Assay of lectin binding sites in colon cancer-associated mucins: comparison of fluorescence microscopy with a quantitative chromatographic technique.
We have developed a technique for quantitation of binding of fluorescent lectins to glycoconjugates in specimens of tumors derived from cultured human colorectal cancer cells. Tumor cells were injected subcutaneously into nude mice, giving rise to xenografts that resemble primary human colorectal cancers. The tumors were extracted with saline and were subjected to dialysis and lyophilization. Standardized amounts of the tumor extract were then incubated with fluorescent lectins and subjected to gel permeation liquid chromatography to separate lectin bound to high molecular weight glycoproteins from free (unbound) lectin, and were quantitated using a spectrofluorometer. This assay permitted quantitative measurement of the lectin bound to high molecular weight glycoconjugates such as mucin. The results of this assay were compared with the standard histochemical assessment of tissue labeling by fluorescent lectins. A close correlation between the two techniques was found, especially when little or no labeling was present. Greater variations were observed at higher levels of labeling. The quantitative assay confirms that lectins bind to high molecular weight mucin-type glycoconjugates on fixed sections of tumors, and supports the use of semi-quantitative histochemical assessments of tissue labeling.
D000230:Adenocarcinoma / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002850:Chromatography, Gel; D003110:Colonic Neoplasms / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006651:Histocytochemistry; D006801:Humans; D037102:Lectins / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008856:Microscopy, Fluorescence; D009077:Mucins / Q000378:metabolism*; D011975:Receptors, Mitogen / Q000032:analysis*; D013861:Thiocyanates
D005452:Fluoresceins; D037102:Lectins; D009077:Mucins; D011975:Receptors, Mitogen; D013861:Thiocyanates; D016650:Fluorescein-5-isothiocyanate