Dear Frances,
we faced the same problems when we wanted to determine the antibody
concentration of our hybridoma cell culture supernatants. We often got some
conflicting results when we compared densitometry, or the BIORAD or Pierce
protein assays.
We now use a quantitative ELISA method, originally used to screen new
hybridomas for antibody production. Using commercially available Ig (G, Aor
M) as a standard we can easily determine the concentration of the antibody
we are looking for. Even in impure soolutions containing other proteins
(like albumin or anything else). This ELISA (HYBSCREEN) is also commercially
available.
I hope this might help
Wolf
Dr. Wolf Splettstoesser, M.D.
FAF Medical Academy
Inst of Microbiology
Munich, Germany
Frances schrieb in Nachricht <35FFC2AC.3495A5AC at ski.mskcc.org>...
>Please help -
>>We are currently quantitating our antibody concentration by two methods
>-
>(1) Comassie Blue G250/densitometery/Computer analysis (BioRAD's
>Molecular Analyst Program) - and - (2) the a Bradford based assay
>(BioRAD
>Protein Assay) - and the two methods do not always give the same
>antibody
>concentraion -
>>When the monoclonal antibody is impure (25-45% from bioreactor
>supernatant) the two approached agree very well when I correct the
>bradford asssay numbers for the puritiy - determined by densitomety -
>But
>as we purify the antibody to 95% pure - we progressivly get less
>agreement
>between the two methods - to the point where the bradford assay gives
>values that are at least 1/2 the value calculated from the comassie
>stained
>gel - We use dilutions of the same rat or bovine IgG prep (from BioRAD)
>as
>standards for BOTH assays -
>>I tend to trust the comassie staining more - because my understanding is
>>that comassie staining is protien composition INDEPENDENT and the
>bradford system is dependent on the number of tyrosines in the protien
>solution -
>>Therefore - does anyone know of another way to quantitate the
>concentration on a purified protien that is independent of the protien
>composition - or am I wrong and should trust the bradford system more -
>>Thanks for any advise or comments you can offer -
>>Frances
>>*************************************
>>Frances Weis-Garcia, Ph.D.
>Manager, Monoclonal Antibody Core Facility
>>Memorial Sloan-Kettering Cancer Center
>1275 York Avenue - Box 341
>New York, New York 10021
>>Phone: 212 - 639 - 2054
>FAX: 212 - 794 - 4019
>e-mail: f-weis-garcia at ski.mskcc.org>>>>
