| Objective:In 2020,there were 590,000 new thyroid cancer cases worldwide,ranking ninth in the global incidence of new cancers.95%of thyroid cancers has low malignancy,but the rate of early local lymph node metastasis in thyroid cancer is as high as 40%-60%.Rapid intraoperative diagnosis of lymph node metastasis is very important for accurate operation scope,complete removal of tumor and postoperative treatment.At present,the clinical detection methods of lymph node cancer cell metastasis are intraoperative frozen section and postoperative histopathological examination.Due to the high false negative rate of intraoperative frozen section,the limited number of test samples and the long time-consuming of postoperative histopathology,the surgical efficiency of thyroid cancer is greatly affected,Therefore,there is an urgent need for a method to objectively detect the metastasis of thyroid lymph node cancer cells during operation.Methods:Colloidal gold immunochromatography detection technology is a new solid phase chromatography technology that combines a variety of detection methods such as nanotechnology,colloidal gold labeling,immune response and chromatography analysis.It has lots of advantages such as rapid detection,low cost,high sensitivity and strong specificity,making it widely used in point-of-care testing.This project is mainly committed to establishing a detection method for rapid and objective judgment of lymph node metastasis of thyroid cancer based on colloidal gold immunochromatography technology,which is mainly completed through the following aspects:1.Preparation of immunochromatographic strip（ICS）for detection of Cyfra21-1content in lymph nodes:In this study,the preparation and optimization of ICS were described in terms of preparation conditions of colloidal gold particles,optimal PH value of colloidal gold particles and antibody coupling and minimum antibody dosage,colloidal gold release rate,screening and pairing of Cyfra21-1 antibody and other aspects.2.Detection and optimization of the ICS:By the gradient dilution of Cyfra21-1recombinant protein,the gray integration of T line and C line at different concentrations was read by chromatographic reading instrument and converted into T/C ratio,then the standard curve of the ICS was draw,the ICS detection threshold of low value was tested by Probit model analysis;Epithelial cell markers such as CK8,CK18,EMA and ESA were detected by ICS to determine the specificity of ICS.ICS were stored at room temperature for half a year to determine the detection efficiency.3.Application of the ICS in clinical samples:Clinical samples were collected for post-processing,then the ICS was used to detect lymph nodes,and it’s result was compared with postoperative histopathology.The expression of Cyfra21-1 in the above lymph nodes was measured by immunohistochemistry and compared with the results of pathology and ICS.Result:1.The synthesized colloidal gold particles have regular morphology,with an average diameter of 33.50 nm.The optimal p H of colloidal gold binding CYFRA21-1antibody was 7.8 and the optimal antibody concentration is 2μg/ml.The colloidal gold particles successfully bind the antibody.2.The detection threshold of the ICS developed this time is 0.55-1.14 ng/ml,and the standard curve equation of the ICS is Y=0.003708×X+0.1101;R~2=0.9800;The fastest reaction time of the ICS in detecting lymph nodes is less than 12min,and it can be stored at room temperature for half a year;3.In this study,a total of 40 lymph nodes were detected by the ICS.Compared with the postoperative histopathology,the sensitivity and specificity of the ICS were100%and 95%,respectively.Compared with the immunohistochemical results,the sensitivity and specificity of the ICS were 100%.Conclusion:The thyroid cancer lymph node detection method established in this study is portable,easy to operate,inexpensive,fast,objective,high sensitivity and good specificity,suitable for the detection of large-scale lymph node samples,which can reduce the anesthesia time of patients,improve the efficiency of thyroid cancer surgery,reduce the burden of social diseases,and have a wide range of clinical application prospects. |
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