title: 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.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Wei
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Li
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Huicui
authors.initials: H
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Kaihong
authors.initials: K
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China. Electronic address: yongganghu@mail.hzau.edu.cn.
authors.forename: Yonggang
authors.initials: Y
authors.lastname: Hu
mesh_terms: D000189:Actinobacillus Infections / Q000175:diagnosis / Q000662:veterinary; D016977:Actinobacillus pleuropneumoniae / Q000276:immunology*; D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis* / Q000097:blood; D001426:Bacterial Proteins / Q000276:immunology*; D015374:Biosensing Techniques / Q000379:methods* / Q000706:statistics & numerical data; D004797:Enzyme-Linked Immunosorbent Assay; D055767:Immobilized Proteins; D007118:Immunoassay / Q000379:methods; D057230:Limit of Detection; D008280:Magnetics; D013050:Spectrometry, Fluorescence / Q000379:methods; D034421:Sus scrofa; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis
chemical_list: D000907:Antibodies, Bacterial; C122177:ApxIVA protein, Actinobacillus pleuropneumoniae; D001426:Bacterial Proteins; D055767:Immobilized Proteins