In vivo electroporation and non-protein based screening assays to identify antibodies against native protein conformations.
In vivo electroporation has become a gold standard method for DNA immunization. The method assists the DNA entry into cells, results in expression and the display of the native form of antigens to professional cells of the immune system, uses both arms of immune system, has a built-in adjuvant system, is relatively safe, and is cost-effective. However, there are challenges for achieving an optimized reproducible process for eliciting strong humoral responses and for the screening of specific immune responses, in particular, when the aim is to mount humoral responses or to generate monoclonal antibodies via hybridoma technology. Production of monoclonal antibodies demands generation of high numbers of primed B and CD4 T helper cells in lymphoid organs needed for the fusion that traditionally is achieved by a final intravenous antigen injection. The purified antigen is also needed for screening of hundreds of clones obtained upon fusion of splenocytes. Such challenges make DNA vaccination dependent on purified proteins. Here, we have optimized methods for in vivo electroporation, production, and use of cells expressing the antigen and an in-cell Western screening method. These methods resulted in (1) reproducibly mounting robust humoral responses against antigens with different cell localizations, and (2) the ability to screen for antigen eliminating a need for protein/antigen purification. This process includes optimized parameters for in vivo electroporation, the use of transfected cells for final boost, and mild fixation/permeabilization of cells for screening. Using this process, upon two vaccinations via in vivo electroporation (and final boost), monoclonal antibodies against nucleus and cytoplasmic and transmembrane proteins were achieved.
Department of Microbiology and Immunology, University of Miami, Miami, Florida, USA. pdaftarian@med.miami.edu
Pirouz
P
Daftarian
Raquibul
R
Chowdhury
Philip
P
Ames
Changli
C
Wei
Alan D
AD
King
Juan Pablo
JP
de Rivero Vaccari
Lloye
L
Dillon
Justin
J
Price
Howard
H
Leung
Brittany
B
Ashlock
Enrique
E
Mesri
Victor
V
Perez
Stephan
S
Züchner
Jochen
J
Reiser
Vance
V
Lemmon
Robert W
RW
Keane
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D015153:Blotting, Western / Q000379:methods; D053468:CARD Signaling Adaptor Proteins; D019556:COS Cells; D002522:Chlorocebus aethiops; D003598:Cytoskeletal Proteins / Q000096:biosynthesis / Q000276:immunology; D018274:Electroporation / Q000379:methods; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D007106:Immune Sera; D039841:Leukocyte L1 Antigen Complex / Q000096:biosynthesis / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008840:Microfilament Proteins / Q000096:biosynthesis / Q000276:immunology; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000276:immunology; D010047:Ovalbumin / Q000096:biosynthesis / Q000276:immunology; D011487:Protein Conformation; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis / Q000276:immunology; D055293:Receptors, Urokinase Plasminogen Activator / Q000096:biosynthesis / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D019444:Vaccines, DNA
D000911:Antibodies, Monoclonal; D053468:CARD Signaling Adaptor Proteins; D003598:Cytoskeletal Proteins; D007106:Immune Sera; C485646:KLHL2 protein, human; D039841:Leukocyte L1 Antigen Complex; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; C402202:PYCARD protein, human; D043562:Receptors, G-Protein-Coupled; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; D019444:Vaccines, DNA; D010047:Ovalbumin