Use of Luminex xMAP-derived Bio-Plex bead-based suspension array for specific detection of PPV W and characterization of epitopes on the coat protein of the virus.
A panel of monoclonal antibodies (MAbs) directed against the N-terminus region of the coat protein (CP) of strain PPV W (isolate 3174) was generated by immunizing mice with recombinant peptides. The best performing MAbs were identified as 2C3 and 10G7. MAb 2C3 was selected for comparison of a standard TAS-ELISA protocol with a Luminex xMAP technology-derived bead-based suspension array system described as a triple antibody sandwich-microsphere immunoassay (TAS-MIA). TAS-MIA was as sensitive as TAS-ELISA for the specific detection of PPV W in herbaceous and woody hosts. It was completed in 4h, and used less reagents. Epitope recognition analysis was carried out using a set of overlapping synthetic pin-bound peptides (Mimotopes). Peptides (2)DEEDD(6) and (46)MFNPV(50) were the epitopes recognized most commonly by the best performing MAbs. Linear epitope prediction of B-cell recognition sites confirmed that both peptides fall within highly antigenic and accessible regions. The second glutamic acid residue of the epitope is crucial for MAb recognition, and the context of the epitope is as important as the sequence of the epitope. The results obtained in ELISA, Western blot, and TAS-MIA correlated with B-cell recognition prediction. This is an effective approach to identify suitable antigenic epitopes that generate antibodies for use in reliable diagnostic procedures. This is the first report of the detection of a plant virus using the Luminex xMAP bead-based suspension array system.
Croft
Heather
H
Sidney Laboratory, Canadian Food Inspection Agency, 8801 East Saanich Road, Sidney, British Columbia, Canada V8L 1H3.
Malinowski
Tadeusz
T
Krizbai
Laszlo
L
Mikec
Ivan
I
Kajic
Vesna
V
Reed
Christopher
C
Varga
Aniko
A
James
Delano
D
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D036022:Capsid Proteins / Q000737:chemistry / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D018985:Epitopes, B-Lymphocyte / Q000737:chemistry / Q000276:immunology; D007118:Immunoassay / Q000379:methods; D051379:Mice; D008863:Microspheres; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D017801:Plum Pox Virus / Q000276:immunology / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D036022:Capsid Proteins; D018985:Epitopes, B-Lymphocyte; D010455:Peptides; D011994:Recombinant Proteins; C095320:coat protein, Plum pox virus