Rapid detection device for Sudan III hapten, coupled antigen, antibody and colloidal gold and its application
A detection device and antigen-coupling technology, which is applied in the direction of measuring devices, food testing, material inspection products, etc., can solve the problems of high technical requirements for operators, failure to display results immediately, complicated operation of instruments and equipment, etc., and achieve convenient and economical use Fast, low-cost results
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Embodiment 1
[0058] Embodiment 1. Preparation of Sudan Red III Hapten
[0059] Add 3.53g of Sudan Red III, 1.52g of bromoacetic acid, and 0.1g of sodium carbonate to a 100mL three-necked flask in sequence, and react overnight in acetonitrile solution at 90°C. After rotary evaporation was completed, ethyl acetate was extracted and purified through a column to obtain Sudan III hapten.
[0060] figure 2 The mass spectrum of the thus obtained Sudan III hapten is given, and it can be known that the molecular formula of the hapten is as follows:
[0061]
Embodiment 2
[0062] Example 2. Synthesis of Sudan Red III coupled antigen
[0063] The Sudan III hapten obtained in Example 1 was used to prepare the immune antigen. Dissolve 0.1 mmol of Sudan Red III hapten in 2 mL of DMF, and add 27.5 mg of dicyclohexylcarbodiimide (DCC) and 14.4 mg of N-hydroxysuccinimide (NHS) with stirring. Magnetic stirring was carried out overnight at 4°C. After centrifugation, the supernatant was liquid A. Weighed 140 mg of bovine serum albumin (BSA) and hemocyanin (KLH), and dissolved them in 10 mL of 0.1 mol / L PBS (pH 8 .0). Add 1 mL of dimethylformamide (DMF), stir and dissolve to prepare liquid B, gradually drop liquid A into liquid B under magnetic stirring, and react at 4°C for 12 hours. After centrifugation, the supernatant was taken, dialyzed with normal saline at 4°C for 3 days, and the dialysate was changed 3 times a day, so as to obtain Sudan Red III-coupled antigens respectively coupled with bovine serum albumin and hemocyanin. The obtained Sudan Red...
Embodiment 3
[0064] Example 3. Preparation of Sudan Red III Monoclonal Antibody
[0065] The Sudan Red III conjugated antigen prepared in Example 2 was used to prepare the Sudan Red III monoclonal antibody, as follows: 4 6-week-old Kunming mice were immunized with the identified Sudan Red III conjugated antigen, and after three booster immunizations, blood was collected Measure the potency. When the serum titer no longer rises, mice are immunized with twice the dose of antigen without adjuvant. Three days later, the mice are killed by decapitation. The spleen is taken under sterile conditions to prepare spleen cells, which are compared with vigorously growing mouse myeloma cells. Mix in a 50mL centrifuge tube at a ratio of 8:1, add 30mL of serum-free IPMI 1640 medium, centrifuge at 1100r / min for 5 minutes, discard the supernatant, shake the cell mass gently, and place in a 37°C water bath. Slowly add 1mL of 50% PEG-4000 to the cells, drop it within 1 minute, and gently stir the sediment a...
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