Antibody titration and storage - Reply

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From:Richard Cartun <> (by way of histonet)
To:histonet <>
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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

 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

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