In article <3614289A.5A26 at ix.netcom.com>,
todd33 at ix.netcom.com wrote:
> Leonard Pattenden wrote:
>> > I have shown the
> > Virology 230 papers were not about "non-isolation" or it's
> > "impossibility". It was about microvesicle contamination.
>> Leonard, maybe you can help me with a question I've never gotten
> a good answer to. With all this microvesicle contamination that
> comes along with "HIV" when it is purified on sucrose gradients
> (at least until recently, when a paper you know about found that
> protease treatment of the gradient fraction containing "HIV"
> could remove up to 95% of the contaminants, mostly actin), how
> did Dr Gallo generate antibodies to "HIV" in a rabbit back in
> 1984 in the _Science_ papers that made him so famous? There is
> no information about what was used as an antigen, but I think
> it is probably safe to assume that it was density gradient
> purified material, which as one of those recent _Virology_ papers
> shows, is at best half "HIV" and half cellular proteins. The
> coomassie stained PAGE in this paper of the gradient purified
> material is not very impressive either, as far as using this as
> an antigen -- there are proteins that run the entire gamut of
> molecular weights!
>> How could he claim that the antibodies from the rabbit were
> specific for "HIV" given that the antigen could not possibly
> have been pure "HIV" and that apparently, at that time, no one
> had really investigated methods of trying to further purfiy
> "HIV"?
>
It doesn't matter at all whether the vaccination happens with a pure isolate
- it all depends on the selection method that's used to characterize the
resulting antibodies. Immunization with something that 'is at best half
"HIV"', and the rest consisting of cellular material from a human cell line,
will work just fine. Of course you'll generate a lot of B-cell lines
producing antibodies specific for common, cellular, proteins. But in addition
there will be B-cell lines that produce an antibody against HIV-proteins.
Even if these proteins haven't been characterized by molecular cloning, you
can identify the antibodies that recognize them. For instance, you screen the
collection of antibodies you generated against uninfected cells and infected
cells. Immunofluorescence will do the trick. Antibodies specific for common
cellular antigens will bind to both infected and uninfected cell lines.
Antibodies specific for an HIV-encoded protein, for instance the envelope
proteins, will only bind to the infected cell lines.
Frank Raaphorst
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