Characterization of phage peptide interaction with antibody using phage mediated immuno-PCR.
Real-time immuno-PCR (RT-IPCR) is a powerful technique that combines ELISA with the specificity and sensitivity of PCR. RT-IPCR of phage-displayed peptides exploits the unique physical associations between phenotype (the displayed peptide) and genotype (the encoding DNA) within the same phage particle. Previously, we identified phage peptides specific for recombinant antibodies (rAbs) prepared from clonally expanded plasma cells in multiple sclerosis (MS) cerebrospinal fluid (CSF) and subacute sclerosing panencephalitis (SSPE) brain. Herein, we applied phage-mediated RT-IPCR to study reactivity of these specific phage peptides for the rAbs. Compared to standard ELISA, which required greater than 10(4) or 10(5) phage particles to detect binding to rAbs, RT-IPCR detected binding with as few as 100 phage particles. RT-IPCR was also superior to ELISA in determining relative affinities of rAbs for phage peptides and was effective in screening MS CSF for IgG reactivity to phage peptides. Phage-mediated RT-IPCR is a rapid, high-throughput technology that avoids the requirement for synthetic peptides and will facilitate the identification of candidate peptides that react with the IgG in MS CSF.
Yu
Xiaoli
X
Department of Neurology, University of Colorado Health Sciences Center, 4200 E. 9th Avenue, Mail Stop B182, Denver, Colorado 80262, USA. xiaoli.yu@uchse.edu
Burgoon
Mark P
MP
Shearer
Andrew J
AJ
Gilden
Donald H
DH
D001323:Autoantibodies / Q000235:genetics / Q000378:metabolism*; D017104:Bacteriophage M13 / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis / Q000134:cerebrospinal fluid; D009103:Multiple Sclerosis / Q000134:cerebrospinal fluid / Q000276:immunology; D010455:Peptides / Q000276:immunology / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D025941:Protein Interaction Mapping; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000276:immunology / Q000378:metabolism*
D001323:Autoantibodies; D007074:Immunoglobulin G; D010455:Peptides; D011994:Recombinant Proteins; D014764:Viral Proteins