Development And Evaluation Of Up-converting Phosphor Technology-based Lateral Flow Assay For Quantitative Detection Of Serum PIVKA-?

Objective:The test card for detecting serum Prothrombin induced by vitamin K absence or antagonist II?PIVKA-II?was developed in this study by combining Up-converting Phosphor Technology?UPT?with Immune lateral flow test?LF?.After the optimization of preparation of biological probes,the construction of test strip and the detection system of test card,the test card has the great potential to distinguish the cohorts of Hepatocellular carcinoma?HCC?from those of Liver benign lesion?LBL?and Healthy control?HC?.In addition,the detection and diagnostic performance of UPT-LF test card were evaluated to further verify the good performance for the detecting of serum PIVKA-II.Methods:1.Preparation of biological probesThe up-converted phosphor nanoparticles?UCPs?NaYF₄:Yb³⁺,Er³⁺were synthesized by thermal decomposition method,coated with Nano-SiO2 and functionalized by APES.The anti-PIVKA-II monoclonal antibodies were combined with the activated UCPs to form the biological probes under appropriate reaction conditions.2.Development of UPT-LF test cardThe nitrocellulose?NC?membrane,conjugated pad,sample pad and absorbent paper were pasted regularly on the laminated card in order of priority,then cut into test strip with 4 mm width,and packed into the special shell of test card.3.Optimization of UPT-LF test cardThe constructing conditions of probe,test strip and detection system of UPT-LF test card were optimized to improve the detection performance.4.Performance evaluation of UPT-LF test cardThe UPT-LF test card was used to evaluate the detection performance,including limit of detection,linearity,thermal stability and validity period,precision,recovery and interference,and so forth;The 228 cases in HCC group and 170 cases in LBL group and 100 cases in HC group were detected respectively.The 178 cases in the HCC group,60 cases in the LBL group,and 35 cases in HC group were simultaneously tested by UPT-LF assay and chemiluminescence immunoassay?CLEIA?,then the diagnostic performance and correlation of the two methods for HCC were analyzed.Results:1.The UCPs synthesized and modified in our study have a homogeneous crystal phase and dispersed uniformly in solution.The probes formed by binding of activated UCPs with labeled antibodies have favourable and stable luminous intensity.2.The sample was added to test card and after 15 minutes of immune reaction,which can be successfully scanned by the biosensor and the test results can be obtained.3.After the optimization of the test card,the ratio of T/C value?P/N?of positive sample?P?to negative sample?N?has been significantly improved compared with that of unoptimized.4.The limit of detection and linear range of UPT-LF test card for serum PIVKA-II were2.66 and 4.8-20000 ng/ml,respectively;The test card stored at room temperature for 60 days and 4?for 18 months within validity period has a lesser variability?CV%?.However,there was a slight variation with CV%was less than 15%after storage at 37?for 7 days;The CV%of the intra and inter assay was less than 10%;The recovery rate of serum samples with low,medium and high levels of PIVKA-II were 93.1%,95.6%and 99.2%,respectively;Strong specificity was observed in the detection of serum samples with 8 common interfering substances in HCC serum;Using 25.3 ng/ml as the cutoff of HCC diagnosis,The sensitivity,specificity and AUC were 71.5%,88.9%,0.85,respectively;A good linear correlation was observed between UPT-LF assay and CLEIA assay for the quantitative determination of serum PIVKA-II,and the correlation coefficient?R²?was 0.901.Conclusions:UPT-LF assay provides a reliable,rapid and convenient test for quantitative detection of serum PIVKA-II as well as diagnosis of HCC by a point of care testing way.