胶体金免疫层析法检测血清癌胚抗原

建立一种快速、简易的检测血清中癌胚抗原(CEA)的胶体金免疫层析法(GICA)。采用柠檬酸三钠还原法制备胶体金颗粒，标记抗CEA单克隆抗体4A3。将单克隆抗体3B5划线包被于硝酸纤维素膜上，制成免疫层析检测试纸条。血清中CEA与试纸条上胶体金标记物结合后沿着硝酸纤维素膜移动，与膜上的包被抗体结合形成肉眼可见的红色线条。用GICA与ELISA试剂盒对比检测了637份血清标本，其灵敏度为96．69％，特异性为99．38％，两法符合率为98．74％。GICA检测血清中的CEA特异性强、灵敏度高、简便快速，无需特殊仪器设备，有广泛应用价值。     

人心肌肌钙蛋白T胶体金免疫层析法的建立

目的 建立一种简便,快速,准确检测人心肌肌钙蛋白Ｔ（ｃＴｎＴ）的胶体金免疫层析法（ＧＩＣＡ）。方法 制备的胶体金标记抗ｃＴｎＴ单克隆抗体３Ｆ７,生物素标记另一株抗体２Ｈ８,链霉亲和素结合于硝酸纤维素膜上,制成免疫层析试纸条,血清中ｃＴｎＴ与测试条两种抗体结合后,沿硝酸纤维素膜移动,与链霉亲和素交联形成肉眼可见的红色线条。结果 测试条灵敏度可达０．５ｎｇ／ｍｌ。检测３０例急性心肌梗塞（ＡＭＩ）患者血     

玉米赤霉烯酮单克隆抗体的制备及胶体金免疫层析法的建立

目的 制备并鉴定玉米赤霉烯酮(ZEN)单克隆抗体(mAb),并初步建立ZEN的胶体金免疫层析检测方法.方法 采用活泼酯法制备人工抗原ZEN-OVA和ZEN-BSA,以ZEN-OVA作为免疫原制备ZEN mAb.ELISA法鉴定抗体效价、亚类、特异性等.ZEN mAb-胶体金复合物包被于胶体金结合垫上,检测原ZEN-BSA和山羊抗小鼠lgG分别结合于硝酸纤维膜上作为检测线(T线)和质控线(C线),建立ZEN的胶体金免疫层析检测法.结果 经紫外分光光度法和SDS-PAGE鉴定,ZEN人工抗原合成成功.经3次克隆化后,筛选出1株能稳定分泌ZEN mAb的杂交瘤细胞株1G4,腹水效价为1∶1.6×105;抗体亚类为IgG2b;对ZEN的IC50为10.2 ng/mL,检测限为0.58 ng/mL.mAb对ZEN具有高度的特异性,除与β-玉米赤霉烯醇的交叉反应率为12.8％外,与其他ZEN代谢物α-玉米赤霉醇、β-玉米赤霉醇、玉米赤霉酮及其他相似毒素呕吐毒素、伏马毒素、赭曲霉素A等几乎无交叉反应.制备的胶体金免疫层析试纸条在5 min内即可肉眼观察到结果,对ZEN的最低检测限为100 ng/mL.结论 成功制备出1株ZEN mAb,并初步建立了其胶体金免疫层析检测方法,最低检测限为100 ng/mL.     

快速乙肝表面抗原胶体金免疫层析测定法的建立及应用

建立乙肝表现抗原胶体金免疫层析测定法，用于乙肝病毒感染检测。采用亲和力强、特异笥高的自制抗－ＨＢｓ单克隆抗体及胶体金标记和硝酸纤维膜包被技术，制备乙肝表面抗原胶体金免疫层析快速测定试条，并进行了初步临床应用。该法的灵敏度为５ｎｇ／ｍＬ，与目前广泛使用的ＥＬＩＳＡ法对照检测５１３份血清标本，符合率为９９．２％。该法快速、准确、特异、灵敏、简便，基本符合临床应用要求，可用于乙肝表面抗原的检测。     

铜绿假单胞菌胶体金免疫层析法的研制和应用

为建立一种简单、快速、准确的铜绿假单胞菌检测方法,结合抗重组铜绿假单胞菌外膜蛋白单克隆抗体（mAb）、胶体金标记技术,采用双抗体夹心法,建立了检测铜绿假单胞菌的胶体金免疫层析法,并对该方法的特异性、灵敏度和稳定性进行了评价,对临床痰标本进行了检测。结果显示,该胶体金免疫层析法能在3～15min内完成检测,方法的特异性高、稳定性强,检测灵敏度能达到105CFU/mL。与传统细菌培养方法相比,胶体金免疫层析法的相对灵敏度和特异性分别是80%和90.6%,两种方法的总符合率为86%。结论：利用抗铜绿假单胞菌单克隆抗体建立的胶体金免疫层析法,适合现场快速、特异检测铜绿假单胞菌。     

金标免疫层析法测定前列腺抗原方法的应用

目的：采用前列腺抗原（ＰＳＡ）单抗标记胶体金的方法。测定ＰＳＡ抗原。方法：采用柠檬酸三钠还原法制备胶体金、金标免疫层析方法（ＧＳＬＩＡ）测定。结果：将金标方法与ＥＬＩＳＡ方法进行比较灵敏度、特异性、其两者无显著性差异。金标方法可在室温放置１年以上。结论：金标免疫层析法测定前列腺抗原，是一种快速、简便、灵敏的筛选前列腺癌患者的诊断方法。     

胶体金免疫层析法在检测粪便血红蛋白中的应用

胶体金免疫层析技术是把免疫层析技术与胶体金标记技术结合而建立的一种新的免疫学方法，具有灵敏度高，特异性强，简便、快速等特点，有广阔的应用前景。本文就胶体金免疫层析技术的基本原理及其在检测粪便血红蛋白中的应用进行综述。     

免疫层析法同时检测孕妇TORCH—IgM抗体的研究

目的建立一种免疫层析法用于同时检测孕妇血清中TORCH—IgM抗体。方法用胶体金标记羊抗人IgM（斗链）抗体，将巨细胞病毒（CMV）、风疹病毒（RV）、弓形体（Tox）、单纯疱疹病毒Ⅱ型（HSV-Ⅱ）四种重组抗原同时包被在硝酸纤维膜上，研制出同时检测TORCH—IgM抗体免疫层析试纸条。结果自制的免疫层析法与ELISA法对比检测阴阳性血清标本，各单项指标抗-CMV—IgM、抗-RV—IgM、抗-Tox—IgM、抗-HSV-Ⅱ-IgM阳性符合率分别为85．7％、100％、100％、100％，两法总符合率分别为97．9％、100％、100％、100％。结论本方法操作简便，显示结果直观，并且特异、稳定、快速、重复性好，可用于孕妇优生优育检测。     

胶体金免疫层析法在检测粪便血红蛋白中的应用

胶体金免疫层析技术是把免疫层析技术与胶体金标记技术结合而建立的一种新的免疫学方法,具有灵敏度高,特异性强,简便、快速等特点,有广阔的应用前景.本文就胶体金免疫层析技术的基本原理及其在检测粪便血红蛋白中的应用进行综述.     

Development of a lateral-flow immunochromatographic test device for the rapid detection of difloxacin residues

In this study, a rapid competitive immunochromatographic test strip has been developed for specifically testing residues of difloxacin (DIF). 1H₁₀-2B₂, one of the three hybridoma lines that were tested and selected by enzyme linked immunosorbent assay, was used in the test strip. The monoclonal antibody has a good sensitivity with an IC₅₀ of 2.2 ng/mL to DIF, 0.24% cross-reactivity towards danofloxacin and no cross-reactivity towards other related compounds and other drugs. The calibration curve of DIF test strip presented a typical sigmoidal curve. In practice, the lower limit of detection using a strip reader was 0.5 ng/ml while 2 ng/ml by unaided visual assessment. Samples of milk were spiked at 2–8 ng/mL, presenting recoveries between 94.5 and 107%, and the coefficient of variation (CV,%) (1.6–9%). The data above suggests that the strip meets the requirements of high sensitivity, good specificity, simplicity and speed, as well as the characteristics of reproducibility and accuracy.     

Development and validation of an immunochromatographic test strip for rapid detection of doxycycline residues in swine muscle and liver

A one-step lateral flow immunochromatographic technique using colloidal gold-labelled monoclonal antibody (Mab) was developed for the rapid detection of doxycycline (DOX) residues in swine tissues. For this purpose, a Mab against DOX named as 2.3/3A6 with low cross-reactivity (CR) to other tetracyclines (TCs) was produced. The sensitivity of the test strip was as low as 7 ng/mL for DOX and the half maximal inhibitory concentration (IC₅₀) was calculated to be 22±2 ng/mL by relative optical density. The test can be completed within 10 min and the detection limit was 20 ng/mL in unaided visual assessment. Recoveries in samples spiked with DOX at concentrations of 20, 40 and 80 ng/g, were demonstrated to be from 81% to 95% for muscle samples and from 81% to 92% for liver samples. The intra-assay and inter-assay coefficients of variation (CVs) ranged from 3% to 6% and from 4% to 8%, respectively. Comparing with high performance liquid chromatography (HPLC) method in sensitivity and accuracy for the detection of DOX in swine tissues, the test strip showed good agreement. Therefore, the test strip is suitable for rapid and reliable determination of DOX residues in swine tissues semi-quantitatively or qualitatively.     

An immunochromatography test strip for rapid,quantitative and sensitive detection of furazolidone metabolite, 3-amino-2-oxazolidinone,in animal tissues

3-amino-2-oxazolidinone (AOZ) is the metabolite of furazolidone, which has been banned as a veterinary drug due to the potential harmful effects on human health. In this study, a novel immunochromatography test strip (ICTS) based on colloidal gold–McAb probe for detection of AOZ within tissue samples was developed. With this method, the visual cut-off levels for the test strip was achieved at 10?μg/L. Using a test strip reader, the 50% inhibition was 1.3?μg/L. The limit of detection and limit of quantification in four kinds of animal tissues were 0.15?μg/kg and 0.31?μg/kg, respectively. When used to analyze fortified samples of animal tissue, acceptable recovery rates of 76.3–98.4% were obtained. The fortified samples were detected by ICTS and confirmed by LC-MS/MS. The results of the two methods were in good agreement. The proposed ICTS was demonstrated to be a rapid and simple analytical method for detecting AOZ in animal tissue.     

Development of colloidal gold-based immunochromatographic assay for the rapid detection of ricin toxin in food samples

A rapid, sensitive immunochromatographic (IC) assay was developed to detect ricin toxin in assay buffer and food samples. The assay was based on the sandwich format using a monoclonal antibody (mAb) and a polyclonal antibody (pAb) of two distinct specificities. The ricin-containing sample was added to the membrane and allowed to react with pAb-coated particles. The mixture was then passed along the porous membrane by capillary action past the mAb in the detection zone, which would bind the particles that had ricin bound to their surfaces, giving a red colour within the detection zone with an intensity proportional to the ricin concentration. In the absence of ricin, no immunogold was bound to the solid-phase antibody. With this method, 30 ng/ml of ricin toxin was detected in less than 10 min, and the assay also showed no cross-reactivity with abrin toxin.     

心肌肌钙蛋白I试纸条检测系统软件设计

目的:今年来,急性心肌梗死的死亡人数急剧增多,并且发病年轻逐渐提前,最令人担忧的是很多患者不能在第一时间被诊断因而得不到及时治疗。本系统就是针对心肌梗死标志物心肌肌钙蛋白I(cardiac troponin I,cTnI)设计的快速检测方法。方法:首先对检测原理以及胶体金试纸进行了阐述,进而分成四个部分对系统的运作进行详细介绍。结果:基于Windows平台,应用了VC++以及access能得到心肌肌钙蛋白I的浓度。结论:实验证明,该方法能够不仅较能检测出心肌肌钙蛋白I,还能计算出其浓度,提供了医生定量分析的数据。     

Molecular relationships between type Asia 1 new strain from China and type O Panasia strains of foot-and-mouth-disease virus

The complete genome of Asia 1/HNK/CHA/05 strain of foot-and-mouth disease virus (FMDV) was sequenced, which was isolated from Chinese Hongkong in 2005. It is 8187 nt long in size and contains 5′-UTR, polyprotein region, and 3′-UTR. Polyprotein region can be divided into four parts of L, P1, P2 and P3. In this report, these six parts of the whole genome of the strain were compared with 12 reference strains using DNAStar and Simplot softwares. The comparison of P1 confirmed that Asia 1/HNK/CHA/05 has a high identity with nine type Asia 1 reference sequences from 85.9 to 92.6% (Ind/491/97 strain is the highest) but from 69.6 to 69.7% with three type O Panasia sequences. The identities of 5′-UTR, L, P2, P3 and 3′-UTR with three Panasia strains are from 89.0 to 90.6%, 92.5 to 93.4%, 94.8 to 95.5%, 96.0 to 96.7% and 90.7 to 92.5% separately, but with nine type Asia 1 strains are from 83.5 to 85.9%, 87.7 to 90.7%, 87.0 to 91.6%, 91.6 to 92.8% and 86.0 to 100% separately, which illuminates the closer relationship between Asia 1/HNK/CHA/05 and Panasia strains in 5′-UTR, L, nonstructural region and 3′-UTR although they do not belong to the same serotype. The SimPlot software was used to examine the authentic relationships of Asia 1/HNK/CHA/05 with 12 reference sequences. It was found that Asia 1/HNK/CHA/05 strain has a highest similarity with three Panasia strains especially Tibet/CHA/99 in 5′-UTR, L, nonstructural region and 3′-UTR but has a highest similarity with Asia 1/Ind/491/97 strain in P1 region, which suggested that the gene recombination had occurred around nucleotide position 1811 and 3971in the polyprotein region between Tibet/CHA/99 and Ind/491/97 to recombine the Asia 1/HNK/CHA/05 strain. The nucleotide sequence data of Asia 1/HNK/CHA/05 strain reported in this article has been submitted to the Genbank nucleotide sequence database and has been assigned the accession number EF149010.     

O型口蹄疫病毒VP1表位重组蛋白疫苗的构建、表达和纯化

目的 为了克服灭活口蹄疫病毒疫苗可能存在的传播病毒的潜在危险,构建一种能预防O型口蹄疫病毒感染的VPI表位重组蛋白疫苗。方法采用O型口蹄疫病毒（FMDV）表面VP1蛋白上的B细胞表位肽和T细胞表位肽,以PCR扩增和克隆连接等方法构建VP1表位六聚体重组蛋白（vP1epi）基因,并在大肠杆菌中进行诱导表达,镍亲和层析纯化。用ELISA法检测VPlepi免疫豚鼠血清中抗FMDV抗体的水平。结果构建了一种由FMDV表面vP1中B细胞表位肽重复六次的蛋白质多肽,采用pET28原核表达系统,在大肠杆菌BL21（DE3）中获得了高效表达,表达产量约占菌体蛋白的30％左右,经镍亲和层析后获得纯度高于90％的VP1表位重组蛋白。VP1epi免疫的豚鼠血清中含有一定量的抗FMDV抗体。结论VP1epi重组蛋白能诱导机体产生抗O型FMDV的抗体,说明这种重组蛋白可能成为预防O型FMDV感染的基因工程蛋白质疫苗。     

血清人心肌肌钙蛋白I快速测定方法的建立

目的：建立一种简便、快速、准确检测人心肌肌钙蛋白Ｉ（ｃＴｎＩ）的胶体金免疫层析法。方法;应用ｃＴｎＩ抗体细胞株６株。其中３Ｆ１０作为金标抗体,３Ａ７作为生物素化抗体,链霉亲合素结合于硝酸纤维素膜、制成免疫层析测试条,依据测试条上出现肉眼可见的红色线条判定结果。结果：检测灵敏度可达０．４ｎｇ／ｍｌ。测定３０例急性心肌梗塞（ＡＭＩ）患者血清ｃＴｎＩ,并与国外同类产品比较,两者符合率为９３．６％。结论：