TY - JOUR
T1 - Development of a non-denaturing electrophoresis system for characterization of neutralizing epitopes on HPV virus-like particles
AU - Studentsov, Yevgeniy Y.
AU - Burk, Robert D.
N1 - Funding Information:
This work was supported in part from NIH grant CA78527 (RD Burk) and NIH training grant T32AI07506 (Y.Y. Studentsov). The authors thank Dr. N. Christensen (Penn State University) and Dr. Richard Roden (The Johns Hopkins University) for providing monoclonal antibodies for this study. We also thank Dr. Richard Stockert (Albert Einstein College of Medicine) for critically reading the manuscript.
PY - 2007/2
Y1 - 2007/2
N2 - The precise structure of the HPV16 major neutralizing epitope recognized by H16.V5 monoclonal antibody is unknown. This paper describes a novel polyacrylamide gel electrophoresis (PAGE) for separation of HPV virus-like particles (VLPs) using cetyltrimethylammonium chloride (CTAC) as a solubilizing agent. CTAC PAGE employs KOH/CH3CO2H (pH 4-5.4) as a buffer system, K+ as the leading ion and 3-aminopropionic acid as a trailing ion. The unique characteristics of a cationic electrophoresis system allow separation of VLPs without heat denaturation. HPV VLP gel migration patterns were dependent on pre-treatment conditions: (1) thiol-agent reduction alone resulted in a 174 kDa band (interpreted as a L1 trimer), a 53 kDa band (size of the L1 monomer), as well as higher Mr aggregates consistent with a pentamer size; (2) both heat denaturation and thiol-agent reduction resulted in a 53 kDa band. Western blot analysis showed that the 174 kDa L1 trimer was strongly immunoreactive with H16.V5 and HPV16 VLP ELISA positive human sera, whereas no reactivity was seen with the monomeric L1 unit. These data suggest that a structure consistent with the migration pattern of a L1 trimer contains the major neutralizing epitope recognized by the H16.V5 MAb and human sera.
AB - The precise structure of the HPV16 major neutralizing epitope recognized by H16.V5 monoclonal antibody is unknown. This paper describes a novel polyacrylamide gel electrophoresis (PAGE) for separation of HPV virus-like particles (VLPs) using cetyltrimethylammonium chloride (CTAC) as a solubilizing agent. CTAC PAGE employs KOH/CH3CO2H (pH 4-5.4) as a buffer system, K+ as the leading ion and 3-aminopropionic acid as a trailing ion. The unique characteristics of a cationic electrophoresis system allow separation of VLPs without heat denaturation. HPV VLP gel migration patterns were dependent on pre-treatment conditions: (1) thiol-agent reduction alone resulted in a 174 kDa band (interpreted as a L1 trimer), a 53 kDa band (size of the L1 monomer), as well as higher Mr aggregates consistent with a pentamer size; (2) both heat denaturation and thiol-agent reduction resulted in a 53 kDa band. Western blot analysis showed that the 174 kDa L1 trimer was strongly immunoreactive with H16.V5 and HPV16 VLP ELISA positive human sera, whereas no reactivity was seen with the monomeric L1 unit. These data suggest that a structure consistent with the migration pattern of a L1 trimer contains the major neutralizing epitope recognized by the H16.V5 MAb and human sera.
KW - Cationic detergent
KW - Conformational-dependent epitopes
KW - Gel electrophoresis
KW - Human papillomavirus
KW - Neutralizing antibodies
KW - Virus-like particles
UR - http://www.scopus.com/inward/record.url?scp=33846034806&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33846034806&partnerID=8YFLogxK
U2 - 10.1016/j.jviromet.2006.10.002
DO - 10.1016/j.jviromet.2006.10.002
M3 - Article
C2 - 17137641
AN - SCOPUS:33846034806
SN - 0166-0934
VL - 139
SP - 208
EP - 219
JO - Journal of Virological Methods
JF - Journal of Virological Methods
IS - 2
ER -