A Study On The Development And Application Of Artificial Blood--Purification And Modification Of Human Hemoglobin

Objective To set up an effective method to extract human natural stroma-free hemoglobin from waste whole blood and blood products such as whole blood with lack of plasma, concentrated erythrocytes and red cells washed. Method The First, visible components such as fragments of cell membranes etc. in blood were effectively wiped out by way of toluene (methylbenzene) extraction. Thesecond, the human hemoglobin was isolated and purified with the ultra-filter system (MILLIPORE company) in order to get depuration products. The third, it was tried to refine the pure products of human hemoglobin using Triton x-100. Then, the reasonable experimental methods (such as electrophoresis test, scanning of ultraviolet-visible spectrophotometry, reverse high pressure liquid chromatography, CURLTER blood analysis system, ABO blood group test, asepsis and pyrogen experiment etc.) were set up and used for determining the rate of production, concentration and purity of the refined hemoglobin products, and the criteria of quality control for purified hemoglobin products were prepared. Results The average concentration of stroma-free hemoglobin (4 batches) measured was 14.6g/dl by above mentioned methods. Blood type coagulate test and asepsis, pyrogen experiment were negative. The purity of products was 98.3%. The scanning atlas with ultraviolet-visible spectrophotometry and peak spectrum from reverse-high pressure liquid chromatography were coincided with control standard. It was indicated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis that hemoglobin products should be homogeneous distrbution with 86.5% and 10.2% of the mobility ratio of electrophoresis similar to marker (14.4kD and 31.0kD). They are subunits and bi-polymers of hemoglobin.The results from polyacrylamide gel electrophoresis showed that subunits and their molecular weights of hemoglobin were determined, and the mobility ratio of product electrophoresis was fully identified with control standard. Conclusion It can be prepared the stroma-free hemoglobin products with high purity and concentration.