| With the widespread use of antiviral drugs, quantitative detection of hepatitis B surface antigen (HBsAg) used for therapeutic effect monitoring need to be developed in time. Amplified Luminescent Proximity Homogenous Assay(AlphaLISA) is a homogeneous detection technique based on microspheres, which can be used for fast and quantitative detection. It is especially suitable for the quantitative detection of HBsAg. Compared with conventional immunoassay based on the microtiter plate, the technique has the advantages of high sensitivity(pg grade), low background (blank count<100), short reaction time (the whole analysis process is not more than35minutes), stable property, simple operation, easy miniaturization, automation, wide application range, low prices and other advantages. In this study, we developed an AlphaLISA HBsAg quantitative diagnostic kit which was supported by the National Science and Technology Major Project of China. Time-resolved fluorescence immunoassay (TRFIA) is a sensitive high-end quantitative immunoassay technology by using rare earth ion to label antigens or antibodies. Due to the unique fluorescence properties of lanthanide rare earth ions, the technique has advantages of high signal to noise ratio, high sensitivity, easy labeling process, long storage time, no radioactive contamination, good repeatability, short operation process, wide range of detection, not sensitive to natural fluorescence interference and very wide range of applications. TRFIA has higher clinical application value compared with ELISA and RIA, which is widely used in the market for clinical detection of various antigens and antibodies. It is necessary to develop high sensitive HIV diagnostic kit using time-resolved fluorescence immunoassay method.Method:I. Development of hepatitis B virus surface antigen AlphaLISA reagent using double antibody sandwich method.1. Prepare standards and controls with HBsAg antigen, The concentration of standards was:0IU/mL,0.04IU/mL,0.8IU/mL,4IU/mL,20IU/mL,100IU/mL.2. Biotinylate antibody:1mg antibody was added to microspin column, and was centrifuged for5-6min at8000rpm. Subsequently, the antibody was washed six times using0.1mol/L carbonate buffer (pH9.5) repeatedly. NHS-D-Biotin was dissolved in DMSO immediately prior to use in a dark place at a concentration of40mg/mL. After that,2μL NHS-D-Biotin solution was added to200μL antibody solution with gentle stirring, and incubated at room temperature for4hours. The unreacted NHS-D-Biotin was dialyzed with bag filter. 3. Coupling of antibody to beads:0.2mg of antibody was added to microspin column, and was centrifuged for5-6min at8000rpm. Then, the antibody was washed six times using0.13mol/L sodium phosphate buffer pH8.0repeatedly. The solution was added to1mg acceptor beads with10μL of25mg/mL NaBH3CN and1.25μL of10%Tween-20and then incubated at37℃for48hours. To block non-conjugated sites, a fresh carboxy-methoxyl amine (CMO) solution (65mg/mL) was prepared in a0.8M NaOH.10μL of CMO solution was added to the reaction, and incubated for an hour at37℃. The reaction solution was centrifuged, and the supernatant was removed.4. Determination of optimal concentration of biotinylated antibody and donor beads coated with antibody using chessboard titration method.5. Screening of proper sample volume (10μL,20μL,50μL), incubation temperature(25℃,30℃,37℃), and incubation time(5min,15min,30min).6. Antibody screening according to identify ability of adr and ay subtype antigen.7. Determination of reference value:504healthy human serum samples were assayed by the proposed method. The statistical software is used for the analysis of distribution of data.8. Evaluation of assay performance8.1Sensitivity:The zero calibration was detected for10times repeatedly, and the mean signal and the standard deviation were calculated. The signal value, which was obtained by the mean signal value of zero calibrations plus double standard deviation, was substituted into the standard curve equation to acquire the corresponding concentration.8.2Accuracy:Serial dilutions of the WHO international reference materials were determinated. 8.3Linearity:A linear standard curve was obtained on a log-log plot with six concentrations. The correlation coefficient of the dose-response curve was determinated.8.4HOOK effect:HBsAg was added into healthy human serum to formulate into a series of solutions of different concentrations. A linear dose-response curve was obtained on a log-log plot of the series of concentrations.8.5Precision:The control serum was measured10times repeatedly. Each mean concentration and standard deviation were achieved. Thus, the coefficients of variation were calculated.8.6Specificity:A certain amount of interference substances were detected.8.7Coincidence rate of positive and negative reference materials:Reference materials of national institutes for food and drug control were determinated.8.8Interference:The interferent effect of hemolysis, lipemia, and bilirubinemia was assessed by adding hemoglobin, bilirubin and triglyceride to the serum samples of the serum sample.8.9Stability:Reagent performance was evaluated after stored at37℃for7days.8.10Clinical trial:Clinical study was designed as a blinded, controlled trial test, finished in three hospitals. A similar chemiluminescence reagent produced by Beyond Diagnostics Inc. was used as a control reagent. Statistical analysis was performed, including positive and negative coincidence rate, total coincidence rate, adjusting consistency, correlation coefficient index,etc.Ⅱ. Synthesis of photosensitive compounds, preparation of photosensitive microspheres coated with streptavidin.1. Tetra-tert-butyl-di-(tri-n-hexylsilyl)-silicon-phthalocyanine was synthesized and characterized with mass spectrum and ultraviolet spectra. 2. Photosensitive compound was dropped into the polystyrene microspheres.3. Photosensitive microspheres were coated with streptavidin covalently.4. Home-made donor beads were compared with PerkinElmer’s donor beads.Ⅲ. Development of Human immunodeficiency virus antibody TRFIA reagent using double antigen sandwich method.1.Healthy human serum was used as negative control, and normal human serum diluted Goat anti of HIV-1diluted with healthy human serum was used as positive control.2. H67antigen was diluted with coating buffer and coated into microplate. The plates were incubated at4℃overnight, closed, vacuum pumped, and stored at-20℃. Compare the performance of antigen handled with and without β-mercaptoethanol.3. Preparation of antigen labelled with Eu+:Purified antigen was mixed with europium labeling reagent at a mass ratio of2:1, incubated at4℃overnight, and purified over gel chromatography column at the next day. The labelled antigen was diluted with dilution buffer properly. The europium protective solution was added, and then the labelled antigen solution was filtered and lyophilized.4. Determination of optimal concentration of coating antigen and labelled antigen using chessboard titration method.5. Determination of reference value:409healthy human serum samples were assayed by the proposed method. The statistical software is used for the analysis of distribution of data.6. Evaluation of assay performance6.1Reference materials of national institutes for food and drug control were determinated, the coincidence rate of positive and negative reference materials, sensitivity, precision were assayed.6.2Interference analysis:The interferent effect of hemolysis, lipemia, and bilirubinemia was assessed by adding hemoglobin, bilirubin and triglyceride to the serum samples of the serum sample.6.3Specific analysis:Samples of potential cross-reactants were tested with home-made kit and control kit. The cross-reactivity was evaluated.6.4Comparison of serum and plasma for detection:HIV specimens were processed to serum, sodium citrate plasma, EDTA plasma samples and assayed to determine the difference.6.5Stability:Reagent performance was evaluated after stored at37℃for7days.6.6Clinical trial:Clinical study was designed as a blinded, controlled trial test which performed in three hospitals. HIV antibody enzyme-linked immunoassay kit produced by Bio-Merieux Inc. was used as a control reagent. Statistical analysis was performed, including positive and negative coincidence rate, total coincidence rate, adjusting consistency, correlation coefficient index, etc.Results:Ⅰ. Development of hepatitis B virus surface antigen AlphaLISA reagent using double antibody sandwich method.1. Considering the background signal values and signal to noise ratio factor, choose1:100as the optimal dilution of donor beads coated with antibody, choose1:200as the optimal dilution of biotinylated antibody.2.20μL was optimal sample volume,37℃was optimal incubation temperature,15min was optimal incubation time. 3. B028showed highest binding efficiency to adr subtype. S015showed highest binding efficiency to ay subtype. The combination of these two antibodies showed good binding efficiency to both subtypes.4.504healthy human serums were assayed. The average (X) was0.0049IU/mL; the standard deviation (SD) was0.00578IU/mL; the average plus double standard deviation was equal to0.0163IU/mL.99.40%samples had concentration value below0.04IU/mL. So that the HBsAg concentration cut-off was set at0.04IU/mL.5. Performance of HBsAg-AlphaLISA kit:5.1The sensitivity was0.01IU/mL. The adr0.1IU/mL, adw0.1IU/mL and ay0.2IU/mL of national reference material were all tested positive.5.2The ratio of measured concentration and expected concentration of WHO international reference materials was between97%-103%. Serial dilutions of a high concentration sample of3874IU/mL were analyzed, the coefficient of recovery was between96.4%-111.1%.5.3The correlation coefficient of the dose-response curve was0.9994within the linear range.5.4No HOOK effect was found in samples with concentration below5000IU/mL However, in higher concentration samples, lower signals were found.5.5The coefficient of variation was=6.04%.5.6Meaured HBsAg concentrations of HBeAg(160PEI U/mL) and HBcAg(150ng/mL) were both not greater than0.04IU/mL.5.7The coincidence rate of positive and negative national reference materials were20/20and3/3separately.5.8No interferences were detected from triglycerides (5%), bilirubin (0.32mg/mL) and hemoglobin (3mg/mL). However, Interference was found in samples with hemoglobin concentration higher than6mg/mL. So severe hemolytic sample should not be tested with this HBsAg AlphaLISA kit.5.9The reagent showed good stability proved by performance evaluation after stored7days at37℃.6. Clinical trial results:1067cases of specimens, including606positive cases, were detected in home-made reagent and control kit; the positive coincidence rate was99.01%; the negative coincidence rate was99.34%; the total coincidence rate was99.2%; the correlation coefficient was0.921(p<0.01). The clinical trial result showed that the home-made kit was reliable, accurate, safe, simple, stable, with a high clinical value.Ⅱ. Synthesis of photosensitive compounds, preparation of photosensitive microspheres coated with streptavidin.1.10.3g of dark blue solid was obtained with60.2%yield. Mass spectrometry was deteminated after the final product was dissolved in methanol, the AV=3, NL:2.26E7, RT:0.72-0.77. m/z:1364.36(M+H),1063.93(M-OSi (hexyl)3).UV analysis of the final product in methanol solution:λmax=674nm, A=0.259, molar absorptivity:[a]18methanol=1.6E5(160460(λmax=674nm). UV analysis of the final product in toluene solution:λmax=676nm, A=0.199, molar absorptivity:[a]18toluene=3.6E5(360687)(Xmax=676nm). Mass spectrometry and ultraviolet spectroscopy results were consistent with those reported.2. The evaluation of home-made donor beads:The diameter of microspheres was241.4nm. The beads produce fluorescence value of approximately50%compared with the PerkinElmer’s in a HBsAg AlphaLISA assay. Ⅲ. Development of Human immunodeficiency virus antibody TRFIA reagent using double antigen sandwich method.1. The plate coated with antigen dealt with β-mercaptoethanol buffer showed higher signal to noise ratio than the plate coated with unhandled antigen.2. Considering the background signal values and signal to noise ratio factor, choose0.5μg/mL as the optimal coating concentration, and choose1:300as the optimal dilution of labelled antigen.3. Reference range:409healthy human serums were assayed. The ratio of Anti-HIV signal to negative control was calculated. The average ratio(X) was1.06; the standard deviation (SD) was0.21; the average plus double standard deviation was equal to1.48.99.0%samples had a ratio below2.1. So that the cut-off was set at2.1×fluoresence of negative control.4. Performance of the reagent:4.1National reference materials were tested in Anti-HIV TRFIA diagnostic kit. The coincidence rate of negative and positive national reference materials were18/20and20/20separately. P11and p12were both tested strong positive, the fluorescence of P12was higher than P12. CV%=4.3%. S2-S6were all tested positive, and S1was tested negative.4.2No interferences were detected from triglycerides(5%), bilirubin (0.32mg/mL) and hemoglobin (18mg/mL).4.3Each10cases of HBsAg positive, Anti-HCV positive, Anti-TP positive, rheumatoid factor positive and antinuclear antibody positive samples were all tested Anti-HIV negative.4.4The result of serum samples and plasma samples were consistent both in home-made reagent and control reagent. 4.5The reagent showed good stability proved by performance evaluation after stored7days at37℃.5. Clinical trial results:1016cases of specimens were detected in home-made reagent and control kit; the sensitivity was100%; the specificity was98.9%; the accuracy was99.4%. The clinical result showed that the home-made kit was reliable, accurate, safe, simple, stable, with a high clinical value.Conclusion:These results demonstrated that the HBsAg AlphaLISA reagent and Anti-HIV TRFIA reagent developed in this study had good performance, including accuracy, sensitivity, precision, specificity, etc., which could meet the requirements of clinical application. So these reagents are expected to replace ELISA kits or expensive imported kits. The donor beads coated with streptavidin showed similar performance with the imported products, and are expected to be used in production by further optimization. |
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