OUTLINEThis 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

  1. prepare platelets from control and immunized mice
  2. distribute 2.5x106 platelets per well on F-bottomed MaxiSorp Nunc-Immuno microplates (GibcoBRL, Roskilde, Denmark), centrifuge at 3000 rpm, 7 min, 10ѓC.
  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 platelets) by PBS-BSA 1% and coat another half by glycine (x1) - BSA for 1 hour at 37ѓC
  6. wash 3 times in PBS-tween 20, dry
  7. 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)
  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 INFOThis 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.

ELISA相关仪器试剂设备

  • 酶标仪
  • 洗板机
  • 化学发光检测仪
  • ELISA检测试剂盒
  • ELISPOT检测
  • ELISA试剂盒
  • 抗体
  • 酶标板