Non-Specific Ab binding

[Anyname]

The goal:

The goal is to detect HIV-1 Gag and Env proteins being expressed 
from a plasmid which is being transfected into HeLa and HeLa-pTev 
(stably expresses HIV-1 Tev protein) cell lines.  The method of 
detection being used is western blotting and enhanced 
chemoluminescence (ECL; [Amersham]).  The primary Ab used is a 
pooled sera from HIV-infected patients.  The secondary Ab is 
anti-Human IgG-POD (Boehringer-Mannheim).

The problem:

The human sera appears to be picking up HeLa cellular proteins in 
general.  This has been observed with plain HeLa cells, as well, 
when they are used as negative controls.  There is no background 
on the nitrocellulose membrane itself.  The membrane is blocked 
prior to Ab incubation with 5% dried milk in PBS-1% Tween20.  Ab 
have been incubated using PBS-Tween20 alone and with 5% dried 
milk.  Results were the same with both.  Different Ab titres have 
been tried as well.  Also, there is no evidence that the proteins 
of interest are being detected at all or they at least don't show 
up through the background.  We know that they are there because 
p24 ELISA's are positive and mRNA has been detected by RT-PCR. 

The positive control consists of lysate from HIV-1-infected MT4 
cells.  For these, when run in tandem with the above test samples, 
gives no non-specific background and the HIV-1 p24 and p17 
proteins are clearly evident.

Suggestions?:

1.  A suggestion that was given was to pre-adsorb the sera with 
HeLa protein lysate.  Aside from the protocol in Maniatis, are 
there any other protocols that you have tried?  Our preliminary 
results are not promissing.

2.  We have p24-, p17, and gp120-specific Ab (from NIH through 
NIAID), however, the secondary Ab is on backorder for one month.  
Has anyone used Ab from NIH with any success?  We have heard that 
they aren't that great.  I can give Cat. numbers if necessary.

3.  Personnel at the Ontario Ministry of Health HIV lab have told 
us that the detection of gp120 is practically impossible with 
western blotting unless the protein is actually purified.  Our 
lab's past experience was not promissing.  Any 
experience/suggestions on this or on how to detect Env proteins.  
We are currently trying immunoflourescence with the above gp120 
Ab.

All replies will be greatly appreciated and credit will be given 
where necessary.

Thank you.

Rocco Lombardi
Dept. of Microbiology
U. of Toronto
r.lombardi at utoronto.ca