兔抗阴道毛滴虫重组蛋白AP65多克隆抗体的制备、纯化及鉴定

目的制备、纯化并鉴定兔抗阴道毛滴虫重组蛋白AP65的多克隆抗体。方法日本大耳兔多次免疫重组蛋白AP65．间接ELISA法检测抗体效价，用饱和硫酸铵法和nprotein Asepharose4FF柱纯化多克隆抗体，Western blot检测免疫反应性。结果第3次加强免疫后，间接ELISA法检测血清效价达到1：25600，用硫酸铵法纯化多克隆抗体纯度为56％，而后采用亲合层析柱nprotein Asepharose 4FF再次纯化。纯度达到80％。Western blot鉴定兔抗重组蛋白AP65多克隆抗体能识别重组蛋白AP65。结论成功制备了高效价、高纯度的抗阴道毛滴虫重组蛋白AP65多克隆抗体。     

兔抗霍乱弧菌O139血清群rMSHA多克隆抗体的制备和鉴定

目的制备兔抗霍乱弧菌O139血清群rMSHA多克隆抗体。方法用经Ni-NTA亲和层析法纯化的重组蛋白(rMSHA)免疫日本大耳兔,收集免疫兔血清。Western Blot检测重组蛋白的免疫原性;ELISA测定该抗体的效价。结果兔抗rMSHA抗体能与重组蛋白发生特异性反应;间接ELISA法测定该抗体效价为1∶25 600。结论成功地制备了兔抗rM-SHA抗体,该抗体能够识别原核表达的重组蛋白rMSHA,为研制霍乱弧菌疫苗及相关诊断试剂盒奠定了基础。     

人巨细胞病毒PP150的原核表达及其ELISA诊断方法的建立

采用基因工程方法组建含有人巨细胞病毒（HCMV）PP150蛋白强抗原决定簇基因的重组抗原克隆，进行诱导表达及纯化，在此基础上，以纯化重组PP150蛋白作为包被抗原，对各种条件进行优化，确定判定标准，建立检测HCMV IgM抗体的间接ELISA方法。用此方法检测266份血清样品，并与ELISA试剂盒检测结果相比较。结果重组表达质粒pMAL-P2-PP150可在E.coliDH5a中有效表达，表达产物经SDS-PAGE电泳后，凝胶成像系统分析显示相对分子质量约为64000，约占菌体蛋白的10％。Western blot检测，阳性识别率为80％（12／15）。用此蛋白包被ELISA板，检测正常孕妇血清，与ELISA试剂盒的诊断符合率为98．1％，敏感性达88．2％，特异性100％。应用本方法检测569份正常人血清样品HCMV-IgM，阳性率为4．1％。提示该重组蛋白具有良好的抗原性，可望替代HCMV感染检测中的全病毒ELISA试剂盒。     

抗淋病奈瑟菌外膜蛋白I多克隆抗体的制备及其ELISA双抗夹心法的建立

目的 制备抗淋病奈瑟菌外膜蛋白I多克隆抗体,建立检测外膜蛋白I的双抗夹心法。方法 以淋病奈瑟菌及其外膜蛋白I作为抗原,分别免疫健康性家兔及雌性BALB/C小鼠,制备兔抗淋病奈瑟菌和鼠抗外膜蛋白I多克隆抗体,并采用ELISA法检测抗体效价。结果与讨论 经ELISA证明兔抗淋病奈瑟菌多抗可与淋病奈瑟菌外膜蛋白I呈高效价反应,以此建立的ELISA双抗夹心法检测外膜蛋白I可获满意效果。     

抗淋病奈瑟菌外膜蛋白I多克隆抗体的制备及其ELISA双抗夹心法的建立

目的 制备抗淋病奈瑟菌外膜蛋白I多克隆抗体,建立检测外膜蛋白I的双抗夹心法。方法 以淋病奈瑟菌及其外膜蛋白I作为抗原,分别免疫健康雄性家兔及雌性BALB/C小鼠,制备兔抗淋病奈瑟菌和鼠抗外膜蛋白I多克隆抗体,并采用 ELISA法检测抗体效价。结果与讨论 经ELISA证明兔抗淋病奈瑟菌多抗可与淋病奈瑟菌外膜蛋白I呈高效价反应,以此建立的ELISA双抗夹心法检测外膜蛋白I可获满意结果。     

重组刚地弓形虫MIC8 CTD蛋白及其多克隆抗体的制备

目的制备弓形虫微线体蛋白8羧基端胞质尾(MIC8 CTD)重组蛋白及其多克隆抗体。方法以弓形虫基因组为模板,PCR扩增MIC8 CTD基因片段,构建MIC8 CTD/pGEX-4T-1原核表达系统;IPTG诱导表达GST-MIC8CTD融合蛋白;用纯化的融合蛋白加免疫佐剂免疫新西兰兔,制备多克隆抗体,亲和层析纯化并分析抗体的特异性及效价。结果构建了MIC8 CTD原核表达系统,表达并纯化了GST-MIC8 CTD融合蛋白;获得了抗该蛋白的兔源性抗血清,纯化后的多克隆抗体能特异识别MIC8 CTD,ELISA测定抗体效价为1∶8 000。结论制备的GST-MIC8 CTD融合蛋白具有免疫原性,用该抗原免疫动物可获得高效价的多克隆抗体。     

抗淋病奈瑟菌外膜蛋白Ⅰ多克隆抗体的制备及其ELISA双抗夹心法的建立

目的制备抗淋病奈瑟菌外膜蛋白Ⅰ多克隆抗体,建立检测外膜蛋白Ⅰ的双抗夹心法.方法以淋病奈瑟菌及其外膜蛋白Ⅰ作为抗原,分别免疫健康雄性家兔及雌性BALB/C小鼠,制备兔抗淋病奈瑟菌和鼠抗外膜蛋白Ⅰ多克隆抗体,并采用ELISA法检测抗体效价.结果与讨论经ELISA证明兔抗淋病奈瑟菌多抗可与淋病奈瑟菌外膜蛋白Ⅰ呈高效价反应,以此建立的ELISA双抗夹心法检测外膜蛋白Ⅰ可获满意结果.     

沙眼衣原体T3SS效应蛋白CT622的表达与多克隆抗体制备以及对HeLa细胞增殖的影响北大核心CSCD

目的原核表达沙眼衣原体(Chlamydia trachomatis,Ct)CT622蛋白,制备CT622多克隆抗体,初步探讨CT622蛋白对HeLa细胞增殖的影响,为进一步阐明CT622蛋白在沙眼衣原体致病中的作用提供实验依据。方法以Ct D型基因组为模板构建原核表达重组载体pGEX-6p-1/CT622;重组载体经IPTG诱导表达,Glutathione Sepharose 4B beads纯化GST-CT622融合蛋白,PreScission Protease酶切除GST标签后获取纯化的CT622蛋白;将纯化的CT622蛋白去内毒素处理后,经皮下免疫新西兰兔获取抗CT622抗体,采用抗原亲和纯化层析柱进行抗体纯化,BCA测定抗体浓度,SDS-PAGE鉴定多克隆抗体纯度,ELISA检测抗体效价;用不同浓度的CT622蛋白处理HeLa细胞24 h,CCK-8细胞活性检测试剂盒检测细胞存活率(%)。结果成功表达并纯化了CT622蛋白,该蛋白最佳表达条件为IPTG终浓度0.8 mol/L,30℃、180 r/min条件下诱导4 h。制备兔源性抗CT622多克隆抗体,纯化后抗CT622抗体浓度为0.75 mg/ml,纯度>70%,ELISA检测免疫兔血清抗体效价为1∶512000左右;经1~15μg/ml浓度梯度的CT622蛋白处理HeLa细胞后,其细胞存活率高于对照组。结论成功表达、纯化了沙眼衣原体CT622蛋白,制备了高效价兔源性抗CT622抗体。在一定浓度范围内,CT622蛋白能促进HeLa细胞的增殖。     

重组人神经肽Y融合蛋白的原核表达、纯化及其多克隆抗体制备与鉴定

目的：构建人神经肽Y（NPY）基因的原核表达载体,诱导表达重组蛋白,制备兔抗人NPY抗血清。方法：取已构建好且经测序确认无误的再组质粒pET28a—NPY转化大肠杆菌BL21（DE3）,IPTG诱导表达融合蛋白,并经SDS—PAGE检测和Western印迹鉴定,表达产物包涵体经Ni^2＋-NTA亲和层析纯化,以纯化后的融合蛋白pET28a—NPY为抗原免疫家兔,获得抗血清,Western blotting、ELISA法鉴定获得的抗血清。结果经IPTG诱导含有pET28a—NPY重组质粒的DE3菌,表达出重组人NPY融合蛋白。重组蛋白经Ni^2＋-NTA亲和层析进行纯化后,得到了较高纯度的融合蛋白,用纯化的融合蛋白免疫家兔制备了多克隆抗体,Western blotting、ELISA法证实多克隆抗体制备成功。结论：成功表达了人NPY蛋白并获得了其多克隆抗体,为进一步研究人NPY蛋白的功能奠定了基础。     

弓形虫三磷酸核苷水解酶-II的克隆、表达及初步应用

目的探讨弓形虫RH株速殖子NTPase-II重组蛋白的免疫反应性及其在抗体检测中的应用。方法通过PCR获得NTPase-II基因,克隆入pGEM-T Easy载体,经酶切与测序鉴定后亚克隆至表达质粒pBAD-HisB,构建原核表达重组质粒,在大肠杆菌中以包涵体形式获得高效表达。SDS-PAGE和Western-blot分析蛋白表达产物。用纯化的表达产物作为诊断抗原,通过对反应条件的优化,初步建立检测抗NTPase-II抗体的间接ELISA方法。结果PCR扩增得到特异的弓形虫NT-Pase-II基因序列,经测序鉴定无基因突变。重组质粒诱导表达产物相对分子量约70kD,与理论值相符。经Western-blot证实,重组蛋白可刺激机体产生相应的抗体。具有良好的抗原性和免疫原性。用表达的重组NTPase-II蛋白初步建立了用于NTPase-II抗体检测的间接ELISA方法。结论重组NTPase-II蛋白具有较强免疫原性,可作为诊断抗原用于急性弓形虫感染诊断试剂的研发。     

辛伐他汀抑制糖基化终产物诱导心肌微血管内皮细胞炎性反应及机制探讨

目的观察羟甲基戊二酰辅酶A还原酶抑制荆辛伐他汀对糖基化终产物(AGEs)诱导心肌微血管内皮细胞(CMECs)表达单核细胞趋化因子1(MCP-1)、细胞间黏附分子1(ICAM-1)的抑制作用,探讨辛伐他汀在早期糖尿病心肌微血管炎性病变中的保护机制。方法将培养的CMECs用辛伐他汀预孵育6 h,加入400mg/L的外源性糖基化牛血清白蛋白共培养72 h,分为AGEs组和BSA对照组,分别采用ELISA法测定McP-1的表达;流式细胞仪测定ICAM-1的表达;RT-PCR法测定糖基化终产物特异性受体mRNA的表达;Western blot法检测内皮细胞表面特异性受体(RAGE)蛋白表达。结果与BSA对照组比较,AGEs组MCP-1、ICAM-1、RAGE mRNA及其蛋白表达明显增强,差异有统计学意义(P<0.05)。辛伐他汀预孵则显著抑制AGEs诱导的MCP-1、ICAM-1的表达,并在mRNA及蛋白水平下调RAGE蛋白的表达。结论辛伐他汀可能通过抑制AGEs-RAGE信号途径来发挥对糖尿病心肌微血管病变的保护作用。     

Synergistic action of advanced glycation end products and endogenous nitric oxide leads to neuronal apoptosis in vitro: A new insight into selective nitrergic neuropathy in diabetes

Aims/hypothesis We have previously shown that in diabetes nitrergic neurones innervating the urogenital and gastrointestinal organs undergo a selective degenerative process. This comprises an initial insulin-reversible decrease in neuronal nitric oxide synthase (nNOS) in the axons, followed by apoptosis of the nitrergic neurones, a process that is not reversible by insulin. Since apoptosis was independent of serum glucose concentrations, and advanced glycation endproducts (AGEs) have been implicated in the pathogenesis of diabetic complications, we have now measured AGEs in the serum and penis, pyloric sphincter and pelvic ganglia of diabetic animals at different times after streptozotocin treatment. Furthermore, we have studied their effect in vitro on human neuroblastoma (SH-SY5Y) cells in the presence or absence of nNOS expression.Methods Serum AGEs were measured using fluorometry and ELISA. Accumulation of AGEs in the tissues was evaluated with immunohistochemistry. The viability, apoptosis and oxidative stress in SH-SY5Y cells were measured upon exposure to AGEs or high concentrations of glucose.Results AGEs increased gradually in the serum and tissues of streptozotocin-induced diabetic rats; this process was not affected by delayed insulin treatment. In SH-SY5Y cells, AGEs, but not high glucose concentrations, increased the reactive oxygen species and caspase-3-dependent apoptosis in a synergistic fashion with endogenous nitric oxide (NO). Apoptosis was prevented by treatment with a NOS inhibitor, a pan-caspase inhibitor, a soluble receptor of AGEs or an anti-oxidant, but not an inhibitor of soluble guanylate cyclase.Conclusions/interpretation The synergistic actions of NO and AGEs account for the irreversible nitrergic degeneration in diabetes.Abbreviations AGEs advanced glycation endproducts - eNOS endothelial nitric oxide synthase - HSA-AGEs advanced glycated human serum albumin - iNOS inducible nitric oxide synthase - L-NAME N^G-nitro-L-arginine methyl ester (NOS inhibitor) - MPG major pelvic ganglion - NAC N-acetyl-L-cysteine - NO nitric oxide - NOS nitric oxide synthase - nNOS neuronal nitric oxide synthase - ODQ 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (sGC inhibitor) - RA retinoic acid - ROS reactive oxygen species - sRAGE soluble receptor of advanced glycation endproduct - STZ streptozotocin - Z-VAD-FMK Z-Val-Ala-Asp(OMe)-CH₂F (pan-caspase inhibitor)     

格列喹酮对糖基化终产物诱导的人肾系膜细胞RANTES表达的影响

目的探讨格列喹酮对糖基化终产物（AGEs）诱导的人肾系膜细胞（HRMC）正常T细胞表达分泌的调节活化蛋白（RANTES）表达的影响。方法运用糖基化修饰的牛血清白蛋白（AGE-BSA）和格列喹酮干预体外培养的HRMC,用半定量RT-PCR检测细胞中RANTES mRNA水平,用ELISA法测定细胞培养上清中RANTES蛋白水平。结果格列喹酮干预组HRMC RANTES的mRNA表达和蛋白分泌低于AGE-BSA组（P〈0.05）,且降低水平呈浓度和时间依赖性。结论格列喹酮能抑制糖基化终产物诱导的HRMC RANTES的表达和分泌。     

Increased levels of advanced glycosylation end products in the kidney and liver from spontaneously diabetic Chinese hamsters determined by immunochemical assay

Increased levels of advanced glycosylation end products (AGEs) have been reported in tissues in association with diabetes mellitus. Thus, we measured tissue AGE levels and detected an accumulation of AGEs in the kidney and liver from spontaneously diabetic Chinese hamsters (CHAD) to determine the relationship between AGEs and diabetes mellitus. Diabetic CHAD aged 12 to 13 months were studied together with age-matched nondiabetic CHAD. We used an AGE-specific noncompetitive enzyme-linked immunosorbent assay (ELISA) with polyclonal anti-AGE-bovine serum albumin (BSA) antibody to measure tissue AGE levels. The samples extracted from the kidney and liver obtained from diabetic and nondiabetic CHAD reacted with anti-AGE-BSA antibody. When the absorbance of standard AGE-BSA (0.1 microg/mL) was expressed as 1 U, AGE levels in the kidney and liver from diabetic CHAD were significantly increased as compared with nondiabetic CHAD (kidney, 0.26 +/- 0.05 v 0.10 +/- 0.03 U/microg protein, P< .01; liver, 0.20 +/- 0.03 v 0.09 +/- 0.02 U/microg protein, P< .01). Positive AGE staining was observed in the renal cortex, especially in the tubules of diabetic CHAD, but little AGE staining was observed in the glomerulus by the immunohistochemical study. AGE staining was diffuse in the hepatocytes. These AGE levels were significantly correlated with fasting plasma glucose and glycated hemoglobin (P < .01, respectively). In conclusion, we have confirmed that AGE structures were expressed in the kidney and liver from CHAD, and these AGE levels were increased in diabetic CHAD. AGE staining was observed in the renal tubules and hepatocytes. Tissue AGE levels were positively correlated with glycemic control in CHAD.     

Osmotic flows across the blood-joint barrier.

The effective osmotic pressure across the blood-joint barrier is a key factor controlling synovial fluid volume and joint effusions. The effect of plasma colloid osmotic pressure (COP) on trans-synovial flow was studied directly in rabbit knees expanded by intra-articular fluid infusion. The synovial microcirculation was perfused with blood of varying COP. Absorption of fluid from the joint cavity was a linear function of COP; but albumin COP was only 78% effective across the blood-joint interface. Hyperosmolar solutions of small solutes (e.g., glucose) generated transient osmotic flows across the blood-joint barrier, but far less effectively than albumin. The hydraulic permeability of synovium increased at pathological intra-articular pressures--a phenomenon of potential importance to effusion kinetics.     

Detection of antibodies to a halothane metabolite hapten in sera from patients with halothane-associated hepatitis

Sera from 40 patients with a clinical diagnosis of halothane-associated hepatitis were tested for the presence of antibodies to the trifluoroacetate (TFA) halothane metabolite hapten using an ELISA assay, with TFA-albumin as the antigen. Positive results were obtained in 30% of cases of which 3/4 with encephalopathy were positive and 9/36 non-fulminant cases were positive. Antibody specificity to the TFA hapten was confirmed in each positive result by a 'hapten inhibition' experiment in which TFA albumin binding was blocked by preincubation of serum with TFA-lysine. Most probably this assay detects a relatively low affinity cross-reaction with the TFA hapten of antibodies in the patients' sera which are directed against specific TFA-labelled liver proteins. Anti-TFA-albumin antibodies were not detected in 28 normal subjects, 5 subjects with fulminant hepatic failure secondary to other causes, 6 subjects with a history of 2 or more exposures to halothane but with no evidence of liver disease and 28 patients with a variety of chronic liver diseases. It is concluded that ELISA testing using trifluoroacetylated rabbit serum albumin (TFA-RSA) as antigen is a quick and convenient assay for the confirmation of halothane-associated hepatitis in fulminant hepatic failure secondary to halothane, but is less sensitive when the illness follows a milder course.     

Structural modifications of human albumin in diabetes

AimAlbumin, a major protein in the blood circulation, can undergo increased glycation in diabetes. From recent studies, it has become evident that glycation has important implications for albumin actions and impact on cell functioning. This study compares the structural and functional properties of albumin glycated by glucose and methylglyoxal (MGO) with those of albumin purified from diabetic patients.MethodsHuman serum albumin (HSA) was purified from diabetic patients and control subjects using affinity chromatography, and oxidation parameters in various albumin preparations were determined. Tryptophan and 1-anilino-8-naphthalene sulphonic acid (ANSA) probe fluorescence, redox state, antioxidant and copper-binding capacities of the different preparations of albumin were also determined and compared.ResultsOccurrence of oxidative modifications was enhanced in albumin whether purified from diabetic patients, or glycated by glucose or MGO, after determination of their fructosamine and free thiol and amino group contents, carbonyl content and antioxidant activities. Whereas more quantitative changes in oxidative and structural parameters were observed in the glucose- and MGO-modified albumins, significant impairment of albumin function (free-radical-scavenging and copper-binding capacities) were demonstrated in the HSA purified from diabetics. These findings reveal different structural and functional features of diabetic HSA compared with in vitro models.ConclusionThis study provides new information supporting albumin as an important biomarker for monitoring diabetic pathophysiology. In addition, it reconfirms the influence of experimental conditions in which advanced glycation end-products (AGEs) are generated in tests designed to mimic the pathological conditions of diabetes.     

Immunoassays of factor IX antigen using monoclonal antibodies

Monoclonal antibodies to factor IX were produced by immunization of balb/c mice with purified factor IX and fusion of spleen cells with SP-1 murine myeloma cells. Antibody producing hybrids were detected by an enzyme linked immunoassay (ELISA) and by coagulation inhibitor (Bethesda type) methods. Monoclonal antibodies with titres of greater than 1 X 10(5) tested by the ELISA and 5000-8000 inhibitor units were obtained. A new ELISA method was developed using one of these monoclonal antibodies to quantitate factor IX antigen (IXAg) in plasma samples from patients with hereditary factor IX deficiency. In addition a two-site solid phase immunoradiometric assay (IRMA) for factor IX was established. The results of these new methods were compared with those obtained on the same plasma samples using conventional factor IX coagulation assays and the Laurell rocket method. The lower limit for the detection of IXAg by the ELISA was approximately 0.01 unit per ml whilst that for the IRMA was about 0.001 unit per ml (normal plasma = 1 unit per ml). The lower detection limit for IXAg using the Laurell rocket method was about 0.06 units per ml. The improved sensitivity of the new immunoassays enabled quantitation of low levels of IXAg in patients with moderately severe factor IX deficiency and confirmed the presence of excess IXAg compared to IX activity (IXC) in a relatively high proportion of cases (28 out of 51 tested). Results of testing plasma from obligate carriers confirm the suggestion that measurements of IXAg and IXC may improve the classification of carrier status in these kindred.     

Risk factors for thrombosis in nonembolic cerebrovascular disease

Thirty-seven young patients (less than 42 years of age) presenting with sudden onset of idiopathic nonembolic cerebrovascular disease were evaluated for underlying prothrombotic factors. Activated protein C resistance (APC-R) was measured by Dahlback's method and the modified method using factor V-deficient plasma. Activities of antithrombin (AT) III, protein C and S were measured. Anticardiolipin antibody was estimated by ELISA and lupus anticoagulant by kaolin clotting tests. APC-R was the most common defect (21.62%) followed by AT III deficiency and presence of anticardiolipin antibodies (5.6% each). The latter two were present together in one case. It is thus concluded that APC-R is the most common defect underlying idiopathic nonembolic cerebrovascular infarction in young individuals.     

Activation of receptor for advanced glycation end products in osteoarthritis leads to increased stimulation of chondrocytes and synoviocytes

OBJECTIVE: The major risk factor for osteoarthritis (OA) is aging, but the mechanisms underlying this risk are only partly understood. Age-related accumulation of advanced glycation end products (AGEs) could be one of these mechanisms. We undertook this study to investigate the role of the receptor for AGEs (RAGE) in mediating the cellular effects of AGEs on chondrocytes and fibroblast-like synoviocytes (FLS). METHODS: AGE levels in human cartilage were determined by fluorescence, browning, and pentosidine levels. Chondrocyte activation by AGEs was assessed as the release of proteoglycans and the synthesis of matrix metalloproteinase 1 (MMP-1) and type II collagen messenger RNA (mRNA). The activation of FLS by AGEs was measured by MMP-1 production and invasion through matrix proteins. RESULTS: Patients with focal degeneration of cartilage showed increased AGE levels in their healthy cartilage compared with the levels in healthy cartilage from donors without cartilage degeneration (P < 0.01 for both fluorescence and browning; P not significant for pentosidine content). Stimulation of bovine chondrocytes with glycated albumin increased the release of proteoglycans by 110% (P < 0.001) and the production of MMP-1 mRNA by 200% (P = 0.028). In addition, OA FLS produced 240% more MMP-1 when stimulated with glycated albumin (P < 0.001). Glycated matrix or albumin increased the catabolic activity of OA FLS, which was assessed as invasive behavior, by 150% and 140% (P = 0.001 and P = 0.010), respectively. Effects of stimulation with AGEs were blocked by a neutralizing antibody against RAGE, but not by an isotype control. CONCLUSION: This study shows that AGEs trigger RAGE on chondrocytes and FLS, leading to increased catabolic activity and therefore to cartilage degradation. AGEs, via RAGE, could therefore contribute to the development and/or progression of OA.