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This method is based on the following publication;
K Campbell, K Rishi, G Howkins, D Gilby, R Mushens, C Ghevaert, P Metcalfe, WH Ouwehand, G Lucas. A modified fast MAIPA for the detection of HPA antibodies: a multi-centre evaluation of a rapid monoclonal antibody specific immobilisation of platelet antigen (MAIPA) assay. Vox Sanguinis 2007, 93, 289-297.
Reagents
Coating Buffer
Na2CO3 1.59 g
NaHCO3 2.93 g
dH2O to 1 litre, adjust pH to 9.6 using 0.5M HCl. Store at 4oC.
Goat Anti-Mouse IgG
‘Jackson Immunoresearch’ goat anti-mouse IgG, Fcγ fragment specific (affinity purified/minimum cross reactivity) (catalogue code;115-005-164). Store at 4oC
Dilute 1 in 500 in coating buffer. Discard any un-used diluted reagent
10X TBS Stock Buffer
Tris 12.1g
NaCl 85g
dH2O 900mL
adjust pH to 7.4 with 0.5M HCl and make up to IL with dH2O. Store at 4oC
20% Bovine Serum Albumin
Bovine Serum Albumin (Sigma A-7030) 20g
Phosphate Buffered Saline 100ml. Store at 4oC
TBS/BSA Buffer
10X TBS Stock Buffer 50mL
dH2O 445mL
20% BSA 5mL Store at 4oC
Solubilisation Buffer
Make 1L isotonic saline by dissolving 9.0g NaCl in 1 litre deionised water
Dissolve 1.21 g Tris in 950 mL isotonic saline, pH to 7.4 using 0.5M HCl. Add 5 mL Triton X100 and mix well. Make up to 1 litre with isotonic saline.
1M Calcium chloride solution
CaCl2.2H2O 14.7g
dH2O 100mL
Tween wash buffer
10X TBS Stock Buffer 100mL
Nonidet P40 substitute 5mL
(Fluka BioChemika code 74385)
Tween 20 0.5mL
1M CaCl2 0.5mL
dH2O make up to 1L Store at 4 oC
Add 1mL 20% BSA per 100mL before use.
Peroxidase conjugated Goat anti-human IgG (GAH:HRP)
Jackson ImmunoResearch (affinity purified/min. cross reactivity) (catalogue code; 109-035-098) Store at 4oC
Reconstitute with water, dilute with equal volume Glycerol, aliquot (e.g. 20µL) and store at -20oC. Before use dilute this stock solution to working concentration, e.g. 1 in 3,000 in Tween wash buffer (= 1/6000 final concentration). Discard un-used diluted reagent.
OPD (Dako S2045) Substrate Solution
OPD. 2HCl Store at 4oC 4 tablets
dH2O 12mL
30% H202 Store at 4oC. Add 5µL immediately before use.
Discard un-used reagent.
Note: To ensure operator safety, gloves must be worn when preparing and dispensing OPD.
Stop Solution 0.5M H2SO4 Store at room temp.
Platelet preparation
1. Take blood from group O donors into EDTA or citrate.
2. Centrifuge at 1800 rpm for 10 min in bench-top centrifuge
3. Remove the top 3/4 of the PRP from the top of the tube and transfer to 10mL conical centrifuge tube.
4. Add PBS/EDTA buffer to 10mL and centrifuge at 3,800 rpm approx for 5 mins.
5. Decant supernatant and re-suspend cells gently in 2mL buffer and repeat step 4. twice more.
6. Re-suspend platelets in PBS/EDTA at 100x 109/L approx. Store at 4oC for up to 2 weeks.
Or if using cryopreserved platelets, recover using local method and resuspend at 100x 109/L.
Method
1. Preparation of Coated F-Well Microplate
1.1. Prepare goat anti-mouse IgG as above and aliquot 100mL/well into F-well microplate supplied.
1.2. Incubate microplates at 4oC for at least 3 hours. Store sealed coated plates for up to 2 weeks at 4oC.
2. Incubation of platelets with serum
2.1. Use panel platelets at 100x 109/L
2.2. Add 100µL platelets per well of a U-well plate according to the relevant plate plan.
2.3. Centrifuge microtitre plate 1400g for 3mins.
2.4. Discard supernatant and blot plate dry on paper towel.
2.5. Resuspend platelet pellet using a vortex mixer or plate shaker.
2.6. Add 50µl TBS/BSA buffer to each well.
2.7. Dispense 25µL test/control plasma to the wells of the plate according to the relevant plate plan.
2.8. Attach a microplate sealer to the plate if using a waterbath for incubation or a microtitre plate lid if using a dry air incubator.
2.9. Incubate at 37oC for 30min in a waterbath or 40min in a dry air incubator.
3. Removal of unbound immunoglobulins
3.1. Centrifuge microtitre plate at 1400g for 3mins.
3.2. Discard supernatant and blot plate dry on paper towel.
3.3. Resuspend platelet pellet using a vortex mixer or plate shaker.
3.4. Add 200µL TBS/BSA to each well.
3.5. Attach plate sealer to microtitre plate.
3.6. Centrifuge microtitre plate at 1400g for 3mins.
3.7. Discard supernatant and blot plate dry on paper towel.
3.8. Resuspend platelet pellet using a vortex mixer or plate shaker.
3.9. Repeat steps 3.4 to 3.8 leaving the plate dry after the second and final wash (total of 2 washes).
4. Incubation of platelets with monoclonal antibody
4.1. Dilute monoclonal antibodies in TBS/BSA, as determined by prior experiment.
e.g. GPIIbIIIa: PAB-1 and PAB-6, 1 in 10;
GPIbIX: PAB-5, 1 in 10;
GPIaIIa: P16, 1 in 10;
β2 microglobulin: W6/32, 1 in 5;
CD109: 15E10, 1 in 100.
4.2. Add 50µl TBS/BSA buffer to each well.
4.3. Add 40µl of diluted Mab to the designated wells according to the relevant plate plan.
4.4. Attach plate sealer or plate lid as in 2.8
4.5. Incubate at 37oC for 30min in a waterbath or 40min in a dry air incubator.
5. Block coated plates
5.1. Remove and discard capture antibody from plate prepared in step 1.
5.2. Add 125µL Tween wash buffer to all wells.
5.3. Remove and discard wash buffer, blotting excess on a paper towel.
5.4. Repeat steps 6.2 and 6.3 twice (total of 3 washes).
5.5. Add 125µL Tween wash buffer to all wells and leave at 20oC/room temperature for use in step 8.1.
6. Removal of unbound monoclonal antibody
6.1. Centrifuge microtitre plate from step 4.5 at 1400g for 3mins.
6.2. Discard supernatant and blot plate dry on paper towel.
6.3. Resuspend platelet pellet using a vortex mixer or plate shaker.
6.4. Add 200µL TBS/BSA to each well.
6.5. Attach plate sealer to microtitre plate.
6.6. Centrifuge microtitre plate at 1400g for 3mins.
6.7. Discard supernatant and blot plate dry on paper towel.
6.8. Resuspend platelet pellet using a vortex mixer or plate shaker.
6.9. Repeat steps 5.4 to 5.8 twice, leaving the plate dry after the third and final wash (total of 3 washes).
7. Solubilisation of platelet membranes
7.1. Add 130µL of the solubilisation buffer to each well of the U-well plate from step 6.9 above and mix 3 times with a multichannel pipette.
7.2. Attach a plate sealer or plate lid.
7.3. Incubate at 20oC for 15 minutes.
7.4. Centrifuge the plate at 1400g for 15 minutes to pellet cell stroma.
8. Transfer of platelet lysates to F-well plate
8.1. Take blocked F-well plate from step 5.5.
8.2. Remove and discard Tween wash buffer, blotting excess on a paper towel.
8.3. Transfer 100µL lysate supernatant from step 7.4 to corresponding wells of the coated, blocked F-well plate.
8.4. Attach plate sealer to plate or plate lid as in 2.8.
8.5. Incubate plate at 37oC for 30min in a waterbath or 40min in a dry air incubator.
9. Removal of unbound lysate proteins.
9.1. Discard supernatant and blot plate dry on paper towel.
9.2. Add 125µL of Tween wash buffer to each well.
9.3. Discard supernatant and blot plate dry on paper towel.
9.4. Repeat steps 9.2 and 9.3 five times (total of 6 washes).
10. Addition of peroxidase labelled Goat anti-human IgG
10.1. Prepare GAH:HRP as detailed in ‘Reagents’.
10.2. Add 100µL diluted GAH:HRP to each well of the F-well plate.
10.3. Attach plate sealer or plate lid as in 2.8.
10.4. Incubate at 20oC for 60 minutes.
11. Removal of unbound peroxidase labelled Goat anti-human IgG
11.1. Discard supernatant and blot plate dry on paper towel.
11.2. Add 125µL of Tween wash buffer to each well.
11.3. Discard supernatant and blot plate dry on paper towel.
11.4. Repeat steps 11.2 and 11.3 five times (total of 6 washes).
12. Addition of OPD substrate
12.1. Prepare OPD solution as detailed in ‘Reagents’ above.
12.2. Add 100µL OPD solution to all wells
12.3. Incubate at 20oC in the dark for sufficient time to allow adequate colour development e.g. 10 minutes. Clear definition between positive and negative controls should be obtained.
13. Addition of acid to ‘stop’ colour development.
13.1. Add 100µL 0.5M H2SO4 to all wells.
14. Reading of microtitre plate
14.1. Read the plate in a microplate reader at 490nm using a suitable reference wavelength (630-650nm).
14.2. Record OD’s after subtraction of reagent blank OD