The level of concentration of soluble interleukin 2 receptor SIL-2R is closely related to many diseases, such as adult T-lymphoblastic leukemia AIDS, B-cell slow gonorrhea, hairy cell leukemia, and when acute rejection occurs after renal transplantation or when graft-versus-host occurs in allogeneic bone marrow transplants, the level of SIL-2R in the serum of the patient is significantly elevated. (Source: Basic Medical Immunology Laboratory Guide, edited by Jin Boquan and Li Enshan, 1st edition, Beijing: World Book Press, 1990).
Operation method
Sandwich ELISA
Principle
Anti-Tac specific monoclonal antibody Ab1 is adsorbed onto a solid phase carrier and recognizes and binds to Tac in specimens such as cell culture supernatants or serum. A horseradish peroxidase-labeled monoclonal antibody Ab2 specific for another Tac epitope recognizes and binds to Tac immobilized in the solid phase carrier. The level of free Tac in the specimen to be examined is reflected by the enzyme-catalyzed color development of the substrate.
Materials and Instruments
Antibody PHA Move 1. Stimulation of peripheral blood mononuclear cells (PBMC) Caveat 1. The activity of the encapsulated antibody should be high, otherwise the sensitivity of this experiment will be affected. 2. After screening and comparison, 1% BSA-PBS is preferred. 3. The enzyme conjugate should be titrated before application to select the best working concentration. 4. The substrate should be a sensitive hydrogen donor, such as ABTS. 5. If available, the enzyme-labeled McAb can be replaced by biotin-McAb and affinity protein-HRP systems to increase the sensitivity of the assay.If available, the enzyme-labeled McAb can be replaced by biotin-McAb and affinity hormone-HRP systems to increase the sensitivity of the test. For more product details, please visit Aladdin Scientific website.
Buffer Wash Solution Diluent Terminator
Plastic Plate Detector Water Bath Refrigerator CO2 Incubator Sampler
Take 10 ml of peripheral blood, heparin anticoagulation, routinely isolate single nuclear cells, add to 24-well cell culture plate, 2×106 /well, RPMI1640-10% FCS + PHA 3 μg/ml. Wellcome company, 37 ℃, 5% CO2 culture for 72 hours, collect the supernatant, -20 ℃ storage to be measured. The supernatant was collected and stored at -20 ℃ for measurement. The control group without PHA was set up at the same time. Patient's serum can be used as the specimen to be tested.
2. Ab1 coating
Dilute anti-Tac specific monoclonal antibody (Ab1) with 0.05 M pH9.6 carbonate coating buffer to 5 μg/ml. Dilute anti-Tac-specific monoclonal antibody (Ab1) to 5 μg/ml with 0.05 M pH9.6 carbonate coating buffer, add to ELISA plate, 100 μl/well, and incubate at 4 ℃ for 72 hours.
3. Blocking
1 % BSA-PBS blocking, 350 μl/well, incubate at 37 ℃ for 2 hours.
4. Sampling
The ELISA plate was washed 3 times with PBS-T, and the supernatant of PHA-stimulated mononuclear cell culture and the supernatant of control group or patient serum were diluted to 1:1024 in multiplicity. The PHA-stimulated mononuclear cell culture supernatant and control supernatant or patient serum were diluted to 1:1024, and 100 μl of each dilution was added to the ELISA plate. 102B2 cell culture supernatant was used as positive control, and RPMI1640-10% FCS culture medium was used as negative control. 37 ℃, incubate for 2 hours, wash. Incubate at 37 ℃ for 2 hours and wash.
5. Addition of ELISA antibody
Add horseradish peroxidase labeled another anti-Tac specific monoclonal antibody Ab2 to each reaction well at the optimal dilution for potency titration. Dilute 100 μl of Ab2 antibody at the optimal dilution for potency titration. incubate at 37 ℃ for 2 hours and wash.
6. Addition of substrate for color development
Add 100 μl of freshly prepared ABTS substrate solution to each reaction well and incubate at 37 ℃ for 15 minutes. incubate for 15 minutes at 37 ℃.
7. Terminate the reaction
Add 50 μl of 2% sodium fluoride termination solution to each well.
8. Result Determination
Firstly, observe the color in the reaction wells with naked eyes and whether the dilution gradient is uniform. Positive and u The color of the reaction wells was firstly observed by naked eye, and the dilution gradient was uniform. Then measure the OD value (410 nm) of each well on the ELISA detector.