Immunoscreening of phage library

[Yanghy at my-dejanews.com]

Hi, Dear friends, I got some interests in looking for antigens which is in the
RA patients synovium, and by constructing a synovial tissue expression
library, I began to use the patients serum to screen it. because there will be
plenty of human IgG gene in the library and that will give a lot false
positive signals if you add the human serum( anti-IgG), I am trying to use the
secondary ab(Rabit anti-human IgG conjugated with Alkaline phosphatase) alone
to first prescreening the library. but got some very weird results!: 1) the
positive signal appear on the opposite site of the NC membrane contacting
side(with plaques onthe agar plate). 2) there are no signals on the contact
side. but If  I use the serum as primary ab and then add secondary ab-AP I get
the signal on the right side. I don't what is the reason for this. and I try
to seek help from you guys. hopefully some of you can give me some
advice/suggestion/comments about this. esp. if some one has this problem and
has solved it.

Thanks!!!

YANG

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