Establishing Rapid And Sensitive Fluorescence Immunoassay For Fruit Tree Virus Detection Using Bacterial Magnetic Particles

The effect of incubation factors on immobilization efficiency of antibodies, using goat anti-rabbit IgG as an illustration, onto bacterial magnetic particles (BMPs), focusing on the concentration of immunoglobulin G (IgG) and BMPs, buffer selection and its suitable conditions for the conjugation, and the other incubation factors such as, temperature and duration, shaking or stationary condition, BMPs quality, freeze-dry treatment were carried out in this study. New antigen detection method was established by using BMPs while rabbit IgG was applied as a sample, and later on the established detection method used for determination fruit tree virus- Prunus necrotic ring spot virus (PNRSV) and grape fanleaf virus.The resultant data showed that, many factors could influence goat anti rabbit IgG (antibody) immobilized onto BMPs; different linkage rate of antibody (LRA) had varied antigen (rabbit IgG) detection sensitivity; high LRA can obtained high detection sensitivity; and the BMPs based fluorescence assay had much higher detection sensitivity than the method of ELISA in the detection of rabbit IgG and fruit tree virus.Many buffers can be the conjugation buffers for goat anti-rabbit IgG onto BMPs. 12 buffers tested under the normal buffer concentration and pH, the LRAs obtained in the range of 35-95 μg/mg. The selected three buffers, 2 organic (Tris-Cl and HEPES) and 1 inorganic (Na3PO4) were used to investigate the effect of buffer pH and concentration on immobilization efficiency. The pHs and concentrations were different when the LRAs reached the peak value. The suitable pH and concentrations were pH 5.6, 1-5 mM for buffer Na3PO4; pH 5.6, 10-20 mM for Tris-Cl; and pH3.5, 20 mM for HEPES. Therefore, the lower pH benefits LRA, and the concentration of organic buffer was higher than inorganic buffer. Temperature was also affect on LRA, which could reach the peak point faster with higher temperature when 4℃ to 37℃ were used. High quality with appropriate amount of BMPs and enough amount of antibody quantity would lead high LRA. When 0.1 mg of BMPs and 300 μg of antibody mixed into 1 ml of conjugation buffer, it resulted the LRA of 762.6 μg /mg. Freeze-dry BMPs caused LRA decreased, and different pre-treatment of freeze-dry BMPs had significant difference of LRA, which was much higher of pre-treatment in liquor nitrogen than that in -70℃ and -20℃. Resuspending or shaking the conjugation reactants could accelerate the LRA peak appeared, and much higher than that in a stationary stage.Comparing to the different detection methods of ELISA and BMPs based fluorescence assay, the later one had much higher detection sensitivity. The detection sensitivity of the later method was 10 times higher than the former one for rabbit IgG detection and fruit tree virus determination.