Cytokeratin Monoclonal Antibodies Preparation Of Immunoassay Methods To Establish And Preliminary Applications

Cytokeratins(CKs) constitute a diverse group of intermediate filament(IF) proteins,whose fascinating functions in tissue cells especially the relationship between CKs and the change of cellular behaviors have become a research hotspot once again in the last decade . In this paper, we focused on establishing immunoassays for CKs using the monoclonal antibodies(McAbs) to them which were prepared by ourselves. Also, the preliminary clinical applications of these immunoasays were studied.The CKs fraction named TP 21 of human colorectal carcinoma cells (HT-29) was extracted and purified through DE52 ion-exchange chromatography in 8.0 M urea . Using TP 21 as antigen , fusing the spleen cells from BALB/c mice with SP2 /0 murine myeloma cells by 50% PEG(MW 3700), then after many times of cloning , subcloning and thousands of screening . four stable hybridomas(named 1-6, H13, H16, CY19) were obtained finally. Injecting each of these four hybridomas into pristane-primed BALB/c mice, ascitic fluids containing TP21 McAbs could be collected from the mice 10-15 days after inoculation. From these ascitic fluids,the IgG fractions were obtained by Protein G-Sepharose affinity chromatography.Each of the McAbs was characterized. The affinities of 1-6, H13, H16 and CY19 for TP21 are 2.3 ×10~7 L/mol, 1.8×10~7 L/mol, 1.21× 10~8 L/mol, 1.36×10~8 L/ mol respectively. Three of them belong to IgG1 except CY19(IgG2a) . but each of them has λ light chains. Using immunohistochemical analysis, CY19 and H16 showed similar staining features.H13. CY19 and H16 showed specificity for epithlium cells. 1-6 recognized the tissue of muscle origin specifically.Two of these four McAbs (H16 and CY19)were screened to develop a two-site sandwich immunoradiometric assay(IRMA) for TP21 in human serum. Based on IRMA. another two-site sandwich immunoassay called enzyme-linked...