| Immunochromatographic strip (ICS) assay using optical sensor can realize a rapidquantitative detection with the advantages of simplicity, rapidity, on-site monitoring.In recent years, a number of papers have been reported the strip based quantitativeassays. However, few of them described the effects of various factors on thequantitative strip by analyzing immunological kinetics reaction. In the present study,the feasibility of T/C ratio based ICS used for CLE quantitative analysis in pork urineand muscle was evaluated and the main factors that influence quantitative assay werediscussed. The purposes of this research aim to provide the basic data and theoreticalbasis for establishing ICS based quantitative detection method.Firstly, the processing parameters of the CLE-strip were optimized by anorthogonal L9(3)3analysis, the optimal parameters were as follows:1.0μg/mLanti-CLE antibodies labeled colloidal gold;3μL/cm jetted density of colloidal goldprobe on conjugate pad and0.5mg/mL of CLE-BSA sprayed on test line. Theimmunological kinetic curves of antibody on conjugate pad and antigen (CLE-BSAon test line and goat anti-mouse IgG on control line) interaction was obtained byploting the development of OD values on the test and control lines against runningtime, and it was used to evaluate the effects of immunoreaction time, operationtemperature and sample conditions on the quantitative analysis. The resultsdemonstrated that the T/C ratio based ICS quantitative method can reduce thedetection time (10min), and offset the influence of environment temperature on theT/C value of the strip assay in the range of10℃to37℃. Moreover, the saltconcentration (0~0.5M) and different pH values (pH6to9) in the sample solutionwas no significant effect on the T/C value, whereas T/C value was significantlydecreased as pH value lower than5.0in the sample solution.The calibration curve for CLE quantitative analysis was established bydetermination of a serial of CLE standard solutions which were prepared by spikedCLE stock solution in the blank mixed swine urine. The linear range of ICS methodwas between0.1ng/mL to2.0ng/mL. The half maximal inhibitory concentration(IC50) was0.36ng/mL (n=5) and the limit of detection (LOD) for real swine urinewas0.22ng/mL. The intra and inter-assay precision at0.5~1.5ng/mL CLEconcentrations showed coefficient of variation (CV) lower than10%. This new developed ICS system for CLE quantitative detection in spiked urine samplesexhibited a coefficient of correlation at0.97(N=50) with the traditional ELISA kit.The performance of ICS method was further evaluated by analyzing90different CLEspiked real urines, the results showed that96%of revovies were in the range of70%to130%. The above results indicated that the ICS system can be used for quantitativeanalysis of clenbuterol in swine urine, and this new method greatly improves thedetection sensitivity of ICS and broads the application scope of on-site detection.Simultaneously, the T/C ratio based ICS was also developed for rapid andquantitative detection of clenbuterol residues in pork. Five of sample pretreatmentmethods were evaluated. The results showed the recovery of the pretreatment methodin which samples extracted with0.02mol/L HCl solution containing2.8%NaCl fortwice, was achieved at76.7%with a coefficient of variation at7.4%. The linear rangeof the new quantitative method was from0.1to1.5ng/g with a detection limit at0.19ng/g in pork samples. For the spiked samples, the results showed that the averagerecoveries were60.4±12.8%,70.24±4.2%,75.9±4.9%and71.1±5.0%at CLEconcentrations of0.5,1,2and3ng/g, respectively. The new quantitative system forCLE detecting in spiked pork samples exhibited a coefficient of correlation withconventional ELISA method (R2=0.9136).Besides, immunomagnetic beads for CLE purification were prepared bystreptavidin (SA)-biotin system. Under the optimized conditions,77.2μg biotinylatedantibodies were used to bind with1mg magnetic beads which coated with23.9μgstreptavidin. The optimal adsorption and elution conditions of immunomagnetic beadsfor CLE purification were as follows:1g of homogenized pork was extracted with4mL of HCl solution (0.15mol/L) by vigorous shaking. After centrifuged at16000rpmfor5min,0.25mg magnetic beads solution was added and incubated. Theimmunomagnetic beads were separated with magnetic shelf, and the CLE was elutedfrom the immunomagnetic beads using100μL of methanol. The performance ofimmunomagnetic beads was evaluated by using CLE spiked samples at concentrationof0.25,0.50,1.0,2.0and5.0ng/g, respectively. The results indicated that theadsorption efficiency of immunomagnetic beads were at61.2%~109.1%with relativestandard deviations of7.8%~15.7%. |
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