Preparation of peroxidase-anti-peroxidase complex

reagents
and preparation
1. HRP and rabbit (or sheep) anti-HRP: Anti-HRP is obtained with HRP immune rabbit (or sheep).
2. 0.075mol/L sodium acetate -0.15 mol/L ammonium acetate equivalent mixture
3. 0.1mol/LHCl and 0.01mol/LHCl
4. Saturated ammonium sulfate solution
5. 0.01mol/L NaOHoperation method
1. Take rabbit (or sheep) anti-HLP
serum
5ml, 16000r/min centrifugation 20min, to precipitate;
2. Add HLP (2mg/ml) solution to the collected upperqing solution 1 ml, slow stirring at room temperature 1h, omnisor centrifugation;
3. The sediment is washed 3 times with cold physiological saline water and discarded
4. Add 4 ml of HLP (2mg/ml) solution to the precipitation, mix well, move to a small
smoulate
;
5 Add 0.1mol/LHCl (about 10 drops) to the stirring side and adjust the pH to about 2.4 with 0.01mol/LHCl;
6. When the precipitation is completely dissolved and the solution clears, Immediately add 0.01mol/LNaOH to bring the pH back to 7.4;
7. Same centrifugation, to precipitate;
8. Add 1/10 volume of 0.075mol/L sodium acetate-0.15 mol/L ammonium acetate equivalent mixture to the collected upperqing and mix well;
9. Add the same amount of ammonium sulfate solution step by bit under electromagnetic stirring, and continue stirring 10min, put 4 degrees C20min;
10. Same centrifugation, go to the clear, and then wash centrifugation 2 times with the same amount of saturated ammonium sulfate solution;
11. Dissolve the sediment in 5 ml of physiological saline water and load it into
dialysis bag
;
12. Use ph6.75 acetate buffer (13) .5L physiological saline, 1.5L distilled water, 75ml 1.5 mol/L sodium acetate and 75ml 3mol/L ammonium acetate) 4 oC dialysis for 3 days, 2 fluid changes per day;
13. same centrifugation, taken clean for identification. Then add the equivalent amount of 60% glycelycer oil, a small amount of sub-pack, -20 degrees C to save.Note:
1. The equivalent band between HRP and anti-HRP should be pre-selected when preparing PAP.
2. When applying anti-HLP McAb to prepare PAP, the method is simpler. According to Cheng Ming reported, with mice anti-HLP McAb ascertain, according to a certain HLP / IgG molar ratio (4:1 as appropriate) mixed, placed 37 degrees C water bath 2h, that is, rat PAP complex. And since both HRP McAb and HRP-forming compounds are soluble, there is no need to add excessive amounts of enzymes.