ELISA with Platelet

PROTOCOL

1. prepare platelets from control and immunized mice
2. distribute 2.5x106 platelets per well on F-bottomed MaxiSorp Nunc-Immuno microplates (GibcoBRL, Roskilde, Denmark), centrifuge at 3000 rpm, 7 min, 10ѓC.
3. fix with PBS-PF 2 % for 5 min, RT
4. wash 3 times in PBS-tween 20, dry
5. block half of the plate (with platelets) by PBS-BSA 1% and coat another half by glycine (x1) - BSA for 1 hour at 37ѓC
6. wash 3 times in PBS-tween 20, dry
7. incubated at 37ѓc for 1 hour with a 1:1000 dilution of rat IgG2a anti-mouse kappa chain monoclonal antibody conjugated to horse-radish peroxidase (LO-MK1, LO/IMEX)
8. reveal with o-phenylenediamine dihydrochloride (OPD) in revelation solution.

SOLUTIONS

1. PBS-PF (paraformaldehyde), 2%
2. PBS-tween 20, 0.05%
3. glycine (x1) - BSA, 1 mg/ml
4. revelation solution (citrate/phosphate buffer), pH 5, 1 L, water solution = citrique acid, 5.1065 g + Na2HPO4 x 2H20, 9.08 g
5. glycine buffer (coating solution), pH 9.2, water solution = glycine 1 M + NaCl 1.5 M

REFERENCES

Mizutani, H., Engelman, R.W., Kurata, Y., Ikehara, S. and Good, R.A. (1993). Development and characterization of monoclonal antiplatelet autoantibodies from autoimmune thrombocytopenic purpura-prone (NZW x BXSB)F1 mice. Blood 82(3): 837-44.

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• 抗体
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