Deciphering the Molecular Nature of Ovarian Cancer Biomarker CA125

The ovarian cancer biomarker CA125 has been extensively investigated over the last 30 years. The knowledge about the exact molecular nature of this protein, however, remains fragmented. This review provides an overview of the structural research regarding CA125, and presents an orthogonal verification method to confirm the identity of this molecule. The need for independent identification of CA125 is exemplified by several reports where mutually exclusive data concerning the existence of isoforms and the glycan moieties is presented. Mass spectrometry can overcome the pitfalls of a single detection/identification method such as antibody probing. Independent verification of CA125 identity in characterization studies will help establish a refined model of its molecular structure that will promote the development of new approaches for diagnosis, prognosis and therapy of ovarian cancer.     

重组人干扰素βSer~(17)的结构及活性分析

目的分析大肠杆菌表达的重组人干扰素βSer17(rhIFN-Ser17)的分子结构及其生物学活性。方法通过West-ernblot、氨基酸末端测序、质谱及生物学活性分析等,对大肠杆菌表达的rhIFN-Ser17分子进行鉴定。结果大肠杆菌表达的rhIFN-βSer17的N末端20个氨基酸及C端2个氨基酸序列与文献报道一致,产物的相对分子质量为19877·9,比活性超过2×107IU/mg蛋白。结论大肠杆菌表达的rhIFN-βSer17分子结构与理论推测值完全一致。     

新型癌抗原125化学发光免疫试剂盒的制备

目的制备新型癌抗原125(CA125)化学发光免疫试剂盒,并进行验证。方法用1株针对CA125蛋白特异性位点的单克隆抗体作为包被抗体,1株针对CA125分子糖链的碱性磷酸酶(ALP)标记单抗和另1株针对蛋白特异性位点的ALP标记单抗分别作为酶标抗体,采用双抗体夹心法,金刚烷增敏化学发光体系作为酶底物制备试剂盒,检测CA125,并对试剂盒进行验证。结果新型CA125试剂盒检测灵敏度达0.1U/ml,测定范围在0.1~430U/ml之间,与传统CA125试剂盒一致;与CA199、CA153和CA50糖蛋白无交叉反应;精密性和准确性良好。与传统CA125试剂盒比较,检测结果有显著性差异,特别是在卵巢癌标本检测方面,糖链单抗酶标抗体制备的试剂盒与普通抗体酶标抗体制备的试剂盒检测结果的比值与其他标本两者的比值之间差异具有统计学意义。结论已制备了新型CA125化学发光免疫试剂盒,其与传统试剂盒检测标本结果的比值更适用于对卵巢癌进行特异性诊断。     

Surface composition of polystyrene

The physical and chemical properties of bulk polymers are well understood and have been measured exhaustively for numerous systems, but the properties of polymer surfaces are quite often different from those observed in the bulk and are usually not as easily measurable. Since many polymer properties vary with molecular weight, it is of interest to determine whether or not there is any segregation in a homopolymer system based on molecular weight. In particular, does the surface of a polymer sample have the same molecular weight composition as the bulk? The current work answers this question for a polystyrene system. Through the use of secondary ion mass spectrometry and tagged polystyrene, it has been shown that surface and bulk molecular weight composition are indistinguishable within the limits of the experimental method's sensitivity. The sensitivity of the technique is documented using samples artificially created with different surface and bulk molecular weight compositions.     

Selection of thermal,spectroscopic, spectrometric,and chromatographic methods for characterizing historical celluloid

Celluloid in museum collections is very unstable; therefore, heritage professionals carry out research studies dedicated to understanding its decay and prolonging its lifetime. This paper addresses the need to compare and select suitable analytical methods for that purpose. Thermogravimetric analysis coupled with Fourier transform infrared spectroscopy, evolved gas analysis–mass spectrometry, double shot – gas chromatography/mass spectrometry, and gel permeation chromatography (GPC) were employed to characterize the emission of gasses (decay products) and measure the molecular weight and camphor (plasticizer) content from unaged, artificially, and naturally aged celluloid samples. A pioneer GPC set-up for the quantification of camphor was introduced for the first time in this study. Results demonstrated that GPC was the most suitable method for assessing material changes due to degradation. Both set-ups, for measuring molecular weight and quantifying camphor, appear promising for assessing the effect of conservation treatments and investigating the heterogeneous degradation of celluloid objects in future studies.     

酿酒酵母钙通道膜蛋白单克隆抗体制备及鉴定

以酿酒酵母电压门控钙通道膜蛋白（Cch1p）、牵张敏感性钙通道膜蛋白（Mid1p）和瞬时受体电位钙通道膜蛋白（Yvc1p）为研究材料，制备其单克隆抗体。采用生物信息学方法确定3种膜蛋白抗原表位，根据分析结果克隆抗原基因，并进行原核表达和表达产物分析鉴定，通过Ni²⁺-NTA树脂亲和层析技术获得重组抗原蛋白，免疫小鼠后细胞融合技术制备单克隆抗体，酶联免疫吸附测定（ELISA）检测抗体效价，免疫印迹技术检测单克隆抗体对重组纯化抗原和天然酿酒酵母钙通道膜蛋白的反应性和特异性。生物信息学分析结果表明，Cch1p、Mid1p和Yvc1p抗原表位可能分别位于1~300位氨基酸残基、359~548位氨基酸残基、1~236位氨基酸残基；克隆目的基因条带大小分别为926bp、570bp和708bp，与预期结果一致；原核表达抗原蛋白分子量分别为60000、25000和30000，Western blot检测条带正确；重组纯化抗原免疫BALB/c小鼠，细胞融合技术制备单克隆抗体，ELISA检测显示单克隆抗体效价分别高达1∶256000、1∶128000和1∶64000，Western blot检测到3种重组纯化抗原和天然酿酒酵母钙通道膜蛋白Cch1p、Mid1p和Yvc1p。这些结果说明本文制备的单克隆抗体可以成功用于检测酿酒酵母钙通道膜蛋白Cch1p、Mid1p和Yvc1p表达的相关研究。     

绿色荧光蛋白兔多克隆抗体的制备

目的制备绿色荧光蛋白(GFP)兔多克隆抗体。方法选取GFP免疫原性较强、保守性低的98～117位氨基酸序列,利用F-moc法固相合成该段序列,经高效液相色谱纯化,MALDI-TOF-MS鉴定合成多肽的分子量后,与匙孔血蓝蛋白(KLH)偶联,免疫新西兰雄兔,制备抗血清,并进行Western blot鉴定。结果合成的GFP多肽经MALDI-TOF-MS分析,分子量为2497.9,与理论值相符。1∶200稀释的抗血清与转染pEGFP-N1质粒的HeLa细胞表达的增强型GFP在相对分子质量27000处有清晰的杂交信号出现。结论已成功制备了特异性较好的GFP兔多克隆抗体。     

质谱分析导向的稳定同位素标记技术进展

介绍了近年来发展的一系列具有代表性的质谱分析导向的低残留稳定同位素标记技术:二甲氧基甲砜基嘧啶衍生化、低残留稳定同位素标记季铵化衍生化、新型胍剂化合物的衍生化等技术与方法。一些在常规电喷雾或基质辅助激光解吸电离条件下无法有效离子化的目标分子,在衍生化后产生新的正电荷中心或成为容易被电离物种而被质谱检测,从而提高了这类化合物的质谱学检测灵敏度。该类研究显著优势在于所开发的衍生化试剂本身不是离子型化合物,新产生的电荷中心或易于离子化的基团由衍生化反应引入。过量的衍生化试剂易于除去,不会对随后的质谱分析产生明显干扰。这一系列研究成果在代谢组学、食品安全分析、质谱成像以及单细胞分析等方面具有广泛的应用价值。     

HIV-1病毒样颗粒的纯化及其免疫原性

目的纯化HIV-1病毒样颗粒,并检测其免疫原性。方法稳定表达HIV-1结构蛋白Gag和Env的重组293细胞经大规模培养后,收集培养上清,经30%蔗糖垫两次纯化,免疫BALB/c小鼠,Western blot检测小鼠血清中抗体水平。结果重组293细胞培养上清的纯化产物经SDS-PAGE分析,可见目的蛋白的表达。Western blot检测显示,纯化蛋白具有良好的反应原性。免疫小鼠后能够诱导产生针对目的蛋白的抗体,其抗体水平呈剂量依赖关系。结论纯化的HIV-1病毒样颗粒能够诱导小鼠产生抗体,具有良好的免疫原性。     

HIV跨膜蛋白gp36和gp41的截短及融合表达

目的将HIV-1的跨膜蛋白gp41和HIV-2跨膜蛋白gp36进行截短,并在大肠杆菌中进行融合表达。方法用PCR将gp41和gp36的编码基因进行截短,回收的PCR产物纯化后克隆到连接载体pGEM-T上,然后用BamHⅠ、EcoRⅠ和SalⅠ切下目的基因,并构建到表达载体pGEX-4T-3,导入宿主细胞BL21,用IPTG诱导表达。结果酶切鉴定显示,截短的HIV-1gp41和HIV-2gp36跨膜蛋白基因大小与预期的一致,表达产物经SDS-PAGE分析显示在相对分子质量66000处出现融合表达条带,Westernblot分析显示,与相应抗体出现特异性反应。结论已成功对gp41和gp36跨膜蛋白进行截短,并构建表达载体进行表达,为跨膜蛋白的进一步应用研究奠定基础。     

分子量及其分布的测量方法在生物高分子领域中的应用

分子量是生物高分子性质的一项重要参数,决定了生物高分子的许多理化特性和生物效应.准确测定生物高分子的分子量有利于了解其性质,是其有效应用的前提.文章主要简述了近年来常用的测定生物高分子分子量的方法,如黏度测量法、凝胶渗透色谱法、基质辅助激光解暖-飞行时间质谱法、电喷雾电离质谱法,并对其进行分析和比较,为微生物合成生物高...     

New integrated elemental and molecular strategies as a diagnostic tool for the quality of water soluble quantum dots and their bioconjugates

Herein, we demonstrate that both qualitative molecular and quantitative elemental data obtained from size exclusion chromatography coupled online for the first time to both molecular fluorescence and elemental mass spectrometry, respectively, turned out to be critical to evaluate the quality of coatings of quantum dots. Moreover, such an instrumental approach also allowed us to study quantitatively the appropriated bioconjugation of quantum dots to antibodies, a critical step for QDs future use in quantitative fluorescence immunoassays.     

Analyzing molecular weight distribution of whey protein hydrolysates

Process parameters on enzymatic hydrolysis and molecular weight (MW) distribution of whey protein hydrolysates were investigated. Whey protein hydrolysates were first gained by the alkaline protease alcalase for 7 h at temperature (50 °C), pH (8.0) and E/S (3%). The diversification of the hydrolysis degree and dissociative amino acid content was investigated during the whey hydrolysis. The dissociative amino acid content was 56.09 μmol/mL with the hydrolysis degree of 20.04%. The results of Sephadex G25 washing and high performance liquid chromatography–electrospray ionization–mass spectrometry (HPLC–ESI–MS) indicated the molecular weight distribution of whey protein hydrolysates ranged from 300 to 1400 Da, and most of whey peptide was under 1000 Da.     

人67KDa层连蛋白受体前体蛋白的基因克隆、表达及其免疫原性

［摘 要］目的 克隆、表达人67KDa层连蛋白受体前体蛋白（67KDa laminin receptor precursor,LRP）并检测其免疫原性。方法 应用RT－PCR从胃癌细胞9GC7901中扩增编码LRP的cDNA,将其按照T-A克隆法重组入pUCm－T载体并进行DNA序列测定。采用DNA重组技术构建原核表达载体pQE30-LRPP;用M15/pQE系统表达6×组氨酸与 LRP的融合蛋白,并以 SDS－PAGE和Western blot鉴定所获融合蛋白。用含融合蛋白的聚丙烯酸胺凝胶颗粒免疫小鼠,以 Westem blot检测小鼠血清的抗体活性。结果 DNA测序结果证实所扩增的cDNA片段与 LRP的基因序列完全相符。SDS－PAGE检测显示,经IPTG诱导2h后,M15/pQE30－LRP总蛋白中出现一条34KDa的新生蛋白带,Western blot进一步证实该新生蛋白为融合蛋白。其免疫小鼠血清经 Western blot检测仅识别SGC7901细胞中一条大小约为42KDa的蛋白带;其血清即使被稀释2000倍,仍然显示与靶蛋白有较强的结合活性。结论 成功地克隆、表达了人LRP,并具有良好的免疫原性。     

Reliability of molecular weight determination methods for oligomers investigated using certified polystyrene reference materials

Using polystyrene certified reference materials (CRMs) whose molecular weights range from 500 to 2400, we investigated the reliability of molecular weight determination by size-exclusion chromatography (SEC), SEC coupled with multi-angle light scattering detection (SEC-MALS), conventional static light scattering (SLS), matrix-assisted laser desorption/inonization time-of-flight mass spectrometry (MALDI-TOFMS), and ¹H NMR. Average molecular weights determined by these methods were compared with the certified values which were determined by supercritical fluids chromatography with relative standard uncertainty less than 1%. The comparison showed that recent SEC with calibration constructed by uniform polystyrenes can provide just the same average molecular weights as certified ones within the standard uncertainty. ¹H NMR was also found to be a powerful technique to determine number-average molecular weight accurately. Average molecular weights measured by SEC-MALS and SLS nearly agreed with certified values except for lower molecular weights. Although MALDI-TOFMS provided average molecular weights in agreement with certified values, the polydispersity given by MALDI-TOFMS were found to be very small for all the polystyrenes.     

防龋卵黄抗体的研制

应用本室构建的龋齿疫苗工程菌株及 GTF抗原免疫母鸡 ,取鸡卵黄以水 -硫酸铵 -离子交换层析法提取纯化蛋黄抗体 (Ig Y ) ,以放免法测定血清及蛋黄中的抗体。结果用上述 2种抗原免疫的鸡 ,在血清及蛋黄中均能检出抗 GTF抗体。提纯的蛋黄抗体 ,经 SDS- PAGE电泳检测 ,相对分子质量约为 2 2 0 0 0 0 ,纯度达到 97.9% ,经 2 -巯基乙醇处理后 ,分为重链和轻链 ,相对分子质量分别约为 76 0 0 0和 2 80 0 0。经免疫印迹分析 ,具有良好的免疫学特异性。     

Triacylglycerols of human milk: Rapid analysis by ammonia negative ion tandem mass spectrometry

Human milk traicylglycerols (TAG) were analyzed by ammonia negative ion chemical ionization tandem mass spectrometry. The deprotonated molecular ions of triacylglycerols were fractionated at the first mass spectrometry (MS) stage. Twenty-nine of the deprotonated TAG ions were further analyzed based on their collisionally activated (CA) spectra. The tandem MS analysis covered eleven major acyl carbon number fractions, two of which contained odd carbon number fatty acids. Fatty acids of 28 different molecular weights were recorded from the daughter spectra. Hexadecanoic acid was present in all CA spectra, octadecenoic acid in the CA spectra of all mono- and higher unsaturated TAG, and octadecadienoic acid in the CA spectra of all di- and higher unsaturated TAG. The major fatty acid combinations in triacylglycerols were: with 0 double bonds (DB), 12∶0/12∶0/16∶0; with 1 DB, 12∶0/16∶0/18∶1; with 2 DB, 16∶0/18∶1/18∶1; with 3 DB, 16∶0/18∶2/18∶1; with 4 DB, 18∶2/18∶1/18∶1; and with 5 DB, 18∶2/18∶2/18∶1; hexadecanoic acid typically occupied thesn-2 position. The most abundant TAG was shown to besn-18∶1–16∶0–18∶1, comprising about 10% of all triacylglycerols.     

HE4 in the Differential Diagnosis of a Pelvic Mass: A Case Report

Neoplasms of the ovary present an increasing challenge to the physician. Neoplastic ovarian cysts can resemble endometriomas in ultrasound imaging and need to be carefully considered in the differential diagnosis. We report the case of a woman with a strong family history of hereditary breast and ovarian cancer, who presented with a pelvic mass. The young girl refused oncogenetic counseling and genetic testing, even though she had a 50% a priori probability of being a BRCA1 mutation carrier. Pelvic magnetic resonance imaging (MRI) and a comparative analysis of the serum concentration of HE-4 and CA125 biomarkers provided accuracy and sensitivity in the diagnosis of a benign ovarian pathology. Based on this experience, we propose that the sensitivity of a screening program based on a HE4 and CA125 assay and MRI in high risk patients with mutations in the BRCA1 and BRCA2 genes may be considered a useful pre-operative tool for the differential diagnosis of pelvic masses.     

热解温度及AAEM元素对生物质快速热解焦油的影响

生物质热解受热解温度、热解速率和碱金属及碱土金属(AAEM)元素影响显著。利用热裂解气相色谱质谱联用法(Py-GC/MS)针对热解温度及AAEM元素对生物质快速热解焦油的影响展开深入研究,通过样品热解前后的失重情况分析了热解温度及AAEM元素对生物质(稻壳和木屑、酸洗稻壳和酸洗木屑)热解特性的影响规律,利用气相色谱质谱仪(GC/MS)对热解焦油组分及含量进行了在线半定量分析,并对热解焦油组分分子量分布情况展开了讨论。结果表明生物质Py-GC/MS快速热解实验,酸洗脱除AAEM元素致使热解失重率减小。500~900℃范围内随温度的升高,大分子焦油成分逐渐减少,逐渐转化为轻质组分。AAEM元素限制了焦油前体的聚合,进一步抑制了含氧杂环类碳环(糠醛等)的生成。稻壳的热解焦油的相对分子质量主要分布在110~129。木屑快速热解焦油产率明显高于稻壳,且热解焦油中分子量分布广泛,含有更多较大分子量(150~209)的化合物成分。     

Electrochemical immunoassay for CA125 based on cellulose acetate stabilized antigen/colloidal gold nanoparticles membrane

A novel separation-free electrochemical immunosensor for carcinoma antigen-125 (CA125) was proposed based on the immobilization of CA125 antigen on colloidal gold nanoparticles that was stabilized with cellulose acetate membrane on a glassy carbon electrode. A competitive immunoassay format was employed to detect CA125 antigen with horseradish peroxidase (HRP) labeled CA125 antibody as tracer, o-phenylenediamine and hydrogen peroxide as enzyme substrates. After the immunosensor was incubated with a mixture of HRP labeled CA125 antibody and CA125 sample at 35 °C for 50 min, the amperometric response decreased with an increasing CA125 concentration in the sample solution. The decreased percentage of the electrocatalytic current was proportional to CA125 concentration ranging from 0 to 30 U ml⁻¹ with a detection limit of 1.73 U ml⁻¹ (S/N = 3). The proposed immunosensor showed good stability, acceptable accuracy, and would be applicable to clinical immunoassay of CA125.