Antibody titration and storage - Reply
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From: | Richard Cartun <Rcartun@harthosp.org> (by way of histonet) |
To: | histonet <histonet@magicnet.net> |
Reply-To: | |
Content-Type: | text/plain; charset="us-ascii" |
I was told two days ago that we ran out of mAb B72.3 (tumor-associated
glycoprotein 200). Before I ordered new antibody I searched our
refrigerator hoping to find a sample that we could use until the
replacement arrived. I found a large (by antibody standards) bottle
containing 20 mLs of mAb B72.3 that we received on 6/20/1989 (almost 10
years ago!) from a company in Stoughton, MA. I decided that I would
evaluate it in case it still worked. Therefore, I prepared the following
dilutions:
1:10 0.9 mL diluent + 100 microliters of mAb
1:100 0.9 mL diluent + 100 microliters from 1:10
1:1,000 0.9 mL diluent + 100 microliters from 1:100
1:10,000 0.9 mL diluent + 100 microliters from 1:1,000
1:100,000 0.9 mL diluent + 100 microliters from 1:10,000
1:1,000,000 0.9 mL diluent + 100 microliters from 1:100,000
and applied them to sections of our positive control (lung adenocarcinoma)
and allowed the slides to incubate overnight at RT. Normally, I would
evaluate a mAb at just three dilutions: 1:100; 1:1,000 and 1:10,000, but I
seemed to remember that this antibody had a high titer. Today, when I
finished the slides with LSAB+ and DAB+ (DAKO Corporation) I was surprised
to see that not only did the antibody still work, it gave some
immunoreactivity at 1:1,000,000! However, I selected 1:1,000 as the
optimal dilution for overnight incubation. This translates into a
concentration of .02 micrograms per mL according to the label which states,
"20 micrograms/mL".
Please remember that the performance characteristics of this antibody are
very unusual, but I thought you might find this story interesting. Oh, by
the way, does anyone need any B72.3 mAb?
Rich Cartun
rcartun@harthosp.org
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