[Detection of antibody against Helicobacter pylori UreB by fluorescence polarized immunoassay using single epitope synthetic peptide as antigen].
OBJECTIVE
To develop a new method for antibody detection based on fluorescence polarization (FP) technique.


METHODS
BALB/c mice were immunized with single epitope synthetic 8 branches peptide antigen (SVEVGKVADL)8 of Helicobacter pylori (Hp) UreB protein. FP values of the corresponding linear peptide antigen labeled with FITC in different concentration were measured to determine the optimal concentration of the antigen. The antigenicity of the synthetic linear peptide was identified by FP assay. Then, the antibody-positive and negative murine sera were used in FP assay to determine the optimal dilution factor of serum samples. To apply FP technique in Hp antibody detection, 126 human serum samples were detected either by FPIA (fluorescence polarized immunoassay) using the single epitope linear synthetic peptide as antigen or by a commercial ELISA kit. Then, receiver operating characteristic (ROC) curve analysis was performed on the FPIA results by MedCalc software.


RESULTS
The UreB single epitope peptide had strong antigenicity. 1.0 nmol/L of the synthetic linear peptide labeled with FITC was the optimal concentration of the antigen and the optimal dilution factor of serum sample was 1:25. In 77 Hp antibody-positive serum samples detected by ELISA, 66 samples were positive by FP detection method. The sensitivity and specificity of the FPIA assay was 85.7% and 98%, respectively.


CONCLUSIONS
The single epitope synthetic peptide antigen can be used in FP method for rapid detection of serum antibody against Hp UreB. The established FP assay for antibody detection may be used in clinical diagnosis in the future.
Zhang
Wen-hong
WH
Institute of Genetic Diagnosis, Fourth Military Medical University, Xi'an 710032, China. lifesong@fmmu.edu.cn
Wang
Yan
Y
Guo
Hui-fang
HF
Bai
Yu-jie
YJ
Yan
Xiao-jun
XJ
D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis* / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology* / Q000378:metabolism; D001426:Bacterial Proteins / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology* / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D016231:Fluorescence Polarization Immunoassay / Q000379:methods*; D016480:Helicobacter pylori; D016014:Linear Models; D051379:Mice; D010455:Peptides / Q000138:chemical synthesis* / Q000276:immunology / Q000378:metabolism
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D000939:Epitopes; D010455:Peptides; D016650:Fluorescein-5-isothiocyanate