氧化高密度脂蛋白和氧化低密度脂蛋白单克隆抗体的制备

目的 制备抗氧化高密度脂蛋白(Ox-HDL)和氧化低密度脂蛋白(Ox-LDL)单克隆抗体(McAb).方法 以Ox-HDL和Ox-LDL为抗原,免疫BALB/C小鼠,用杂交瘤技术建立稳定分泌抗相关抗原的McAb.用protein A柱亲和层析法纯化腹水中McAb,用Dot-ELISA和ELISA等鉴定其生物学活性.结果 获得3株不同结合特性的抗Ox-HDL和Ox-LDL的单克隆杂交瘤细胞(IC4Gl2,6H9A4A11和8C1A6),2株为IgG1亚型,1株为lgG2b亚型;腹水效价大干10',Dot-ELISA和ELISA检测证实1C4G12为具有对Ox-HDL和Ox-LDL交叉反应性的McAb,其与Ox-HDL单抗6H9A4A11可构成双抗体夹心体系测定0x-HDL水平,而与Ox-LDL单抗8C1A6构成的体系可测定Ox-LDL水平.结论 本实验通过构建3株不同特性的交叉反应性McAb,可用于测定Ox-HDL和Ox-LDL免疫方法 的建立.     

采用自身抗体建立氧化脂蛋白(a)水平的ELISA检测法

目的提取和鉴定人血清中氧化脂蛋白（a）[ox-Lp（a）]自身抗体，建立ox-Lp（a）ELISA检测法并进行临床研究。方法采用溴化氰活化的sepharose4B交联ox-Lp（a）制备免疫吸附层析柱，从正常人血清中提取抗ox-Lp（a）自身抗体，分析其免疫反应性。分别建立以抗ox-Lp（a）的自身抗体、抗氧化LDL（ox-LDL）多克隆抗体为包被抗体，酶标抗载脂蛋白（a）[apo（a）]为检测抗体的ox-Lp（a）ELISA检测法，并对100例冠心病患者和100名健康体检人群进行分析。结果8份正常人血清中均存在抗ox-Lp（a）自身抗体，与ox-Lp（a），ox-LDL均具有良好的反应性；再经亲和层析除出抗ox-LDL自身抗体后，2份标本同ox-Lp（a）仍具有较高的反应性。同对照组相比，本组冠心病患者血浆Lp（a）水平显著升高[（279．6±162．7）mg／L比（206．3±126．4）mg／L，P〈0．01]；ox-Lp（a）水平亦明显增加（P〈0．01），采用自身抗体和抗ox-LDL抗体建立的ELISA法测定结果分别为：（24．3±33．4）μg/ml比（8．4±9．3）μ/ml和（13．0±13．8）μ/ml比（7．3±9．7）μ/ml；两种ELISA法间结果高度相关（r=0．78，P〈0．01）。结论提取的人血清ox-Lp（a）自身抗体可识别apo（a）和apoB氧化位点，采用自身抗体建立的ox-Lp（a）检测法能更真实、准确地反应体内Lp（a）的氧化状态，冠心病患者ox-Lp（a）水平显著升高。     

免疫PCR检测乳腺癌患者血清p185蛋白抗体

目的 为乳腺癌诊断提供血清学新标志.方法 免疫PCR方法检测血清抗p185蛋白抗体,酶免疫组化方法检测组织p185蛋白表达.结果 乳腺癌患者血清抗p185蛋白抗体阳性率为30%,而非癌患者和正常人血清抗p185蛋白抗体均为阴性,乳腺癌患者血清中抗p185蛋白抗体显著高于非癌患者和正常人(P<0.01).p185蛋白阳性表达的乳腺癌患者抗p185蛋白抗体阳性率为69.2%,明显高于p185蛋白阴性表达组,血清p185抗体测定与p185蛋白表达密切相关(P<0.01).结论 检测血清抗p185蛋白抗体是检测组织p185蛋白理想的替代工具.抗p53蛋白抗体可以作为乳腺癌血清学诊断新标志,用于乳腺癌的普查和早期诊断.     

自身免疫性肝炎患者血清 RF、ANA、ASMA及AMA检测与分析

目的分析自身免疫性肝炎(AIH)患者血清中类风湿因子(RF)与抗核抗体(ANA)、抗平滑肌抗体(ASMA)和抗线粒体抗体(AMA)的相互关系,为临床诊断AIH筛选和确立相应的实验辅助诊断指标.方法收集34例AIH患者血清,采用免疫浊度法检测血清中RF,间接免疫荧光法检测血清ANA、ASMA和AMA.结果患者血清RF的阳性率为67.6%(23/34),ANA、ASMA和AMA的阳性率分别为52.9%(18/34)、17.6%(6/34)和11.7%(4/34),3种自身抗体检出的总阳性率为58.8%(20/34).统计分析显示,RF阳性率与3种自身抗体检出的总阳性率之间无明显差异(P＞0.05).此外,20例自身抗体阳性血清均为RF阳性.进一步分析显示,血清RF含量与3种自身抗体阳性率之间具有密切相关性.结论 AIH患者血清中RF阳性率与其ANA、ASMA和AMA 3种自身抗体的阳性率密切相关,提示RF与ANA、ASMA和AMA均可作为实验诊断AIH的辅助参考指标.     

氧化、丙二醛修饰低密度脂蛋白的ELISA检测法及临床应用

目的 建立血浆氧化、修饰低密度脂蛋白(LDL)ELISA检测法并进行临床研究.方法 采用自制的多克隆抗体建立ELISA分别测定铜离子氧化型(Ox)和丙二醛(MDA)修饰型LDL,并对方法进行考核;对冠心病(CHD)患者、键康对照人群Ox-LDL、MDA-LDL水平进行分析.结果 抗Ox-LDL与MDA-LDL几乎没有反应性;Ox-LDL和MDA-LDL测定平均批内变异(CV)分别为6.1%和6.8%,平均批间CV分别为9.7%和10.1%;Ox-LDL和MDA-LDL测定平均回收率分别为95.1%和93.7%;两法检测限均为0.05～1.5 mg/L.CHD患者较正常人血浆Ox-LDL[(178.1±73.8)mg/L vs.(82.7±29.1)mg/L,P＜0.01]、MDA-LDL[(48.7±25.6)mg/L vs.(39.2±32.9)mg/L,P＜0.05]水平均升高;Ox-LDL作为预测动脉粥样硬化发生指标的价值优于MDA-LDL.Ox-LDL、MDA-LDL分别同TC、TG、LDL-C呈正相关,而与HDL-C呈负相关.结论 建立的方法特异、灵敏、准确,适于临床检测.高Ox-LDL、MDA-LDL与CHD密切相关.     

育龄期多囊卵巢综合征患者血清自身抗体研究

目的：探讨血清非器官特异性自身抗体与多囊卵巢综合征（PCOS）的关系。方法选择就诊于本院的育龄期PCOS患者69例为研究组,同期健康育龄期妇女69例为对照组。采用 ELISA 法检测血清抗核抗体（ANA）、胶体金斑点渗滤法检测血清抗双鋣 DNA（ds-DNA）抗体、免疫印迹法检测血清抗可提取性核抗原（ENA）抗体谱。结果研究组与对照组比较,血清中ANA 和抗 ds-DNA 阳性率显著升高,差异有统计学意义（P ＜0．05）,抗 ENA 自身抗体谱中各种抗体差异无统计学意义（P ＞0．05）。结论PCOS 患者中存在血清自身抗体阳性。     

心肌重构对抗β1受体自身抗体及免疫功能的影响

目的检测心肌重构患者血清中抗β1受体自身抗体和相关免疫指标的变化情况,探讨其变化特点、相互关系及临床意义.方法选择心室壁增厚、心腔与心壁比值增大、拟行手术的患者28例,正常人10例作为对照.应用链霉亲和素-酶联免疫吸附测定技术(SA-ELISA),测定血清中抗β1受体自身抗体;应用流式细胞技术,测定CD3+、CD4+、CD8+含量及CD4+/CD8+比值,IgG、IgA、IgM及C3、C4含量.结果当心肌发生重构时,患者产生抗β1受体自身抗体,随着术后好转,该自身抗体也会随之降低.当抗β1受体自身抗体存在时,机体的细胞免疫CD4+/CD8+比值明显增高,体液免疫5项指标均有先下降后上升的趋势,与细胞免疫基本一致.结论心肌重构后,患者抗β1受体自身抗体的变化能反映术后疗效及心脏功能的恢复情况,可用来指导临床治疗.免疫功能的变化,说明心肌重构早期,机体免疫功能受到抑制,应注意围手术期采用综合性预防感染的措施.     

液相芯片系统在检测抗核抗体谱中的应用

目的探讨抗核抗体谱联合检测在液相芯片检测系统中的性能,提高自身免疫性疾病的诊疗效果。方法收集已确诊为自身免疫性疾病患者血样120例(病例组),体检正常血清90例(健康对照组),分别以线性免疫印迹法(LIA法)和液相芯片系统联合检测抗Ro52、抗SSA、抗SSB、抗Sm、抗RNP、抗Scl-70、抗Jo-1、抗Rib、抗His、抗PCNA、抗CB、抗PM、抗M2、抗NUC、抗双链DNA抗体15种自身抗体,评估其灵敏度、特异度和交叉反应。结果健康对照组与病例组患者总体阳性率比较,差异无统计学意义(P0.05)。液相芯片系统与LIA法对15种自身抗体的检测结果具有良好的一致性(P0.05),但灵敏度优于LIA法。抗体在液相芯片系统中无交叉反应。结论液相芯片系统对抗核抗体联合检测具有良好的性能,其对抗核抗体谱的检测具有较好的灵敏度和特异度。     

免疫斑点法测抗钙调素自身抗体及其与其他8种自身抗体的关系

目的 探讨抗钙调素（ＣａＭ）自身抗体与自身免疫病及其他自身抗体的相互关系。方法 采用ＳＰＡ－免疫斑点法（ＳＰＡ－ＩＤＡ）对３４０例自身免疫病患者,４８例肿瘤患者,５２例正常人血清进行抗ＣａＭ抗体测定,并随机取自身免疫病患者血清同时检测其他８种自身抗体。结果 各类延寿央免疫病患者体内不同程度地存在着抗ＣａＭ自身抗体,４８例肿瘤患者血清中仅１例阳性,５２例正常人血清中无１例阳性。经统计学分析,抗ＣａＭ     

丙型肝炎病毒不同编码区重组抗原在抗体检测中的应用

目的　探讨丙型肝炎病毒 (HCV)不同编码区重组蛋白片段的免疫反应性及其在抗体检测中的应用。方法　分别以 4种HCV单片段抗原 (C、NS3、NS4或NS5 )检测抗 HCV抗体阳性血清 ;以 4种单片段抗原的混合物 (MIX)检测抗 HCV抗体阳性血清、大学生体检血清和抗 HCV抗体阴性质控血清。结果　 90份抗 HCV抗体阳性血清中共有 75份可与一种或多种单片段抗原反应 ,阳性率分别为 70 .0 % (C)、6 1.1% (NS3)、5 2 .2 % (NS4 )、4 4 .4 % (NS5 ) ,其中仅与C、NS3、NS4和NS5一种抗原发生反应的分别有 8份、1份、2份和 3份血清标本 ;90份抗 HCV阳性血清经MIX抗原检测 ,阳性率为 83.3% (75 / 90 ) ;10 0份大学生血清和39份阴性质控血清经混合抗原检测均阴性。结论　HCV不同编码区重组蛋白片段有不同的免疫反应性 ,在发展抗 HCV抗体诊断试剂时 ,应采用各种不同编码区的混合抗原。     

Relationship between the sialic acid content of low-density lipoprotein (LDL) and autoantibodies to oxidized LDL in the plasma of healthy subjects and patients with atherosclerosis.

To determine whether the sialic acid (SA) content of the low-density lipoprotein (LDL) is related to the plasma concentration of autoantibodies to oxidized LDL (oxLDL), we measured the SA content of LDL and the concentrations of oxLDL and autoantibodies to oxLDL in plasma of 20 apparently healthy subjects and 20 patients with advanced coronary atherosclerosis. In the healthy subjects the SA content of LDL correlated positively with plasma concentration of autoantibodies to oxLDL. In agreement with the literature the decreased SA content of LDL was associated with an increased fraction of oxLDL; a decreased fraction of oxLDL was associated with an increased plasma concentration of autoantibodies to oxLDL. In the patients the SA content of LDL and plasma concentrations of oxLDL and autoantibodies to oxLDL were not related. We conclude that the SA content of LDL correlates positively with plasma concentration of autoantibodies to oxLDL in healthy subjects. However, this association may vary depending on the stage of atherogenesis. Although our results suggest dependence of LDL SA content on the clearance of oxidatively modified (desialylated and oxidized) LDL from blood by autoantibodies to oxLDL, the mechanisms regulating the SA content of LDL await further studies.     

CETP and oxidized LDL levels increase in dyslipidemic subjects

OBJECTIVES: To explore the possible associations among cholesteryl ester transfer protein (CETP), contents of lipids in low-density lipoprotein (LDL), and in vivo oxidized LDL (Ox-LDL). DESIGN AND METHODS: CETP and Ox-LDL were both detected by ELISA. Their levels and the lipid contents of LDL were investigated in 200 subjects with various dyslipidaemias. RESULTS: Compared to the control, CETP levels were significantly increased in subjects with mixed hyperlipidaemia and hypercholesterolaemic. Ox-LDL levels were only significantly increased in mixed hyperlipidaemia subjects. Triacyglycerols to cholesterol ratio in LDL was significantly increased in various dyslipidaemias subjects, of which, hypertriglyceridaemic subjects exhibited the most significant change, while hypercholesterolaemic subjects the least. Multiple linear regression analysis showed that total cholesterol and triacyglycerols levels in very low-density lipoprotein were significantly related with CETP (R(2)=0.066), and triacyglycerols and total cholesterol levels were significantly related with Ox-LDL (R(2)=0.094), respectively. CONCLUSIONS: High CETP promotes the transport of lipids among lipoproteins, which changed the lipid composition of LDL, resulting in the increase of in vivo Ox-LDL level, and subsequently contributing to the atherogenic process in dyslipidaemias subjects.     

Epitaxial deposition of calcium oxalate on uric acid rich stone matrix is induced by a 29 kDa protein

BACKGROUND: Association of macromolecules particularly the role of proteins in urolithiasis has been studied for last few centuries, but still a complete profile of stone matrix proteins that mediate co-precipitation of uric acid and calcium oxalate has not been characterized. We isolated and characterize proteins from uric acid rich stone matrix, which have oxalate binding activity. METHODS: Matrix proteins were isolated from uric acid rich stone matrix using EDTA as a demineralizing agent. The radiolabelled solubilized proteins were fractionated with increasing ionic concentration by DEAE cellulose column chromatography to identify the oxalate binding protein. It was purified using Sephadex G-200 column chromatography. Amino acid composition was determined and monoclonal antibody was produced against the oxalate binding uric acid rich stone matrix protein. Urinary uric acid binding proteins were isolated from stone formers urine, their oxalate binding activity assayed and cross reactivity with the produced monoclonal antibody were checked using ELISA and Western blotting. RESULTS: Matrix on DEAE column chromatography elution yielded 3 protein peaks and they were named as fraction I, II and III among which fraction I had higher oxalate binding activity which was further purified with Sephadex G-200 column which yielded 2 protein peaks designated as Ia and Ib. Fraction Ib with molecular weight 29 kDa exhibited the maximum oxalate binding activity. Forty percent of this 29 kDa protein is comprised of basic amino acids. Monoclonal antibody (IgG1) was produced against the 29 kDa stone matrix protein. Urinary uric acid binding proteins were isolated from stone formers, 4 protein peaks were obtained named as fraction I to IV. Among them, fraction IV having molecular weight of approximately 29 kDa cross reacted up to 85.6% with 29 kDa stone matrix protein. Moreover, urinary 29 kDa protein exhibited oxalate binding activity of 94.16 +/- 6.08 pmol/mg protein at pH 5.5. CONCLUSION: The 29 kDa protein isolated from uric acid rich stone matrix and urine are one and the same, thereby insinuating that 29 kDa protein might play a major role in epitaxial deposition of calcium oxalate over uric acid core, consequently favoring the lithogenic events like uric acid and calcium oxalate nucleation, aggregation and retention.     

Minimally modified electronegative LDL and its autoantibodies in acute and chronic coronary syndromes

OBJECTIVES: To determine the minimally modified electronegative LDL (LDL-) and its autoantibodies in coronary syndromes. DESIGN AND METHODS: LDL(-) and its autoantibodies were determined by ELISA in patients with acute (ACS, unstable angina; AMI, acute myocardial infarction) and chronic coronary syndromes (stable angina, SA) and compared to subjects without coronary disease (controls). Results are expressed as median of LDL- (microg/mL) and anti-LDL(-) IgG (OD405 nm). RESULTS: The concentrations of LDL(-) were higher in patients with coronary disease (ACS: 40.7 microg/mL; SA: 35.0 microg/mL) as compared to controls (21.6 microg/mL). The highest LDL- concentrations were found in patients with AMI (41.8 microg/mL). Anti-LDL(-) IgG was elevated in ACS (1.143) in relation to CCS (0.527) and controls (0.467). A positive correlation was observed between anti-LDL- IgG and CRP levels (r = 0.34, p <0.01) in the studied groups. CONCLUSIONS: LDL(-) and anti-LDL(-) autoantibodies may be useful markers to follow patients with high risk for coronary events.     

Association of plasma lipoproteins with postheparin lipase activities.

Studies were designed to explore the association of lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) activities with lipoproteins in human postheparin plasma (PHP). The major peak of LPL activity after gel filtration of PHP eluted after the triglyceride-rich lipoproteins and just before the peak of low density lipoprotein (LDL) cholesterol. When PHP contained chylomicrons, an additional peak of LPL activity eluted in the void volume of the column. Most HTGL activity eluted after the LDL and preceded the elution of high density lipoprotein cholesterol. LPL activity in preheparin plasma eluted in the same position, relative to lipoproteins, as did LPL in PHP. Gel filtration of purified human milk LPL mixed with plasma or isolated LDL produced a peak of activity eluting before LDL. During gel filtration of PHP in high salt buffer (1 M NaCl) or after isolation of lipoproteins by ultracentrifugation in high salt density solutions, most of the lipase activity was not associated with lipoproteins. LPL activity was removed from PHP by elution through immunoaffinity columns containing antibodies to apolipoprotein (apo) B and apo E. Since lipoproteins in PHP have undergone prior in vivo lipolysis, LPL activity in PHP may be bound to remnants of chylomicrons and very low density lipoproteins.     

Human-human hybridoma autoantibodies with both anti-DNA and rheumatoid factor activities.

Human hybridomas have been produced by fusing peripheral blood lymphocytes from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) with the GM 4672 human cell line. 262 hybridoma clones from the fusions of four RA and five SLE patients were screened for binding to denatured DNA (dDNA), native DNA, and the Fc fragment of human IgG (HIgG). Of the 17 hybridoma antibodies (nine RA, eight SLE) selected for strong binding to denatured DNA, Fc, or both, five reacted with dDNA only, one with Fc only, and eight with both dDNA and Fc. Hybridoma supernatants exhibiting dual reactivity were absorbed over HIgG and bovine serum albumin (BSA)-Sepharose immunoabsorbent columns. The reactivities to both DNA and HIgG were completely removed by the HIgG column but unaffected by passage over the BSA column, and both DNA binding and rheumatoid factor activities were recovered in the acid eluates from the Sepharose-IgG column. The binding of dual reactive hybridoma autoantibodies to the Fc fragment of HIgG was specifically competed by dDNA and HIgG, providing additional evidence that one antibody may be capable of reacting both as a rheumatoid factor and as an anti-DNA antibody.     

Proton magnetic resonance spectroscopy of fractionated plasma lipoproteins and reconstituted plasma from healthy subjects and patients with cancer.

Proton nuclear magnetic resonance spectra at 500 MHz of plasma and the very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) fractions isolated by KBr gradient ultracentrifugation were analysed in 16 cancer patients, six pregnant and nine non-pregnant healthy subjects. In spectra with narrow plasma composite aliphatic peaks (methylene at 1.2-1.4 p.p.m. and methyl at 0.8-0.9 p.p.m., respectively), a relative increase in either VLDL, LDL, or both, or a decrease in HDL signals was observed. The mechanism for line-width narrowing seemed different in cancer patients (less signals from HDL relative to VLDL) compared with pregnant women (more signals from LDL). By reconstitution of plasma samples from both healthy subjects and patients with malignant disease, decreased concentration of VLDL or HDL resulted in broadening or narrowing of the composite peaks, respectively. The effects of VLDL and HDL on the plasma line width were moderated by the signals from LDL. Within lipoprotein fractions, the methylene and methyl resonances were shifted to a higher field with increased observation temperature, the change in shift being greatest for HDL. The line width of composite peaks in plasma varied with the observation temperature, depending on the relative concentrations of individual lipoproteins. The correlation coefficient (r) for the relation between total plasma triglyceride level and the average of the line-width of the composite methylene and methyl peaks was -0.78 (p less than 0.001). For spectra of individual lipoproteins, statistical significant relationships were found between line-widths and triglyceride content of the LDL fraction (methyl line-width, r = -0.63) (p less than 0.001) and between methylene line-width and cholesterol of HDL (r = 0.54) (p = 0.003). In summary, the shape and width of the composite aliphatic peaks of plasma were affected by the relative concentration, chemical shift and transition temperature of both VLDL, LDL, and HDL, and by the total triglyceride level. Comparing pregnancy and malignant disease, the lipoprotein resonances contributed differently in giving narrow composite signals.     

Simple and practical sandwich-type enzyme immunoassay for human oxidatively modified low density lipoprotein using antioxidized phosphatidylcholine monoclonal antibody and antihuman apolipoprotein-B antibody

OBJECTIVES: To develop a simple and practical enzyme immunoassay (EIA) for oxidatively modified low density lipoprotein (Ox-LDL) in human blood, a biological marker of atherogenesis. DESIGN AND METHODS: A sandwich EIA suitable for the measurement of human Ox-LDL was developed using the mouse monoclonal antibody FOH1a/DLH3. This antibody, specific for oxidized phosphatidylcholine, was used as the capture antibody, and a horseradish peroxidase (HRP)-labeled goat anti-human apolipoprotein-B (Apo-B) IgG was used for detection. Copper-oxidized human LDL, prepared under controlled conditions, was used as a standard and the results of the EIA were expressed in arbitrary units (U/mL). RESULTS: This EIA meets all the requirements for use in routine clinical assays in terms of sensitivity (detection limit: 1 U/mL), reproducibility (total CV: 2.3-7.7%), accuracy (recovery: 90.6-103.8%), simplicity and rapidity (<4 h). Clinical performance of the assay was assessed by measurement of the Ox-LDL in the plasma of normal subjects (10.8 +/- 2.8 U/mL, mean +/- SD) and patients with coronary heart disease (CHD) (19.7 +/- 10.2). The present EIA had a sensitivity of 79% and a specificity of 75% for CHD. CONCLUSIONS: We have developed a new assay, suitable for the measurement of Ox-LDL in human blood, which meets the requirements for routine clinical assay.