sepharose and Precursor-Cell-Lymphoblastic-Leukemia-Lymphoma

The present work aims at the development of plant asparaginase-based asparagine biosensor for leukemia. It is a novel diagnostic for monitoring asparagine levels in patients suffering from acute lymphoblastic leukemia (ALL). Various immobilization strategies have been applied to improve the stability of the asparaginase. The latest and updated information including some new techniques of immobilization related to L-asparaginase such as gelatin, agarose, agar, and calcium alginate methods are described in detail along with response time studies and comparative data. Furthermore these immobilization techniques have been applied for the detection of asparagine in normal and leukemia serum samples.

Topics: Agar; Alginates; Asparaginase; Asparagine; Biosensing Techniques; Color; Gelatin; Glucuronic Acid; Hexuronic Acids; Humans; Immobilized Proteins; Limit of Detection; Plant Proteins; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Sepharose; Time Factors

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.

Topics: Cell Line, Tumor; Cell Separation; Chromatography, Affinity; Flow Cytometry; Humans; Jurkat Cells; Lectins; Microscopy, Fluorescence; Plant Lectins; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Sepharose; Soybean Proteins; Spectrometry, Fluorescence; T-Lymphocytes