HELP: Viral hemagglutination

[Graeme Price]

In article <44aafn$gvr$1 at mhadf.production.compuserve.com>, George M.
<75763.12 at CompuServe.COM> wrote:

> Could anyone recommend a recent reference with excellent methods
> outlining the standardized viral hemagglutination assays utilizing
> the microtiter technique as developed by Hierholtzer (1969). 
> 
> Technical advise is also welcome.
> 
> Thank you in advance.
> 
> -- 
> George M. 

For our purposes (HA assays of influenza A virus), we use 1% human
erythrocytes in 0.85% saline. It is important to remove all the serum and
buffy coat, as serum contains non-specific inhibitors of HA - this is
easily done by spinning the whole blood and washing it in saline 3-4
times. We use spun haematocrits to estimate the final erythrocyte
concentration.

The virus is double diluted in saline across the rows of a V or U bottom
(not flat bottom) microtitre plate to give 50 microlitres in each well. We
always set up replicates of each dilution (this is easily done in the
plate using a multichannel pipette). An equal volume of erythrocytes is
then added and the plate is agitated briefly. The cells are then left to
settle for about 45 minutes before reading. Some workers suggest that HA
assays for influenza are best left at 4 C whilst waiting for the cells to
settle (viral neuraminidase can free the virus from the cells otherwise),
but it depends on the NA activity of the virus (I have never had any
problems with doing the assay at room temperature for the strains I use). 

Hope this is of some use to you (most of the points above are pretty
obvious though).

Regards

Graeme

-- 
Graeme Price
Microbial Molecular Genetics and Cell Biology Group, 
School of Biological Sciences, Biology West Building,
University of Birmingham,
Edgbaston, Birmingham,
West Midlands, B15 2TT.
United Kingdom.

Tel. (+44) (0)121 414 6555
Fax. (+44) (0)121 414 6557
E-mail g.e.price at bham.ac.uk