Development of an Indirect Enzyme-Linked Immunosorbent Assay for Seromonitoring Contagious Bovine Pleuropneumonia Using Recombinant Lipoprotein LppQ of Mycoplasma mycoides subsp mycoides SC as Antigen

Mycoplasma mycoides subsp mycoides SC （MmmSC） is the etiological agent of contagious bovine pleuropneumonia （CBPP）. The lipoprotein LppQ encoded by lppQ gene is specific to MmmSC and is found in the type strain and in field strains isolated in Europe, Africa, and Australia, as well as in vaccine strains. No serological cross-reactions were observed with the related mycoplasmas of the Mycoplasma mycoides cluster. The N-terminal domain of the mature lipoprotein LppQ is hydrophilic, and it induces a strong, specific, early, and persistent immune response in naturally and experimentally infected animals. Mycoplasma-specific TGA （Trp） codons are utilized as stop codons in most other organisms. The lppQ N-terminal fragment from MmmSC HVRI X strain, the Chinese strain for CF antigen production, was mutated with one-step overlapping extension PCR. Sequence analysis confirmed the successful mutation from A to G in codon 198 in the lppQ gene. The fragment containing the mutation site was subcloned into the pET32a expression vector. The recombinant protein with molecular weight of 42 kDa was purified using the Ni-NTA His.Bind purification kit, with a purity of up to 95%. Western blot indicated that the standard positive serum of CBPP could react with the recombinant protein. The purified protein was diluted to 0.35 μg mL^-1, and coated to microtiter enzyme-linked immunosorbent assay （ELISA） plates. Indirect ELISA reaction conditions were optimized. The value of P/N was determined to be 4.8 （0.934/0.193）, the sensitivity to be 95.8% （46/48）, and the specificity to be 98.9% （161/163）. 3 817 cattle serum samples from three different provinces were detected by the indirect ELISA and CFT. The Kappa value is 0.63, which is middle or high agreement between the two methods.

Microcystin-LR detection based on indirect competitive enzyme-linked immunosorbent assay

Microcystins(MCs)are a group of closely related toxic cyclic heptapeptides produced by common cyanobacte-ria,which cause lots of accidents and threatens human health.In this paper,an indirect competitive enzyme-linked immu-nosorbent assay(ic-ELISA)was established and used to detect microcystin-LR(MC-LR)in drinking and surface waters.The concentration of coating antigen was 5 mg/mL,the dilution of monoclonal antibody MC10E7 was 1:3000,the dilution of enzyme tracer(goat anti-mouse IgG-peroxidase)was 1:3000,the standard concentration of MC-LR ranged from 0.001 mg/L to 30 mg/L,and o-phenylenediamine was used as substrate.The assay showed high relativity with high performance liquid chromatography(HPLC)with a correlation coefficient of more than 99%.The relative standard deviation was less than 10%,the detection limit was achieved down to 0.01 mg/L and up to 5.1 mg/L.The quantitative detection range was from 0.03 mg/L to 3 mg/L,and the antibody had high specificity for[4-arginine]microcystins.It performed well in spite of the influence of the real samples.

Development of ELISA and immunochromatographic assay for ofloxacin

Two rapid, sensitive and reliable immunoassay methods, namely competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) and colloidal gold-based immunochromatographic assay (CGIA), were developed to detect ofloxacin (OFL). The linear range of the CI-ELISA was from 0.5 to 128 ng/mL with a limit of detection (LOD) of 0.35 ng/mL. Good recoveries were obtained in analyzing simulated swine urine samples. The CGIA could accurately estimate OFL at concentrations as low as 10 ng/mL in less than 10 min, and test results were read visually without any instrument.

基于免疫磁珠净化间接竞争酶联免疫吸附试验检测氟喹诺酮类药物

本研究旨在建立一种基于免疫磁珠进行分离、富集和净化前处理,检测鸡肉、鸡肝和鱼肉中氟喹诺酮类药物残留的间接竞争酶免疫吸附试验(indirect competitive enzyme-linked immunosorbent assay,icELISA)的研究方法。通过对合成免疫磁珠及免疫磁珠净化过程中单克隆抗体添加量、偶联时间、缓冲液pH、抗原添加量、抗原捕获时间、温度及包被条件等进行优化,初步建立了基于免疫磁珠净化的icELISA检测方法。结果显示:1)在1 mg的磁珠中,沙拉沙星(sarafloxacin,SAR)单克隆抗体最佳偶联量为15μg,偶联时间60 min,pH为4.4;2)最佳抗原添加量为1 ng·mL^(-1),捕获时间40 min,缓冲液为0.01 mol·L^(-1)PBS,IC50为0.73 ng·mL^(-1),线性范围为1.0~3.2 ng·mL^(-1);3)氟喹诺酮类药物在鸡肉、鸡肝、鱼肉的检测限均不超过1.33、2.17、2.31μg·kg^(-1),回收率为76.83%~98.70%,批内变异系数批间变异系数均不超过15%,经验证,鸡肉样本检测结果与高效液相色谱法(HPLC)结果一致。结果表明,与传统仪器检测方法相比,该方法提高了检测氟喹诺酮类药物的简便性、选择性以及检测效率,为氟喹诺酮类药物的残留检测提供了新思路。

Preparation of high-affinity rabbit monoclonal antibodies for ciprofloxacin and development of an indirect competitive ELISA for residues in milk

A convenient competitive enzyme-linked immunosorbent assay(ELISA) for ciprofloxacin(CPFX) was developed by using rabbit monoclonal antibodies(RabMAbs) against a hapten-protein conjugate of CPFX-bovine serum albumin(BSA).The indirect competitive ELISA of CPFX had a concentration at 50% inhibition(IC50) of 1.47 ng/ml and a limit of detection(LOD) of 0.095 ng/ml.The mAb exhibited some cross-reactivity,however,not so high with enrofloxacin(28.8%),ofloxacin(13.1%),norfloxacin(11.0%),fleroxacin(22.6%),and pefloxacin(20.4%).And it showed almost no cross-reactivity with other antibiotics or sulfonamides evaluated in this study.The competitive ELISA kit developed here could be used as a screening tool to detect and control illegal addition of CPFX in food products.This kit had been applied to milk detection and the recovery rates from samples spiked by CPFX were in a range of 63.02%-84.60%,with coefficients of variation of less than 12.2%.

基于高特异性单克隆抗体的间接竞争ELISA检测食品中胭脂红的研究

该研究设计合成了一种胭脂红半抗原,分别采用重氮化法和戊二醛法将半抗原与载体蛋白偶联制备人工抗原,通过免疫Bal b/c小鼠及杂交瘤技术成功筛选制备了胭脂红高特异性单克隆抗体,与苋菜红、柠檬黄等结构类似物无交叉反应。基于该抗体建立了间接竞争酶联免疫分析方法用于检测食品中胭脂红残留。该方法对胭脂红的半抑制浓度(IC_(50))和检出限(IC_(10))分别为10.1 ng/mL和0.98 ng/mL,线性范围为2~50 ng/mL。将该方法应用于山楂条和雪碧中胭脂红的快速检测,样品加标回收率分别为93.0%~113%和93.0%~100%,相对标准偏差分别为9.7%~12%和3.2%~3.9%。结果与HPLC-UV方法一致,表明该方法可用于食品中痕量胭脂红的快速筛查。

Immunodiagnostic efficacy of detection of Schistosoma japonicum human infections in China:a meta analysis

Objective:To assess the diagnostic efficacy of the currently most widely used indirect hemagglutination assay(IHA) and enzyme-linked immunosorbent assay(ELISA) for detection of Schistosoma japonicum human infections.Methods:A comprehensive search was undertaken from China National Knowledge Infrastructure,Wanfang Database.VIP Database,PubMed. Cochrane Library,Science Citation Index Expanded.Proquest,and the inclusion and exclusion criteria were strictly settled.The funnel plot was used to assess the publication bias.Cochran’s Q test was employed to measure the homogeneity between studies,a summary receiver operating characteristic(SROC) curve was used to compare the diagnostic accuracy between the IHA and ELISA qualitatively by means of the Weighted Least Square method,the Ordinary Least Square method and the Robust regression method,and the diagnostic odds ratio(DOR) was drawn to compare the accuracy quantitatively.Results:Out of 785 publications,19 papers were eventually selected for analysis.Literature finality assessment indicated that minor publication bias existed in studies pertaining IHA test,but no bias was found in literatures regarding ELISA test.The heterogeneity test showed a heterogeneity between studies was present(χ~2 =466.07 and 34.67. both P values【0.0001).The areas under the SROC curves of IHA were all higher than that of ELISA test using the three methods(Weighted Least Square method:0.766 vs.0.695.Ordinary Least Square method:0.826 vs.0.741.Robust regression:0.815 vs.0.715).The TPR* values for IHA and EUSA were 0.710.0.759.0.749.and 0.650.0.686 and 0.666.respectively,and OR values were 5.997.9.937.8.893.and 3.432.4.784 and 3.959.respectively.The DOR of IHA was 9.41(95% CI:4.88-18.18).and 4.78(95%CI:3.21-7.13) for ELISA.Conclusions:All above results revealed that the diagnostic performance of IHA is better than that of ELISA.However,taking into account their unsatisfactory diagnostic value in areas with low infection intensity,a search for a better diagnostic test that can be applied in field situations in China should be given high priority.

Production and Characterization of Monoclonal Antibodies to Bluetongue Virus

In the present study,a total of 24 MAbs were produced against bluetongue virus (BTV) by polyethyleneglycol (PEG) mediated fusion method using sensitized lymphocytes and myeloma cells. All these clones were characterized for their reactivity to whole virus and recombinant BTV-VP7 protein,titres,isotypes and their reactivity with 24 BTV-serotype specific sera in cELISA. Out of 24 clones,a majority of them (n = 18) belong to various IgG subclasses and the remaining (n = 6) to the IgM class. A panel of eight clones reactive to both whole BTV and purified rVP7 protein were identified based on their reactivity in iELISA. For competitive ELISA,the clone designated as 4A10 showed better inhibition to hyperimmune serum of BTV serotype 23. However,this clone showed a variable percent of inhibition ranging from16.6% with BTV 12 serotype to 78.9% with BTV16 serotype using 24 serotype specific sera of BTV originating from guinea pig at their lowest dilutions. From the available panel of clones,only 4A10 was found to have a possible diagnostic application.

氟喹诺酮药物格林沙星抗体制备及其icELISA方法建立

目的：制备格林沙星多克隆抗体并建立间接竞争酶联免疫吸附分析方法（icELISA）。方法：采用碳二亚胺法将格林沙星偶联于牛血清白蛋白（BSA）作为免疫原,免疫新西兰大白兔获多克隆抗体;采用活泼脂法分别将格林沙星、克林沙星、加替沙星、培氟沙星和沙拉沙星偶联于卵清白蛋白（OVA）,制备5种包被抗原并做性能比较;建立icELISA方法并对其灵敏度和特异性进行评价。结果：成功制备了免疫原和包被抗原及抗体,抗体效价最高达64 000倍。以克林沙星-OVA作异源包被,建立的icELISA方法灵敏度最高,半抑制药物浓度（IC50）为9.14 ng/mL,检测限（IC10）为0.9 ng/mL,检测范围（IC20~IC80）为2.3~36.4 ng/mL,与其他喹诺酮类药物无明显交叉反应。结论：首次制备出格林沙星抗体并建立间接竞争酶联免疫吸附检测方法,该法灵敏度高、特异性强,为分析生物样品中格林沙星含量的测定提供了1种新的方法。

Effect of Puerarin on the Pharmacokinetics of Baicalin in Gegen Qinlian Decoction (葛根芩连汤) in Mice

Objective： To study the pharmacokinetics of puerarin（PUE） in Gegen Qinlian Decoction（葛根芩连汤, GQD）, and the effects of PUE dosage variations on the pharmacokinetics of baicalin（BAL） in mice. Methods： GQD is composed of the concentrated granules of four Chinese herbs. Three dosages with different levels of PUE, including GQD, GQD co-administered with PUE, and GQD co-administration with two times the amount of PUE, were used to research the pharmacokinetics of PUE and BAL in mice. The indirect competitive enzyme-linked immunosorbent assay（ic ELISA） methods based on an anti PUE-monoclonal antibody（MAb） and BAL-MAb were employed to determine the concentration of PUE and BAL in mice blood. Results： After the co-administration of GQD with PUE, the area under the curves（AUC0-14 h） of PUE increased 2.8 times compared with GQD. At the dose of GQD co-administration at two times that of PUE, the AUC0-14 h of PUE was almost equal to that of GQD co-administration of PUE, showing non-linear pharmacokinetics. The AUC0-48 h of BAL showed a good dose-related increase of PUE（r=0.993） in the range from 100 to 300 mg/kg, indicating that PUE dramatically affects the absorption of BAL in mice. There was no significant difference in the other pharmacokinetic parameters, such as the first time of maximum concentration（Tmax）, the second Tmax, or the mean residence time. Conclusions： The ic ELISA methods were successfully applied to pharmacokinetic studies of PUE and BAL in GQD in mice. The dosage variability of PUE of the main ingredient in GQD affects its own pharmacokinetic characteristics and the absorption characteristics of BAL.