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).
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
- 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
- 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.
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
