Interaction of soybean agglutinin with leukemic T-cells and its use for their in vitro separation from normal lymphocytes by lectin-affinity chromatography.
A procedure for separation of leukemic T-cells from normal lymphocytes, using lectin-affinity column chromatography, is described. CNBr-activated Sepharose 6MB was used as a non-mobile phase. The gel was covalently coupled with soybean agglutinin (SBA), then served as an affinity probe for fractionation of mixture of normal lymphocytes and leukemic cells. Leukemic cell lines, derived from acute lymphoblastic leukemia (Jurkat, MOLT-4, RPMI-8402), were tested. The elution of normal lymphocytes was carried out by PBS(-). The leukemic T-cells, interacting with SBA, were removed by N-acetyl-D-galactosamine or low-concentration acetic acid. The type and viability of the separated cell fractions were analyzed by flow cytometry and fluorescent microscopy, using adequate fluorescent antibodies. The interaction of leukemic T-cells with free SBA, as well as with SBA-conjugated Sepharose beads, was examined fluorimetrically and visualized by fluorescent microscopy, using FITC-SBA as a marker. The rate of cell elution on SBA-affinity column decreased in order: normal > leukemic T-cells. Both normal lymphocytes and leukemic T-cells were removed in a mixture from SBA-free Sepharose 6MB by PBS(-) and were not fractionated discretely. The leukemic T-cells specifically interacted with SBA as well as with SBA-affinity adsorbent. In contrast, the normal lymphocytes did not interact with free SBA as well as with SBA-conjugated Sepharose beads in the concentrations applied. The method potentially combines a discrete cell fractionation with manifestation of a specific target cytotoxicity of SBA against leukemic T-cells, without any influence on normal lymphocytes.
Bakalova
R
R
Natural Substance-Composed Materials Group, Institute for Structural and Engineering Materials, Kyushu, 807-1 Shuku-machi, Tosu, Saga-ken 841-0052, Japan. r.bakalova-zheleva@aist.go.jp
Ohba
H
H
D045744:Cell Line, Tumor; D002469:Cell Separation; D002846:Chromatography, Affinity; D005434:Flow Cytometry; D006801:Humans; D019169:Jurkat Cells; D037102:Lectins; D008856:Microscopy, Fluorescence; D037121:Plant Lectins / Q000494:pharmacology*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000473:pathology*; D012685:Sepharose; D030262:Soybean Proteins / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D013601:T-Lymphocytes / Q000187:drug effects* / Q000473:pathology; D010835:Phytohemagglutinins
D037102:Lectins; D037121:Plant Lectins; D012685:Sepharose; D030262:Soybean Proteins; C028824:soybean lectin; D010835:Phytohemagglutinins