黄鳝IgM的分离纯化及兔抗黄鳝IgM抗血清的制备

目的分离纯化黄鳝血清免疫球蛋白,制备其兔抗血清,并检测抗血清的特异性。方法用Protein A亲和层析的方法纯化黄鳝血清免疫球蛋白,通过SDS-聚丙烯酰胺凝胶电泳检测蛋白的纯度,免疫大耳白兔制备抗血清,利用免疫双扩散检测抗血清的效价,通过western blotting检测抗血清的特异性。结果纯化了黄鳝血清免疫球蛋白,免疫双扩散法测定兔抗黄鳝免疫球蛋白血清效价为1∶32,western blotting结果显示抗血清具有很好的特异性。结论成功纯化了黄鳝免疫球蛋白,制备了兔抗黄鳝IgM抗血清,为建立黄鳝的血清学检测系统奠定了基础。     

一种制备特异性人体甲胎蛋白抗血清的简便方法

单相特异性抗甲胎蛋白抗血清是临床检测血清甲胎蛋白不可缺少的一种试剂。以往制备这种抗血清,除用甲胎蛋白粗制品免疫的需用正常人混合血清吸收外,均需经分离和纯化甲胎蛋白的步骤,因此操作繁复,周期较长。最近,我们用聚丙烯酰胺凝胶-并列抗体凝胶交叉免疫电泳(详见《细胞生物学杂志》一九七九年第一卷第二期),取得纯化的抗原抗体复合物,并用以免疫动物,可获得较大量的单相特异性的甲胎蛋白抗血清。现将方法简述如下。     

免疫耐受性在人免疫球蛋白诊断血清制备上的应用

免疫球蛋白(简称 Ig)由于在结构、所带电荷和生物活性方面极不匀一,故分离和纯化比较困难。如用作抗原以免疫动物,获得的抗血清总带有非特异性的抗体成分(IgG 诊断血清除外),必须用脐血清或相应的抗原经液相或固相方法吸收后才能除去,因而抗原消耗大操作也繁琐。近年来,我们运用特异性免疫抑制即免疫耐受性原理,制备了纯度理想的人 Ig 诊断血清。现以人 IgM 诊断血清为例,将制备方法和检测结果报道如下。     

人肝癌特异性γ－GTⅡ纯化及抗血清制备

以生化技术从人肝癌组织中提取、纯化出肝癌特异性γ－谷氨酰转肽酶（γ－ＧＴⅡ），并以此酶为抗原免疫新西兰兔制备抗γ－ＧＴⅡ血清。应用免疫双扩散法对该抗血清的特异性进行检测。结果显示此抗血清与人肝癌γ－ＧＴⅡ抗原及原发性肝癌病人血清均具有很好的特异性免疫反应。     

人血清中补体第一成份亚基的分离

参照本室改良Lepow 等的方法从人血清中分离微量C1q,免疫家兔,制备出抗人C1q 抗血清。将此抗血清经DEAE-纤维素柱层析纯化后,偶联到过碘酸活化的不溶性载体Se-phadex G-75上,制备成亲和层析柱。吸附人血清中相应的依赖于Ca~(2+)结合在一起的C1成     

人肝癌特异性γ—GTⅡ纯化及抗血清制备

以生化技术从人肝癌组织中提取，纯化出肝癌特异性γ－谷氨酰转酞酶（γ－ＧＴⅡ），并以此酶为抗原免疫新西兰家兔制备抗γ－ＧＴⅡ血清。应用免疫双扩散法对该抗血清的特异性进行检测，结果显示此抗血清与人肝癌γ－ＧＴⅡ抗原及原发性肝癌病人血清均具有很好的特异性性免疫反应。     

卡波氏肉瘤相关疱疹病毒ORF65抗体的制备及其特异性检测

目的：制备高效价卡波氏肉瘤相关疱疹病毒（KSHV）病毒ORF65衣壳蛋白抗体并检测其特异性。方法将人工合成的ORF65蛋白抗原肽经弗氏佐剂乳化,在家兔背部、颌下等皮下多点免疫注射,间隔2周免疫,共4次。末次免疫后1周取兔血清,ELISA法检测兔免疫血清抗体IgG抗体水平。进一步采用免疫荧光、Western blot检测制备的抗血清中与ORF65结合的特异性。结果家兔在全程免疫后产生了高效价的特异性抗体,最高滴度达1∶12800。免疫荧光检测显示,与未经佛波酯（TPA）刺激的BCBL‐1细胞相比,兔抗血清主要结合于BCBL‐1细胞胞质,与ORF65蛋白在细胞内表达位置一致。Western blot结果显示在21 kD处有特异性蛋白条带,其大小与预期的ORF65蛋白大小相符,同时经T PA刺激的BCBL‐1细胞中ORF65蛋白表达量高于对照组,与ORF65蛋白裂解期蛋白的特性相符。结论用ORF65衣壳蛋白抗原肽段免疫家兔,可成功制备高效价的特异性抗血清,该抗体可与KSHV病毒ORF65蛋白特异性结合。     

人肝脏微粒体蛋白凝胶免疫多克隆抗体的制备

目的制备人肝微粒体蛋白多克隆抗体,为进一步研究微粒体蛋白的功能及制备单克隆抗体打下基础.方法用含人肝微粒体高丰度蛋白的蛋白凝胶免疫家兔,再用ELISA和Western blot方法检测血清效价和抗体特异性.结果 2只家兔均产生了高效价的抗血清,Western blot结果显示,抗血清可以与相应蛋白结合.结论用含人肝微粒体高丰度蛋白的蛋白凝胶免疫的2只家兔均获得了高效价的抗血清,为深入研究该蛋白的定性、定量及定位奠定了基础,并为将来进行单克隆抗体的制备做前期技术支持.     

人血清转铁蛋白酶联免疫测定法的研究

建立人血清转铁蛋白的竞争性酶联免疫测定法，促进Ｔｆ的实验研究及临床检测。方法：采用纯化人Ｔｆ免疫家兔制备抗血清，进行间接竞争性酶联免疫抑制试验，并进行灵敏度，精确度准确性、特异性试验。     

抗人载脂蛋白E多抗的制备与纯化

利用纯化的人载脂蛋白Ｅ（ａｐｏＥ）作为抗原，常规免疫纯系大耳白家兔，获得高效价、高特异性的抗血清，抗体滴度经ＥＬＩＳＡ法检测达１：１２８００倍稀释度，抗体特异性检验证明抗体只与ａｐｏＥ反应。抗血清经ＰｒｏｔｅｉｎＡ－ＳｅｐｈａｒｏｓｅＣＬ－４Ｂ亲和层析纯化，分离为碱性洗脱峰Ⅰ和酸性洗脱峰Ⅱ，峰Ⅱ经ＳＤＳ－ＰＡＧＥ检测在分子量为２７ｋｄ和５４ｋｄ处呈现两条带，分别相当于抗体的重链和轻链的分子量，证明峰Ⅱ为纯化的抗体，这就为建立测定人血清ａｐｏＥ浓度的免疫学方法奠定了基础。     

The detection of transmissible gastroenteritis viral antibodies by immunodiffusion.

Precipitating antibodies against transmissible gastroenteritis viral antigens were detected by the immunodiffusion test in two transmissible gastroenteritis viral hyperimmune antisera and in antiserum prepared against haemagglutinating encephalomyelitis virus but not in sera from several species of normal animals, in antisera prepared against a variety of othet viruses and bacteria or sera from swine with bacterial enteritis. When the immunodiffusion test was compared with the virus neutralization test for the detection of transmissible gastroeneritis viral antibodies in 20 swine sera certain samples which contained high titres of virus neutralizing antibodies failed to produce precipitation while other sera were positive in the immunodiffusion test although their virus neutralizing antibody titres were relatively low. Precipitating antibodies were also detected by immunodiffusion in several samples of milk whey from a sow which had been vaccinated with inactivated transmissible gastroenteritis virus.     

ACTIVATION OF THE ALTERNATIVE COMPLEMENT PATHWAY IN PATIENTS WITH EPIDEMIC HEMORRHAGIC FEVER

The levels of Factor B (B), properdin (P) and the third complement component (C3) in serial serum samples obtained from 36 hospitali- zed patients with epidemic hemorrhagic fever (EHF) were measured by single radial immuno- diffusion. These proteins of the alterna.tive activating pathway of C were activated and their circulating levels reduced. Good correlations were observed between the B, P a.nd C3 levels in patient sera The alternative C pathway was activated in these patients. The num.ber of the positive cases and positive sera was increa.sed with severity of illness, strongly suggesting the correlation of activatio,n of this pathway with the disease condition. In addition, activat.ion of the classical C pathway in the same patients was again observed through determination of activa- tion of the first complement component (CI) by assessing the ratio of the subunit Clr to subunit Cls (the Clr:Cls ratio) according to a method previously described. Some discrepancies were found in activation between the 2 pathways. A variety of biologically act.ive peptides produced during enormous activation of the C system via the alternative and classical pathwaysmay co-ntribute to a number of serious conse- quences in EHF, including increased vascular permeability9 hypotension, shock, hemorrhage and renal damage.     

A microtitration agglutination test for detecting group E streptococcus infection in swine

A microtitration agglutination test was developed and evaluated for detecting infection of swine with group E streptococci type IV, the most common causative agent of streptococcic lymphadenitis of swine.Whole cell agglutinogens representing group and type antigens of group E streptococci were tested in the microtitration agglutination test against reference antisera to Streptococcus groups A, B, C, D, E, F, G. H, K, L, M, N, O, P, Q, R, S and U, as well as specific antisera to types II, IV and V of group E. Group E specific agglutinogens were unsatisfactory in the microtitration agglutination test because of cross reactions with group P and U antisera and because of poor reproducibility of the test. Type specific agglutinogens of group E streptococci reacted only with their respective homologous antisera and not with any heterologous group antisera. None of the group E streptococci agglutinogens reacted with 52 normal swine sera.Agglutinogen made from group E streptococci type IV was selected for further evaluation in the microtitration agglutination test because group E streptococci types II and V are considered to be of minor importance in the etiology of streptococcic lymphadenitis of swine. Swine experimentally infected with a type IV strain developed significant titers in the microtitration agglutination test. All swine tested negative before exposure and seroconverted (titer >/=4) two to six weeks postexposure.The microtitration agglutination test was used by two different laboratories to test 187 duplicate samples of serum from infected swine. A total of 94.1% of the tests were read at either the same titer (48.1%) or a difference of not more than one dilution (46.0%) at the two laboratories. There was disagreement between the two laboratories in the test-positive test-negative status of 19 of the sera (10.2%). Titers of two of the sera differed by two dilutions (<4 at one laboratory and 8 at the other). The remaining 17 sera differed in titer by only one dilution (<4 at one laboratory and 4 at the other).     

1000例非孕经产妇淋巴细胞毒抗血清的筛选

为鉴定中国人白细胞抗原(HLA)系统,本文采用微量淋巴细胞毒试验方法,筛选武汉地区非孕经产妇血清1000份,获得淋巴细胞毒反应强度20％以上的阳性血清136份,其中70％以上16份,总阳性率13.6％,强阳性率1.6％。136份阳性血清中98份阳性血清进行了相关集群,共集得16组显著相关的淋巴细胞毒抗血清,希望通过集群获得HLA分型血清。本文对筛选结果和试验方法进行了讨论。     

乙型肝炎补体Cl激活的研究

作者测定88例 HBV 感染的急肝、慢肝、重型肝炎和肝炎肝硬化患者的 Clg、Clr、Cls 和 CiINH,并探讨其变化之意义。发现患者 Cl 明显激活,血清 Cls 减少与病情轻重有关,Cls 水平随肝脏损伤加剧而下降。CiINH 消耗增多、合成减少,不能有效地调控Cl 活化。认为测定血清 Cls 对研究补体经典途径活化、探讨乙肝发病机理、判断患者肝脏损害程度及估计预后都有一定的意义。     

用多价抗血清间接荧光抗体法快速鉴定艰难梭菌的研究

用四株不同分离来源的艰难梭菌免疫家兔制备的抗血清,经索氏梭菌吸收后混合成多价抗血清,通过间接荧光抗体法,对从138份腹泻标本中分离出的10株艰难梭菌进行快速鉴定并对其特异性及敏感性进行了讨论。     

生物素-亲和素酶联免疫吸附试验系统检测兔抗伤寒杆菌抗体的实验研究

以国产生物素(B)及亲和素(A)试剂,初步建立了检测免疫血清中抗伤寒杆菌H及O抗体水平的BA—ELISA系统。检出抗H及抗O抗体的敏感性(以GMT计),较常规ELISA高6～7倍;较传统的肥达氏试验高11～22倍。除与副伤寒甲、乙及伤寒Vi血清有轻度交叉反应外,与其他各种菌的抗血清均呈阴性.丁不同天对8份阳性血清经6次重复试验,所得变异系数均<11％,提示本试验系统稳定性良好。     

The bovine immune response to Brucella abortus I. A water soluble antigen precipitated by sera of some naturally infected cattle.

Selected sera from cattle naturally infected with Brucella abortus precipitate water soluble antigens extracted by sonication from B. abortus. One of these antigens resembles antigen E (Baughn and Freeman) as it is excluded from Sephadex G-200 gels, migrates anodally when electrophoresed at pH 8.6, resists heating at 100 degrees C for ten minutes and appears to be susceptible to papain digestion. Precipitins specific for this antigen remained in sera from which all detectable Brucella agglutinating antibody had been removed by adsorption with live or heat killed B. abortus. The antigen has been extracted from smooth and rough strains of B abortus. Precipitins specific for this antigen have been detected in antisera produced against Brucella canis.     

Serological cross-reactivity of porcine reference antisera to Mycoplasma hyopneumoniae, M. flocculare, M. hyorhinis and M. hyosynoviae indicated by the enzyme-linked immunosorbent assay, complement fixation and indirect hemagglutination tests

The enzyme-linked immunosorbent assay (ELISA) indicated significant cross-reactivity between the antigens of Mycoplasma hyopneumoniae ( HyoP ) and M. flocculare (Floc), another porcine mycoplasma of wide distribution but uncertain pathogenic significance, when porcine antisera of each specificity were tested against HyoP antigen. The titers of the anti-Floc sera ranged from threefold to 13-fold less than the titer of the anti- HyoP reference serum at different times after immunization. These values ranged from onefold less than to fourfold greater than the minimal positive titer of 80. The antisera to the other porcine mycoplasmal antigens [i.e. M. hyorhinis ( HyoR ) and M. hyosynoviae ( HyoS )] reacted less strongly to HyoP antigen but titers only slightly less than to slightly greater than the minimal positive titer were noted for some sera. Cross-reactivity was also detected by the complement fixation test, although the titers for this test were generally lower than for the ELISA, presumably reflecting lower sensitivity of the complement fixation test. Positive indirect hemagglutination titers to HyoP antigen were also observed for both anti-Floc sera obtained at one or more times during the immune response. With two exceptions (one anti- HyoR serum with a complement fixation titer of 16 and one anti- HyoR serum with an indirect hemagglutination titer of 10), none of the anti- HyoR or anti- HyoS sera had detectable indirect hemagglutination or complement fixation titers to HyoP antigen at any time after immunization. The levels of cross-reactivity detected by the complement fixation test and indirect hemagglutination and, especially, the ELISA would be of significance for the development of any practical sero-diagnostic test for mycoplasmal pneumonia of swine.     

Humoral antibody responses of swine infected experimentally with group E streptococcus. I. Whole cell agglutinin response

Reference streptococcal antisera and sera collected from swine infected experimentally (by intranasal inoculation or contact exposure) with group E Streptococcus (GES) were studied in a tube agglutination system using whole GES cells.Specificity studies revealed common group specific antigen among GES serotypes I and III, GES strains devoid of type specific antigen (untypable by ring precipitin testing) and group P and group U Streptococcus. The group specific antigens were not agglutinated by GES type specific antisera or by group specific antisera against Streptococcus groups A, B, C, D, F, G, H, K, L, M, N, or O. Results of the study suggested that GES serotypes I and III are invalid; i.e., they are devoid of type specific antigen.Groug E Streptococcus type specific antigens II, IV, and V were agglutinated significantly only by their homologous antisera.Experimentally infected swine developed significant titers against both the group and type specific antigen of GES. Antibodies appeared from three to eight weeks postexposure and persisted for the duration of the experiment (six months). The potential utilization of the whole cell agglutination (WCA) test for detection of GES carrier swine is discussed.