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: [email protected] [mailto:[email protected]] On Behalf Of Liuchuxin Sent: Tuesday, April 12, 2011 9:58 PM To: [email protected] 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 _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
