OUTLINE

This modification of qualitative ELISA (Enzyme-Linked Immunosorbent Assay) is used for either screening detection of anti-plaet antibodies or for detection of plaet-associated Ig (PAIg) (shown here).

 PROTOCOL

1. prepare plaets from control and immunized mice
2. distribute 2.5x106 plaets per well on F-bottomed MaxiSorp Nunc-Immuno microplates (GibcoBRL, Roskilde, Denmark), centrifuge at 3000 rpm, 7 min, 10?C. 
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 plaets) by PBS-BSA 1% and coat another half by glycine (x1) - BSA for 1 hour at 37?C
6. wash 3 times in PBS-tween 20, dry
7. 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)
8. reveal with o-phenylenediamine dihydrochloride (OPD) in revelation solution.

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, 1 L, water solution = citrique acid, 5.1065 g + Na2HPO4 x 2H20, 9.08 g
5. 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-plaet 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

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