Establishment Of A Quantitative Detection Method For Anti- TAAs And Evaluation Its Value In The Diagnosis Of HCC

Purpose and SignificanceAutoantibodies against tumor associated antigens (Anti-TAAs) are a group of biomarkers that have the potential to serve as early diagnostic biomarkers for hepatocellular carcinoma (HCC). It is of great significance to identify optimal anti-TAAs for the diagnosis of HCC. Although thousands of candidate TAAs were screened out by high-throughput methods, it is necessary to further verify and evaluate them with population sera. ELIS A is a very commonly used method in serological validation of anti-TAAs, However, it encounters a bottleneck to proceed the validation and evaluation of anti-TAAs, because the comparability are poor when results finished by classical ELISA from different people or different time. It was hard to establish effective and reliable quantitative ELISA to detect those candidate anti-TAAs identified recently from human sera.The purpose of of this study was to verify those potentially valuable candidate anti-TAAs identified from high-throughput screening methods, based on the quantitative measurement method established, and to further identify a mini-array with high value for the diagnosis of HCC, in combination with currently available anti-TAAs, and to promote clinical translation of anti-TAAs as serological biomarkers for HCC diagnosis.Methods1. Cloning, expression and purification of 18 candidate TAAs and validated TAAs.(1) Construction of recombinant plasmids for those no plasmids that can express the target proteins: Target genes were inserted into the expression plasmid pET-28a(+) respectively.(2) Construction of prokaryotic expression system for target proteins: The recombinant plasmids were transformed into E.coli (BL21) to construct expression systems for target proteins respectively. Then the induction concentration of IPTG was optimized, and the main expression form of each recombinant protein was checked by soluble analysis.(3) Expression and purification of target proteins:Thirteen Histidine-tagged proteins, including NPM1,14-3-3 (?), MDM2, p53, Koc, IMP1, AHSG, FTL, c-Myc, Survivin, RalA, HCC1.4 and p62, were purified by His-tag protein purification protocol, and five GST-tagged proteins, including p 16, Cyclin E, Cyclin B1, Cyclin D1 and p90, were purified by GST-tag protein purification protocol.(4) Bradford method was used to measure the concentration of purified proteins, and Western Blot was used to verify the purified proteins.2. Establishment of standardized quantitative ELISA for the detection of anti-TAAs.(1) Selection of those anti-TAAs to be developed quantitative ELISA:Classical ELISA was used to verify 10 candidate anti-TAAs to determine which one was probably of high diagnostic value. In combination with other 13 anti-TAAs validated in previous studies, to determine which anti-TAAs would be selected to develop quantitative detection technique.(2) Establishment of positive standard and negative standard for each anti-TAA:For each anti-TAA, Sera with high or low absorbance were screened out by classical ELISA and Western blot from a group of cancer sera and normal ones.(3) Optimization of the key reaction parameters by chessboard titration method for each anti-TAAs, respectively.(4) Establishment of quantitative ELISA for anti-TAAs measurement based on the optimization of parameters.1) According to the characteristics of IgG structure, and its specific binding capacity of Fc fragment with Staphylococal Protein A, the quantitative principle of IgG type autoantibodies detection was proposed.2) The saturated coating concentration of quantitative reference (human IgG) was measured, and the linear response range of standard curve for quantitating was assessed.3) Two protocols, Bradford method and direct ELISA, were tested to measure the actual amount of quantitative reference absorbed on the ELISA plate.4) The stability of the quantitative standard curve was assessed through multiple repetition.3. Evaluation of mutiple anti-TAAs in the diagnosis of HCC.(1) The concentration of each anti-TAAs were measured in sera from 97 HCC cases,83 liver cirrhosis cases,89 chronic hepatitis cases and 104 normal individuals by using quantitative ELISA established in this study.(2) Determination of the cut-off value for each anti-TAA.The cut-off value for each anti-TAA was determined through comparing the positive percentages of HCC, liver cirrhosis, chronic hepatitis and normal groups by three different methods, as were mean plus three times of the standard deviation, ROC curves and P95 percentile, respectively.(3) The diagnostic value of anti-TAAs array was evaluated for HCC, while chronic hepatitis and live cirrhosis were taken as controls.1) The optimal anti-TAAs array for the diagnosis of HCC was acquired by constituting various anti-TAAs with parallel strategy.2) Binary Logistic regression model was used to set predictive model for HCC with multiple anti-TAAs as independent variables, and the diagnosis value of the model was evaluated by ROC analysis.(4) The diagnostic value of anti-TAAs array was analyzed for AFP negative HCC patients.The positive rate was calculated by anti-TAAs array for AFP negative HCC patients, and the increased rate was calculated for HCC when combined anti-TAAs array with AFP.Results1. Prokaryotic expression systems of NPM1,14-3-3 (?) and MDM2 were successfully constructed. The expression and purification protocols for NPM1,14-3-3 (?), MDM2 and p90 were established, and 18 candidate TAAs or validated TAAs were purified successfully.2. A standardized quantitative measurement method was successfully established for detecting IgG type anti-TAAs.(1) Six kinds of candidate anti-TAAs, as were NMP1, MDM2,14-3-3 (?), p90, HCC1.4 and FH respectively, from 10 kinds of identified candidate anti-TAAs, might be valuable for the detection of HCC, and further validation was needed. While PBP, GLUD1, NDRG1 and PRDX1 were possible worthless for the detection of HCC. Combining multiple anti-TAAs validated in previous studies,18 kinds of indicators were selected to develop quantitative ELISA.(2) For 16 of the total 18 anti-TAAs, positive and negative standards were established for each anti-TAAs, and key reaction parameters was optimized for each anti-TAA in ELISA detection.(3) The linear response range of standard curve for quantitating was from 0～300 ng/ml; The actual amount of quantitative reference absorbed on the ELISA plate was acquired; the quantitative standard curve showed good repeatability.3. The evaluation results of 16 anti-TAAs in the diagnosis of HCC.(1) The percentile P95 of control group, including chronic hepatitis and liver cirrhosis, was the most appropriate method for defining cut-off value.(2) Anti-TAAs array constituted with parallel strategy showed a higher sensitivity: The sensitivity was increased to 82.5%, and specificity 79.7% when an anti-TAAs array including 14 indicators constituted with parallel strategy; 8 anti-TAAs were entered into the regression equation when combining anti-TAAs by logistic regression predictive model, the sensitivity of detection was increased to 70.8% at the specificity 73.8% when the cut-off value was determined by ROC analysis.(3) It was of great significance of anti-TAAs array with parallel combination, especially for AFP negative HCC patients:For 29 AFP negative HCC patients,26 cases (89.7%) was positive when detected by anti-TAAs array; for all the 87 HCC cases detected both anti-TAAs and AFP, the positive percentage was 96.6% (84/87) when combined AFP and anti-TAAs array.Conclusions1. A quantitative ELISA method was established for detecting all the IgG type anti-TAAs, and it can serve as a good tool for the verification and validation of candidate anti-TAAs in methodology.2. Combination of multiple anti-TAAs can increase the diagnostic value for HCC effectively, and it might be taken as an effective method to detect HCC.