软海绵酸胶体金免疫检测法的建立与应用

目的 建立并应用软海绵酸(OA)胶体金免疫层析检测法.方法 通过纯化抗OA单抗、制备胶体金、标记单抗、制备胶体金试剂条、样品模拟提取与加标回收检测、贝类染毒提取及检测,建立和优化金标免疫层析法.结果 选择30 nm胶体金颗粒用于单抗标记,金标单抗浓度为200 μg/mL,金标单抗包被量为2.5 μL/cm,二抗包被浓度为1.0 mg/mL,反应时间为3～5 min,对加标贝肉和染毒贝类的检测灵敏度为25 ng/mL.结论 建立了可用于OA检测的胶体金免疫层析检测法,满足规定的贝类OA含量安全阈值,为腹泻性贝毒检测与食品安全检验提供了新的技术方法.     

滴金法同步检测甲、乙、丙型肝炎病毒免疫标志物方法学建立及评价

目的　建立同步检测甲、乙、丙型肝炎病毒免疫标志物的金免疫渗滤法 (GIFA)的实验方法 ,并对其性能作评价。方法　采用胶体金作为免疫示踪物 ,标记羊抗人IgG单克隆抗体 ,将相应的病毒抗原HAV Ag、HBc Ag及HCV Ag包被在硝酸纤维素薄膜表面 ,标本中所含抗体与相应的抗原结合后 ,加入金标记抗体 ,即可在薄膜表面形成肉眼可见的红色斑点。结果　自制的GIFA纸条法检测 64 5份血清标本 ,与ELISA、RIA法作对比 ,符合率分别为 97.4%、96.9%、10 0 .0 1% ,最低检测浓度分别为 2ng/ml、1ng/ml、1ng/ml。 结论　本实验方法具有较高的特异性和灵敏度 ,操作简便。     

化学发光免疫法与免疫层析法测定肌钙蛋白Ⅰ结果的比较

目的用定量与定性的方法测定血清肌钙蛋白Ⅰ,对两种方法测定的结果进行比较分析.方法为了比较两种方法的敏感性,我们把患者分为低浓度组(cTn Ⅰ≤10ng/ml)和高浓度组(cTn Ⅰ＞10ng/m1),把化学发光免疫法测定的定量结果＞1.5ng/ml作为阳性样本个数,这样有利于两种方法之间的比较.结果用定量的化学发光免疫法测定肌钙蛋白Ⅰ的灵敏度比免疫层析法的快速诊断卡高,两种不同的方法,测定的结果存在较大差异(P＜0.01).结论定量的方法灵敏度高,即临床诊断率高;免疫层析法快速简便,但在低浓度时,不能及时检出.如用两种方法同时检测,这样可以提高心肌损伤病人的诊断率,做到快速准确.     

血清胃蛋白酶原和抗Hp IgG抗体对消化性溃疡的临床意义

目的研究血清胃蛋白酶原(PG)和抗幽门螺杆菌(Hp)IgG抗体对消化性溃疡的临床价值。方法选取2007年10月至2012年12月期间,在上海华东疗养院体检时发现的消化性溃疡115例作为溃疡组,其中胃溃疡65例,十二指肠溃疡50例。对照组为同期健康体检者,共90名,其中男女比例及年龄均与溃疡组匹配。采用胶乳增强免疫透射比浊法检测PGⅠ、PGⅡ,并计算两者比值(PGⅠ/PGⅡ,PGR),利用酶联免疫吸附试验(ELISA)对受检者血清进行抗Hp IgG抗体检测。结果溃疡组中,PGⅠ和PGⅡ水平分别为(180.14±20.56)和(24.98±10.14)ng/m L,PGR为7.87±1.29,PGⅠ、PGⅡ及PGR明显高于对照组(P0.05)。溃疡组和对照组抗Hp IgG抗体阳性率分别为77.4%和23.3%,差异具有统计学意义(P0.05)。Hp阳性组的PGⅠ与PGⅡ分别为(165.35±22.14)及(20.75±11.26)ng/m L,PGR为7.64±2.03,明显高于Hp阴性组(P0.05)。结论异常血清PG和抗Hp IgG抗体与消化性溃疡密切相关,并且可以作为消化性溃疡早期的筛查指标。     

化学发光免疫法与免疫层析法测定肌钙蛋白Ⅰ结果的比较

目的 用定量与定性的方法测定血清肌钙蛋白Ⅰ,对两种方法测定的结果进行比较分析.方法 为了比较两种方法的敏感性,我们把患者分为低浓度组（cTn Ⅰ≤10ng/ml）和高浓度组（cTn Ⅰ＞10ng/m1）,把化学发光免疫法测定的定量结果＞1.5ng/ml作为阳性样本个数,这样有利于两种方法之间的比较.结果 用定量的化学发光免疫法测定肌钙蛋白Ⅰ的灵敏度比免疫层析法的快速诊断卡高,两种不同的方法,测定的结果存在较大差异（P＜0.01）.结论 定量的方法灵敏度高,即临床诊断率高;免疫层析法快速简便,但在低浓度时,不能及时检出.如用两种方法同时检测,这样可以提高心肌损伤病人的诊断率,做到快速准确.     

胶体金免疫层析技术同步检测甲、戊型肝炎病毒IgG抗体的研究

[目的]探索并建立胶体金免疫层析(GICA)同步检测血清中的甲、戊型肝炎病毒的IgG抗体的方法。[方法]采用柠檬酸三钠还原法制备胶体金颗粒，标记鼠抗人IgG单克隆抗体，制成免疫层析测试条。血清中甲、戊型肝炎的特异性IgG抗体与测试条上金标抗体结合后沿着硝酸纤维素膜移动与膜上固相包被的抗原(HAAg、HEAg)、抗体(羊抗鼠IgG)结合形成肉眼可见的红色线条。[结果]自制的GICA试条与EIA对比检测血清标本188份，各单项指标抗-HAV IgG、抗-HEV IgG两法总符合率分别为98．9％、98．9％，检测灵敏度可达1ng／ml。[结论]用于同步检测血清中的甲、戊型肝炎病毒特异性IgG抗体的GICA试条的制备条件已基本建立；其检测结果特异性强、灵敏度高、操作方法简便、快捷，无需特殊仪器设备，有广泛的应用价值。     

细胞因子诱导的杀伤细胞(CIK)培养方案的优化

目的选择最优的细胞因子组合方案体外制备CIK细胞,为基于CIK细胞的肿瘤免疫治疗提供有价值的数据。方法采用Ficoll分离法获取健康人外周血单个核细胞(PBMC),PBMC加入激发型鼠抗人抗-CD3单抗(1μg/m L)包被的96孔板(2×106孔),各孔中分别按浓度梯度加入激发型抗-CD28单抗0.25μg/m L、0.5μg/m L、1μg/m L,IL-2 0.02万U/m L,0.1万U/m L,0.5万U/m L,IFN-γ50 ng/m L、100 ng/m L、200 ng/m L,于1、2、3、4、5、6、7 d通过细胞计数计算各孔细胞数量,以确定能获得最佳增殖效果的最低浓度,该浓度即为CIK诱导的合适浓度。获得最适浓度后,采用CCK-8掺入法检测不同诱导方案下CIK细胞体外增殖能力,采用流式细胞术观察在活化的于1、3、5、7 d不同诱导方案下CIK上CD4+、CD8+、CD56+的表达。结果 PBMC在细胞因子作用下均能增殖,加入细胞因子后培养至4-5 d时,可见细胞体积逐渐增大,培养至7 d细胞的胞体和胞核均增大,胞浆增多,胞核密度加强。细胞增殖曲线提示,在抗-CD3单抗浓度为1μg/m L下抗-CD28单抗、IL-2、IFN-γ的最适浓度分别为0.5μg/m L,、0.1万U/m L、100ng/m L。CCK法和流式细胞术分析显示,各组CIK细胞随着培养时间的延长增殖愈显著,且组间呈现统计学差异,各组中以抗-CD3+抗-CD28+IL-2+IFN-γ联合培养组CIK细胞体外增殖能力最强(P0.05),CD4+、CD8+和CD56+细胞比例培养前分别为(44.8±11.3)%、(21.1±8.5)%、(3.4±0.7)%,培养后分别为(72.1±13.8)%、(51.1±17.9)%、(10.7±4.4)%,比较均有差异性(P0.05)。结论采用激发型抗-CD3+抗-CD28+IL-2+IFN-γ联合培养方案获得的CIK细胞体外增殖能力最强,表型最为成熟。     

血清胃蛋白酶原Ⅰ/Ⅱ检测对胃癌诊断及预后判断的意义

目的探讨血清胃蛋白酶原Ⅰ/Ⅱ测定对胃癌诊断及预后判断的意义。方法用酶联免疫吸附实验分别测定胃溃疡、胃癌患者及健康人胃蛋白酶原Ⅰ/Ⅱ含量,并与单抗7相关抗原进行比较。结果健康人血清胃蛋白酶原Ⅰ/Ⅱ浓度分别为(98.77±31.24)ng/mL、(38.68±13.20)ng/mL;胃溃疡患者血清胃蛋白酶原Ⅰ浓度为(250.08±71.29)ng/mL,明显高于健康人(P<0.05),胃蛋白酶原Ⅱ浓度为(31.23±6.83)ng/mL,与健康人比较差异无统计学意义(P>0.05);胃癌患者血清胃蛋白酶原Ⅰ/Ⅱ浓度分别为(31.09±10.24)ng/mL、(9.67±7.23)ng/mL,均明显低于健康人(P<0.05);胃癌术后3年以上无复发或转移者血清胃蛋白酶原Ⅰ/Ⅱ浓度分别为(129.43±51.00)ng/mL、(23.83±13.68)ng/mL,明显高于新诊断胃癌患者(P<0.05);胃溃疡、胃癌患者血清单抗7相关抗原水平离散系数均较大,与胃蛋白酶原Ⅰ水平无相关性,两者在胃癌诊断中的特异性和灵敏度分别为81.1%、56.7%及91.9%、76.7%。结论血清胃蛋白酶原水平的测定可以为胃癌的诊断、疗效观察及预后判断提供依据,并优于血清单抗7相关抗原测定。     

检测反流性食管炎患者血清胃蛋白酶原和幽门螺杆菌抗体的临床意义

目的 探讨血清胃蛋白酶原(PG)含量和幽门螺杆菌(Hp)感染与反流性食管炎(RE)的关系.方法 经胃镜检查诊断RE患者31例,其中0级10例,Ⅰ级8例,Ⅱ级7例,Ⅲ级6例,正常对照组20例.用乳胶增强免疫比浊法测定血清中的PGⅠ和PGⅡ含量,同时计算出PGⅠ/PGⅡ比值(PGR).另外采用酶联免疫法分析血清抗幽门螺杆菌IgG抗体.结果 RE分级越高组的患者PGⅠ的含量越低(依次为112.2±14.4 ng/ml;88.2±9.8 ng/ml;76.1±10.2 ng/ml;62.4±13.5 ng/ml);RE各分级组间PGⅠ含量和PGR值差异均有统计学显著性意义(F=128.71,P<0.01;F=88.45,P<0.01),PGⅡ含量(依次为45.1±5.1 ng/ml;46.3±4.2 ng/ml;47.5±4.4 ng/ml;49.7±5.7 ng/ml)相比差异无统计学显著性意义(F=1.72,P>0.05);血清抗Hp抗体阳性的RE患者血清PGⅠ含量(78.2±10.4 ng/ml)及PGR(55.2±11.6 ng/ml)比血清抗Hp抗体阴性的RE患者低,两组相比均差异有统计学显著性意义(t=23.14,P<0.01;t=48.26,P<0.01).结论 PG含量变化和Hp感染与RE病变程度有关,联合检测PG和Hp对诊疗RE具有积极的意义.     

用对流免疫电泳(CIE)测定人血浆中C_3裂解产物(C_3SP)

用CIE技术检测人血浆中C_3SP是一种可估价C_3在体内转化的筛选方法。其原理乃因C_3SP所带负电荷较天然C_3分子多,在硷性环境中电泳时,其电泳速度较天然C_3分子快,故可出现偏阳极侧的第二条沉淀线。试验采用EDTA二钠抗凝剂,以阻止补体在体外继续活化。静脉取血后2小时内分离血浆。按常法制板、打孔,待测样品加在阴极孔,阳极孔加含抗C_3C—C_3d的抗C_3血清。CIE完毕,板置湿盒37℃扩散1～2小时观察结果。检测同时用菊糖活化血清或聚合人IgG活化血清作阳性对照。     

Purification and use of the C3d subunit C3

A method is described for the preparation of C3d from fresh-frozen CPD plasma. A redissolved EDTA euglobulin precipitate formed from defibrinated plasma is chromatographed on DEAE cellulose. The C3-rich peak is further chromatographed by stepwise elution from hydroxy-apatite. The highly purified C3 and C3b so obtained are then treated with commercial C3b inactivator. G-200 sephadex filtration of this material produces a low molecular weight peak containing C3d greater than 95 per cent purity. The purified C3d, which contains negligible carbohydrate, has a molecular weight of 28,000 (based on SDS polyacrylamide gel electrophoresis). When added at a final concentration of less than 70 mug per ml, it completely inhibits the anti-C3d hemagglutinin reactivity of a potent anti-C3d antiglobulin serum but does not inhibit sera with reactivities against C3c, C4b, and C5b. Two of three rabbits hyperimmunized with purified C3d produced antisera exhibiting only anti-C3d precipitin activity. After absorption of heterologous antibodies, the antisera strongly agglutinated C3d-coated RBC, failed to agglutinate RBC glutinated C3d-coated RBC, failed to agglutinate RBC coated only with C4, and reacted weakly against strongly anti-Rh coated RBC. The latter reactivity was readily absorbed by whole IgG. Preliminary studies with 125I-labeled C3d indicate its potential value in assessing potency of anti-C3d reagents.     

Human Lymphocytes Bear Membrane Receptors for C3b and C3d

Human peripheral blood lymphocytes have membrane receptors for EAC43b (sheep erythrocytes sensitized with antibody and complement) and also for EAC43d, obtained by treating EAC43b with C3b inactivator. Human granulocytes bind only EAC43b, C3 fragments obtained by limited trypsin digestion of purified human C3 display both C3b and C3d sites, since they inhibit rosette formation of lymphocytes with EAC43b and EAC43d. These findings raise the possibility that C3b and C3d receptor sites may be selectively distributed among normal subpopulations of B lymphocytes as well as among leukemic leukocytes.     

Partial characterization of physiologically generated C3 components expressing C3d but not C3c epitopes

Techniques for the quantification of C3d are shown to estimate the sum of 4 different plasma protein components possessing C3d but not C3c epitopes. All 4 components were C3-derived polypeptides as shown by activating serum containing 125I-labelled C3, isolating the anti-C3d reactive material in 14% PEG supernatant, followed by analysis on SDS-PAGE and autoradiography. Identical results were obtained by radiolabelling 14% PEG plasma supernatants followed by analysis of the anti-C3d reactive material. The components are referred to as d1, d1', d2 and d3 based on their relative electrophoretic mobilities (alpha 1, alpha 1, alpha 2 and alpha 2 respectively) judged by crossed immunoelectrophoresis. Their apparent molecular weights by SDS-PAGE were 129K (d1), 110K (d1'), 46K (d3) and 45K (d2). The possibility that one or more of the C3d containing components represented a complex of a C3 fragment with another plasma protein was investigated. The role of these components in the scheme of the physiological breakdown of C3 and the importance of the individual C3d components as indicators of complement activation in clinical materials is discussed. It is proposed that the 45K d2 component represents a final physiological breakdown product of C3 in human serum.     

The role of membrane receptors for C3b and C3d in phagocytosis

In this paper we re-examine the roles of particle-bound IgG and C3 in phagocytosis of sheep erythrocytes (E) by monolayers of purified human monocytes and polymorphonuclear leukocytes (PMN). We conclude that two fragments of the C3 molecule, that is, C3b and C3d, can function as opsonins if the phagocyte has the appropriate membrane receptors. Monocytes, that bind both C3b and C3d, respond to both as opsonins. PMN, which do not bind C3d, respond only to particles opsonized with C3b. C3 and IgG have separate roles in phagocytosis. IgG, through its Fc fragment, directly stimulates particle ingestion, but is relatively inefficient at inducing particle binding. On the other hand, C3 primarily mediates the binding of the particle via complement receptors. A marked synergy exists between C3 and IgG in inducing phagocytosis. Thus, opsonization of the particle with C3 can be a necessary condition for particle ingestion, although by itself C3 does not trigger phagocytosis. The opsonic effect of C3 can be mimicked by a variety of nonimmunologic agents which enhance binding of the particle to the phagocyte without directly stimulating ingestion. The contact- inducing agents used include centrifugation of particle and phagocyte, high molecular weight dextran, protamine, and treatment of E with neuraminidase. These results suggest that the role of C3 in opsonization is mainly or exclusively one of establishing contact between particle and phagocyte.     

Anti-C3d Antiglobulin Reagents. II. Preparation of an Antiglobulin Serum Monospecific for C3d

Three approaches are described to the preparation of a potent antiglobulin reagent monospecific for the C3d component of C3: 1) attempts to absorb out the anti-C3c component of a bisperific anti-C3c, C3d serum, 2) attempts to isolate by affinity chromatography purified C3d to be used for animal immunization, 3) animal immunization with a relatively crude filtration fraction of aged serum which contained C3d but lacked C3c. Problems encountered with the first two approaches are described and discussed. Preparation of a potent anti-C3d serum by the third approach is documented, as are methods employed to assess the specificity and potency of the reagent.     

Differences in specificities of anti-C3d sera raised to C3d antigens prepared in different ways

Anti-C3d sera were raised to different C3d antigens or to the same C3d antigen by different methods. Although identical by immunoprecipitation studies, the various anti-C3d sera showed differences in specificities against bound C3d antigen. Such differences were observed wih red blood cells coated with C3d in vivo and in vitro. Antisera made to the C3d− KAF antigen detected fewer molecules/cell on C3d−tryp cells than did antisera made to the C3d−tryp antigen. The converse was true for C3d− KAF cells. "Saturation" experiments indicated that different anti−C3d detected different "subpopulations" of bound C3d. C3d bound to red blood cells in vivo was, in at least one case, detectable by some anti- C3d sera but not by others. Such differences in anti-C3d specificity may be important in determining the optimal characteristics of anti-C3d antiglobulin serum for routine laboratory use.     

Red blood cell-bound C3d in selected hospital patients

A radioactive anti-antiglobulin technique was used to measure C3d bound to the red blood cells of 227 hospitalized patients in 129 patients, with a wide variety of diseases, normal levels of RBC-bound C3d were found. Seventy-two patients had moderately elevated RBC-bound C3d; they generally did not have autoimmune hemolytic anemia but had diseases in which complement is thought to be activated. Patients with markedly elevated RBC-bound C3d (26 patients) usually had autoimmune hemolytic anemia with a positive antiglobulin test. Some patients with only moderately elevated levels of RBC-bound C3d had autoimmune hemolytic anemia and a negative antiglobulin test in individual patients the level of RBC-bound C3d correlated with both the severity of disease and the response to treatment. RBC-bound C3b was detected in two patients with a very high level of RBC-bound C3d. This study provides background data for assessing the significance of complement activation and fixation to RBC in health and disease.     

Comparative study of assays detecting complement activation. Complement-split product C3d (rocket immuno-electrophoresis) and C3d neodeterminants (ELISA).

The aim of the study was to investigate the correlation between two types of assay measuring specific products of complement C3 activation and their clinical application. Complement C3d split product was estimated using double-decker rocket immunoelectrophoresis (DD-RIE) and measurements of C3d neodeterminants exposed after C3 activation was carried out with an enzyme-linked immunosorbent assay (ELISA). A total of 595 blood samples were measured in parallel in the DD-RIE and the ELISA test systems. The samples originated from blood donors (44), uraemic patients undergoing dialysis (135), serial samples from rheumatoid arthritis (RA) patients during steroid treatment (88) and 328 randomly collected patient samples. The mean values for DD-RIE and ELISA (+/- 1 SD) for the 595 samples were 48 (+/- 20) mU/l and 48 (+/- 28) mU/l respectively. The interassay coefficient of variation (CV) in the ELISA was 18%. The Spearman rho-correlation coefficient between the two assays was 0.63 for all 595 samples. The mean values using ELISA and DD-RIE were practically identical for the 328 successively incoming samples, the samples from the 135 dialysis patients and the 44 donors. In RA patients a higher mean value was found for the 88 samples using DD-RIE than ELISA. In the majority of patient samples there was a good correlation between the two assays. However, the ELISA appears to be more sensitive in detecting acute complement activation and to give lower levels in RA patients with chronic complement activation.