1.Alteration of CK8 Both in Its Expression Level and Its Glycan Parts Related to HCC Metastatic Ability
Zhi DAI ; Yinkun LIU ; Kun GUO ; Qiangling SUN ; Wenjing LU ; Pengyuan YANG
Progress in Biochemistry and Biophysics 2006;0(02):-
A glycomic method was used to screen the aberrantly ?1-6 fucosylated glycoproteins related to HCC metastasis and analyze the alteration of CK8 both in its expression level and its glycan parts associated with metastatic ability. Based on the approach, 2-DE coupled with lectin affinity blot, lectin affinity precipitation followed by MALDI-TOF-MS/MS, the lens culinaris agglutinin (LCA) affinity glycoprotein profiles from MHCC97-L and MHCC97-H cells, two higher metastatic HCC cell lines, were obtained, in which a differentially displayed protein spot was indicated when compared with Hep3B, in the region within 55~60 ku in molecular mass and 4~6 in isoelectric point. The identification result was CK8 by MALDI-TOF-MS/MS. To confirm the relation between increased core-fucosylation of CK8 and HCC metastasis, LCA affinity precipitation was used to extract the ?1-6 fucosylated glycoproteins, followed by Western blot. And it was found that CK8 was highly fucosylated in both MHCC97-L and MHCC97-H cells compared to Hep3B. Immunofluorescence analysis and Western blot were used to detect its intracellular localization and its protein expression levels, indicating that CK8 distributed in cytoplasm and increased protein expressions in MHCC97-L and MHCC97-H cell lines. And further lectin binding studies found that CK8 has a high affinity for Con A in both MHCC97-H and Hep3B cells, indicating that CK8 was a glycoprotein with high-mannose type N-glycans. But the amount of the lectin RCA-1 binding to CK8 was greater in MHCC97-H than Hep3B, suggesting that CK8 contained the increased terminal galactose residues ?-1, 4-linked to GlcNAc in MHCC97-H. All the results suggested that the increase of CK8 in its protein expression level, core-fucosylation and terminal gal ?1,4 GlcNAc disaccharides might be related to HCC metastatic ability.
2.Application of SELDI-TOF-Ms technology in research of molecular markers of lung cancer
Huifang SHA ; Jianding YE ; Qiangling SUN ; Xiaohua YANG ; Guoliang BAO ; Jiuxian FENG ; Leluo GONG
Journal of Shanghai Jiaotong University(Medical Science) 2009;29(10):1178-1181,1195
Objective To explore the changes of proteomic spectra from plasma of patients with lung cancer or benign lung diseases and health controls in order to establish a primary diagnosis model of lung cancer. Methods The proteomic spectra from plasma of 108 patients with lung cancer, 40 patients with benign lung diseases and 22 healthy individuals were analysed by surface-enhanced laser desorption/ionization time of flight mass spectrometry ( SELDI-TOF-MS). The best decision tree model was established by cluster analysis and principal component analysis. Then the model was blindly validated by the protein of 21 patients with lung benign diseases and 47 patients with stage I lung cancer. Results Twenty-three significantly differentially expressed protein peaks were successfully detected (P <0.001). Blinded validation suggested that the accuracy for diagnosing lung cancer was 72. 06%, the sensitivity and specificity were 72. 34% and 71.43%, respectively, and the positive predictive value and negative predictive value were 85. 0% and 78. 95%, respectively. Conclusion SELDI-TOF-MS protein chip technology provides a new tool for the early diagnosis of lung cancer.
3.Analysis of Differential Gel Electrophoresis of Paclitaxol Resistant and Sensitive Lung Adenocarcinoma Cells' Secretome
SUN QIANGLING ; YANG XIAOHUA ; LU JING ; XIE YINYIN ; CHU TIANQING ; SHA HUIFANG
Chinese Journal of Lung Cancer 2009;12(7):735-740
Background and objective Paclitaxol (PTX) resistance is one of main factors which affect the outcome of chemotherapy of lung adenocarcinoma. The aim of this study is to compare the secreted protein expression profiles between Paditaxol (PTX) resistant and sensitive lung adenocarcinoma cells by proteomic research method, so as to provide evidence of choosing individual chemotherapy drugs in clinical treatment. Methods Total secreted proteins extracted from a PTX sensitive cell line A549 and a PTX resistant cell line A549-Taxol were separated by fluorscent differential gel electrophoresis (DIGE). High quality 2-DE profiles were obtained and analyzed by Decyder 6.5 analysis software to screen differentially expressed protein spots. Those spots were identified by mass spectrometry. Results 2-DE patterns of lung adenocarcinoma cells with high-reso-lution and reproducibility were obtained. 76 significantly differentially expressed protein spots were screened, 19 proteins were identified by mass spectrometry. The identified proteins could be classified into different catogories: metabolic enzyme, extracel-lular matrix (ECM) degradation enzyme, cytokine, signal transducer, cell adhesion, and so on. Conclusion Multiple secreted proteins related to chemoresistance of A549-Taxol cells were identified in this study for the first time. The results presented here would provide dues to identify new serologic chemoresistant biomarkers of NSCLC.
4.Establishment of double antibody sandwich ELISA for detection of 2019-nCoV antigen
Yuanyuan QU ; Wei WU ; Qiangling YIN ; Chuan LI ; Jiandong LI ; Mifang LIANG ; Lina SUN ; Shiwen WANG
Chinese Journal of Experimental and Clinical Virology 2021;35(3):357-360
Objective:To explore an early, rapid and accurate detection method of severe acute respiratory syndrome coronavirus 2 (2019-nCoV) antigen and improve the detection rate of patients with 2019-nCoV.Methods:We detected the characteristics of monoclonal antibodies (mAbs) against 2019-nCoV generated previously and established a double antibody sandwich ELISA against S protein of the virus.Results:All 12 mAbs against 2019-nCoV screened in our previous study were able to recognize purified virion and S protein. They were good candidates for detecting antibody to 2019-nCoV antigen. A double antibody sandwich ELISA established in this study could rapidly and effectively detect 2019-nCoV S protein with high sensitivity. The detection limit of 2019-nCoV S protein was lower than 1 ng/ml.Conclusions:This method could specifically detect 2019-nCoV antigen, and provided a meaningful reference for early, sensitive and specific diagnosis of COVID-19 infection.
5.Establishment of 2019 novel coronavirus neutralizing antibody detection method based on quantum dotimmunochromatography
Qiangling YIN ; Hengqiang WANG ; Tingyou CHEN ; Yuanyuan QU ; Lina SUN ; Wei WU ; Tao HUANG ; Xiao XU ; Tingting TIAN ; Chuan LI ; Dexin LI ; Mifang LIANG ; Shiwen WANG
Chinese Journal of Experimental and Clinical Virology 2022;36(2):205-213
Objective:To establish a simple, rapid and low-cost 2019 novel coronavirus (2019-nCoV) neutralizing antibody detection method.Methods:The 2019-nCoV RBD specific immunoglobulin G (RBD-IgG) detection method was established based on the principle of quantum dot immunochromatography(QDs), and the detection was evaluated by using of sera from coronavirus disease 2019 (COVID-19) convalescent patients ( N = 97), vaccinated donors ( N = 82) and healthy donors ( N = 299). The suitability of fingertip blood was evaluated by matching blood samples with peripheral blood ( N=54). Results:The 2019-nCoV RBD-IgG detection method based on QDs was successfully established. The detection result of QDSs had strong correlation ( Spearman r > 0.73, P < 0.000 1) and good consistency ( Kappa=0.93, P < 0.01) with the result of micro-neutralization test(MNT). The sensitivity and specificity were 92% and 99%, respectively. There was high correlation ( Spearman r =0.932 6, P < 0.000 1) and no significant difference ( P=0.102 6) between result of fingertip blood and peripheral blood. Fingertip blood can be used as a surrogate sample for testing. Conclusions:The 2019-nCoV neutralizing antibody detection method established in this study can provide an immediate, efficient and low-cost method selection for the assessment of herd immunity status, and provide technical support for the herd immunity monitoring of 2019-nCoV vaccinated population and the prevention and control of the epidemic.