Preparation And Identification Of The Monoclonal Antibody Against Breast Tumor Marker CA15-3and Its Clinical Application

My breast cancer has become the main killer of the threat to women’s health in China. The key to reduce the incidence of breast tumors and mortality is early diagnosis, early examination, early intervention.The most convenient and fast way is to detect the serum markers of breast cancer. CA15-3levels in the serum of breast cancer patients are extremely higher than benign tissue, and positively correlated with the histological grade, tumor size and axillary lymph node metastasis. Breast cancer is a high incidence of malignant tumors whose survival period is longer. It has instructional clinical value in treatments that postoperative monitoring CA15-3level is to facilitate early detection of cancer recurrence and metastasis.At present, CA15-3as serum markers of breast cancer is the most commonly used in vitro rapid diagnostic applications.In this study, I discussed the preparation and application of high sensitivity monoclonal antibody against CA15-3, which I research on the purity, titer testing, subtype identification and HRP-labeled modification of. Meanwhile, I made a research on the double antibody sandwich detection of CA15-3system, what was the detection limits, precision and specificity, and preliminary clinical application. The processes of my research are showed as below.1.The monoclonal antibody against CA15-3was preparation. Balb/C mice were immunized by CA15-3antigen, and detected the serum of the third immunization mice. Using classic hybridoma technology, I fused with myeloma cell SP2/0in PEG solution. After a large scale screening and hybridoma subclones, I have selected5hybridoma which are strong positive signal, high affinity, specificity and secretion, and named#3-1-3,#5-2-2,#11-2-2,#12-1-3and#16-1-3mAbs.2.The obtained five monoclonal antibodies were researched on the functional analysis. The characteristics of mAbs are:(1) antibodies titer:10-7～10-9(g/mL);(2) purity:>95%;(3) light chains:the same κ chain, heavy chain:#3-1-3mAb for IgG2a,#5-2-2mAb and#12-1-3mAb for IgG2b,#11-2-2mAb and#16-1-3mAb for IgG3. I obtained2antibodies against different epitopes with#16-1-3mAb by competitive ELISA, respectively#11-2-2mAb,#12-1-3mAb.3.CA15-3double-antibody sandwich detection system was used in clinical trial. CA15-3double-antibody sandwich detection system was respectively established with#11-2-2mAb and#16-1-3mAb-HRP,#12-1-3and#16-1-3mAb-HRP. The linear correlation coefficient of#11-2-2mAb and#16-1-3mAb-HRP system was0.9768<0.99, which had the effect of the HD-HOOK.The linear correlation coefficient of#11-2-2mAb and#16-1-3mAb-HRP system was0.9928>0.99. The titer of#11-2-2mAb was1.0×10-7(g/mL) and#12-1-3mAb was5.4×10-8(g/mL).Therefore, I selected the double antibody sandwich system of#12-1-3and#16-1-3mAb-HRP. Its sensitivity was0.59U/mL and the precision was about6%, which was not cross-react with the tumor marker CA19-9, CA50, CA72-4.1randomly selected150serum samples from breast cancer patients and other cancers and50serum samples from healthy individuals in clinical trial. The results which were contrasted with the hospital test results showed that:KAPPA=0.879, correlation coefficient r=0.965, P=0.38in t-test. CA15-3double-antibody sandwich detection system which was established in this experiment had a high level of consistency and equivalence with control trial from Roche.