| Lateral flow immuno-chromatographic assay (ICAs), employing colloidal gold as label, has unique advantages including rapidness, simplicity and strong capacity of resisting disturbance in sample matrix, and consequently acts as the first choice of rapid screening at point of care. However, ICAs results based on visual determination are qualitative and less sensitive, moreover, read by naked eye can introduce errors. In this study, we utilized newly developed fluorescent silica microspheres in ICAs and established a quantitative detection model based on T/C. This system provides scientific basis for the rapid and quantitative analysis and stretches the scope of application of the ordinary immuno-chromatographic strips.The fluorescent microspheres used in this study was synthesized by the hydrolysis of organic silicon doping with Ru (4,7-Ph2-phen)3. The maximum excitation wavelength and emission wavelength of the synthesized fluorescent microspheres are at 450 nm and 570 nm, respectively. The hard diameter is about 77 ± 6 nm determined by transmission electron microscopy and the average hydrodynamic diameter is about 240 nm with zeta potential of-25.34 mV at pH 5.0 measured by nano-particle size analyzer. The saturation amount of antibodies for conjugation is about 120 μg(Ab)/mg(Bead). The fluorescent microspheres maintain good fluorescent stability in pH range from 4 to 10 and the salt concentration below 1 mol/L.A fluorescent microspheres based immuno-chromatographic system aiming at clenbuterol (CLE, a small molecule hapten) detection is successfully established. The strip was manufactured as follows:1) Microsphere-antibody coupling:30 μg antibody was added per milligram of fluorescent microspheres; and then the conjugates was blocked with 1% BSA (w/v) dissolved in 0.02 mol/L PB; 2) Strip assemble:the conjugates were sprayed onto the conjugate pad at a spraying rate of 5 μg/cm and then the pad was dried at 30℃ in a vacuum dryer, the concentration of CLE-BSA sprayed at test line was 0.5 mg/mL while the donkey-anti-mouse antibody at control line was 1.0 mg/mL. Results from the immunological kinetics analysis at the detection zone showed that the fluorescence intensities at both lines increased as the reaction time expanding, and reached stable values at about 40 min after the sample addition. However, it takes only 5 to 10 min for the T/C ratio values to reach a constant number. This phenomenon indicated that the T/C ratio could eliminate the differences caused by the dynamics of the immunological reaction. Thus, the quantitative detect system for CLE was based on the consistency of T/C ratio. The immune competitive inhibition curve in this immuno-chromatographic system was in highly accordance with the law of the immunological reaction. The IC50 value was 1.43 ng/mL (N= 5) and the concentration range for quantitative determination was from 0.3 to 5 ng/mL with linear equation coefficient (R2) of 0.9957. Cross reactivity study showed that the strip has a cross reactivity of about 0.2% with salbutamol and less than 0.01% with ractopamine, cimaterol, cimbuterol, terbutaline and other structural analogues. The recovery of spiked CLE in pig urines testing by the strips was between 97.7% and 114.5%. The coefficient variation detected within the same batch was 10.4% to 16.8% in intra-assays while it reached 12.4% to 14.2% in inter-assays.It indicated that the test strips had good accuracy and precision. Urine samples from 50 pigs with different origins were chosen as real samples for quantitative analysis and verification of the correlation between the strips and commercialized ELISA kit. Results showed a good correlation (R2=0.9792) between the two methods, indicating the method of using T/C model based ICA strips can be used for quantitative detection of CLE residues in pig urines.To confirm the above theory, a similar study was performed using enrofloxacin (ENR) as the analyte. Similarly, the strip was manufactured as follows:1) Microsphere-antibody coupling:20 μg antibody was added per milligram of fluorescent microspheres; and then the conjugates was blocked with 1% BSA (w/v) dissolved in 0.02 mol/L PB; 2) Strip assemble:the conjugates were sprayed onto the conjugate pad at the spraying rate of 4 μg/cm, the concentration of ENR-BSA sprayed at test line was 1.2 mg/mL while the donkey-anti-mouse antibody at control line was 1.0 mg/mL. To investigate the effects on antigen-antibody reaction kinetics, detection sensitivity and quantitative analysis, two different patterns including the test line or the control line settled at former position were investigated. Result showed that the control line-former mode had a lower sensitivity compared with the test line-former mode. Data from the antigen-antibody reaction kinetics test showed that the reaction takes about 10 to 20 min to reach a T/C ratio steady in control line-former mode needed, while 5 to 10 min in test line-former mode. The following experiment was completed using the test line-former mode for quantitative detection of ENR. The immune competitive inhibition curve was also in good accordance with the law of the immunological reaction. The IC50 value was 1.2 ng/mL (N= 5) and the concentration range for quantitative determination was 0.5 ng/mL to 2.5 ng/mL with a linear equation coefficient (R2) of 0.9907. The cross reactivity with ciprofloxacin, norfloxacin, ofloxacin, sarafcoxacin were less than 6%, while less than 0.01% with enoxacin, danofloxacin and lomefloxacin. The recovery of spiked ENR in positive samples testing by the strips was between 92.4% and 107.9%. The coefficient variation detected within the same batch was 7.9% to 15.6% in intra-assays while it reached 9.6% to 13.4% in inter-assays.It indicated that the test strips had good accuracy and precision.In summary, this study has successfully established a T/C ratio based quantitative ICA detection system using fluorescent microspheres as labels for testing clenbuterol and enrofloxacin. Real sample analysis CLE showed the ICA strips we developed had good accordance with the results from the corresponding commercial ELISA kits. |
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