Dinitrophenyl (DNP) hapten sandwich staining (DHSS) procedure : A 10 year review of its principle reagents and applications

Over the past 10 years an immunoperoxidase method using dinitrophenyl (DNP) hapten-labelled primary or secondary probes has been devised. Its widely successful application in research and diagnostic work has depended upon the development of certain key reagents. These include a novel non-deleterious DNP labelling compound, a unique multivalent monoclonal bridge antibody, and an efficient DNP hapten substituted or anti-DNP linked marker enzyme. In this article the development of these reagents and various modifications of the basic technique are reviewed in conjunction with the special applications accruing from their use.     

A Biosensor-based Immunoassay for Rapid Screening of Deoxynivalenol Contamination in Wheat

A surface plasmon resonance (SPR) based indirect inhibitive immunoassay was developed for the rapid quantification of concentrations of the trichothecene mycotoxin deoxynivalenol (DON). A DON-biotin conjugate was synthesized and immobilized to a streptavidin coated SPR sensor surface to measure free anti-DON antibody added to a sample. For analysis, ground wheat was extracted with 10% (v/v) methanol in water with 6% (w/v) polyvinyl-pyrrolidone, filtered and cleaned up with MycoSep™ columns. Extracts were mixed with a polyclonal anti-DON antibody and pre-incubated prior to injection into a BIAcore® device. The sensor surface was regenerated with a rinse of 6 M-guanidinechloride in 10 mm-glycine (pH 2.9). The assay had a working range between 0.13 and 10.0 μg ml -1 of DON, a 50% inhibition concentration (IC 50 ) of 0.72 μg ml -1 , and a detection limit of 2.5 pg μl -1 . Recovery of DON in spiked wheat was 104 ±15%. The system enables analysis of a sample to be completed in 15 min including sample preparation (10 min) and quantification (5 min). The assay was applied to the analysis of wheat samples with different levels of DON contamination. Statistical analysis revealed a positive, linear correlation between concentrations of DON measured with the new biosensor and GC/MS or HPLC as reference methods. Coefficients of correlation of R 2 = 0.9464 (GC/MS data) and R 2 = 0.9066 (HPLC data) were found.     

Direct Versus Competitive Biosensor Immunoassays for the Detection of (Dihydro)Streptomycin Residues in Milk

A monoclonal antibody (MAb) against dihydrostreptomycin (4G8) was developed and its performance compared with a previously developed MAb against streptomycin (4E2) in biosensor immunoassays (BIAs) using a surface plasmon resonance (SPR)-based biosensor (BIACORE 3000). Direct BIAs for the detection of dihydrostreptomycin (DHS; 583 Da) and streptomycin (STREP; 581 Da) were developed by immobilising the MAbs on the sensor chip (CM5). These direct BIAs were compared with competitive inhibition BIAs, using a STREP- protein conjugate immobilized on the chip. The sensitivities of the direct and competitive BIAs for both drugs in buffer were comparable (10-20 ng ml- 1 at 50% binding or inhibition). With milk, interferences, probably due to the nonspecific binding of proteins to the sensor chips, were observed in both BIAs. These interferences could be largely reduced using ultra filtration (UF) as sample pre-treatment. Another option was the use of a reference flow channel to correct for nonspecific binding. Using this option with five times diluted milk, MAb 4G8 was found to be suited for the direct BIA of both drugs with a limit of detection (LOD) of 20 ng ml- 1 and both MAbs could be applied in the competitive BIA format with similar LODs.     

雷公藤多甙片治疗小儿哮喘疗效及其免疫作用机理的研究

作者对37例小儿哮喘病患者经用雷公藤多甙片(Tabellon Multiglycosidorum Triptergii)治疗12周后,随访8个月,其中有效者33例,总有效率为89.18%,各类型病者间有效率无显著差异(P>0.05);对其中27例病儿服药前后外周血T细胞亚群及抑制性细胞活性进行检测,结果显示:病儿CD_?~+亚群从25.36±5.16%上升为28.96±3.03,(P<0.05),CD_3~+、CD_4~+亚群改变不大(P>0.05),而抑制率(SR)从-20.86±39.10上升为11.93±30.83(P<0.001),同时发现,抑制率的恢复与临床疗效间有一定关系。由此,作者认为,该药对小儿哮喘的治疗机理可能与机体免疫抑制功能恢复有关。     

犬同种带瓣大动脉移植后的免疫学研究

目的：检测同种带瓣大动脉（conduit valved homograft．CVH）移植后的免疫反应,探讨其作用机制及临床意义。方法：①建立液氮冻存和4℃保存犬CVH（各14例）移植于异性受体犬腹主动脉实验模型：②在移植术后不同时点切取植入的CVH标本：免疫组织化学染色法检测细胞免疫及体液免疫反应,用光镜、扫描电镜及透射电镜进行观察。结果：术后7d即可出现细胞免疫反应：1～3月达高峰：6～12月呈持续的低水平状态;表现为CVH内膜、动脉外膜、瓣下心肌组织中T细胞（以CD8^＋细胞和CD4^＋细胞为主）浸润,IL-2Ra持续表达体液免疫反应仅发生于术后7d～3个月,IgG沉积于CVH内膜和动脉中层平滑肌细胞周围;CVH瓣下心肌细胞、动脉中层平滑肌细胞间有散在的CD21^＋细胞浸润。结论：①CVH具有免疫原性,内皮细胞、平滑肌细胞、心肌细胞是其免疫原性表达的主要部分：②CVH移植术后早期（3个月以内）行免疫排斥反应的监测及防治具有临床意义。     

A Cloth-based Enzyme Immunoassay for Detection of Peanut Proteins in Foods

A dot blot enzyme immunoassay was developed for detection of peanut proteins in foods based on the use of polyester cloth as the solid phase. Samples were spotted on polyester cloth pre-coated with anti-peanut antibodies (chicken IgY), and bound peanut proteins were detected by sequential reactions with peroxidase-conjugated anti-peanut antibodies and chromogenic substrate. This assay detected peanut proteins in a variety of foods, in some instances giving positive results with samples containing less than 1 ppm peanut protein.     

宫颈癌组织中人乳头瘤病毒16型E7蛋白致癌机理初探

目的 研究宫颈癌组织中人乳头瘤病毒（ＨＰＶ）１６－Ｅ７蛋白对视网膜母细胞瘤基因（Ｒｅｔｉｎｏｂｌａｓｔｏｍａ）Ｒｂ蛋白及Ｅ２Ｆ－１的作用的机制,探讨ＨＰＶ１６－Ｅ７蛋白与宫颈癌发生的关系。方法 采用聚合酶链反应检测宫颈癌及正常宫颈组织中ＨＰＶ１６感染等,用蛋白印迹技术对ＨＰＶ１６ ＤＮＡ阳性的宫颈癌组织中是否存在ＨＰＶ１６－Ｅ７蛋白和Ｒ６蛋白－Ｅ２Ｆ－１形成的复合物进行检测。正常宫颈组织作为对照,     

Fluid therapy directly interferes with immunoassay for cardiac troponin I

Objective: To investigate interference in cardiac troponin I (cTNI) immunoassay induced by some widely used loading fluids. Setting: A biochemistry unit of a university hospital. Measurements and results: Human serum with a cTNI concentration of 0.32 μg/l was diluted at 10, 20, 40, and 80 % with saline serum (SS), Plasmion (P) (a modified fluid gelatin), hydroxyethyl starch (HES), and 20 % human albumin (Alb). Serum with a cTNI concentration of 1.29 μg/l was diluted at 20, 40, 60, and 80 %. Four samples with increasing cTNI concentrations (from 0 to 8.14 μg/l) were diluted at 80 % with the four fluids. Differences (delta-C) between expected concentrations resulting from the dilutional effect and those measured with the Access cTNI immunoassay were expressed in μg/l. Statistical analysis was performed using a nonparametric test. No false positivity was observed. At a low cTNI concentration (0.32 μg/l), interference was observed with SS and HES at 40 and 80 % dilution and with P and Alb at 80 %. When cTNI was 1.29 μg/l, interference was observed with each fluid at a dilution of 20 % and increased with the increase in dilution. The highest interference was observed with HES, the lowest with P. When the dilution was at 80 % for increasing concentrations of cTNI, the higher the initial cTNI was the higher the interference appeared, mostly with SS and HES. Conclusions: SS, P, Alb, and HES interfere in this cTNI immunoassay. This interference is higher with SS and HES and is higher when the percentage of hemodilution or real cTNI concentration increases. Received: 21 September 1998 Final revision received: 23 February 1999 Accepted: 6 April 1999     

实时荧光PCR检测乙肝病毒DNA的临床意义

目的用实时荧光定量PCR检测乙型肝炎患者HBV—DNA结果进行分析,以探讨其临床诊疗意义。方法对850例临床标本用时间分辨荧光免疫技术定量测定乙肝病毒血清学指标,用荧光定量PCR法检测血标本中的HBV—DNA,并依据乙肝两对半结果进行归类分组。结果302份HBsAg（＋）、HBeAg（＋）、HBcAb（＋）标本中有261份HBV—DNA为阳性,阳性率为86．4％,其PCR定量拷贝数为（2．21±0．76）×10^7／ml;321份HBsAg（＋）、HBeAb（＋）、HBcAb（＋）标本有96份HBV—DNA阳性,阳性率达到29．91％,PCR定量拷贝数为（1．69±0．98）×10^6／ml;32份HBsAg（＋）、HBcAb（＋）标本中有9份HBV—DNA为阳性,阳性率为28．13％,28份HBeAb（＋）、HBcAb（＋）标本有4份PCRHBV—DNA阳性,阳性率14．29％;11份HBcAb（＋）标本有6份PCRHBV—DNA阳性,阳性率为42．9％;74份HBsAb（＋）、HBeAb（＋）、HBcAb（＋）标本有5份HBV—DNA阳性,阳性率6．75％;13份HBsAb（＋）、HBeAb（＋）阳性标本有2份PCRHBV—DNA阳性,阳性率为14．3％;12份HBsAb（＋）、HBcAb（＋）标本有1份HBV—DNA阳性,阳性率7．69％;4份HBsAb（＋）的标本HBV—DNA均为阴性,PCR定量拷贝数为〈1．00E×103;24份HBsAg（＋）、HBsAb（＋）、HBeAg（＋）、HBcAb（＋）标本中有21份HBV—DNA为阳性,阳性率为87．50％;1份HBsAg（＋）标本HBV—DNA均为阳性,阳性率为100％;10份HBsAg（＋）、HBsAb（＋）、HBeAb（＋）、HBcAb（＋）标本有3份HBV—DNA均为阳性,阳性率30．00％,其余38例全阴性结果和各少见模式基本见有HBV—DNA的检出。结论PCR定量测定HBV—DNA可以真实反映体内乙肝病毒感染和复制及病毒载量情况,更有利于临床治疗和疗效观察。     

免疫传感器的发展概述

介绍了免疫传感器的概念及基本原理,根据换能器的不同进行了分类比较,并分别阐述了各种免疫传感器的由来及发展过程。在免疫传感器的制作上,浅谈了固定和再生性两个问题。另外,还对免疫传感器的应用和发展趋势予以了简要的回顾和展望。