| Part Ⅰ Construction and Performance Evaluation of RBP4 Protein KitObjective:To establish a detection method for serum RBP4 protein ELISA kit,and to evaluate its performance based on the standards of"In Vitro Diagnostic Reagent Analysis Performance Evaluation Guidelines",constructing a serum RBP4ELISA test kit that meets with the criteria of in vitro diagnostic kits in order to provide auxiliary testing methods for clinical diagnosis and population epidemiological screening.Methods:1.Discussing the optimization of reaction conditions for the detection method of RBP4 combined ELISA kit;taking the maximum of P/N value as the standard to determine the coating temperature and time of the optimal capture antibody（37℃2 h,25℃overnight,4℃overnight）.Using the checkerboard square titration method,optimize the concentration of anti-RBP4 capture antibody and anti-RBP4 detection antibody,according to P value closed to 1,N value closed to 0.2,and standard with the largest P/N value determined its optimal working concentration.2.Using the maximum P/N value as the standard to determine the optimal detection antibody incubation time（1 h,1.5 h,2 h）.Using the checkerboard matrix titration method,the maximum P/N value was used as the standard to determine the optimization of HRP working concentration and time;and the maximum P/N value was used to judge and optimize the TMB substrate coloring time.3.According to the optimized RBP4 combination kit,determining the standard curve according to whether the standard curve had"hook effect"and goodness-of-fit.4.The performance of the established RBP4 protein ELISA kit was evaluated in accordance with the"Guidelines for Performance Evaluation of In Vitro Diagnostic Reagents".The sensitivity evaluation was obtained by finding their（X+2S）.Precision evaluation:the intra-batch variation coefficient and the inter-batch variation coefficient were obtained.5.Accuracy assessment:by adding different concentrations of the standard samples into the sample to be tested,prepared a recovered sample,and calculated its recovery rate.Interfering substance evaluation:by adding different concentrations of hemoglobin and vitamin C interfering substances to the sample to be tested,the interference of the sample test was evaluated.Stability assessment:Judging by comparing the newly prepared kit and the consistency between the two groups at the end of the stability period（matching t test method）.Finally,the serum RBP4 levels of 22 normal and liver cancer patients were detected.Results:1.Selecting the 150 pg/m L RBP4 protein standard with an OD450nm value of 1.06 as the standard positive standard P,and useing the diluent as the standard negative standard N.2.The optimal conditions for the capture antibody coating were the overnight coating conditions at 4℃.The reaction concentrations of capture antibody and detection antibody were 2μg/m L and 0.25μg/m L,respectively.3.The detection antibody incubation time was 1 h.According to the checkerboard method to determine the HRP working concentration and time,the dilution of HRP labeled streptavidin was 1:150,the incubation time was 20 min.The substrate coloring solution incubation time was 15 min.Based on the above optimized conditions,the detection range of the final standard curve was 1500pg/m L～23.44 pg/m L,goodness of fit>0.999.4.Sensitivity evaluation:The sensitivity of RBP4 ELISA kit was 16.58pg/m L.Precision evaluation:The intra-batch variation coefficient was between1.41%and 5.42%,and the inter-batch variation coefficient was between 2.45%and 5.08%.5.Accuracy assessment:The results showed that the average recovery rate was between 99.34%and 100.32%,and the systematic error of the ratio was between 0.32%and 0.66%,both of which were not greater than 5%.Interference evaluation:the interference effect of interferon vitamin C was in the range of 0.99%～3.50%,the interference effect of hemoglobin was in the range of-4.27%～-1.53%,and the interference effect of two interfering substances were both≤±10%.6.Stability:The results showed that the consistency between the two groups was good（P>0.05）,no difference in stability.The levels of serum RBP4in normal controls and liver cancer patients were 43.42（37.72,53.16）μg/m L,and 23.05（18.47,41.19）μg/m L,respectively（P<0.05）.The serum RBP4 level in patients with liver cancer were significantly reduced（p<0.05）.Conclusion:1.In this study,the serum RBP4 protein ELISA detection kit was successfully established.2.The constructed serum RBP4 protein ELISA kit had high sensitivity,high precision,high accuracy,strong anti-interference,and good stability.All of the indicators meet with the requirements of applying in vitro diagnostic kits.3.The constructed serum RBP4 protein ELISA kit could be used for the detection of serum RBP4 protein level.Part Ⅱ RBP4 Protein Expression and Monoclonal Antibody PreparationObjective:To express RBP4 recombinant protein through E.coli expression system,optimize the condition of protein expression and purify RBP4 protein.RBP4recombinant protein was used as immunogen to prepare RBP4 monoclonal antibody,and its affinity and specificity were identified.Methods:1.RT-PCR amplified RBP4 gene fragment.After double enzyme digestion of RBP4 gene and p ET30a plasmid,connected and constructed the p ET30a-RBP4 recombinant vector.Finally,the bacterial clones showed positive from PCR and double enzyme digestion were selected and sent to sequencing to verify p ET30a-RBP4 vector.2.Useing E.coli BL21 to induce protein expression and analyze the protein expression form.Inducing at different induction temperatures for 4 h to determine the optimal induction temperature.Adding IPTG inducers with different concentrations corresponding to the optimal induction temperature and determine the optimal IPTG concentration.3.Using different imidazole concentration elution to determine the optimal concentration of imidazole for protein purification by Ni column.Using Ni agarose gel chromatography for the preliminary purification,combined with Kcl negative staining and gum recovery method,so high-purity RBP4 recombinant protein was obtained.By Western blot,identifying RBP4 recombinant protein.4.Immunize BALB/c mice with high-purity RBP4 recombinant protein as the immunogen,and detected the serum titers of the mice by indirect ELISA.When the serum titers of the mice was greater than 1:10000,performing cell fusion.Positive hybridoma cells were selected by indirect ELISA,and positive hybridoma cells were subcloned by limiting dilution to obtain monoclonal positive hybridoma cells.5.A large amount of RBP4 monoclonal antibody was prepared by ascites induction method.The ascites was purified by r Protein G column and RBP4monoclonal antibody was obtained.RBP4 monoclonal antibody subtype was detected by Sigma kit.Determination of affinity constant of monoclonal antibody by indirect ELISA.6.Indirect ELISA and Western blot for specific identification of RBP4monoclonal antibody.Results:1.A single band which matched with the length of expected RBP4 target gene appeared at about 600 bp.The sequencing results were consistent with the target gene sequence of RBP4.This indicated the successful construction of p ET30a-RBP4 recombinant vector.2.RBP4 recombinant protein existed as precipitated inclusion bodies.The expression of RBP4 recombinant protein in a large amount under the induction condition of 1.0 m M IPTG at 28℃3.The optimal imidazole concentration of Ni column was 150 m M.After secondary purification,RBP4 recombinant protein with a purity of more than96%could be obtained.Western blot results showed that there was a clear protein band at 29 k D,indicating that the recombinant protein expressed was RBP4 recombinant protein4.The serum titer of mice detected by indirect ELISA was 1:729000,which reached the standard of cell fusion.Through three subcloning screens,5 strains（1D2,1G2,2F4,10G2,and 10B10）of positive hybridoma cells capable of stably secreting RBP4 monoclonal antibodies were obtained,with ascites titers greater than 1:2187000.The subclass identification result of monoclonal antibodies showed that they were all Ig G subtypes.5.The affinity constant of RBP4 monoclonal antibody measured by indirect ELISA was 3.98×108 L/mol,which belonged to high affinity antibody.Specific identification showed that the prepared RBP4 monoclonal antibody was only compatible with the natural RBP4 protein expressed by liver cancer cells Hep3B and Huh7,recombinant RBP4 protein,RBP4 protein standard（R&D company）and specifically binded,but it had no cross reaction with SHBG-His recombinant protein,recombinant SAA4-His protein,recombinant NEK2-His protein,and BSA albumin.Conclusion:1.pET30a-RBP4 recombinant vector was successfully constructed.The RBP4 recombinant protein induced by E.coli existed in the form of inclusion bodies.After secondary purification,the high-purity RBP4 recombinant protein was obtained.2.High-purity RBP4 recombinant protein could obtain high-efficiency serum antibodies.The prepared RBP4 monoclonal antibody belonged to the Ig G subtype with a strong stability.Its high-efficiency,high affinity,and strong specificity could be applied to RBP4 ELISA detection reagents.The construction of the kit lays out the foundation for the independent research and development of the RBP4 ELISA test kit. |
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