Clenbuterol hydrochloride micromolecule hapten high-sensitivity detection test paper based on nano antibody, and preparation method thereof

[0062] (2) Preparation of anti-clenbuterol hydrochloride nanobody

[0063] (2.1) Alpaca immunity and blood collection

[0064] At the first immunization, 400 μg of BSA-CL mixed with complete Freund's adjuvant was injected through the cervical lymph nodes of the alpaca. Two weeks later, BSA-CL was mixed and emulsified with Freund's incomplete adjuvant, and the second immunization was carried out as above, and a total of 4 immunizations were carried out. Blood was collected from the neck vein, the antibody was detected by Western blot, and the antibody titer was determined by indirect ELISA established with OVA-CL as the coating antigen. Peripheral blood lymphocytes were isolated, total RNA was extracted, mRNA was purified, and first-strand cDNA was synthesized by reverse transcription.

[0065] (2.2) Preparation of phage peptide library

[0066] The PCR reaction was carried out by using the primers that specifically amplified the variable regions of alpaca IgG2 and IgG3 heav...

[0108] For the detection of small molecules of clenbuterol hydrochloride in pig urine samples, the urine samples were collected in a clean container and can be detected directly. If there is turbidity, centrifuge at 3000r / min for 5min, and take the supernatant for testing. Use clenbuterol hydrochloride small-molecule hapten high-sensitivity detection test paper to detect and judge the results according to the above-mentioned operation method (13) to determine whether the pigs eat clenbuterol hydrochloride small-molecule drug. Two reddish-brown bands (test line and quality control line) appear on the test paper. "||" is negative for clenbuterol hydrochloride, which means that the pigs to be tested have not consumed the small-molecule drug clenbuterol hydrochloride; only one reddish-brown band appears The band (quality control line) "|" is positive for clenbuterol hydrochloride, indicating that the pigs to be tested have eaten the small molecule drug clenbuterol hydrochloride; t...

[0110] For the detection of small molecules of clenbuterol hydrochloride in raw pork samples, take about 5 grams of meat slurry samples and place them in a centrifuge tube, boil them for 10 minutes, and take the supernatant for detection. If there is turbidity, centrifuge at 3000r / min for 5min, and take the supernatant for testing. Use clenbuterol hydrochloride small-molecule hapten high-sensitivity detection test paper to detect and judge the results according to the above-mentioned operation method (13) to determine whether the pigs eat clenbuterol hydrochloride small-molecule drug. Two reddish-brown bands (test line and quality control line) "||" on the test paper are negative for clenbuterol hydrochloride, indicating that the pigs to be tested have not consumed the small-molecule drug clenbuterol hydrochloride; one reddish-brown band ( Quality control line) "|" is positive for clenbuterol hydrochloride, indicating that the pigs to be tested have eaten the small molecule dr...