RE: Antibody cocktails

From:"Johnson, Teri"

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I've made up antibody cocktails for cytokeratin by using a cocktail with a final dilution of CAM5.2 1:8 and AE1/AE3 1:200.  Note I said FINAL dilution.  When used separately, we would use 1:8 and 1:200 respectively on our tissue sections.  Were we to use equal parts of the two, our final dilutions would be CAM5.2 at 1:16, and AE1/AE3 at 1:400.  I would make it up using the following recipe (for 2 ml total):
 
250 microliters CAM5.2 (BD predilute)
1740 microliters antibody diluent
10 microliters AE1/AE3 (concentrated Ab)
 
They were both mouse anti-human antibodies, so cross-species reactivity was no problem.  Biocare Medical has done a great job of providing commercially available cocktails.  They're really helpful for the screening process, in reducing the occurrences of "false" negatives, because you're targeting more antigens at one time. We would only charge for 1 antibody stain, because it was done on 1 slide.  Reporting is simple, "Antibody staining with a cocktail of x-antibody and y-antibody yielded...".
 
Teri Johnson
Managing Director Histology Core Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, Missouri  64110
tjj@stowers-institute.org


-----Original Message-----
From: Van Eyck, Deb [mailto:deb.vaneyck@phci.org]
Sent: Monday, October 14, 2002 12:14 PM
To: 'http://ls.cytopathnet.org'
Subject: 



Here is another question for all you immuno wizards.  At several of the immuno workshops at the NSH I heard several people say they made up antibody cocktails by mixing equal amounts of optimally diluted antibodies.  (For example HMB45, Tyrosinase and Mel A or Mart 1)---Not too worry proteins seem to like each other!  In the immuno world this just seems a little too easy, plus you can charge for the use of the separate clones?  How would you report this????? Please explain ---aren"t there some rules like same species, same diluents, etc?


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I've made up antibody cocktails for cytokeratin by using a cocktail with a final dilution of CAM5.2 1:8 and AE1/AE3 1:200.  Note I said FINAL dilution.  When used separately, we would use 1:8 and 1:200 respectively on our tissue sections.  Were we to use equal parts of the two, our final dilutions would be CAM5.2 at 1:16, and AE1/AE3 at 1:400.  I would make it up using the following recipe (for 2 ml total):
 
250 microliters CAM5.2 (BD predilute)
1740 microliters antibody diluent
10 microliters AE1/AE3 (concentrated Ab)
 
They were both mouse anti-human antibodies, so cross-species reactivity was no problem.  Biocare Medical has done a great job of providing commercially available cocktails.  They're really helpful for the screening process, in reducing the occurrences of "false" negatives, because you're targeting more antigens at one time. We would only charge for 1 antibody stain, because it was done on 1 slide.  Reporting is simple, "Antibody staining with a cocktail of x-antibody and y-antibody yielded...".
 

Teri Johnson
Managing Director Histology Core Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, Missouri  64110
tjj@stowers-institute.org

-----Original Message-----
From: Van Eyck, Deb [mailto:deb.vaneyck@phci.org]
Sent: Monday, October 14, 2002 12:14 PM
To: 'http://ls.cytopathnet.org'
Subject:

Here is another question for all you immuno wizards.  At several of the immuno workshops at the NSH I heard several people say they made up antibody cocktails by mixing equal amounts of optimally diluted antibodies.  (For example HMB45, Tyrosinase and Mel A or Mart 1)---Not too worry proteins seem to like each other!  In the immuno world this just seems a little too easy, plus you can charge for the use of the separate clones?  How would you report this????? Please explain ---aren"t there some rules like same species, same diluents, etc?

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