Complement-mediated cytotoxicity test ã€principle】 Wooden Series Paper Wooden Series Paper,Notebook Cover Wooden Series Paper,Vintage Wooden Series Paper,Vintage Wooden Paper DONGGUAN SHENGYUAN INDUSTRIAL CO,.LTD. , https://www.elementpuleather.com
The principle of the complement dependent cytotoxicity test is that after specific antibodies bind to the corresponding antigen on the cell membrane, cell membrane damage and cell death can occur with the participation of complement; cells without the corresponding antigen still survive. Dye rejection experiments were used to determine the state of lymphocytes. The living cells were not colored, had refractive properties, and were normal in size. Dead cells are stained and the volume increases. The following uses HLA-A, B, C antigen detection as an example.
ã€material】
1. Terasaki plastic plate (60-well or 96-well), Fisher centrifuge tube, Fisher centrifuge, horizontal centrifuge, inverted microscope, blood cell counting plate, general microscope, hair suction dropper, large test tube, pilot tube, rubber nipple, Thermostat, spark vacuum detector.
2. Heparin anticoagulant (1000U / 0.5ml / tube), Hanks solution (pH7.2), McCoy medium, lymphocyte separation solution (specific gravity 1.077), 50 g / L Eosin Y, 34-40% neutral Formaldehyde, saline, thrombin.
3. Rabbit complement Fresh sera from more than 20 rabbits are mixed and divided into sterile penicillin vials. In the absence of HLA antiserum, there should be no lymphocyte toxicity; 1,2 dilution should still be able to score the typed serum reaction score of 8 points (see the results of this experiment) Do not dilute, store at -70 ℃ for future use. Half an hour before use, remove at room temperature and thaw, and discard the remaining liquid after use. Do not freeze and thaw repeatedly.
4. Lymphocyte suspension (1) Take heparin anticoagulated peripheral blood 10 ~ 15ml, centrifuge at 1800rpm for 10 ~ 15min.
(2) Take the leukocyte layer (1.0 ~ 1.5ml) and add it to the medium test tube containing 1.5ml Hanks solution.
(3) Mix with a hair suction pipette, gently add it along the wall of the tube to a centrifuge tube containing 1.5 ml of lymphocyte separation solution, so that it overlaps on the lymphocyte separation solution, and the layering is good.
(4) Centrifuge at 2000 rpm for 20 minutes.
(5) Pipette the mononuclear cell layer cells into a Fisher centrifuge tube and centrifuge with a Fisher centrifuge at 4000 rpm for 5 minutes.
(6) Discard the supernatant, add Hanks solution, and centrifuge at 1500 rpm for 10 min to remove platelets.
(7) Discard the supernatant (with platelets in it), add Hanks solution and mix well, add a drop of thrombin.
(8) Rotate the Fisher centrifuge tube to promote coagulation until platelet clot is generated (about 2 min).
(9) Centrifuge at 1500 rpm for 10 minutes to precipitate platelet clot.
(10) Transfer the upper suspension to a new Fisher tube and centrifuge at 1500 rpm for 10 minutes.
(11) Discard the supernatant (remove thrombin), add Hanks solution to resuspend the cells, centrifuge at 1500 rpm for 15 min, discard the supernatant, and repeat this step once.
(12) Discard the supernatant, suspend the cells with McCoy solution, and adjust the cell concentration to 1.5 ~ 2´106 / ml.
(13) If the cell suspension also contains red blood cells, anti-A, anti-B, and anti-H treatments can be added and removed.
5. HLA serum plate (1) Take clean Terasaki plastic plate, add a certain amount of paraffin oil to each well.
(2) Using a clean micro syringe, add 1 ml of various known control sera and specific antisera to the corresponding wells.
(3) Store in the refrigerator at -70 ℃ or -80 ℃ for standby (storage period is 6 ~ 12 months).
6. Control sera The negative control is best selected from AB male sera with no history of blood transfusion in healthy men, inactivated at 56 ° C for 30 minutes, or a mixture of healthy people inactivated at 56 ° C for 30 minutes. The positive control used horse anti-human lymphocytes.
7. Specific antibodies: Use maternal serum, serum of multiple recipients, serum of volunteers planned to be immunized, serum of renal transplant rejection or monoclonal antibodies.
ã€method】
1. Remove the HLA serum plate from the -80 ° C refrigerator and melt at room temperature.
2. Mark the serum plate and indicate the cell number to be tested.
3. Mix the T lymphocytes or total peripheral lymphocytes to be tested (1.5´106 ~ 2´106 / ml).
4. Add 1ml of cell suspension to each well by soft drop method and mix it thoroughly with serum (mixed with spark vacuum detector) at room temperature (25 ° C) for 30min.
5. Add 5ml of rabbit complement to each well and incubate at room temperature (25 ° C) for 60min.
6. Add 5ml eosin staining solution to each well. After 2 minutes, add 5ml of neutral formalin to each well.
7. Place 50´75mm2 slides on the serum plate to flatten the droplets from each well. Check the percentage of cell survival under an inverted microscope.
ã€result】
The result record is "6" or "8" as the exact positive reaction. Cell death is stained by dyes. The cells are gray and dark, and the volume increases, indicating that there is an antigen corresponding to the specific HLA antibody on the surface of the cell; if there is no antigen corresponding to the specific HLA antibody on the surface of the lymphocyte, the cell is not stained (stained The number of cells is <10% ~ 19%). Or the cells are refractive and normal in size.
<10% cell death, 1 point, negative 11% ~ 19% cell death, 2 point, negative 20% ~ 29% cell death, 4 point, positive suspicious 30% ~ 80% cell death, note 6 points, for a positive reaction> 80% cell death, score 8 points, a strong positive reaction in the negative control and positive control results are correct, according to the "majority response principle" to determine the test cell antigen. If the result is abnormal, you must carefully check whether each step of the operating procedures and reagents meet the requirements, and redo if necessary. For beginners, record a score of 4 points, it is best to have experienced people review and determine the results, do not easily draw conclusions.
ã€discuss】
1. Precautions (1) The key to this test is to obtain various HLA standard antisera. Since HLA antibodies are not naturally formed, they do not need to be stimulated by allogeneic antigens. At present, the main sources of standard typing sera are those who have undergone maternal and planned immunization.
(2) Pay attention to the existence and activity of lymphocytes, because granulocytes and monocytes can engulf the dye non-specifically and cause the illusion of cell damage; obvious contamination of granulocytes and platelets can cause false negative reactions because they are associated with HLA Antibody binding reduces antibody binding to lymphocytes; red blood cell contamination is difficult to distinguish from small lymphocytes under an inverted microscope. To this end, the test is treated with thrombin, etc., and try to avoid other cells (red blood cells, platelets, granulocytes, and monocytes) contaminated.
(3) If the HLA-DR and DQ antigens are measured, the operation steps (4) except that the lymphocytes are changed to B cells and the standard typing serum is changed to HLA-DR, DQ typing serum and anti-B lymphocytes as positive controls (5) The test conditions were changed to 37 ° C for 60min and 25 ° C for 2h, and the others were the same as HLA-A, B and C antigen determination.
2. Methodological evaluation The method is simple and easy to use. The amount of antiserum and the amount of cells are very small. The results are reliable, reproducible, and no special equipment is required. Therefore, it is a standard method commonly used internationally.
3. Clinical significance HLA typing has very important significance and uses in many fields such as immunogenetics, anthropology, organ transplantation, clinical blood transfusion, paternity testing, and disease-related research. For example, in organ transplantation, in order to improve the success rate of organ transplantation, it is necessary to choose a more ideal donor. In addition to ABO blood group antigens must be compatible, donor HLA antigens should be as close as possible to the recipient, especially HLA-D, DR antigens are more important.