Magnetic Beads-based Enzymatic Spectrofluorometric Immunoassay For The Detection Of Antibody Against ApxIVA Of Actinobacillus Pleuropneumoniae

Porcine Contagious pleuropneumonia is caused by Actinobacillus pleuropneumoniae (APP), belonging to one of pig’s three kinds respiratory diseases, which has broken out in the vast majority of countries and regions, causing huge economic losses of swine industry. Once the pigs were infected with this disease, the acute mortality is up to80%or more. When it became chronic infection, the growth of pigs would be seriously affected. Moreover, the survived pigs have been to pathogen carriers, the disease maybe broke out again under the appropriate conditions. Therefore, in order to effective prevention and control of the outbreak of the disease, the detection, especially early diagnosis has been of great significance.Magnetic particles have been widely studied since the1970s. Because it has own a large surface area, small size (10nm-100μm), bio-affinity and superparamagnetic characteristics, which make it been used as an effective solid-phase carrier for the immunoassay, purification of biological molecules and cell separation in the biological field. In immunoassay aspect, due to the good ability for capturing antibody (antigen) and the immune reaction in three-dimensional space, the disease diagnosis based magnetic bead have a good sensitivity, rapid separation, easy to operate and low cost, making it get the attention of researchers.In this experiment, we have used magnetic beads as a carrier and APP specific exotoxin protein ApxIVA coming from E. coli recombinant expression as the antigen, to build a simple, low cost, easily-operated and enzyme-amplified fluorescence immunoass-ay method using magnetic particles for the detection of antibody against ApxIVA of Actinobacillus pleuropneumoniae. First, ApxIVA was immobilized onto the magnetic bead surfaces. Second, the serum infected with APP is then added to induce primary immunorecognition, followed by Horseradish peroxidase conjugated Rabbit anti-pig IgG antibodies (HRP-IgG) to initiate the second immunorecognition event. Horseradish peroxidase (HRP) can catalyze the substrate4-hydroxyphenylacetic acid (p-HPA) generating fluorescent bi-p, p’-hydroxyphenylacetic acid (DBDA). The ApxIVA antibody was then 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 range1:160-1:5120. The limit of detection (LOD) for the assay was1:10240, considerably lower than that of ApxIVA-ELISA (1:320)(S/N=3). A series of repeatability measurements of using1:320-fold diluted standard positive serum gave reproducible results with a relative standard deviation (RSD) of4.8%(n=11). The ability of the immunosensor to analyze clinical samples was tested on porcine sera. The immunosensor yielded an efficiency of89.7%, sensitivity of90.9%and specificity of89.3%compared with ApxIVA-ELISA.