OUTLINE
This modification of qualitative ELISA (Enzyme-Linked Immunosorbent Assay) is used for either screening detection of anti-platelet antibodies or for detection of platelet-associated Ig (PAIg) (shown here).
PROTOCOL
prepare platelets from control and immunized mice
distribute 2.5x106 platelets per well on F-bottomed MaxiSorp Nunc-Immuno microplates (GibcoBRL, Roskilde, Denmark), centrifuge at 3000 rpm, 7 min, 10?C.
fix with PBS-PF 2 % for 5 min, RT
wash 3 times in PBS-tween 20, dry
block half of the plate (with platelets) by PBS-BSA 1% and coat another half by glycine (x1) - BSA for 1 hour at 37?C
wash 3 times in PBS-tween 20, dry
incubated at 37?c 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)
reveal with o-phenylenediamine dihydrochloride (OPD) in revelation solution.
SOLUTIONS
PBS-PF (paraformaldehyde), 2%
PBS-tween 20, 0.05%
glycine (x1) - BSA, 1 mg/ml
revelation solution (citrate/phosphate buffer), pH 5, 1 L, water solution = citrique acid, 5.1065 g + Na2HPO4 x 2H20, 9.08 g
glycine buffer (coating solution), pH 9.2, water solution = glycine 1 M + NaCl 1.5 M
ADDITIONAL INFO
This protocol may be used for screening of hybridoma supernatants for anti-platelet Ab. In this case, the extra step such as an incubation with a certain SN and additional washing should be added before the step No 7.
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.
