Preparation, Characterization And Application Of AFP And Hp Monoclonal Antibody

Primary liver cancer (PLC) is one of the most common malignant tumors in our country. Due to the difficulty of early diagnosis, liver cancer mortality is higher. Serological markers such as AFP are used in clinical practice of the prognosis and diagnosis of liver cancer. AFP is one of the earliest found serological markers. It can be detected when liver tissue shows biochemical indicator abnormality or liver cell cancerization. Therefor, AFP is the best candidate to detect liver cancer in blood check up and clinical diagnosis. Due to its lack of specificity and sensitivity, Hp is further used to distinguish liver cancer and innocence liver disease. In future liver cancer detection items, the identification of both AFP and Hp at the same time will provide valuable information in early diagnosis and treatment of liver cancer.In this study, the preparation and application of high sensitivity monoclonal antibody was studied based on problems in liver disease detection in China. Meanwhile, AFP and Hp chemiluminescent immunoassay (CLIA) detection system was optimized. The significance and values of AFP and Hp in early diagnosis of liver cancer was discussed. The processes of my research are showed as below:1. The preparation of AFP and Hp monoclonal antibody. Two groups of Balb/C mice were immunized by AFP and Hp antigen respectively. The spleen cell were fused with myeloma cell SP2/0.14and16strains of positive hybridoma cells were screened out by high throughput screening, respectively. Monoclonal cell positive hole rate was100%using subcloning limited dilution. Finally, AFP group selected6strains secreting monoclonal antibody cell strain; Hp group selected7strains secreting monoclonal antibody cell strain, the corresponding monoclonal antibody were obtained by protein A affinity purification.2. Through titer detection and epitope analysis, for screening antibody by ELISA method, titers of two groups of antibody were up to10-9.#8-7-1,#5-1-1AFP groups were aimed for different epitope antibody.#Hp3-1-1and#6-2-2Hp groups were aimed for different epitope antibody. And ELISA double antibody sandwich method was sensitive up to the nanogram level. Four antibodies were idenyified by the subtype identification kit. The results showed that4strains of antibody light chain type are k, except that the heavy chain type of#8-7-1is IgGl, the rest of the antibody heavy chain types are IgG2b.3. Using linear range and reproducibility as index, detecting system is optimized by the CLIA method, and the eventual AFP, Hp detection system were established. AFP group used pH9.0, K2HPO4solution system coating1.5g/mL AFP#8-7-1. Hp group used pH7.2phosphate buffer systems coating a concentration of2g/mL. Confined fluids of two detection system were0.15mol/L PBS,2%BSA,0.1%Proclin300, pH7.2. Dilutions of two detection system samples were1.5%NaCl,0.2%BSA, and pH7.2. Enzyme diluent of two detection system were0.1mol/L TRIS-HCl,10%calf serum,0.1%Proclin300,7.2pH. Response patterns were "1hour+1hour". 4. The detection system was assessed by drawing standard curves and detection quality control. In the research, AFP group showed a linear limit up to800ng/mL, Hp group showed a linear limit up to1000ng/mL, and both linear correlation coefficient are greater than0.99. Two groups of detection systems showed high accuracy, the relative error are less than7%and the minimum detection limit was0.85ng/mL. They showed no cross reaction with CEA, HCG. Comparing AFP detection system with labeling reagent detection,200cases of blood test results showed that two methods have good consistency and the correlation coefficient is more than0.95.5. A hundred cases of healthy human serum were detected by the CLIA method. The normal ranges of two groups’detection system were identified as0-10ng/mL and1000-1500μg/mL. On a random sample of100cases of patients with hepatocellular carcinoma,100cases of serum hepatitis with liver cirrhosis and50cases of normal human serum, AFP, Hp detection results showed the united detection sensitivity was90%and specificity was88%. Separate detection notably increased when compared to the AFP (80%,69%) or Hp (82%,85%). Results are more conducive to the early diagnosis of primary liver cancer.