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Development and Application of A Double Monoclonal Antibody Sandwich ELISA for the Assay of Nodal |
WU Meng-ling, ZHOU Jia-wang, DU Jun |
Department of Microbial and Biochemical Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China |
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Abstract Background:Nodal is a member of the transforming growth factor beta (TGF-beta) super family, essential in maintaining the pluripotency of human embryonic stem cells (hESCs), which is not active in most adult tissues, its reexpression and signaling have been linked to multiple types of human cancer. Recent findings have revealed that Nodal is a critical regulator of melanoma growth, plasticity and tumorigenicity, and holds promise as a new tumor biomarker. Similar observations have been reported in multiple human cancers. To utilize Nodal as a useful biomarker for cancer detection, a double monoclonal antibody sandwich enzyme-linked immunosorbent assay (ELISA) may be a reliable, easy and cheap means. Methodology/principal findings:Recombinant Nodal was used as antigen to immunize BALB/c mice, by cell fusion between plasma cells and SP2/0 cells, 7 specific positive monoclonal antibodies were picked out, which were verified through indirect ELISA, Western blot and flow cytometry analyses. Therefore 15 pairs of antibodies can be used to set up a double monoclonal antibody sandwich ELISA, AF12-DG5 was the most appropriate antibody pairs. Combined with biotin-avidin systerm, DG5-biotin was used as detection antibody while the complex of streptavidin and HRP-biotin (mass ratio 4:1) as detection reagent. The best working concentration of antibodies were AF12 2μg/ml and DG5-biotin 2μg/ml. Linear range of this ELISA is 0~3 000pg/ml, regression equation is y=0.000 3x+0.150 5,R2=0.999, low limit of detection is 68pg/ml, average recovery is 99.6%. The result of serum detection indicates that more Nodal was expressed in 70% of gallbladder carcinoma patients, 92% of nasopharyngeal carcinoma patients and 97% of colorectal carcinoma patients, which were statistically significantly increased compare to normal human.Conclusions/significance:A sensitive and specific Nodal ELISA test has been developed. This test is high sensitive and easy to use and can discriminate cancer patients from normal person specifically. The achieved data suggest that the developed ELISA may be applicable as a research tool for detection of human cancer.
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Received: 26 December 2016
Published: 25 March 2017
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