A two-site lectinoenzymatic assay for determination of tumour marker glycoproteins in rectal secretions.
A method is described for a titre-tray based two-site lectinoenzymatic assay of glycoproteins. WGA lectin, reacting with the core-part of glycans, was combined with lectins PNA and DBA, the latter two reacting with terminal parts of glycans. A standard curve was obtained with bovine submaxillary gland asialomucin, and measurements of human rectal secretion were calibrated against this curve. The assay showed an intra-assay reproducibility of 2.4-7.5%, and inter-assay reproducibility of 3.9-20.8%. Recovery tests showed a linearity close to predicted values. The selected standard was ideal as inhibition of lectin binding by monosaccharides showed similar inhibition profiles for human rectal secretion and for asialomucin standard. Neuraminidase treatment dramatically increased the PNA binding to human rectal secretion immobilized on WGA. Western blotting of human rectal secretion demonstrated a large range of lectin-reactive glycoproteins, the main fraction reacting with all lectins being approximately 250 kDa. The assay described is well suited for studies of the glycan part of tumour marker glycoproteins, and changes occurring in these. It has a high sensitivity by ignoring that the glycans may be present on different molecules. Examination of rectal secretions from various cancer patients showed significantly increased PNA binding, as well as an increased PNA/DBA binding ratio, in patients with colorectal cancer (p<3x10(-3)) and, unexpectedly, in patients with other cancers (p<5x10(-3)).
Orntoft
T F
TF
University Department of Clinical Biochemistry, Aarhus University Hospital, Denmark.
Jepsen
J
J
Hansen
P V
PV
Raundahl
U
U
Langkilde
N C
NC
D000818:Animals; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000032:analysis; D014408:Biomarkers, Tumor / Q000032:analysis*; D002277:Carcinoma / Q000175:diagnosis; D002417:Cattle; D015179:Colorectal Neoplasms / Q000175:diagnosis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D004800:Enzymes, Immobilized; D006023:Glycoproteins / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D006031:Glycosylation; D006735:Horseradish Peroxidase / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D037102:Lectins / Q000378:metabolism* / Q000494:pharmacology; D009005:Monosaccharides / Q000378:metabolism; D009439:Neuraminidase / Q000378:metabolism / Q000494:pharmacology; D019887:Peanut Agglutinin; D037121:Plant Lectins; D012007:Rectum / Q000378:metabolism*; D015203:Reproducibility of Results; D014909:Wheat Germ Agglutinins / Q000378:metabolism
D015295:Antigens, Tumor-Associated, Carbohydrate; D014408:Biomarkers, Tumor; D004800:Enzymes, Immobilized; D006023:Glycoproteins; D037102:Lectins; D009005:Monosaccharides; D019887:Peanut Agglutinin; D037121:Plant Lectins; D014909:Wheat Germ Agglutinins; C036554:dolichos biflorus agglutinin; D006735:Horseradish Peroxidase; D009439:Neuraminidase