Development of a class-specific polyclonal antibody-based indirect competitive ELISA for detecting fluoroquinolone residues in milk

Modified 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC)method was employed to synthesize the artificial antigen of norfloxacin(NOR),and New Zealand rabbits were used to produce anti-NOR polyclonal antibody(pAb).Based on the checkerboard titration,an indirect competitive enzyme-linked immunosorbent assay(icELISA) standard curve was established.This assay was sensitive and had a working range from 0.12 to 68.40 ng/ml,with the half maximal inhibitory concentration(IC50)and limit of detection(LOD)values of 2.7 ng/ml and 0.06 ng/ml,respectively.The produced pAb exhibited high cross-reactivity to fluoroquinolones(FQs)tested,and the IC50 values to enoxacin,ciprofloxacin,and pefloxacin were 3.1,3.4,and 4.1 ng/ml,respectively.It also indicated that the concentrations of NaOH and methanol in assay buffer should not be higher than 10%and 30%.When spiked in milk at 5,20,and 50 ng/ml,the recoveries for NOR,enoxacin,ciprofloxacin,and pefloxacin ranged 90.5%-98.0%,84.0%-95.2%,94.0%-106.0%,and 89.5%-100.0%,respectively.The results suggest that this class-specific pAb-based icELISA could be utilized for the primary screening of FQ residues in animal-original products.

Development of an indirect competitive ELISA for simultaneous detection of enrofloxacin and ciprofloxacin

Modified 1-ethyl-3-(3-dimethylaminopropy) carbodiimide(EDC) method was employed to synthesize the artificial antigen of enrofloxacin(ENR),and New Zealand rabbits were used to produce anti-ENR polyclonal antibody(pAb).Based on the checkerboard titration,an indirect competitive enzyme-linked immunosorbent assay(ELISA) standard curve was established.This assay was sensitive and had a linear range from 0.6 to 148.0 μg/kg(R2=0.9567),with the half maximal inhibitory concentration(IC50) and limit of detection(LOD) values of 9.4 μg/kg and 0.2 μg/kg,respectively.Of all the competitive analogues,the produced pAb exhibited a high cross-reactivity to ciprofloxacin(CIP)(87%),the main metabolite of ENR in tissues.After optimization,the matrix effects can be ignored using a 10-fold dilution in beef and 20-fold dilution in pork.The overall recoveries and coefficients of variation(CVs) were in the ranges of 86%-109% and 6.8%-13.1%,respectively.It can be concluded that the established ELISA method is suitable for simultaneous detection of ENR and CIP in animal tissues.

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.

Study of Immunoassay Methods for Recombinant Human Erythropoietin (rhEPO) Using Competitive ELISA

Two different immunoassay methods, competitive indirect enzyme-linked immuno-sorbent assay (CI-ELISA) and amplificative competitive indirect ELISA (ACI-ELISA) using biotin-avidin complex system were studied to detect rhEPO. The linear ranges were 50-20000 ng/mL and 10-50000 ng/mL for CI-ELISA and ACI-ELISA, respectively. The low detection limits of CI-ELISA and ACI-ELISA were 62.8 ng/mL and 8.5 ng/mL, respectively.

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.

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.

A Novel Chemiluminescence Immunoassay Using Solid-Phase Antigen for Free 17β-Estradiol in Human Serum

A high-performance chemiluminescence immunoassay, with long-term durability, good precision and time- saving, was proposed for the detection of free 17β-estradiol （E2） in human serum. Ninety-six microplates were coated with bovine serum albumin conjugated E2 antigen as solid phase for the immunoassay. The E2-BSA antigen coated on the microplate and the E2 antigen in the sample competed for the binding sites on the horseradish peroxi- dase （HRP） labeled anti-E2 antibody. Chemiluminescence reaction was subsequently carried out by HRP catalyzing luminol-H2O2 substrates, and the chemiluminescence intensity was inversely proportional to the amount of analyte in human sera samples. The concentration of immunoreagents, immunoreaction time, and other relevant variable conditions upon the immunoassay were studied and optimized. The proposed method exhibited detection limit as low as 5.94× 10^-3 μg·L^-1 in a linear detection range from 0.01 to 1.00μg·L^-1 good recoveries between 105% and 108%, and high precision with intra- and inter-assay coefficients between 7.9% and 14.3%.

An immunoassay based on lab-on-a-chip for simultaneous and sensitive detection of clenbuterol and ractopamine

Both clenbuterol(CLB)and ractopamine(RAC)areβ-adrenergic agonists.After long-term excessive intake,there will be adverse reactions such as headache,chest tightness,limb numbness,and serious lifethreatening.Simultaneous detection of CLB and RAC in related samples is of great importance for human health.In this work,we outline a microfluidics-based indirect competitive immunoassay(MICI)system that can sensitively detect residual CLB and RAC in pork,swine blood and swine urine.The rapid detection of multiple samples can be achieved in one chip,which greatly improves the detection efficiency.This method has good stability and reproducibility and the microfluidic chips are easy to manufacture.The linear ranges for CLB and RAC detection by MICI are 0.1-2.5 ng/mL and 0.1-5 ng/mL,and the limits of detection(LODs)are 0.094 ng/mL and 0.091 ng/mL,respectively.This straightforward and portable immunoassay system provides a good platform for rapid detection of harmful substances in food samples.

Synthesis of haptens and production of antibodies to bisphenol A

Three immunizing haptens of bisphenol A(BPA), including two new haptens, were used to produce highly sensitive and specific polyclonal antibodies. The spacer arms of haptens for coupling to the protein carrier were located at different positions in BPA, and different length spacer arms were tested. Highly sensitive polyclonal antibodies were obtained and characterized using indirect competitive enzyme-linked immunosorbent assay(ic ELISA). Under optimized conditions, the half maximal inhibitory concentration(IC50) value of the best polyclonal antibody was 2.1 mg·L^(-1), based on coating heterogeneous antigens, and this optimal polyclonal antibody was highly sensitive toward BPA and displayed negligible crossreactivity with bisphenol B and bisphenol E. A sensitive ic ELISA method utilizing the polyclonal antibody was developed for the determination of BPA in milk. In spiked samples(5, 10 and 20 mg·L^(-1)), the recovery ranged from 80% to 102% with a coefficient of variation(CV) value below 15.8%. The limit of detection of ic ELISA was1.95 mg·L^(-1). These results indicate that the ic ELISA method is suitable for the detection of BPA in milk.