Development Of Human Serum Y-Box Binding Protein1Quantitative Assay Method And Exploration Of It As A Tumor Marker

Objective:1. Establishment of genetic engineering system to prepare recombinanthuman Y-box binding protein1(YB-1); and preparation of polyclonalantibody (pAb) and monoclonal antibody (mAb) against YB-1.2. Development of a chemiluminescence immunoassay (CLIA) methodfor quantifying serum YB-1.3. Quantitatifing serum YB-1with established CLIA, and investigatingthe clinical significance of serum YB-1as an aid-diagnostic biomarkerfor common malignancy tumors.Methods:1. YB-1coding sequence was obtained by RT-PCR using the total RNAof human renal cancer cell line786-0as template. The sequence wascloned into vector pGEX-6P-1and confirmed by double-enzymedigestion, and further identified by DNA sequencing.2. For prokaryotic expression of YB-1, pGEX/YB-1was transformed into E. coli to express fusion protein GST-YB1. GST-affinitychromatography and chromatographic column protease digestion wereapplied to purify the recombinant YB-1protein.3. PAb against human YB-1was raised by injecting a rabbitsubcutaneously four times at2-week intervals with2mg each time ofthe recombinant human YB-1. The IgG fraction of antiserum waspurified by protein A sepharose column chromatography. Anti-YB-1mAbs used in CLIA of human sera YB-1were prepared by hybridomatechnique; then B-lymphocytes epitope prediction were applied toidentify the epitopes recognition by YB-1mAbs.4. The prepared pAb and mAb against YB-1were used to establish CLIAfor quantifying serum YB-1. Methodology evaluation was carried outto analysis the performance of CLIA.5. The concentrations of serum YB-1in patients with malignant cancer(n=157), and benign tumor (n=30) and healthy individuals (n=100) ascontrol groups were determined by CLIA; and preliminary evaluationof clinical significance of serum YB-1as an aid-diagnostic tumormarker was performed.6. Receiver operating characteristics (ROC) was used to compare thediagnostic performance of serum YB-1and alpha fetoprotein (AFP) fordiogmosis of hepatocellular carcinoma (HCC). Results:1. The vector pGEX/YB-1was successfully constructed and identified.The purified YB-1protein was prepared by means of prokaryoticexpression, GST-affinity chromatography and chromatographic columnprotease digestion.2. YB-1pAb was obtained by immunizing rabbits.2hybridoma cell lines(1-D9,3-E8) stably secreting mAb against YB-1were obtained, thesemAbs could specifically recognize exogenous and endogenous YB-1.3. The recognized epitope of the2mAbs might harbor in134-160aa and266-303aa of YB-1protein sequence.4. CLIA was established based on double-antibody sandwich. Thecalibration curve of the CLIA had a coefficient of linear correlation r2>0.99(slope0.0702, intercept5.8112). The minimum detection limit was0.1μg/L; the linear range was from1.0to150.0μg/L; the intra-andinter-assay CV values were≤4.8and≤12.5%at10.0μg/L, respectively.The average recovery was100.6%(93.9%~109.0%). The total time ofthe assay was around90min.5. With CLIA assay, the mean (SD) concentration of YB-1in100healthyserum samples was10.79±2.39μg/L; in30patients with benign tumorwas11.62±2.97μg/L; in157cancer patients was18.69±6.11μg/L,which was significantly higher than the values of healthy and benign tumor groups (P <0.0001).6. ROC analysis demonstrated that the area under a ROC curve (AUC) forthe YB-1curve was0.896(P <0.0001). Serum YB-1had a sensitivityof80%(95%CI73%-86%), a specificity of81%(95%CI73%-87%)at the cut-off value of13.45μg/L for diagnosis cancer.7. For diagnosis of HCC, the AUC for the YB-1curve was0.901(P <0.0001). Serum YB-1had a sensitivity of84%, a specificity of77%atthe cut-off value of14.69μg/L, while the AUC for the AFP curve was0.912(P <0.0001). AFP had a sensitivity of78%, a specificity of84%at the cut-off value of425.0μg/L.Conclusions:1. The recombinant YB-1protein and its pAbs and mAbs are successfullyprepared, and the recognized epitope of the two mAbs (1-D9,3-E8) isconfirmed.2. A high-performance double antibody sandwich CLIA for thequantitative measurement of serum YB-1with the combination ofMAbs1-D9and Anti-YB-1PAbs has been developed.3. Serum concentrations of YB-1might be used as a biomarker ofaided-diagnosis of common malignant tumors.4. The diagnostic performance of serum YB-1is close to AFP for diagnosis of HCC.5. Our primary exploratory study has suggested that quantifying serumYB-1, as an easily accessible circulating biomarker, could offer usefulaid-diagnostic information for patients with cancer; and this study laysfoundations for clinical application of the established CLIA in tumoraid-diagnosis and further evaluation of cancer prognosis.