique for antigen retrieval and immunofluorescent staining of formalin-fixed paraffin-embedded tissues by showing that it works well with several antibodies and buffers. This microwave-assisted method for antigen retrieval and immunofluorescent staining eliminates the need for blocking reagents and extended washes, which greatly simplifies the protocol allowing one to complete the analysis in less than 3 h.     

DTaP-Hib联合疫苗中Hib-TT免疫原性研究

考查DTaP-Hib联合疫苗中Hib-TT的免疫原性,对其剂量、免疫持久性和抗原相容性进行分析。将不同剂量的Hib-TT、DTaP-Hib联合疫苗分别免疫小鼠,设单价的Hib-TT结合疫苗为对照,末次免疫后1、2、4、6、8、10w分别采集血清测定血清中Hib多糖抗体滴度。结果显示,不同剂量的Hib-TT和DTaP疫苗联合后均具有较好的免疫原性,血清中Hib多糖抗体阳转率达100%,并具有剂量效应和较好的免疫持久性。2.5μg剂量Hib-TT的DTaP-Hib联合疫苗免疫小鼠后1~2w诱导产生的Hib多糖抗体水平显著性地低于单价Hib-TT(P<0.05),4~10w,二者的Hib多糖抗体水平无显著性差异(P>0.05)。5μg剂量Hib-TT的DTaP-Hib联合疫苗在免疫小鼠后1w诱导产生的Hib多糖抗体水平与单价2.5μg剂量Hib-TT无显著性差异(P>0.05),免后2~10w则显著性地高于单价2.5μg剂量Hib-TT(P<0.001)。Hib-TT和DTaP疫苗联合后,仍然具有较好的免疫原性、剂量效应和免疫持久性;其抗原性干扰只是暂时的。     

免疫组织化学反应抗原恢复新法在临床病理诊断中的应用

应用蒸馏水在ｐＨ６２, 加热煮沸后能够较好的恢复组织中被封闭的抗原。经２８０４ 张组织切片在进行免疫组织化学反应之前, 用现配ｐＨ６２ 的蒸馏水加热煮沸后微火保温１０ 分钟, 然后再按常规免疫组化染色。经此法恢复抗原处理后, 并显示强度和阳性率与微波辅助盐溶液方法相比结果基本相似, 两者与未抗原恢复处理相比有显著性差异（Ｐ＜ ００１）。经彩色图像分析结果同样有显著性差异（Ｐ＜ ００５）。本法恢复抗原与微波技术相比, 同样能使组织内抗原得以充分的暴露, 提高抗原的检出率, 证明了蒸馏水在ｐＨ６２ 时,可作为一种新的暴露抗原的方法, 且操作简单, 抗原恢复均匀, 很适合各种实验室的应用     

铅螯合物人工抗原的制备与鉴定

以双功能螯合剂异硫氰酸苄基乙二胺四乙酸(ITCBE)螯合铅离子,制备得半抗原Pb-ITCBE,然后再分别与载体蛋白KLH或BSA偶联制备得免疫原Pb-ITCBE-KLH与包被抗原Pb-ITCBE-BSA,ITCBE-BSA.用二喹啉甲酸法测3种抗原的浓度,分析半抗原、抗原与载体蛋白的紫外吸收光谱,利用SDS-PAGE对3种抗原的分子量进行鉴定,用三硝基苯磺酸法检测3种抗原中的赖氨酸残基的ε-NH2被半抗原替换的程度,用石墨炉原子分光吸收法检测抗原中铅的含量.研究结果表明,免疫原与包被抗原制备成功,Pb-ITCBE-KLH、Pb-ITCBE-BSA、ITCBE-BSA的浓度依次为6.47± 0.08 mg/ml,6.68± 0.06 mg/ml,5.57± 0.05 mg/ml;抗原与载体蛋白的紫外吸收光谱的特征各不相同;SDS-PAGE的结果显示3种抗原的分子量均不同于各自的载体蛋白;抗原中载体蛋白ε-氨基的替换程度依次为1.86± 0.74 %、55.53± 1.13%、54.19± 1.34%;铅的含量依次为15.64± 0.11 μg/ml,17.33± 0.15 μg/ml,0 μg/ml.     

不同狂犬病毒株抗原反应性的比较研究

应用ELISA法对285份免疫前及健康人血清和742份以不同毒株制备的狂犬病疫苗(aG株、CaG株、CTN株,PM株)全程免疫后人群的血清标本进行抗狂犬病毒抗体的检测。结果显示CTN株病毒抗原对不同毒株狂犬病疫苗免疫后血清的抗体阳性检出率均能达到90％以上,且与SNT效价的相关性较好;而aG株抗原对除aG株以外其它毒株狂犬病疫苗免疫后血清的阳性检出率仅为50％左右。因此不同毒株狂犬病毒抗原的反应性存在明显差异,选用纯度高、活性好的CTN株病毒或两株病毒以不同比例混合作为ELISA检测用抗原,测定疫苗免疫后人群的抗体水平,可获得满意的结果。     

Cytotoxic enhancement of a bispecific diabody by format conversion to tandem single-chain variable fragment (taFv): the case of the hEx3 diabody

Diabodies (Dbs) and tandem single-chain variable fragments (taFv) are the most widely used recombinant formats for constructing small bispecific antibodies. However, only a few studies have compared these formats, and none have discussed their binding kinetics and cross-linking ability. We previously reported the usefulness for cancer immunotherapy of a humanized bispecific Db (hEx3-Db) and its single-chain format (hEx3-scDb) that target epidermal growth factor receptor and CD3. Here, we converted hEx3-Db into a taFv format to investigate how format affects the function of a small bispecific antibody; our investigation included a cytotoxicity assay, surface plasmon resonance spectroscopy, thermodynamic analysis, and flow cytometry. The prepared taFv (hEx3-taFv) showed an enhanced cytotoxicity, which may be attributable to a structural superiority to the diabody format in cross-linking target cells but not to differences in the binding affinities of the formats. Comparable cross-linking ability for soluble antigens was observed among hEx3-Db, hEx3-scDb, and hEx3-taFv with surface plasmon resonance spectroscopy. Furthermore, drastic increases in cytotoxicity were found in the dimeric form of hEx3-taFv, especially when the two hEx3-taFv were covalently linked. Our results show that converting the format of small bispecific antibodies can improve their function. In particular, for small bispecific antibodies that target tumor and immune cells, a functional orientation that avoids steric hindrance in cross-linking two target cells may be important in enhancing the growth inhibition effect.     

Humanization of Antibodies Using Heavy Chain Complementarity-determining Region 3 Grafting Coupled with in Vitro Somatic Hypermutation

A method for simultaneous humanization and affinity maturation of monoclonal antibodies has been developed using heavy chain complementarity-determining region (CDR) 3 grafting combined with somatic hypermutation in vitro. To minimize the amount of murine antibody-derived antibody sequence used during humanization, only the CDR3 region from a murine antibody that recognizes the cytokine hβNGF was grafted into a nonhomologous human germ line V region. The resulting CDR3-grafted HC was paired with a CDR-grafted light chain, displayed on the surface of HEK293 cells, and matured using in vitro somatic hypermutation. A high affinity humanized antibody was derived that was considerably more potent than the parental antibody, possessed a low pm dissociation constant, and demonstrated potent inhibition of hβNGF activity in vitro. The resulting antibody contained half the heavy chain murine donor sequence compared with the same antibody humanized using traditional methods.