Establishment Of A Method For Detection Of Serum SCCAg Based On Chemiluminescence

Squamous cell carcinoma(epidermoid carcinoma)is a malignant tumor mostly occurrs in the lungs,skin,anus,neck,throat,nose and bladder,where covered with squamous epithelium tissues.Because of the high mortality,the squamous cell carcinoma is very harmful.At present,it is believed that the development and application of effective tumor markers for early diagnosis and disease monitoring is an effective measure to prevent and treat tumor diseases.Squamous cell carcinoma antigen was first found in cervical cancer tissues,including two homologous molecules SCCA1 and SCCA2,which two have a high degree of homology of amino acid and highly similar epitope characteristics,all belong to the family of serine protease inhibitors.Studies have shown that SCCAg is an important tumor marker,and has a close relationship with squamous cell carcinoma of multiple organs.The level of serum SCCAg can be used for clinical diagnosis,disease monitoring and prevention of cervical,head and neck cancer,non-small cell lung cancer and colorectal cancer,and so on.At present,the mainstream of clinical SCCAg detection reagents are mainly foreign tubular chemiluminescence reagents,the development of the SCCAg diagnostic method with independent intellectual property rights is of great significance to reduce the cost of medical testing.Therefore,this study carried out research on the establishment of SCCAg type tube serum chemiluminescence detection method,established the method of efficient soluble expression system of Escherichia coli expressing recombinant SCCAg antigen,prepared SCCAg specific monoclonal antibody,established SCCAg tube type chemiluminescence detection method and carried out performance evaluation.In this study,a simple and efficient method for the preparation of soluble recombinant SCCAg antigen based on Escherichia coli expression system was established.The SCCA1 and SCCA2 full-length genes were cloned into pGEX-6P-1 vector to construct recombinant expression plasmid pGEX-SCCA1 and pGEX-SCCA2,and the expression of antigen was carried out by E.coli ER2566.The results showed that the recombinant SCCA1 and SCCA2 antigen could be expressed in soluble form in the supernatant under different conditions.By optimizing the solubility and induction time of IPTG,the optimal conditions for inducing the expression of soluble SCCA1 and SCCA2 antigen were obtained.SCCA1 and SCCA2 antigen with high purity were obtained by affinity chromatography.In this study,we compared the activity of recombinant antigen with human SCCAg antigen by using the SCCAg detection reagent which is mainly used in clinic.The results showed that the SCCA1 antigen and SCCA2 antigen obtained in this study had the basic reactivity with human SCCAg antigen,which could be used in the follow-up study.In this study,SCCAg specific monoclonal antibodies were prepared and screened for the detection pairing of monoclonal antibodies against SCCAg.The purified recombinant SCCA1 and SCCA2 antigen were used to immunize BALB/c mice,and the serum titer after immunization was tested.The results showed that the recombinant SCCAg antigen obtained in this study had good immunogenicity.80 monoclonal antibodies against SCCAg antigen was prepared by indirect ELISA method.By pairing experiment,linear detection,sensitivity analysis,correlation detection,monoclonal antibodies pair 7F10/4H8-SAE was screened out.Based on this study,we established a SCCAg tube chemiluminescence detection method.In summary,this study established an effective expression system and method for purification of soluble SCCAg expression on Escherichia coli.The recombinant SCCAg antigen with high purity and activity were prepared.The monoclonal antibody paring,which can be used for detection of serum levels of SCCAg were prepared.The establishment of tube type chemical luminescence detection method with considerable performance provides important support for the basic research and application of SCCAg.