| Background and PurposeHepatocellular carcinoma(HCC),the fifth most common malignancy in humans,accounts for 6%of all cancers worldwide,with an estimated half million new cases diagnosed globally per-year,it is the second most common malignancy in China,and currently,imaging techniques are important for the detection of HCC.However,the accuracy of imaging techniques such as computed tomography scanning,ultrasonography or magnetic resonance imaging is relatively insufficient for the diagnosis of small lesions,or dysplastic and cirrfhotic nodules.Therefore,suitable biochemical markers to differentiate HCC fare urgently needed.Currently,?-fetoprotein(AFP)is the most widely used tumor marker for the diagnosis and detection of HCC.However,AFP is significantly increased in the serum of patients with chronic liver disease,15%-58%of patients with chronic hepatitis,liver repair after injury or cirrhosis,11%-47%with cirrhosis.Therefore,specific tumor markers are required for early detection of tumors and prognosis monitoring based on translational medicine.Glypican-3(GPC3)is a member of the glypican family of heparan-sulfate proteoglycans,which are linked to the cell surface through a glycosyl phosphatidyl inositol anchor.The expression levels of GPC3 are high in some embryonic tissues,such as the liver.However,it is absent in the corresponding normal adult tissue.Moreover,GPC3 is expressed in a large proportion of HCC patients.Several studies indicated that GPC3 protein levels increased in the serum of patients with HCC,and was lower in the serum of healthy humans and patients with benign liver diseases.These studies suggest that GPC3 is highly expressed in HCC patients,but not in adjacent normal liver or benign liver lesions.However,there are no specific,economical GPC3 kit can use in the clinic.This is a critical issue for GPC3 is urgently needed.Chemiluminescence immunoassay(CLIA)is a technology combined with highly sensitive of chemiluminescence and high specificity of immune response,for a variety of antigens,haptens,antibodies,hormones,enzymes,fatty acids,vitamins and drugs detection and analysis,its a new immunoassay technology following the radioimmunoassay,enzyme immunoassay,immunofluorescence analysis,and time-resolved fluorescence immunoassay.Which can be used for fast and quantitative detection.Compared with conventional immunoassay,the technique has the advantages of high sensitivity,Non-radioactive and teratogenic substances,low background,short reaction time,stable property,simple operation,easy automation,wide application range,low prices and other advantages.It has higher clinical application value compared with ELISA and RIA,which is widely used in the market for clinical detection.Therefore,we developed the Glypican-3(GPC3)quantitative diagnostic kit(CLIA)Method:?.Optimizing the production methods of Glypican-3(GPC3)quantitative diagnostic kit1.Used Two-step double-antibody sandwich developed GPC3 ChemiluminescenceImmunoassay reagent.2.Preparation of magnetic microspheres coated with antibodies:1.5 mg antibodywas added into ultrafiltration tube(50KD),and centrifuged for 5-6 min at 8000rpm.the antibody was washed five or six times using coating buffer repeatedly.The filtrate was collected from the centrifuge tube.120mg magnetic beads was added into reaction flask,and washed five times using coating buffer repeatedly in the magnetic rack,added activation buffer and suspended the magnetic microspheres 30 minutes.Washed five times,then,added the antibody into the magnetic beads and reaction at room temperature for 12 hours.Using blocking solution blocked it at room temperature for 4 hours.3.Preparation of acridinium ester labeled antibody:0.5 mg antibody was added into ultrafiltration tube(50KD),and centrifuged for 5-6 min at 8000 rpm.the antibody was washed five or six times using coating buffer repeatedly.Then,added 5ul acridinium ester solution and vibrated it at room temperature for 6 hours.Removed acridinium ester by using SephadexTM G-50 Medium.4.Prepare standards,kit calibrators and controls with GPC3 antigen.1)Standards:A:0 ng/ml;B:1.0ng/ml;C:2.5ng/ml;D:10.0ng/ml;E:50.0ng/ml;F:200.0ng/ml.2)Kit calibrators:Calibrator 1:1.0ng/ml,Calibrator 2:50.0ng/ml.3)Controls:C1:5.0ng/ml;C2:15.0ng/ml;C3:60.0ng/ml.5.Screening of sample volume(10?L,20?L,50?L),incubation temperature(25?,30?,37?).?.Evaluate the Performance of Glypican-3(GPC3)quantitative diagnostic kit1.Sensitivity:The zero calibration was detected for 20 times repeatedly,and the mean signal and the standard deviation were calculated.The sensitivity,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.2.Accuracy:GPC3 antigen diluted to the series of concentrations and as samples were measured by the three batch kits.The ratio of measured concentration and expected concentration was between 0.900 to 1.100.3.Linearity:the detection range is 1.0ng/ml?200.0ng/ml,a linear standard curve was obtained on a four parameters plot with six concentrations.The correlation coefficient of the dose-response curve was obtained.4.Repeatability:The low,medium and high controls were detected 20 times,obtained the mean signal and standard deviation,obtained the coefficient of variation according to the formula CV=SD/M × 100%.5.Control values:the low,medium and high controls detected 10 times,calculated the average value of 10 measurements.Detection range of low control in the 3.5?6.5ng/ml,the median 10.5?19.5ng/ml,the high 42.0?78ng/ml.6.The between-run precision:detected the median control using three batch of kits and repeat 10 times,obtained the mean signal and standard deviation from the 30 measurements,obtained the coefficient of variation according to the formula CV=SD/M x 100%.7.Specificity:The AFP,CEA,CA19-9,HBsAg,HBeAg diluted to 200.0ng/ml,detected them using the three batch of kits.8.Interference:Triglycerides,bilirubin,hemoglobin were added in GPC3 controls,detected them using the three batch of kits,calculated the mean.9.HOOK effect:GPC3 antigen diluted to the series of concentrations:Ong/ml,lng/ml,2.5ng/ml,10ng/ml,50ng/ml,200ng/ml,400ng/ml,600ng/ml,800ng/ml,1000ng/ml,1200ng/ml,1400ng/ml,1600ng/ml,1800ng/ml,2000ng/ml.detected them and a linear dose-response curve was obtained on a four parameters plot of the series of concentrations.10.Stability:The validity stability(2?8? refrigerator,0 month,3 months,6 months,9 months,12 months,14 months,16 months),Thermal stability(37 ?incubator 0 day,3 days,7 days),Corkage stability(open the bottles and placed in 2?8 ? 0 day,10 days,20 days,30 days,40 days,50 days),Transportation stability(Simulated the transport eondition 4 days).?.Evaluate the clinical application performance of the kit1.Determined the reference range:Detected the 362 healthy humans serum,the serum of 101 patients with hepatitis B and 100 patients with cirrhosis using GPC3 kit.Got the frequency distribution.Detected the 53 patients with hepatocellular carcinoma serum and then got the ROC curve,Combined the ROC curve and other literature data,ultimately determine the reference values of serum GPC3.2.Detected the pre-operative and post-operative hepatocellular carcinoma serum:Detected the 32 pre-operative and post-operative hepatocellular carcinoma serums using the GPC3 kit and AFP kit.Results:?.Optimizing the production methods of Glypican-3(GPC3)quantitative diagnostic kit1.Double antibody sandwich two-step method had high Luminous value and the linear correlation coefficient 0.9999.2.Magnetic microspheres coated by 12.5?g/mg concentration and vibrated 12h at room temperature,then,used blocking buffer 3#to sealed it at room temperature(18-25?)for 4h.Obtained the best results.3.Acridinium ester 10:1 and incubation at room temperature(18-25?)for 6h,saved with solution 2#.Low background and good linear correlation coefficient.4.80?L volume of sample had a high luminous level,a good linear correlation coefficient.37? was optimal incubation temperature.?.Evaluate the Performance of Glypican-3(GPC3)quantitative diagnostic kit1.The sensitivity was 0.1ng/ml.2.The ratio of measured concentration and expected concentration was 0.968 to 1.080.3.The correlation coefficient of the dose-response curve was 0.9995?0.9999 within the linear range.4.The coefficient of variation was 4.76%to 5.42%.5.Controls were within the allowable range.6.The between-run precision was 6.41%.7.AFP(200ng/ml),CEA(200ng/ml),CA19-9(200ng/ml),HBsAg(200ng/ml)and HBeAg(200ng/ml)were both not greater than 0.1ng/ml.8.No interferences were detected from triglycerides(5%),bilirubin(0.32mg/ml)and hemoglobin(18mg/ml).9.No HOOK effect was found in samples with concentration below 2000ng/ml.10.The reagent showed good stability proved by performance evaluation after stored 12 months at 2?8?,7 days at 37?,opened 30 days at 2?8? or Simulated transport conditions 4 days.?.Evaluate the clinical application performance of the kit1.Determined the reference range:When the concentration was 1.5578 ng/ml,contained 97.5%of the 362 healthy humans.When the concentration was 2.1035ng/ml,contained 97.5%of Hepatitis B and liver cirrhosis patients.The result of ROC curve showed that When GPC3 reference value was 2.2150 ng/ml,the sensitivity reached 35.8%and specificity was 99.1%.Thus,determined<2.2ng/ml was the reference range of GPC3 kit.2.The positive rate of GPC3 was 34.38%(11/32)in the pre-operative hepatocellular carcinoma serums,AFP was 43.75%(14/32).The positive rate of combined detection was 59.38%(19/32).GPC3 and AFP results showed a downward trend in the post-operative positive serum,rate of deeline were 27.65%and 17.50%,respectively.Conclusion:These results demonstrated that the GPC3(CLIA)reagent developed in this study had good performance,including sensitivity,linearity,accuracy,precision,specificity,etc.Which could meet the requirements of clinical application.It is expected to be used in production by further optimization. |
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