一种可同时检测抗双链和单链DNA抗体的快速斑点免疫金渗滤试验

目的 建立一种可同时检测抗双链ＤＮＡ（ｄｓ－ＤＮＡ）和单链（ｓｓ－ＤＮＡ）抗体的快速斑点免疫渗滤试验（ＤＩＣ－ＦＡ）。方法 将ｄｓ－ＤＮＡ和ｓｓ－ＤＮＡ抗原结合在同一检测盒内,采用胶体金标记和快速膜斑点渗滤技术,同时检测抗ｄｓ－ＤＮＡ和ｓｓ－ＤＮＡ抗体。在结果：ＤＩＧＦＡ既可同步又可区别检测两种ＤＮＡ抗体,且检测ｄｓ－ＤＮＡ抗体的敏感性优于酶联免疫吸附法（ＥＬＩＳＡ）,结论：ＤＩＧＦＡ简便,快速,     

斑点金免疫渗滤法检测血清抗精子抗体

目的建立检测血清抗精子抗体的斑点金免疫渗滤法 (dot im munogold filtration assay,DIGFA)。方法　采用经 Sephadex G- 75 -抗人全血清柱亲和层析纯化的人精子抗原和胶体金标记的 SPA,根据免疫渗滤原理 ,建立抗精子抗体(ASAb)的斑点金免疫渗滤法检测法。结果　纯化的人精子抗原具有较好的特异性 ;斑点金免疫渗滤法与 EL ISA法比较 ,符合率为 97.7%。特异性为 96 .2 % (TN/ TN+ FP× 10 0 % ) ,敏感性为 88.9% (TP/ TP+ FN× 10 0 % ) ,阳性预测值 84.2 % (TP/TP+ FP) ,阴性预测值 97.2 % (TN/ TN+ FN) .结论　斑点金免疫渗滤法方法检测血清抗精子抗体有较好的特异性和敏感性 ,重复性好 ,试剂稳定 ,操作简便快速 ,具有临床应用价值     

斑点金免疫渗滤试验检测结核血清抗体方法的建立及初步临床应用

从结核分枝杆菌 (H37Rv菌株 )中提纯了阿拉伯糖甘露糖脂 (LAM)抗原 ,将其用于斑点金免疫渗滤试验以检测结核血清抗体。对痰菌阳性组和痰菌阴性组的活动性肺结核病人血清标本 ,本法的阳性率分别为 91 9% (6 8/ 74)和 5 3 3 % (8/ 15 )。对正常人、肺癌病人和肺炎病人血清标本 ,本法的阴性率分别为 96 4% (80 / 83)、 94 4% (17/ 18)和 10 0 0 % (12 / 12 )。     

斑点金免疫渗滤法检测MCTD患者血清中抗鸡卵核72 000蛋白抗体的研究

目的 探求一种可用于混合性结缔组织病(MCTD)诊断的简便、快速、可靠的方法.方法以硝酸纤维素膜为载体,胶体金标记蛋白为显示剂,建立检测MCTD患者血清中抗鸡卵核72000蛋白抗体的斑点金免疫渗滤试验(DIGFA).结果本实验重复3次,在66例MCTD患者血清中平均有53例检出该抗体,平均阳性率为80.30%(53/66);硬皮病(PSS)42例、皮肌炎和多发性肌炎(DM/PM)51例、系统性红斑狼疮(SLE)93例均未测出有交叉反应;98例正常对照人群检测均为阴性.53例血清中抗鸡卵核72000蛋白抗体阳性的MCDT患者在治疗前的几何平均倒数滴度(GMRT)为496.62±19.96,治疗后为295.68±20.38;该抗体与MCTD活动指数、ANA滴度及抗nRNP抗体滴度呈显著正相关.结论 DIGFA检测MCTD患者血清中抗鸡卵核72000蛋白抗体,对MCTD诊断有一定的价值,在治疗过程中也有一定的疗效提示作用.     

用抗-(抗HBc)抗体在同系小鼠中诱导抗-抗Id抗体

用单克隆抗-(抗HBc)抗体(抗-Id)免疫BALB/c鼠,诱生出具有与抗-HBc相同反应性的抗-抗Id抗体(Ab_3)。这种抗体在ELISA试验中可与HBc-Ag特异性反应,并能抑制单克隆抗-HBc与HBcAg的结合。表明我们建立的抗-H-Bc的抗Id能模拟HBcAg刺激小鼠产生免疫反应。     

点免疫金渗滤法筛选胶体金标记抗人IgG 的研究

目的:应用点免疫金渗滤法筛选适宜胶体金标记的抗人IgG(以下简称二抗),并运用多个评价指标进行比较和分析。方法:选择三个不同公司的二抗用胶体金标记,根据棋盘滴定法确定最佳标记条件;运用点免疫金渗滤法对三种二抗的胶体金标记后检测效能进行比较,采用包虫病人和健康对照血清,用包虫病特异性抗原检测包虫病患者血清中的特异性抗体,评价最优的二抗,并与酶联免疫吸附测定法进行比较。结果:A、B、C 三种二抗标记的最佳标记条件为:pH 均为8.5,加入量分别为38.4、24、19.2 g/ ml,综合评价二抗B 检测效能最优。将其与酶联免疫吸附测定法检测效能进行比较,两种方法检测结果的Kappa=0.895(P<0.05),吻合度较强。结论:应用点免疫金渗滤法,以及本文提出的评价指标,能够筛选出最佳二抗,对检测试剂盒中二抗的选择具有重要的参考价值。     

斑点金免疫渗滤法联合检测糖尿病自身抗体的研究

目的构建联合检测糖尿病自身抗体的斑点金免疫渗滤法(dot immunogold filtration assay,DIGFA)。方法将重组人胰岛素、谷氨酸脱羧酶、蛋白酪氨酸磷酸酶和锌转运体8抗原点样于硝酸纤维素膜上,制备胶体金标记链霉亲和素作为显色探针,建立斑点金免疫渗滤法检测1型糖尿病(type 1 diabetes,T1DM)患者外周血中相应的自身抗体,并对方法性能开展评估。结果DIGFA法检测42例T1DM敏感性66.7%,检测100例健康对照特异性97%。与ELISA法相比对T1DM自身抗体检出率无显著差异(P0.05)。3个批次的免疫渗滤装置检测结果一致,4℃保存10周内检测性能稳定。结论本研究构建的斑点金免疫渗滤法准确、快速、简便,为联合检测糖尿病自身抗体提供了一种高效的新途径。     

A whole blood assay to assess peripheral blood dendritic cell function in response to Toll-like receptor stimulation

We report a practical technique to assess peripheral blood dendritic cell (DC) maturation and function in whole blood (WB), which requires minimal blood volumes and minimizes ex vivo manipulations of clinical specimens. We determined optimal conditions for flow cytometric analysis of markers of DC maturation, including CCR7, CD25, CD80 and CD83, and of the intracellular cytokines, tumor necrosis factor- (TNF-) and interferon- (IFN-), in both myeloid (mDC) and plasmacytoid (pDC) lineages. We demonstrate concentration-dependent production of these cytokines by DC following short-term stimulation with ligands to Toll-like receptors (TLRs) 2/1, 3, 4, 7, 8 and 9. Kinetic studies revealed maximal TNF- and IFN- protein expression at 2 to 3 h after stimulation with certain TLR ligands. Finally, utilizing cells from a cohort of eight healthy donors, we compared DC responses to TLR activation in WB, freshly isolated peripheral blood mononuclear cells (PBMC) and cryopreserved PBMC. We found that TNF- responses were essentially preserved, but IFN- responses were profoundly diminished or entirely abrogated following cryopreservation. In conclusion, we propose that WB analysis of peripheral DC function is a rapid, reliable and simple method to evaluate TLR function in clinical specimens, which obviates artifact-prone cell purification. The major impact of cryopreservation on some DC responses further strengthens the case for a rapid method that uses fresh blood.     

Physiological quality assessment of stored whole blood by means of electrical measurements

The physiological parameters of blood such as extracellular Na⁺, K⁺, Cl⁻, pH, 2,3-DPG and ATP and the complex electrical impedance were measured using whole blood samples from 31 male donors (21 donors form the training set and ten donors were used for testing), on the 0th, 10th and 21st day of blood bank storage. During storage, while the extracellular fluid resistance (R _e) and the intracellular fluid resistance (R _i) decreased progressively with time, the effective cell membrane capacitance (C_m) has increased. Blood bank storage resulted in a rise in K⁺ and a fall in Na⁺, Cl⁻, pH, 2,3-DPG and ATP. Accordingly, all electrical parameters correlated with Na⁺, K⁺, Cl⁻, pH and ATP, at varying levels. By applying the multi-regression analysis, it was demonstrated that R _i, R _e and especially C _m were appropriate for the assessment of Na⁺, K⁺, Cl⁻, pH and ATP until the 21st day of storage.     

Tuberculin-induced lymphocyte proliferation in whole blood: an antigen specific method for assessing immunosuppressive agents

Antigen-stimulated whole blood cultures have not been used to study the effects of immunosuppressive drugs. The aim of this study was to assess the potential usefulness of tuberculin purified protein derivative (PPD)-stimulated lymphocyte proliferation in whole blood for studying the effects of T cell inhibitory agents. We have investigated whether PPD causes antigen specific T cell proliferation, and the role of the major histocompatibility complex class II (MHC class II), co-stimulation and IL-2 in the development of this response. We have also studied the effects of prednisolone and cyclosporin on lymphocyte proliferation. Heparinised blood from healthy volunteers was diluted in culture medium and incubated with PPD. Cell proliferation, measured by liquid scintillation counting, was maximal using 1000 units/ml PPD incubated in 10% whole blood for 6–7 days. A population of large CD4+ lymphocytes appeared in cultures incubated with PPD, suggesting that the major responding population was composed of T lymphocytes. There was no significant response to the negative control antigen KLH, indicating that proliferation was antigen specific. Monoclonal antibodies (mAbs) against MHC, CD2, CD26, CD28 and IL-2 inhibited proliferation. Prednisolone was more potent than cyclosporin in this assay (IC50 values; prednisolone 20 nmol, cyclosporin 278 nmol). For the first time, this report shows that the PPD causes antigen specific lymphocyte proliferation in whole blood, which is dependent on antigen presentation via MHC class II, co-stimulation and IL-2 production. Because the proliferative response is dependent on the major interactions that lead to T cell activation, this simple assay can be used to assess the effects of novel immunomodulators.     

Optical endpoint sensing in an automatic whole blood clotting timer

Most clotting time estimations are performed manually, although attempts have been made previously to automate them. The two major methods for automatically detecting the formation of the gel-like clot are mechanical (viscometric) and optical. The latter is superior in terms of accuracy of timing and freedom from artefacts but can only be performed on blood plasma. This paper describes a device which combines centrifuging to remove red cells and optical sensing of clot formation into a single operation, therepy giving activated clotting times on a par with those obtained mechanically from whole blood. The system offers the advantage over mechanical sensing that no nondisposable parts come in contact with the blood thereby eliminating e major source of timing errors. The timer works with any liquid coagulation activator, and will also time plasma clotting. The two-chambered design of the cuvette allows the activator to be kept separate from the blood until rotor startup The start of centifugal action mixes the blood and activator and starts the time. Timing is stopped auto matically when the rate of increase of optical density in the plasma, owing to fibrin formation, reaches a predetermined fevel. Patents pending     

Performance evaluation of ISFETs and other ISE sensors for whole blood ion assay

Performance evaluations of ion-selective field-effect transistors (ISFETs) have been carried out using both quality-control materials and whole arterial blood samples. Comparison of the results from these evaluations suggests that the whole blood evaluation may be more useful when assessing the value of particular sensors for clinical applications. The effect of outliers on the imprecision estimates is demonstrated for ISFETs and ISEs, both graphically and in the calculation of the estimates with an without the outliers present. Estimates of constant and proportional bias, against an alternative sensor, determined from the intercept and slope of the linear regression vary according to the regression method used. The bias estimates obtained for the K+ISFET against the Radiometer KNA1 using ordinary least squares regression are compared with the Deming/Mandel method and the three-group resistant line method of Tukey. The Thorn EMI ISFETs are demonstrated to have acceptable imprecision and only a small bias compared with direct ISE instruments for whole blood assay and can be considered suitable for incorporation into clinical instrumentation.     

Oxidative burst assessment and neutrophil-platelet complexes in unlysed whole blood

BACKGROUND: Methods currently employed for measuring reactive oxygen species production can lead to both cellular depletion and in artifactual activation. The objective of this study was to design a methodology allowing the measurement of oxidative burst (OB) with minimal sample manipulation. METHODS: To that purpose a flow cytometry technique developed in our laboratory, based on nucleic acid staining to discriminate erythrocytes and debris, was employed. DRAQ5 dye and PECy5-CD45 monoclonal antibody (MoAb) were simultaneously used in FL3 to identify the leukocyte population and the PE-CD14 MoAb emission was detected in FL2 for monocytes. OB was measured by using the fluorogenic probe dihydrorhodamine 123, a marker of hydrogen peroxide production. Phorbol myristate acetate (PMA), Opsonized Zymosan (OZ), fMLP and calcium ionophore A23187 activators were also used in this study. For OB assays, dose-response curves were performed for each activator. In addition, the effect of activator concentration on annexin V binding, as a measure of phosphatidylserine translocation, was evaluated. RESULTS: With this method no-lysis and no-wash steps are required, thus avoiding an unwanted damage to leukocytes. PMA and Zymosan produced an increase in annexin V binding, while fMLP and calcium ionophore did not. CONCLUSIONS: This study reports a feasible and reproducible new flow cytometry assay for assessing OB of neutrophils and monocytes with minimal sample manipulation. In addition, under PMA and OZ conditions, the number of neutrophils showing annexin V binding was strikingly increased. This effect is not related with a phagocytic overstimulation, but with an increased neutrophil-platelet complexes formation.     

Computerized acquisition and evaluation of whole blood aggregometry data

A special-purpose software package has been developed to acquire and analyze analog signals from a whole blood aggregometer. Up to four independent blood samples can be analyzed simultaneously or with arbitrary delays in time. The user menu ensures porper calibration prior to each measurement and provides a real-time graphic display of the signals. From analog/digital converted data, several “derived diagnostic quantities” are calculated. They are constructed with the aim to mimic the process of visual inspection of aggregometry curves and thus place the intuitive diagnostics of blood platelet function on a quantitative basis.     

Re-evaluation of anti-HBc non-reactive serum samples from patients with persistent hepatitis B infection by immune precipitation with labelled HBV core antigen

BackgroundCore antigen (HBcAg) is the most immunogenic component of hepatitis B virus (HBV) and is believed to induce virtually always antibodies (anti-HBc) in immunocompetent infected persons. However, some chronically infected persons do not develop detectable anti-HBc.ObjectiveA more sensitive assay for anti-HBc was to be developed and used to re-evaluate a cohort of chronically HBV infected persons without detectable anti-HBc.Study designAmong 3309 serum samples which had been tested by commercially available (microparticle) enzyme immune assay (M/EIA) 34 samples from 22 patients were identified having reacted positive for HBsAg and negative for anti-HBc. Nine of these patients had immunosuppression or HIV coinfection, 13 patients were immunocompetent, 5 of them were perinatally infected.Anti-HBc was re-tested for in an immune precipitation (IP) assay using ³²P-labelled recombinant HBcAg as reagent and anti-human-IgG-coated magnetic beads as separation system for immunecomplexes containing HBcAg. Specificity was controlled for by competition with unlabelled HBcAg.Results27 serum samples from the 22 patients could be retested. IP was positive in 7 MEIA negative sera, unspecific positive in 4 and negative in 16. Using 5 anti-HBe positive control sera, we found IP to be 1.8-fold (1.3–2.9) more sensitive than MEIA, but IP was 6.5-fold (5.8–7.4) more sensitive with 4 anti-HBe negative, anti-HBc positive sera.ConclusionIP allowed specific detection of anti-HBc in about 25% of MEIA negative chronic HBV patients. The majority of these seem to produce no or very little anti-HBc, however.     

Comparison of platelet aggregation and adenosine triphosphate secretion in whole blood and platelet-rich plasma from normal dogs

Platelet aggregation and adenosine triphosphate (ATP) secretion were measured in whole blood and platelet-rich plasma (PRP) from normal dogs using electrical impedance and turbidimetric techniques. General appearance of the aggregation curves, ATP secretion, and aggregation rate were similar in PRP in response to adenosine diphosphate (ADP) or collagen using both techniques. In response to ADP, aggregation was detected sooner while the maximum aggregation response decreased at a relatively greater rate with decreasing agonist concentrations using the turbidimetric technique. Shape change was consistently detected using the turbidimetric, but not the impedance, technique. Using electrical impedance, there were differences between whole blood and PRP in aggregation and ATP secretion responses which depended on the agonise used to activate the platelets. In response to ADP, aggregation responses were lower in whole blood relative to PRP. In response to platelet-activating factor (PAF), maximum aggregation was greater in whole blood while aggregation rate and ATP secretion were greater in PRP. In response to collagen, aggregation response and ATP secretion were lower in PRP. Dilution of PRP with buffer instead of platelet-poor plasma (PPP) lessened many of the differences between whole blood and PRP samples. These findings suggest that plasma constituents and blood cells other than platelets affect aggregation and secretion in an agonist-dependent manner.