I'm afraid it comes down to trial and error.

First off, we keep certain variables constant. These are things we want
to stay the same when we run the antibody with others in a run. We keep
the detection system the same (as much as possible) the same for all
antibodies for instance.

You will need to then devise a method in which you can test several
variables without changing more than one variable per slide.  We use a
cross-match method in which we do several variations in the same run.

For instance, one one set of slides we keep the dilution the same but
change the pretreatment. On another set of slides we have one
pretreatment but change the dilution. On any run it probably is not
worthwhile to test more than two or maybe three variables since the
number of slides increases dramatically with each variable introduced.

For dilutions we will start with higher concentrations ( 1:20, 1;50,
1:100) and work our way up.

As we gain information about the antibody we fine tune the variables.



Tim Morken, B.S., EMT(MSA), HTL(ASCP)

Infectious Disease Pathology

Centers for Disease Control

MS-G32

1600 Clifton Rd.

Atlanta, GA 30333

USA



email: tim9@cdc.gov

       timcdc@hotmail.com



FAX:  (404)639-3043

----Original Message Follows----
Date: Sat, 09 Jan 1999 10:39:15 -0800
From: Heike Grabsch <h.grabsch@uni-koeln.de>
Subject: antibody testing
To: histonet@pathology.swmed.edu

I hope I will get some input on the following problem:

I got several monoclonal antibodies against different epitopes of the =

VEGF-receptor which were produced by another lab and tested on frozen =

human tissue sections. I have not seen the slides myself. Now, they want
=

our lab to test those antibodies on paraffin embedded material. On
frozen =

sections they used a concentration of 5 =B5g/ml with the Vectastain =

ABC-Kit.

My question is: is there a reasonable way or strategy how to test the =

antibodies or is it just trial and error? As I have no idea which =

concentration or which potential pre-treatment could be succcessful I am
=

not sure where I should start.

Any suggestions are highly appreciated.

thanks for your help,

Heike Grabsch, Germany









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