Abstract
Currently, Graft-versus-Host Disease (GvHD) has been the major barrier to the application of allogeneic bone marrow transplantation for hematopoietic disorders treatment, especially leukemia. GvHD is caused by donor mature T cells’ attack on recipient’s tissues and organs. Recently, T cell depletion using immunomagnetic nanoparticles is one of the most effective methods to prevent GvHD. In this present study, polyclonal antibodies against Jurkat T cell membrane were generated as a component of immunomagnetic particle. Firstly, Jurkat T cell membrane was fractionated by cloud point extraction using Triton X-114. Subsequently, the fractionated membrane was used to subcutaneously immunize rabbits for polyclonal antibodies production with a dose of 50 μg for priming and 25 μg for the next four boostings. Rabbit serum was collected and partially precipitated in 50 percent of saturated ammonium sulfate solution. Next, precipitated polyclonal antibodies were purified by using protein-A affinity chromatography column and the purity was determimed by SDS-PAGE. Afterwards, the purified antibodies were used in immune-fluorescent stainings and the recognition to Jurkat T cells was evaluated via fluorescent microscopic imaging. Finally, the purified antibodies were negatively selected by incubating with TF-1 cell, a hematopoietic stem cell, to eliminate cross-reacting antibodies. The immunocytofluorescent staining results showed that the purified and selected polyclonal antibodies weakly cross-reacted with TF-1 cells and strongly bound to Jurkat T cell
Highlights
Polyclonal antibodies against Jurkat T cell membrane were generated as a component of immunomagnetic particle
Jurkat T cell membrane was fractionated by cloud point extraction using Triton X-114
Precipitated polyclonal antibodies were purified by using protein-A affinity chromatography column and the purity was determimed by SDS-PAGE
Summary
Vật ghép chống chủ (GvHD) vẫn là rào cản lớn nhất trong ứng dụng ghép tủy dị cá thể để hỗ trợ điều trị bệnh ung thư máu. Kháng thể đa dòng kháng tế bào T Jurkat được tạo ra như nguồn nguyên liệu cho việc tạo hạt từ miễn dịch hỗ trợ loại bỏ tế bào T. Màng tế bào T Jurkat được thu nhận bằng phương pháp ly trích điểm sương sử dụng Triton X-114. Phân đoạn màng được sử dụng để kích thích đáp ứng miễn dịch ở thỏ với lượng 50 μg ở lần đầu tiên và 25 μg ở bốn lần nhắc sau. Kết quả cho thấy kháng thể đa dòng thu được có khả năng nhận diện yếu tế bào TF-1 và nhận diện mạnh tế bào T Jurkat. Từ khoá: chọn lọc âm tính, GvHD, kháng thể kháng tế bào T, ly trích điểm sương, vật ghép chống chủ
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