Dear DR, Terry
First, I would tell you that high positive results are expected starting from 3 weeks and more after injecting antigen in mice,
Before starting the test makes sure that RBCs are freshly used,probably washed , probably diluted and in case of using stored RBCs not use stored one more than one week in 4oc . Hemolyzed RBCs should be excluded.
After washing RBCs and dilution to 1% ,in another HA plate add about 25 M normal saline and 25 M of 1% RBCs in all wells ,leave to settle about 20 and use the tested RBCs if typical complete tear (button)shape appears within 20 minutes not more.
make sure that positive( complete tear shape) and negative controls ( lacet shape) typically appear.
in case of still unexpected positive results try to use HAU 2*3 instead of 2*2 or try to use live antigen if you use killed one .
If you use BSA or other proteins in your buffer I would recommend against it.
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