Preparation Of The Specific Keratin Monoclonal Antibody Of Anti-Lingual Squamous Cell Carcinoma

Human oral cavity is the best example to illustrate differentiation dependent expression of cytokeratin (CK). CK expression is differentiation dependent and developmentally regulated. And this expression may be modified during malignant transformation. Oral mucosa carcinogenesis is a multi-factors process. Human oral precancer as well as cancer in the various sites of oral cavity show different levels of differentiation and keratinization and accordingly their CK expression varies. CKs are also being used in the differential diagnosis of various epithelial tumours, and have been proved very good markers in detection of local invasion and micro and distant metastasis of the tumours. Till to-date hundreds of MAb have been developed against various CK polypeptides. Hence, antibodies developed against CK tend to cross-react with many CK polypeptides as also other intermediate filament(IF) proteins. Thus, the need for the development of monospecific MAb arose. We need to analyze the change of cytokeratin proteinogram of oral squamous cell carcinoma. Extract, separate, purify and identify the specific keratin series profile. Then prepare and screen the monoclonal antibody of keratin. This is the fundamental work of basic investigation of cytokeratin.In view of the reasons above, the study was divided into four parts.Part â…  Cytokeratin proteinogram analysis of lingual well-differentiated squamous cell carcinomaObjective: To analyze cytokeratin proteinogram of lingual well-differentiated squamous cell carcinoma. Methods: Specimens of lingual well-differentiated squamous cell carcinoma and normal tongue tissue were investigated by immunohistochemistry, SDS PAGE and Western blotting. Results: CK18, 19 were detectable in lingual well-differentiated squamous cell carcinoma , differentiated cancerous cells. Conclusions: Aberrant expression of CK18 and 19 and non-expression of CK 10 was a common feature in oral squamous cell carcinoma. These observations suggest that tumours recapitulate common patterns of cytokeratin expression during oral carcinogenesis. The changes of cytokeratin proteinogram of lingual squamous cell carcinoma would be the important oncobiological characteristic figure.. Part â…¡ Abstraction and purification of lingual carcinoma keratin series profile Objective: To extract the keratin series from lingual carcinoma tissues. And using keratins as antigen to prepare the monoclonal antibody. Methods: After have definited the expressinglocation of cytokeratin proteinogram in cancer tissue, a great quantity of keratin was extracted by electrophoresis method and verified by SDS-PAGE. Results: A great quantity of keratin was successfully extracted and molecular weight(MW) is consistent with keratins. Conclusions: Electrophoresis method can provide sufficient antigen for preparation of monoclonal antibody. Part m Preparation and identification of keratin monoclonal antibody of anti-lingual squamous cell carcinomaObjective: Using lingual squamous cell carcinoma keratins as antigen to prepare the monoclonal antibody. And to carry out screening and identification. Methods: Immunize BALB/c mice with purified antigen. By mouse B leukomonocyte hybridoma technique, five cloned strain had been established, which could secrete keratin monoclonal antibody of anti-lingual squamous cell carcinoma stably. The positive cloned strain was screened by ELISA. And detected the antibody secretion and subcloned. Then identified subgroup and specificity of the antibody. Results: Cell confluented for 384 holes total and confluence rate was 95%. Obtained five hybridoma cell strains which could secrete keratin monoclonal antibody of anti-lingual squamous cell carcinoma stably. Cloning 3 times by ending dilution method. Positive ratio was 100%. Named from Lckl to Lck5. IgG subgroup determined category of IgGiK chain. Specificity of Lck4 was highest. Conclusions: Set up 5 strains of monoclonal antibody. Lckl-5 would be used in pathological diagnosis and study of cytokeratin constituent as well as basic research of oral mucosa epithelium.Part IV Study and identification of keratin monoclonal antibody of anti-lingual squamous cell carcinoma by immunohistochemical stainingObjective: To evaluate and study the immunohistochemical location of Lck monoclonal antibody. Methods: 18 cases of oral squamous cell carcinoma specimens with lymphonode metastasis were pathologically chosen. The carcinoma tissue, peri-carcinoma tissue, metastasis lymphonode and negative lymphonode stained by S-P microwave immunohistochemical method. Results: Lckl, Lck3, Lck4 and Lck5 could express partly in carcinoma tissue and metastasis lymphonode. Positive cells were located in cancer cells cytoplasm. Specificity and sensitivity of Lck4 was highest. Conclusions: Specificity and sensitivity of Lck4 was highest in 5 strains of monoclonal antibody. Lck4 would be an optimal tumor specific antigen and antibody to be used in the future.