Hybridoma cell strain, type 2 short nude-dinoflatoxin monoclonal antibody secreted by hybridoma cell strain and application of antibody
A hybridoma cell line and monoclonal antibody technology, applied in the field of immunology, can solve the problems of insufficient sensitivity of nucleic acid aptamers, immature immune analysis, and unstable structure
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Embodiment 1
[0028] The preparation of embodiment 1 hapten
[0029] Take 2mg of BTX-2 standard substance, add 5mL of pyridine to dissolve it, and raise the temperature to 90°C;
[0030] Weigh 1 mg of CMO, dissolve it with 3 mL of pyridine, then drop it into the aforementioned BTX-2 standard solution, and react with magnetic stirring for 6 hours;
[0031] The reaction solution was evacuated and dried by rotary evaporation, the pyridine solution was removed, and 10 mL of 0.1M NaHCO was added 3 , to dissolve the residue; extract with 5mL ethyl acetate, discard the organic phase ethyl acetate layer, use 1M hydrochloric acid to adjust the pH to about 3 in the aqueous phase, extract 3 times with 5mL ethyl acetate, combine the organic phase ethyl acetate, ethyl acetate The ester is blown dry with nitrogen to obtain the hapten BTX-2-CMO, see the chemical reaction equation figure 1 .
[0032] Take 2mg of BTX-2 standard substance, add 5mL of pyridine to dissolve it, and raise the temperature to 9...
Embodiment 2
[0035] The preparation of embodiment 2 antigen
[0036] The hapten BTX-2-CMO was dissolved in 0.5mL DMF, then EDC 2mg, NHS 1.2mg were added, and the reaction was stirred at room temperature for 4h. Weigh 5 mg of KLH (BSA 10 mg) and dissolve it in 5 mL of 0.1M PBS (pH 7.4), then drop the activated BTX-2-CMO into the carrier protein solution, and stir for 6 hours at room temperature. Afterwards, the PBS was dialyzed for 3 days to obtain the antigen BTX-2-CMO-KLH / BSA, which was aliquoted and stored at -20°C.
[0037] The hapten BTX-2-HS and the carrier protein BSA were synthesized by the active ester method. As mentioned above, PBS was dialyzed for 3 days to obtain the coated original BTX-2-HS-KLH / BSA, which was stored at -20°C .
[0038] The immunogens BTX-2-CMO-KLH and BTX-2-HS-KLH were identified by UV scanning, see image 3 , the maximum absorption wavelengths of BTX-2-CMO-KLH and BTX-2-HS-KLH are evenly shifted compared with the carrier protein KLH, indicating that the an...
Embodiment 3
[0040] The preparation of embodiment 3 monoclonal antibody
[0041] 3.1 Immunization of mice
[0042]Six 6-week-old Balb / C female mice were immunized with the immunogens BTX-2-CMO-KLH and BTX-2-HS-KLH prepared in Example 2. The immunization program used one basic immunization and two booster immunizations, and the interval between immunizations was 21 days. Eight days after the third immunization, the tail blood was collected, the serum was separated, the serum antibody titer was detected by indirect ELISA, and the specificity of the serum was detected by indirect competition ELISA. The coating was originally BTX-2-HS-BSA, and the dilution factor was 500 times. After coating, the mouse serum was diluted 500 times for measurement. The results are shown in Table 1 and Table 2. It shows that the BTX-2-CMO-KLH immunized mice obtained better immunity. According to the inhibition of the standard substance, the No. 6 mouse was selected for fusion. However, the BTX-2-HS-KLH immuniz...
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