| FL is a transmembrane or soluble protein and is expressed by a variety of cells including hematopoietic and marrow stromal cells. As a potent hematopoietic cytokine, FL is capable of stimulating proliferation and differentiation of stem cells, myeloid and lymphoid progenitor cells, dendritic cells and natural killer cells in combination with other growth factors. The receptor of FL is a member of the typeⅢtyrosine kinase receptor (RTK) family and is expressed by immature hematopoietic progenitor cells. FL induces activation of the receptor and leads to tyrosine phosphorylation of various key adaptor proteins known to be involved in different signal transduction pathways that control proliferation, survival and other processes in hematopoietic cells. At present, as a potential applied valuable cytokine, FL is mainly involved in the therapy for hematopoietic stem cell transplantation and cancer.ObjectiveTo obtain a hybridoma cell line that can stably secret mouse anti-human FL monoclonal antibody and study its biological characteristics. To establish a sensitive, specific, stable ELISA assay for detecting soluble human FL molecule.MethodsB lymphocyte hybridoma technique was applied to obtain a hybridoma cell line secreting anti-human FL antibody. The subclass of the mAb was identified through fast-strip method analysis. Ascites were induced to produce the monoclonal antibody from BALB/c mouse, and were purified with affinity chromatography method. Indirect immunofluorescence was used to test the abiliby of mAb to recognize the FL molecule on different cell membranes. The epitope recognition was identified through antigen competitive inhibition assays. An ELISA kit for detecting sFL was set up. The concentrations of soluble antigen FL in serum samples were analyzed.ResultsA hybridoma named 2D2 was obtained which can stably secreting monoclonal antibody against human FL molecule. The subclass of 2D2 was mouse IgG1 with kappa light chains through fast-strip method analysis. Ascites were induced to produce the monoclonal antibody and 7.4 ml ascites was obtained from each BALB/c mouse on average. The purified 2D2 from ascites was about 1.2-4.7 mg per ml ascites with affinity chromatography method. The 2D2 could specifically recognize membrane FL expressed on cells of FL-L929, U937 and HL-60 by indirect immunofluorescence with the positive percentage of 85.2%, 33.1% and 39.2%, respectively. However, the 2D2 could not detect FL expression on the other cells that were screened. ELISA method for measuring soluble human antigen FL was successfully established. The detecting limit was 7.8pg/ml. The CV of kit is less then±7.6% and the retrievable rate is 97%～110% within three months as the kit stored in 4℃.It evidenced that the kit was of high sensitivity, stability and accutacy. The sFL concentrations in sera from healthy donors group and patients of aplastic anemia and leukemia were 328.6±42.89pg/ml; 2607±286.8pg/ml; 1091±255.2pg/ml respectively.ConclusionsA mouse anti-human FL functional monoclonal antibody was successfully obtained, named 2D2. And the ELISA method for measuring soluble human FL was established with high specifity, sensitivity and accuracy. Establishment of detecting sFL assay may be of significance in basic research and the diagnosis, curative effect evaluation and prognostic estimation of sFL relative diseases. |
PDF Full Text Request