ELISA withplatelets
This modification of qualitative ELISA (Enzyme-LinkedImmunosorbent Assay) is used for either screening detection ofanti-platelet antibodies or for detection of platelet-associated Ig(PAIg) (shown here).
A. PROTOCOL
1. prepare platelets from control and immunized mice
2. distribute 2.5x106 platelets per well on F-bottomedMaxiSorp 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 ofrat IgG2a anti-mouse kappa chain monoclonal antibody conjugated tohorse-radish peroxidase (LO-MK1, LO/IMEX)
8. reveal with o-phenylenediamine dihydrochloride (OPD) inrevelation solution.
B. 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, 1L, water solution = citrique acid, 5.1065 g + Na2HPO4 x 2H20, 9.08g
5. glycine buffer (coating solution), pH 9.2, watersolution = glycine 1 M + NaCl 1.5 M
C. ADDITIONAL INFO
This protocol may be used for screening of hybridomasupernatants for anti-platelet Ab. In this case, the extra stepsuch as an incubation with a certain SN and additional washingshould be added before the step No 7.
D. REFERENCES
Mizutani, H., Engelman, R.W., Kurata, Y., Ikehara, S. and Good,R.A. (1993). Development and characterization of monoclonalantiplatelet autoantibodies from autoimmune thrombocytopenicpurpura-prone (NZW x BXSB)F1 mice. Blood 82(3): 837-44.