ELISA calls for help

Juan Li via methods%40net.bio.net (by jli from custombiologics.com)
Wed Apr 13 13:14:08 EST 2011


Hi Chuxin,

You may want to try different blocking agents or procedure after coating
your plate with antigen, for example 3% BSA or 5% FBS for an hour. Otherwise
all kinds of stuff from mouse serum can bind to your plate non-specifically
and your secondary antibody (anti-mouse Ab, I suppose) will pick up
everything.

Have fun with ELISA,
Juan

-----Original Message-----
From: methods-bounces from oat.bio.indiana.edu
[mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of Liuchuxin
Sent: Tuesday, April 12, 2011 9:58 PM
To: methods from magpie.bio.indiana.edu
Subject: ELISA calls for help

Dear sir/madam,
Sorry to interrupt you! I am a postgraduate in China, and I get in trouble
in 
ELISA.  In ELISA procedure, I am confused at the final step-data processing.
In 
my work, I used luminol as substrate to generate chemiluminescentsignals,
rather 
than TMB to bring color changes. However, in my indirect ELISA to detect 
antibodies in mouse serum, the negative sample or even the blank sample will

always produce quiet strong signals, eg. 22366, 30512, 31290 for the 
chemiluminescence density values of three antiserum samples, 22621 and 22152
of 
the two negative samples, 22435 of the blank one (no coating reagent or 
substrate added). The difference between the antiserum and control serum or 
blank sample is tiny, how can I diminish the background signal and how to
assess 
the antibody titers in this condition? What are the advantages and
disadvantages 
of the chemiluminescence ELISA, or the color ELISA?
                        
I am eager to your reply.
 
Best regards!
 
Chuxin Liu
Huazhong Agricultural University
Wuhan, China
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