Gobrocypris rarus vitellogenin monoclonal antibody and application thereof
A technology of vitellogenin and rare crucian carp, applied in application, anti-animal/human immunoglobulin, instruments, etc., can solve the problems of large individual differences, limited quantity, difficult to repeat preparation, etc., to achieve rapid sample requirements, The effect of reducing analysis costs and wide application prospects
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Embodiment 1
[0029] Example 1 Induction and Purification of Rare Goblinian Vitellogenin Standards
[0030] 10mg 17β-estradiol (17β-estradiol, E 2) was dissolved in 10 mL of methanol to prepare a stock solution with a concentration of 1 mg / mL. According to the standard of 1g fish in 1L water, put rare gobi carp in a glass tank, add an appropriate volume of 17β-estradiol stock solution, so that the final concentration of 17β-estradiol is 100ng / L, and the concentration of methanol is not higher than 0.01%. . Exposure to the rare gobi was done in a semi-static manner with daily water changes. After 21 days of exposure, blood was collected from the tail vein of rare gobi carp, and the serum was obtained by centrifugation at 4°C. ion exchange membrane chromatography (Sartobind TM MA15 Q) Purification of vitellogenin from serum. Serum was diluted with phosphate buffered saline (PBS, 20 mM phosphate buffered saline containing 0.16 M NaCl, pH 6.5), and filtered through a 0.22 μm filter. T...
Embodiment 2
[0031] Example 2 Preparation of monoclonal antibody against vitellogenin of rare gobi carp
[0032] Take an appropriate amount of vitellogenin lyophilized powder, dissolve it in deionized water, and add an equal volume of complete Freund's adjuvant. After emulsification, BalB / C mice (purchased from the Academy of Military Medical Sciences) were initially immunized by subcutaneous multi-point injection (0.2 mL / point). Three weeks later, the mice were immunized again with vitellogenin plus incomplete Freund's adjuvant to emulsify the antigen (dose as above). Three weeks later, without adjuvant, the antigen was injected intraperitoneally in normal saline, and blood was collected seven days later to measure the titer and detect the immune effect. Immunization was boosted two weeks later, and three days later, splenocytes were taken and fused with SP2 / 0 cells (purchased from the Academy of Military Medical Sciences) (for specific methods, see Experimental Techniques of Cellular...
Embodiment 3
[0035] Example 3 Preparation of kit for detecting vitellogenin of rare gobi carp
[0036] The kit for quantitatively detecting vitellogenin of rare squid crucian carp provided by the invention comprises the following components:
[0037] 1) Blank microplate (1 piece), which can be a detachable or non-detachable 96-well microplate;
[0038] 2) Standard product: 1 vial (10 μg freeze-dried powder of rare goblin carp vitellogenin);
[0039] 3) Vitellogenin monoclonal antibody (10 μL), diluted 32000 times before use;
[0040] 4) Horseradish peroxidase-labeled goat anti-mouse IgG antibody (10 μL), diluted 10,000 times before use;
[0041] 5) Coating buffer (A), sample diluent (B), substrate buffer (C), substrate (D, chromogenic reagent), and oxidant (E) each one;
[0042] 6) Stop solution (2mol / L H 2 SO 4 ) 10mL;
[0043] Among them, coating buffer (A) is 0.05M carbonate buffer, pH9.6; sample diluent (B) is 0.2M PBS buffer, pH7.4, containing 0.05% Tween-20; The solution is...
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