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.

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%.

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.

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.