In article <342C89BF.503D at pacbell.net>, Mike O'Hara
<URL:mailto:mjohara at pacbell.net> wrote:
> Colleagues
>> I am looking for a protocol or reference to a protocol that would allow
> me to couple specific antibodies to red cells to be used in a
> haemagglutination assay. Ideally it will include a step that tans the
> red cells so that they last longer and [really asking for too much
> but..] will have methods for validation in particular sensitivity and
> cross-reaction..
>> Help greatly appreciated.
>> Mike O'Hara
>
I would suggest you look at papers by Robin Coombs (R.R.A. Coombs). He
published numerous assays based on red cell agglutionation, including
methods for fixation, stabalization and freeze-drying of coupled red cells.
I had the pleasure of collaborating with him on a couple of projects see
Clark,M.R., Cobbold,S.P., Waldmann,H., & Coombs.R.R.A. (1984), J. Immunol.
Meth. 66, 81-87 Detection of monoclonal antibodies against cell surface
antigens: the use of antiglobulins coupled to red blood cells.
Welsh,C.J.R., Steinitz,M., Clark,M.R., & Coombs,R.R.A. (1987), J. Immunol.
Meth. 104, 271-274 The detection of heat-aggregated IgG (as a model for
immune complexes) by reverse passive haemagglutination using a human
monoclonal rheumatoid factor coupled to erythrocytes.
The major method used for coupling of proteins was based on Chromic
Chloride and fixation was usually with a mild glutaraldehyde treatment. A
certain amount of "cookery" skills are needed to get the methods to work
reproducibly but the assays are incredibly sensitive and have many
advantages (but also some disadvantages) over ELISA assay systems.
Mike Clark, <URL:http://www.path.cam.ac.uk/~mrc7/>
--
o/ \\ // || ,_ o M.R. Clark, PhD. Division of Immunology
<\__,\\ // __o || / /\, Cambridge University, Dept. Pathology
"> || _`\<,_ // \\ \> | Tennis Court Rd., Cambridge CB2 1QP
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