Monoclonal antibody for myxobolus honghuensis polar filament protein and application thereof
A technology of monoclonal antibody and polar filament protein, applied in the biological field, can solve the problems of hindering the prevention and control strategy of laryngosporidiosis and the lack of species identification technology, and achieve the effect of low cost, high specificity and sensitive response
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Embodiment 1
[0028] Example 1 Preparation of Honghu Iodophora antigen
[0029] 1.1 Separation and purification of spores
[0030] Use scissors to cut open the operculum of the host fish body, and peel off the intact cysts from the pharynx with elbow surgical forceps; wash 3 times with phosphate buffer solution (PBS, pH 7.0), centrifuge at 300 rpm for 5 min; resuspend the cysts in a small amount of PBS Use a glass homogenizer to homogenize the slurry by hand until the slurry is uniform. Centrifuge at 1000 rpm for 20 minutes to collect the precipitate and supernatant; resuspend the precipitate with an appropriate amount of PBS and use a sucrose density gradient (1:1, 1:2, 1:3, 1 :4, 1:5) Centrifuge, 1000rpm, 15min to separate mature spores (between 1:1~1:2); mature spores are washed twice with PBS, 3000rpm, centrifugation for 10min, remove the supernatant, the precipitate is Mature spores.
[0031] 1.2 Preparation of soluble protein
[0032] The separated and purified spores are washed with PBS (p...
Embodiment 2
[0035] Example 2 Preparation of monoclonal antibodies
[0036] 2.1 Animal immunity
[0037] 2.1.1 Animals: female, 5 BALB / c mice aged 6-8 weeks (purchased from Wuhan Institute of Virology, Chinese Academy of Sciences).
[0038] 2.1.2 Antigen: The soluble antigen of Iodophora honghuensis prepared in Example 1, 50 μg / head / time.
[0039] 2.1.3 Immunization route and procedure: First immunization: The soluble antigen of Iodophora honghuensis and the complete Freund's adjuvant of equal volume are mixed thoroughly to form water-in-oil, and injected into multiple points in the back of the mouse. For the second immunization 3 weeks later, the Honghu Iodophora soluble antigen and incomplete Freund’s adjuvant were mixed in equal volumes and injected into the back in multiple points. After immunization every 3 weeks (without adjuvant in the abdominal cavity), 20 μL of tail blood was taken from 5-7 days after immunization, and the antibody titer was measured by ELISA. When the antibody titer re...
Embodiment 3
[0053] Example 3 Identification of monoclonal antibodies
[0054] 3.1 Antibody Subclass
[0055] Use mouse monoclonal antibody subclass detection kit (Sigma) to determine. The goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgA and IgM were diluted 1:1000 with coating solution, coated with 100μL / well, and incubated at 4°C overnight or 37°C for 2h; washed with washing solution PBST 3 times, 3min / Add 100μL of the monoclonal antibody cell culture supernatant to be tested to each well, and incubate at 37°C for 1h; wash 3 times as above, and add 1:5000 diluted horseradish peroxidase labeled goat anti-mouse polyvalent immunoglobulin (G , A, M) Antibody, 100μL per well, incubate at 37°C for 1h; wash 3 times as above, add 100μL of freshly prepared TMB substrate solution to each well, incubate at 37°C for 20min in the dark; add 50μL 2M H to each well 2 SO 4 Stop the reaction. Read OD on the microplate reader 450 For the absorbance value, the subtype category of the positive reaction well is th...
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