Magnetic beads-based enzymatic spectrofluorometric assay for rapid and sensitive detection of antibody against ApxIVA of Actinobacillus pleuropneumoniae.

Abstract

In this paper, a simple, easily-operated and enzyme-amplified fluorescence immunoassay method using magnetic particles for the detection of antibody against Actinobacillus pleuropneumoniae (APP) has been presented. The A protein of APP Repeats-in-Toxin IV (ApxIVA) with high specificity to the APP species was immobilized onto the magnetic bead surfaces. Horseradish peroxidase (HRP), which can catalyze the substrate 4-hydroxyphenylacetic acid (p-HPA), generating fluorescent bi-p, p'-hydroxyphenylacetic acid (DBDA), was selected as an enzymatic-amplified tracer. The ApxIVA antibody was detected for the presence of APP infection by measuring the fluorescence intensity of DBDA. Under optimal conditions, the calibration plot obtained for standard positive serum was approximately linear within the dilution range 1:160-1:5120. The limit of detection (LOD) for the assay was 1:10240, considerably lower than that of ApxIVA-ELISA (1:320) (S/N=3). A series of repeatability measurements of using 1:320-fold diluted standard positive serum gave reproducible results with a relative standard deviation (RSD) of 4.8% (n=11). The ability of the immunosensor to analyze clinical samples was tested on porcine sera. The immunosensor yielded an efficiency of 89.7%, sensitivity of 90.9% and specificity of 89.3% compared with ApxIVA-ELISA.

• Authors: Bo Wei, Fang Li, Huicui Yang, Lei Yu, Kaihong Zhao, Rui Zhou, Yonggang Hu
• Journal: Biosensors & bioelectronics (Biosens Bioelectron) Vol. 35 Issue 1 Pg. 390-393 (May 15 2012) ISSN: 1873-4235 [Electronic] England
• PMID: 22503209 (Publication Type: Comparative Study, Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2012 Elsevier B.V. All rights reserved.

Chemical References

• Antibodies, Bacterial
• ApxIVA protein, Actinobacillus pleuropneumoniae
• Bacterial Proteins
• Immobilized Proteins

Topics

• Actinobacillus Infections (diagnosis, veterinary)
• Actinobacillus pleuropneumoniae (immunology)
• Animals
• Antibodies, Bacterial (analysis, blood)
• Bacterial Proteins (immunology)
• Biosensing Techniques (methods, statistics & numerical data)
• Enzyme-Linked Immunosorbent Assay
• Immobilized Proteins
• Immunoassay (methods)
• Limit of Detection
• Magnetics
• Spectrometry, Fluorescence (methods)
• Sus scrofa
• Swine
• Swine Diseases (diagnosis)