Determination method of biological activity of spleen polypeptide preparation
A method for measuring biological activity, which is applied in the field of measuring biological activity, can solve the problems of sheep's physical state and the influence of seasonal changes, inconvenient access to fresh sheep blood, and untraceable test results.
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Embodiment 1
[0038] Example 1. Selection of T cell source
[0039] (1) Human peripheral blood T cells and porcine thymus T cells
[0040] Human peripheral blood T cells and porcine thymus T cells were prepared according to the following methods, respectively.
[0041]Preparation of T cells from human peripheral blood: take a 50mL centrifuge tube, first add lymphocyte separation medium equal to that of the blood sample, carefully draw the blood sample with a straw and add it to the liquid surface of the separation medium, 500 ~ 1100g (maximum centrifugal force does not exceed 1200g) centrifuge for 20-30min. After centrifugation, the centrifuge tube is divided into four layers from top to bottom. The first layer is the plasma layer. The second layer is the ring milky white lymphocyte layer. The third layer is a transparent separation liquid layer. The fourth layer is the red blood cell layer. Use a pipette to carefully draw the second layer of milky white lymphocyte layer into anothe...
Embodiment 2
[0051] Example 2. Extraction of De-E Receptor Thymic T Cells
[0052] For fresh porcine thymus, different E removal methods were used to remove E from T cells, and the experiment was carried out according to the following experimental method, repeated 3 times, and statistical analysis was carried out.
[0053] One-off E removal method: make a suspension of thymic T cells separated with lymphocyte separation medium, wash once, add an appropriate amount of Hank's solution to the sediment and mix well, keep warm in a constant temperature water bath at 45°C for 30 minutes, every 5 minutes Shake once, centrifuge at 1500 rpm for 3 to 5 minutes, discard the supernatant, add appropriate amount of Hank's solution, mix well, then place in a constant temperature water bath at 45°C for 30 minutes, take it out and centrifuge at 1500 rpm for 3 to 5 minutes , discard the supernatant, then add an appropriate amount of Hank's solution, mix well, and count, so that the final concentration is 1×...
Embodiment 3
[0062] Embodiment 3. the selection of experimental temperature and measurement time in the detection step
[0063] preparation
[0064] Take 56 1.5mL centrifuge tubes, 14 of which were added with 500 μL of Hank’s solution and 50 μL of E-depleted recipient thymus T cell suspension obtained by two de-E methods as blank tubes. The other 42 tubes were added to the test solution (spleen aminopeptide oral solution, registered trademark "Jingsheng", manufactured by Beijing First Biochemical Pharmaceutical Co., Ltd., batches: 170307, 170308, 170309) each batch of 14 tubes of 500 μL, each Add 50 μL of the de-E receptor thymus T cell suspension obtained by the de-E method to each tube twice, and use it as a test tube. Incubate at 37°C for 1 hour.
[0065] Determination of the test article
[0066] Take 7 blank tubes and 7 tubes of the test product (batch: 170307, 170308, 170309), put them in the refrigerator at 4°C, and take the remaining 7 blank tubes and the tube of the test ...
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