| Objective and significanceDuring blood donation and transfusing, HBV, HCV, HIV-1, HIV-2 and syphilis testing are compulsorily required in China nowadays. Hepatitis B virus and Hepatitis C virus mostly caused the PTH (post transfusion hepatitis). And the clinical laboratories usually use ELISA (Enzyme linked immunosorbent assay) to analyze those pathogenic microorganisms. Most of these assays are for antibody test and the delay of antibody production (window phase) is inevitably can not be checked out. Morever, the different of testing kits use caused the different testing time.As the Quantum dots (QDs) with different size can be inspired different fluorescence by monochromatic light and the Magnetic Microspheres (MMS) can be enriched in a magnetic field, we may establish and apply testing system with high sensitivity, specificity, efficiency and speed for the simultaneously detection of antigens in liquid. Materials and methods1. The conjugation of MMS and Ab (ready for Reagent 1, R1): Chose the 3μm diameter Carboxyl MMS and use the carbodiimide cross-link technique activate the MMS with EDC and NHS in different concentration, then linked mice anti-HBsAg-IgG and mice anti-HCcAg-IgG on them.To view the effect with different concentration of Ab, bounding time and pH value which act on the conjugation of MMS and Ab. Then examine the ratio of conjugation by folin-phenol method.2. The conjugation of QDs and Ab (ready for Reagent 2, R2): Activate QDs with emission wavelengths at 581nm and 618nm by EDC/NHS at the volume proportion of 1:50. To view the effect with different concentration of Ab, bounding time and pH value which act on the conjugation of QDs and Ab. Then examine the ratio of conjugation by folin-phenol method.3. The single test systems: Use the QD-Ab as the fluorescence labels and the MMS-Ab as carriers to solid-liquid separate. After obtained the optimal value of react time we analyzed the negative serums to get the average of double negative control adds average of blank as cut off values. Then we used R1, R2 to analyze mixed standard antigen at different concentrations of HBsAg and HCcAg to determine the detection limits.4. To establish the simultaneously detection system: On the basis of single test systems we combined the conditions and choose the best react time.Then analyzed the negative serums to get the average of double negative adds average of blank as cut off values. Because of the overlapping of MMS, we use the filters to obtain the different colors of fluorescence partly. Then we used R1, R2 to analyze mixed standard antigen at different concentrations of HBsAg and HCcAg to determine the detection limits.5. 188 unknown samples were analyzed by the simultaneously detection system and compared the results with ELISA.6. We analyzed the individual and mixed standards at concentration of 10ng/mL, 100ng/mL, 1μg /mL as the repetitive experiments.7. The samples with hemolysis, jaundice and lipemia were analyzed as the interference factors and the positive HAV, TP serums as the specificity experiments.8. The fluorescence of prepared reagents and the complexs after reaction were recorded in 8 weeks to investigate the stabilities.Results1. The condition for the conjugation of MMS and Ab: The concentration of activators EDC at 6mg/L and NHS at 4mg/L for mice anti-HBsAg-IgG, bonding time t=120min, pH value 6.6 and the concentration of Ab was 20mg/mL. EDC at 4mg /L and NHS at 4mg /L for rabbit anti-HCcAg-IgG, bounding time at 120min, pH value 6.6 and the concentration of Ab was 20mg/mL. The efficiency ratios of conjugation were 41% and 44%. Add 1% BSA in R1 and store at room temperature for 4 hours and washed with pH7.4 PBS for use.2. The condition for the conjugation of QDs and Ab: Bonding time t=120min, pH value 6.2 and the concentration of rabbit-anti-HBsAg-IgG was 20mg/mL to QD581. Bonding time t=120min, pH value 6.2 and the concentration of rabbit anti-HCcAg-IgG was 10mg/mL to QD618. The efficiency ratios of conjugation were 45% and 45%.3. The single test systems: Serum 50μL; R1, 100μL; R2, 5μL (after enrichment), add in EP tube totally then react under the condition of pH7.4, 37℃for 20min and 30min (HBV and HCV). The cut off value were 122,136. The detecting ranges were 2ng/mL~10μg/mL for HBsAb and 5ng/mL~5μg/mL for HCcAg. The time of detection were shortened from 1h and 3h to 30min and 40min.4. The parallel test system: Serum 100μL; Each R1, 100μL and each R2, 5μL (after enrichment), add in EP tube totally then react at the condition of pH7.4, 37℃for 40min. The cut off value were 133,140 of HBsAg and HCcAg. The detecting ranges were 2ng/mL~10μg/mL for HBsAb and 5ng/mL~5μg/mL for HCcAg.5. The results of 188 unknown samples analysis: Positive HBsAg serums were 36, 35, 34 and HCcAg were 11, 11, 8 by PCR, parallel test system and ELISA. The mixed 5 standards and 2 multiple infection serums were detected correctly. The accordance rates, sensitivity, specificity of parallel test system were 98.4%, 94.4%, 99.3% for HBsAg and 97.9%, 81.8%, 98.9% for HCcAg between PCR. The accordance rates of parallel test system were 95.8%, 98.4% between ELISA for HBsAb and HCcAg.6. Standards analyze results of repetitive experiments show that the average coefficient variation (CV) of within-run and the inter-day-run of single test systems were 9.7%, 9.8% and 10.3%, 10.1% for HBsAb and HCcAg. The CV of within-run were 10.0%, 10.1% and of the inter-day-run were 10.7%, 10.6% for HBsAb, HCcAg of parallel test system7. The analysis of samples with hemolysis, jaundice and lipemia show theses factors do not interfere the results and the test of positive HAV, TP serums show a good specificity.8. It show that the using effect of reagents and the intensity of fluorescence of the complexes after reacted which stored at 4℃did not change obviously after 4weeks.Conclusions1. In this study, by using the speciality of the different QDs can be inspired different fluorescence by monochromatic light, we took HBV and HCV which mostly caused PTH as examples and established the method that can detect different antigens in parallel.This research may helpful to the foundation of the antigens detect system in parallel and ensure the transfusion security. Because of the aiming at antigen detect, this method may avoid the the false-negative caused by window phase.2. We linked the antibody with QDs and MMS by carbodiimide cross-link technique and analyzed the antigens by double antibody method. Since we used the Magnetic Microspheres as carrier, we can analyze the mixed antigens at the concentration of 2ng/mL, 5ng/mL for HBsAb and HCcAg with few requirements of equipments.3. The clinical sample test results show that there were no significant difference between ELISA and the QD-MMS method. However, the measure times were shortened from 1 hour and 3 hours of ELISA to 40min in parallel. Since there was a positively correlation between the intensity of fluorescence and concentration of antigen, we may detect the samples semi-quantitatively by using image analysis software.4. The prepare times of reagents were improved obviously than ELISA and because of the mild degree of photobleaching, the complexes after reacted were easy to store and stable that may help to callback and the long-time monitoring.
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