结核分支杆菌H37Rv株菌体抗原单克隆抗体制备、特性分析及纯化

目的　利用单克隆抗体技术分析分支杆菌多肽抗原的共性和个性 ,建立纯化单克隆抗体的最佳方法。方法　按常规方法制备单克隆抗体。分泌单克隆抗体阳性杂交细胞株的筛选采用ELISA法 ,确认采用免疫印迹法。 2 4种分支杆菌菌株均生长于改良罗氏培养基上 ,H3 7Rv株分别生长于改良罗氏培养基和苏通液体培养基上。单克隆抗体的纯化采用硫酸铵沉淀法、辛酸沉淀法和离子交换法。结果　经筛选获得 14株单克隆抗体杂交细胞瘤。其中 ,C2 、7C12 单克隆抗体所抗的 76 0 0 0、16 0 0 0抗原成份为分泌抗原 ,76 0 0 0抗原存在于 2 4种分支杆菌菌体中 ,为共同抗原。 16 0 0 0抗原仅存在于H3 7Rv ,H3 7Ra,牛分支杆菌 ,BCG株中。单克隆抗体免疫印迹结果显示 ,分支杆菌H3 7Rv株在不同培养条件下 ,其 40 0 0 0 / 380 0 0多肽抗原表达不同。单克隆抗体纯化以硫酸铵沉淀法回收率最高 ,离子交换法获得的单抗纯度最高 ,辛酸沉淀法可以有效地去除白蛋白 ,并具有简单、省时等优点。单克隆抗体的相对亲和力均在 10 -4 ～ 10 -7之间。结论　C2 、7C12 单克隆抗体均抗结核分支杆菌H3 7Rv株的分泌抗原成份。 40 0 0 0 / 380 0 0多肽抗原为结核分支杆菌H3 7Rv株固体培养菌体特有。单抗纯化结果显示离子交换层析法适用于科研分析 ,而辛酸     

四种前处理对分支杆菌生长活力观察

本文报道对抗酸分支杆菌标准菌株17株,临床分离菌株10株,分别作酸、碱、中和及胰酶-新洁尔灭等四种前处理后,接种于改良罗氏或酸性罗氏培养基,经37℃、8周观察细菌生长活力受影响的程度。结果显示,对大部分致病性分支杆菌的生长活力无明显影响,而对大部分非致病性分支杆菌的生长活力有明显影响。     

气相色谱鉴定分支杆菌对比实验

采用毛细管柱色相色谱（ＧＣ）检测分支杆菌细胞脂肪酸（ＦＡ）的方法，通过制备２６种参考菌株ＧＣ图谱（原冻干菌株及其移种改良罗氏培养基生长３￣４周菌株）对临床分离的５４株非结核分支杆菌以ＧＣ法与传统生物学鉴定法进行对比，两者符合率达９４％；观察了结核分支杆菌和牛分支杆菌临床分离株ＧＣ图谱变化与耐药性的关系。认为ＧＣ法鉴定临床分离分支杆菌目前采取同时观察细菌生长速度、产色情况以及在对硝基苯甲酸（ＰＮＢ）     

两种测定分支杆菌药敏试验的方法比较

目的:分支杆菌药敏试验通常采用以罗氏培养基为基础绝对法中的间接法,也就是在固体罗氏培养基中按要求加入各种药物,根据细菌在培养基上的生长情况来判断药敏试验的结果;采用BACTECMGIT 960测定仪检测分支杆菌药敏试验是目前比较先进的方法,其主要是采用连续检测接种标本的培养管所显示的荧光强度来判断分支杆菌生长的情况.     

Tween80培养基对分支杆菌临床分离培养的实验研究

本文应用Ｔｗｅｅｎ８０培养基和改良罗氏培养基，对临床３９０份标本作分离培养结核分支杆菌比较。结果表明在结核菌生长速度，生长量，阳性率等方面，Ｔｗｅｅｎ８０培养基均优于改良罗氏培养基，对提高涂阴标本培阳率尤为显著（Ｐ＜０．０１），并有利于部分劣势生长的分支干菌发育。     

Tween80培养基对分支杆菌临床分离培养的实验研究

本文应用Ｔｗｅｅｎ８０培养基和改良罗氏培养基，对临床３９０份标本作分离培养结核分支杆菌比较。结果表明在结核菌生长速度、生长量、阳性率等方面，Ｔｗｅｅｎ８０培养基均优于改良罗氏培养基，对提高涂阴标本培阳率尤为显著（Ｐ＜Ｏ．０１），并有利于部分劣势生长的分支干菌发育。     

细胞脂肪酸鉴定分支杆菌

细胞脂肪酸鉴定分支杆菌程冬娥蔡玉珍刘奕华梅国华吴采樱曾昭睿方法：取人型结核分支杆菌、牛型结核分支杆菌、耐对氨水杨酸（ＰＡＳ）结核分支杆菌等２３株标准株，及临床分离的人型结核分支杆菌等共３０株。均在改良罗氏培养基上生长丰满，收集后固定、洗涤、真空干燥制...     

一种新型结核杆菌快速培养促生长剂的研制

目的以改良罗氏培养基为基础成分,研制一种快速培养结核杆菌的新型快速促生长剂。方法将结核杆菌H37Rv株和20株结核杆菌临床分离株,分别接种于含促生长剂的改良罗氏培养基和改良罗氏培养基上,观察两者的生长效果。结果结核杆菌H37Rv株在含促生长剂的罗氏培养基上5~7d可形成肉眼可见的米粒状菌落,而在改良罗氏培养基上14d才形成肉眼可见的米粒状菌落;采用结核杆菌临床分离株分别进行20组实验,在含促生长剂的罗氏培养基上的平均生长时间为7d,而在不含促生长剂的培养基上的平均生长时间为14.7d(P<0.01)。结论新型结核杆菌促生长剂能使结核杆菌标准株和临床分离株在7d内快速培养出,此快速促生长剂的研制为结核杆菌的快速培养及结核病的诊断、化疗效果观察、流行病学调查、抗结核药物作用研究等有效的手段。     

变色液体培养基快速培养分支杆菌的临床应用

快速分离临床标本中分支杆菌一直是结核病学科领域中迫切需要解决的课题。结核分支杆菌分裂周期长,生长速度慢,在改良罗氏培养基上一般需要2～8周才能看到可分辨的菌落,难于满足临床需要,我们应用变色液体培养基对临床痰标本进行培养可快速检出生长的分支杆菌,现报告如下。     

酸化离子水配制吡嗪酰胺药敏培养基

目的：探讨满足吡嗪酰胺活性需要的同时，研制使结核菌生长良好的PZA药敏培养基。方法：以改良罗氏培养基为基础，加大镁离子浓度，去掉马铃薯淀粉，用PH2．34，氧化还原电位为1174mv的酸化离子水配制PZA药敏培养基，按《结核病诊断细菌学检验规程》，测试了120株结核分杆菌对PZA的敏感性。结果：门诊分离的60株分支杆菌，PZA耐药率为13．3％，住院患者和深圳市慢性病医院分离15株完全耐药的分支杆菌，在此培养基上对PZA耐药。结论：此培养基配制简单，可与其他药敏试验同步进行，易于推广应用。     

A comparative serological study of antigenic glycolipids from Mycobacterium tuberculosis.

Two fractions of antigenic diacyl trehaloses (DAT1 and DAT2) were isolated from the type strain of Mycobacterium tuberculosis (H37Rv). Phenolic glycolipid (PGL) and polar glycolipid antigens (C1-C4) were isolated from an unusual smooth so-called 'Canetti' strain of M. tuberculosis. These lipids were analyzed by enzyme-linked immunosorbent assay (ELISA) using antisera against a range of mycobacteria and sera from 50 tuberculosis patients and 25 healthy blood donors. All the lipids gave strong reactions with homologous mycobacterial antisera, except the least polar 'Canetti' polar glycolipid (C4). The phenolic glycolipid (PGL) from the 'Canetti' strain gave only a very weak response with antisera against M. tuberculosis H37Rv. The diacyl trehaloses (DAT) from H37Rv gave only weak reactions with the antisera against the 2 Canetti strains of M. tuberculosis. The 3 most polar glycolipid antigens (C1-C3) from the Canetti strain gave strong responses with serum against M. tuberculosis H37Rv. None of the lipids was able to discriminate between patient and control sera at a level suitable for a serodiagnostic test. A combination of results from several lipids appears to be of greater value in this respect. Thus, PGL, DAT2 and C2 were the best combination, reacting with all but 4 of the patient sera and with only 1 of the control sera.     

鸡疟原虫雌配子单克隆抗体的制备和鉴定

用鸡疟原虫雌配子免疫ＢＡＬＢ／ｃ小鼠，取免疫鼠脾细胞与ＮＳＩ鼠骨髓瘤细胞融合，筛选出的６株（３Ｈ１、２Ｈ１、１Ｇ１、１２Ｄ１、６Ｅ２、６Ｃ２）杂交瘤细胞，证明能分泌效价高、特异性强的单克隆抗体（ＭｃＡｂ）。亚类鉴定为ＩｇＧ１（６Ｃ２，３Ｈ１）、ＩｇＧ２ａ（１Ｇ１，２Ｈ１）、ＩｇＧ２ｂ（６Ｅ２）、ＩｇＧ（１２Ｄ１），其中２Ｈ１、６Ｅ２、１Ｇ１、１２Ｄ１株ＭｃＡｂ为抗膜抗体，而３Ｈ１和６Ｃ２株ＭｃＡｂ为非抗膜抗体。除１２Ｄ１株ＭｃＡｂ与蓝氏贾第鞭毛虫滋养体有交叉反应外，其它各株ＭｃＡｂ与阴道毛滴虫滋养体、杜氏利什曼原虫前鞭毛体、鼠疟原虫和鸡疟原虫红细胞内期均无交叉反应。     

人结核分枝杆菌和牛结核分枝杆菌差异蛋白质组学的研究

目的 探讨人MTB标准株H37Rv与牛分枝杆菌标准株卡介苗株的亚细胞蛋白质组学差异.方法 应用密度梯度离心法分离H37Rv和卡介苗株的细胞壁、细胞膜和细胞质蛋白,利用二维液相色谱分离技术进一步获得H37Rv和卡介苗株亚细胞蛋白质组,采用酶联免疫吸附试验分别检测H37Rv和卡介苗各组分与结核病患者和健康人各5例血清的免疫学反应.组间比较采用t检验.结果 共获得26个特异性引发免疫反应的H37Rv菌株蛋白组分.细胞膜组分M3Fr18与结核病患者的血清免疫学反应的吸收光度(A450值)为(721±3)×10-3,明显高于健康人[(356±6)×10-3],也明显高于卡介苗株细胞膜相应组分与健康人的血清免疫学反应强度[(414±7)×10-3],差异均有统计学意义(t值分别为1.852和1.037,均P＜0.01).细胞膜组分MI0Fr21与结核病患者的血清免疫学反应的A450值为(954±6)×10-3,明显高于健康人[(415±6)×10-3],也明显高于卡介苗株细胞膜相应组分与健康人的血清免疫学反应强度[(315±4)×10-3],差异均有统计学意义(t值分别为2.113和2.550,均P＜0.01).结论 二维液相色谱和酶联免疫吸附试验联合应用,有助于检测人MTB感染的相关抗原蛋白.M3Fr18和M10Fr21蛋白质组分可引起结核病患者的特异性免疫反应,提示其可能是人MTB感染的特异抗原.

Abstract：

Objective To compare the proteome of Mycobacterium tuberculosis (MTB) H37 Rv strain with Bovine mycobacterium (Bacillus Calmette-Guerin, BCG) strain at the sub-cellular level. Methods Proteins of the cell wall, membrane and cytolymph of H37 Rv and BCG were extracted by density gradient centrifugation. Sub-cellular proteome of H37 Rv and BCG were analyzed using 2-dimensional liquid chromatography. The immunity reactions of H37 Rv fractions with sera from patients ( n = 5 ) and healthy controls ( n = 5 ), as well as BCG fractions with sera from healthy controls ( n = 5 ) were analyzed by ELISA.Data was analyzed by t test. Results Twenty-six fractions of H37 Rv were found to elicit specific antibody response. Fraction M3Fr18 of H37 Rv reacted with sera from patients. The A450 [ (721 ± 3 ) × 10-3] was higher than that with sera from healthy controls [ ( 356 ± 6) × 10 - 3 ] , as well as the A450 of the corresponding fractions of BCG with sera from healthy controls [ (414 ± 7) × 10-3 ]. The differences between the patient group and the 2 healthy control groups were significant ( t = 1. 852 and 1. 037, all P ＜ 0. 01 ). Moreover,fraction M10Fr21 of H37Rv reacted with sera from the patients. The A450[ (954 ±6) × 10 -3 ] was higher than that with sera from the healthy controls [ (415 ± 6 ) × 10-3 ], as well as the A450 of the corresponding fractions of BCG with sera from the healthy controls [ ( 315 ± 4 ) × 10-3 ]. The differences between the patient group and the 2 healthy control groups were significant ( t = 2. 113 and 2. 550, all P ＜ 0. 01 ).Conclusions The combination of 2-dimensional liquid chromatography and immunology technology is useful in finding antigens associated with MTB infection. Fractions M3Fr18 and M10Fr21 can elicit specific immune reaction among MTB patients, suggesting that they may be specific antigens of MTB infection.     

抗霍乱弧菌O139型的单克隆抗体免疫印迹分析抗原表位及应用

目的制备霍乱弧菌O139型(V ibrio cholerae O139,VC O139)单克隆抗体后应用免疫印迹技术识别抗原表位特性。方法SDS-PAGE结合W estern b lotting以及APAAP方法。结果霍乱弧菌O139型抗原经还原剂处理,SDS-PAGE 11%~20%梯度胶电泳,转印染色后,能分辨出20余条清晰的蛋白带和脂多糖片状图谱。3株抗霍乱弧菌O139型单抗的抗原表位,主要集中于20~70 kD相对分子质量的蛋白带,其中O139的2株单抗O139-14、O139-15分别与68~50 kD、45~25 kD、20~14 kD对应VC O139的蛋白质抗原表位起反应,而另外一株单抗,O139-92只与65 kD、20kD的抗原表位特异性结合。结论VC O139的O139-14、O139-15 M cAb同对应抗原表位点的结合分布较广,有利于对VC O139抗原的快速、敏感、特异检测。     

阴道毛滴虫单克隆抗体的制备和鉴定

用阴道毛滴虫滋养体免疫BALB/c小鼠,取脾细胞与NS_1鼠骨髓瘤细胞融合,选择4株(2A2,2A4,2A12,2H9)杂交瘤细胞,证明能分泌效价高、特异性强的单克隆抗体(McAb)。亚类鉴定为IgG1(2A2,2A4),IgG2b(2A12),IgG3(2H9)。2H9株和其他3株McAb分别能抗阴道毛滴虫细胞核和细胞膜。除2H9株外均可凝集或杀伤虫体,但2A2、2A4和2A12株介导的细胞毒性分别是不依赖和依赖补体的。这4株McAb与杜氏利什曼原虫前鞭毛体、枯氏锥虫锥鞭毛体和蓝氏贾第鞭毛虫滋养体无交叉反应。     

黑龙江立克次体054株单克隆抗体对斑点热群立克次体抗原结合位点及特性的分析

本文利用三株黑龙江立克欢你054株（HLJ－054）的单克隆抗体对四株斑点热群立克次体西伯利亚种（R．s．）246株、精河株（JH－74）,立氏立克次体（R.r．）R株及HLJ-054株进行了血清学反应,对其蛋白组成及单抗结合抗原特性等方面进行了研究。结果表明：JH-74林与246株在上述三个方面完全一致,不同于HLJ－054及R．r．R株;HLJ－054株与R．r．R株与三株单抗血清学反应高度交叉．但二者在蛋白组成及与单抗反应的抗原组成上均存在差异。该结果支持黑龙江立克次体054株为斑点热群立党次作的一个新成员。单抗F7针对的抗原结合表位为蛋白多肽;而单抗2E1、4G2结合的抗原表位可能为精蛋白。     

In vitro bactericidal action of dissolved ozone on different strains of Mycobacterium tuberculosis]

The mycobacterium tuberculosis (MBT) strains H37 Rv (USA and Czechoslovakia), Erdman, and multiresistant clinical strain 2255 having different milliliter levels of microbes were kept in the soluble ozone at the concentrations of 1-4 micrograms/ml for 30 and 60 minutes. The soluble ozone-treated strains were cultured in the Finn-I medium. The soluble ozone at the concentration was found to produce bactericidal and bacteriostatic effects on MBT, including multiresistant strains. The MBT strains resistant to antituberculosis agents were also more resistant to soluble ozone; however, longer exposure to the "therapeutical" concentrations of soluble ozone proved to damage multiresistant strains.     

抗弓形虫McAb的建立及应用研究

应用杂交瘤技术 ,建立了 5株分泌抗弓形虫单克隆抗体的杂交瘤细胞。其中 1E9和 4C10 2株分泌的 Mc Ab效价达 1∶ 12 80 0。该 2株单克隆抗体均属 Ig G1亚类 ,其识别的弓形虫抗原分别为 P2 2、P2 8。1E9和 4C10 Mc Ab混合后与兔抗弓形虫抗体联合 ,进行双抗体夹心 EL ISA测定弓形虫的敏感性分别为 12 .5和 2 5个虫体 / m l。检测 146份有不良孕产史妇女血清 ,11份弓形虫循环抗原 ( CAg)阳性 ( 7.5 3% )。检测 11份自然流产物 ,1份 CAg阳性 ,该流产物转种小鼠后证实确含有弓形虫。 92份育龄妇女血清 1份弓形虫 CAg阳性 ( 1.0 9% )。有不良孕产史的妇女血清弓形虫 CAg阳性率明显高于育龄妇女血清阳性率 ( P<0 .0 5 )。弓形虫经 1E9、4C10 Mc Ab处理后对小鼠腹腔巨噬细胞攻击 ,感染率分别为 7.8%和 7.5 % ,而对照组感染率为 14.5 % ,证明该 2株单克隆抗体对弓形虫有明显的抑制作用     

抗重组日本血吸虫P38抗原单克隆抗体的制备与鉴定

目的?摇在大肠埃希菌中可溶性表达日本血吸虫P38（SjP38）抗原分子，并以其纯化产物为抗原，制备抗SjP38单克隆抗体(McAb)。 方法 将pET32(a)-P38重组质粒转化大肠埃希菌BL21(DE3)，在1 mmol/L异丙基-β-D-硫代半乳糖苷诱导下表达，超声破菌后获得可溶性表达产物，通过镍柱一步法纯化，以纯化的重组SjP38为抗原，免疫BALB/ｃ小鼠，采用杂交瘤技术制备McAb，用ELISA和有限稀释法筛选出高分泌滴度McAb杂交瘤细胞株，测定其免疫球蛋白亚类及其效价，蛋白质印迹法（Western blotting）分析其特异性。 结果 筛选出能稳定分泌抗重组SjP38单克隆抗体的8株杂交瘤细胞株，均为IgG1； Western blotting 分析显示8株单抗能与日本血吸虫虫卵抗原中的天然P38发生特异性结合。 结论 制备的抗P38杂交瘤细胞株能分泌高滴度、高特异性的McAb。     

Variability of the pigmentation of mycobacteria by the composition of medium

1. The pigmentation of mycobacteria was not influenced by the nitrogen source and by the carbon source. Under similar conditions, the pigmentation was very stable. 2. The pigmentation was usually more intensive on egg media than on the Sauton medium agar. In addition, the colouring was influenced by the age of cultures more markedly on egg media than on the Sauton medium agar. 3. The pigmentation of mycobacteria appeared to be more stable on the Sauton medium agar than on egg media, the Loewenstein-Jensen medium and the Ogawa egg medium. It had been recommended that the pigmentation is defined on the fresh cultures grown on the Sauton medium agar. 4. High temperature produced a more intensive pigmentation than low temperature. 5. Aromatic compounds sometimes influenced the colouring of mycobacteria, but this effect was not constant. 6. Sodium thioctate increased the colouring of scotochromogens in old cultures. 7. Development of the colouring ofM. kansasii, i.e. photosynthesis of carotenoids, was done only in the existence of oxygen. 8. Pigmentless mutants induced by ultraviolet irradiation showed the same biochemical characteristics as the original pigmented strains. 9. It has been found that all strains tested (six strains) of scotochromogens contain the same pigment beta-carotine.