Is there a way to deliver antibodies, or their fragments, into viable cells?

We have heard of using liposomes, but wondered if perhaps newer techniques have emerged.

Thanks.

Ray Hester

Univ. of South Alabama

Mobile, AL 36688

rhester@jaguar1.usouthal.edu

I. Active Motif (www.activemotif.com) sells a transfection reagent called Chariot designed to transmit biologically active proteins and peptides into living cells. Delivering antibodies is one of their advertised applications. I have never tried this reagent, nor have any ties to the company, but you might want to check it out.

Dr. Wesley M. Garrett, Ph.D.

Germplasm and Gamete Physiology Lab

USDA/ARS/ANRI

Building 200, Room 101 A

Beltsville, MD 20705

E-mail: wgarrett@anri.barc.usda.gov

Phone: 301-504-7413

FAX: 301-504-5123

II. Antibodies can be introduced into "live" semi-permeabilised cells using the bacterial toxin Streptolysin-O. I have used this toxin to label "live" malarial parasites with fluorescently labeled antibodies, and believe that it can be used to label other cell types.

You may want to have a look at the following web site (http://www.liv.ac.uk/~giles/theory/slo_perm.htm), where Streptolysin-O is used to get oligonucleotides into live cells.

Dr. Alan R. Hibbs

FB Biologie/Zoologie, Philipps University

D-35039 Marburg, Germany.

Phone: 49 6421 282 3404 FAX: 49 6421 282 1543

BIOCON (publishers of the manual "Confocal Microscopy for Biologists",

mailto:orders@biocon.com.au)

7 Walhalla Drive Phone: 61 3 9876 9822 FAX: 61 3 8660 2290

Ringwood East VIC 3135 Director: Dr. Alan R. Hibbs

Australia

III. Or an older method - microinjection!

There is also something called "Chariot" that gets proteins into cells,

but it may be liposome-based. I think it is from Active Motif.

Two other methods if you just need to get your stuff into the cytoplasm, both "tried & true" - physical loading via glass beads or trituration.

Tamara

On Thu, 21 Mar 2002, ray hester wrote:

> Search the CONFOCAL archive at

> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

>

Tamara Howard

Department of Cell Biology and Physiology

University of New Mexico - Health Sciences Center

Albuquerque, NM 87131

thoward@unm.edu

|--------------------------------------------------|

 
IV. One more for the list/worth trying: - not used it personally - a
variation of hypotonic shock. Molecular Probes offers a pinocytotic
loading solution: you expose the cells to a concentrated solution of the molecule you wish to load, which is then taken up by pinocytotsis. The pinocytotic vesicles are then lysed in a hypotonic solution, releaseing the contents to the cytoplasm. Looks neat, but may not work for all cell types obviously.
 
>< >< >< >< >< >< >< >< >< >< >< >< ><
Dr Ian S. Harper, Director
Monash Micro Imaging
The Microscopy & Imaging Research Facility
Building 13C, Monash University,
Clayton, VIC 3800, Australia
 
Email: Ian.Harper@med.monash.edu.au
Tel: +61 3 9905 5635; Fax: +61 3 9905 2733
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General enquiries: microscopy@med.monash.edu.au

V. Hi,
 
We used electorporation with good results several years ago. The cells were in suspension, but I don't know why it could not be adapted to adherent cells.
 
Berglund and Starkey
J. Imm. Meth, 125 (1989) 79-78
 
________________________________________________________
V. Hi Ray,
A colleague passed your question from the cytometry listserve on to me and I thought I'd reply with a novel method I came across recently. Feel free to post it. In a recent Nature Biotechnology article (Morris MC, Depollier J, Mery J, Heitz F, Divita G. A peptide carrier for the delivery of biologically active proteins into mammalian cells. Nat Biotechnol. 2001 Dec;19(12):1173-6.) they linked a couple of different Abs to a Pep1 fragment, a protein transduction domain, and successfully got them into cells. High efficiency, low toxicity.
Regards,
Mark
 
 
C. Mark Lies, Ph.D.
Technical Marketing Scientist
Miltenyi Biotec, Inc.
12740 Earhart Ave.
Auburn, CA 95602
 
Toll free: (800)367-6227
Direct: (530)887-5365
mlies@miltenyibiotec.com
www.miltenyibiotec.com

VI. Greetings,
 
Below is one protocol suggested for the introduction of antibodies into viable cells that wasn't posted to the entire list:
 
............................................................................
............
might try sterile saponin solutions
 
 
Saponin Method: Permeabilization REAGENT for Paraffin sections & Itracellular Cytokine staining
 
Dulbecco's PBS (without Mg2+ or CA2+)
1% heat inactivated FCS (or BSA-do not use animal serum with  Biotin detection systems due to endogenous biotin levels)
0.1% w/v sodium azide
0.1% w/v saponin (Sigma cat#S-7900)
Adjust pH buffer to 7.4-7.6 and filter
 
see ref:
1)Sander, B., J. Andersson and U. Andersson, 1991:Assessment of cytokines by immunofluorsecence and the paraformaldehyde-saponin procedure:Immunol Rev 119:65-93
 
2) Anderson, U. and J. Andersson 1994. Immunolabelling of cytokine producing cells in tissues and suspension. In Cytokine Producing Cells eds. D. Fradelizie and D. Emelie. INSERM, Paris. 32-49.
 
 
for intracellular cytokines, stop golgi process with with either monesin (Sigma cat#M-5273) or Brefeldin A (Sigma cat#B-7551)
 
-ref#Nature 373:255-257
 
-Prussin C and Metcalfe DD. Detection of Intracytoplasmic Cytokine Using Flow Cytometry and Directly Conjugated anti-Cytokine Antibodies.  Journal of Immunological Methods, 188, 117-128 (1995).
 
 
-J. Immunol Meth 159:197-207
 
see antibodies at: http://www.researchd.com/absort.htm
 
Sincerely,
Research Diagnostics Inc
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