Non-radioactive hybridization in microwells using enzyme linked immune sorbent assay for detection of RT-PCR-Am-plified CK19- and CEA-mRNA

Abstract

PCR enzyme linked immune sorbent assay (ELISA) was developed for detection of RT-PCR-amplified cytokeratin 19 (CK19) mRNA and carcinoembryonic antigen (CEA) mRNA. The non-radioactive hybridization was performed in a streptavidin-coated microwell with digoxigenin-labeled PCR products and with biotin-labeled capture probe. PCR ELISA was proved to be expedient, simple, sensitive and safe for identification of CK19-, CEA-RT-PCR products. These results were proven by sequencing.

Keywords

CK19 / CEA / RT-PCR / ELISA / non-radioactive hybridization

References

[1] GerhardM, JuhlH, KalthoffH, et al.. Specific detection of carcinoembryonic antigen-expressing tumor cells in bone marrow aspirates by polymerase chain reaction. J Clin Oncol, 1994, 12: 725-725

[2] DattaY H, AdamsP T, DrobyskiW R, et al.. Sensitive Detection of Occult Breast Cancer by the Reverse-Transcriptase Polymerase Chain Reaction. J Clin Oncol, 1994, 12: 75-75

[3] BaderB L, JahnL, FrankeW W. Low level expression of cytokeratins 8,18 and 19 in vascular smooth muscle cells of human umbilical cord and in cultured cells derived therefrom, with an analysis of the chromosomal locus containing the cytokeratin 19 gene. Eur J Cell Biology, 1988, 47: 300-300

[4] SchreweH, ThompsonJ, BonaM, et al.. Cloning of the complete gene for carcinoembryonic antigen: analysis of its promoter indicates a region conveying cell type-specific expression. Mol Cell Biol, 1990, 7: 2738-2738

[5] LindemannF, SchlimokG, DirschedlP, et al.. Prognostic significance of micrometastatic tumor cells in bone marrow of colorectal cancer patients. Lancet, 1992, 340: 685-685

[6] PantelK, BraunS, PasslickB, et al.. Minimal Residual Epithelial Cancer: Diagnostic Approaches and Prognostic Relevance, 1996Stuttgart, New YorkGustav Fischer Verlag

[7] SavtchenkoE S, SchiffT A, JiangCK, et al.. Embryonic expression of the human 40-kD keratin: evidence from a processed pseudogene sequence. Am J Hum Genet, 1988, 43: 630-630

[8] ThompsonJ. Molecular cloning and expression of carcinoembryonic antigen gene family members. Tumor Biol, 1995, 16: 10-10