Experimental reagent CFD dilution (VBS), 10% trichloroacetic acid, C1q solution
Experimental equipment PEG (MW6 000), centrifuge
Experimental procedure 1. C1q labeling method C1q isotope labeling, generally 125I, 131I can also be used.
There are two methods for 125I labeling, namely chloramine T method and lactoperoxidase method. The following introduces the chloramine T method:
1) In 100μl C1q (1mg / ml), add 100μCi125I, mix well, then add 25μl 0.4mol / L PB (pH 7.4, containing 1mg chloramine T), stirring in an ice bath for 4 ~ 6min;
2) Immediately add 50μg / μl potassium sulfite to stop the reaction, and then add 50μl 1% bovine serum albumin as the carrier protein;
3) Pass Sephadex G25 column (20cm × 0.5cm, pre-equilibrate with pH 7.4, 0.4mol / L PB, and inject 1ml 1% bovine serum albumin) to separate from free 125I;
4) After aliquoting with appropriate concentration, store at -70 ℃. To reduce self-aggregation, add 0.02% Tween20.
2. Take the labeled C1q and dilute it to 1 mg / ml with CFD.
3. Centrifuge at 7 000 r / min for 30 min (4 ° C) to remove insoluble aggregated protein and store the supernatant at 4 ° C.
4. Take 4 test tubes, add 0.2ml of determination serum, 0.2ml of CFD, and label C1q 50μl (1μg / ml). 1, 2 tubes are measuring tubes, 3 and 4 tubes are control tubes.
5. After shaking, put it in a 25 ° C incubator for 60 minutes, remove it and put it in a 4 ° C refrigerator for 60 minutes.
6. Add 3ml of PEG to each tube, and the final concentration is 2.5%. After shaking, put it at 4 ℃ for 2h.
7. Add 10% trichloroacetic acid to tubes 3 and 4 to precipitate all the protein. Centrifuge all 4 tubes at 1 000 r / min for 20 min, remove the supernatant, and place the precipitate in the gamma counter to measure cpm.
8. Calculation
B = (a plus b2 plus c plus d2) × 100
B: labeled C1q binding amount (%). a, b: 1, 2 tubes (measurement tube) cpm. c, d: 3, 4 tubes (control tube) cpm.
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