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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, 10ѓC.
- 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 37ѓC
- wash 3 times in PBS-tween 20, dry
- 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)
- 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
(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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