Do my human mABs bind HIV?

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brix29
brix29's picture
Do my human mABs bind HIV?

Hi,

we got human recombinant antibodies cloned from plasma cells of HIV infected patients.
We like to find out, if they are specific for HIV proteins.

What I have tried so far:
- ELISA coated with recombinant HIV proteins (produced in E.coli) --> no signal
- ELISA coated with lysate of HIV infected cells --> no signal
- Western Blot with Lysate of HIV infected cells --> no signal
- DotBlot with lysate of HIV infected cells --> no signal
- DotBlot with recombinant HIV proteins (produced in E.coli) --> weak signal of single mAB

Positive controlls (HIV serum) were working nicely in all experiments.
Out of security reasons the HIV lysate has been denatured. To me it looks like the antibodies seem to be highly conformational.
I will try to get access to native HIV particles and HIV infected cell lines.
Which method would you prefer for highly conformational (viral) epitopes? Do you have any good protocols?

Thank you very much in advance!

Best regards,
Brix

samm
samm's picture
The only other thing I can

The only other thing I can suggest is using charged beads to crosslink your HIV prtns - (non-denatured if you can get it, but try it both ways) - the bead format sometimes works better for conformation specific Abs.
Crosslinking the non-denatured HIV lysate will also make it safer to handle.
I'll check around at CFAR/CHAVI to see if anyone has a workaround.

brix29
brix29's picture
Hey Samm,

Hey Samm,

thank you very much for your quick reply. I think this is a very good option. I will ask my collaborator to incubate the beads with HIV particles. The infection risk should then be pretty low and we could handle the beads in our lab.
Can you recommend a product? We generally use the Dynabeads System.

If you get any other tips from CFAR/CHAVI, it would be great!

Best regards,
Brix

samm
samm's picture
I've used 3 different

I've used 3 different Spherotech beads (size/charge/lattice) in the past: 2 worked very well, one didn't.
 I'll try and dig out the cat #s. I just use the Dynal mag beads - don't really have much experience with Dynabeads.

samm
samm's picture
I believe GE or Perkin Elmer

I believe GE or Perkin Elmer have come up with some new bead for native protein crosslinking too - it was in some journal I read a couple of weeks back. More digging!

brix29
brix29's picture
Hey Samm,

Hey Samm,

thanks again for your help. I tried to find the "native coupling" beads you were talking about, but I haven't been successful so far.
Looking forward to your reply!

Best regards,
Axel