E L I S A w i t h p l a t e l e t s

互联网2013-09-06

2114

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 2.5 x106 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 37 ѓc for 1 hour with a 1 :1000 dilution of rat IgG2 a 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 + Na2 HPO4 x 2H2 0, 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.