Anti-procalcitonin high-affinity nano antibody and application thereof
A nanobody and procalcitonin technology, applied in the field of peptides, can solve the problems of low molecular weight affinity of nanobodies, hindering the application of nanobodies, short serum half-life, etc., and achieve excellent detection results
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[0034] Example 1 Preparation of anti-PCT Nanobody
[0035] 1.1 Construction and screening of anti-PCT Nanobody phage display library
[0036] 1.1.1 Immunization of alpaca: select a healthy adult alpaca, mix the human recombinant PCT antigen and Freund’s adjuvant at a ratio of 1:1, and use 6-7μg / Kg to immunize with multiple subcutaneous injections on the back Alpacas were immunized four times with a immunization interval of 2 weeks. Afterwards, the peripheral blood of alpaca was collected and used to construct a phage display library.
[0037] 1.1.2 Separation of camel-derived lymphocytes: According to the routine procedures in this technical field, lymphocytes are analyzed from the collected camel-derived anticoagulated whole blood, every 2.5×10 7 Add 1 mL of RNA isolation reagent to each living cell, take 1 mL for RNA extraction, and store the rest at -80°C.
[0038] 1.1.3 Extraction of total RNA: Extract total RNA according to conventional procedures in this technical field, adjust...
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[0060] Example 2. Preparation of anti-PCT Nanobody 6H6
[0061] 2.1 Amplification of the original Nanobody strain TG1 and transformation of the Nanobody recombinant plasmid into E. coli BL 21 (DE 3 )
[0062] The original strain TG1 glycerol bacteria containing Nanobody nucleic acid was inoculated in 5 mL of fresh LB-A medium at a ratio of 1:1000, and cultured overnight at 37°C and 200 rpm. The next day, use Plasmid mini kit (OMEGA) to extract plasmids according to the instructions. After verification, 1 μl of the above plasmid was transformed into 100 μl of competent cells, mixed gently, placed on ice for 30 minutes, heated in a 42°C water bath for 90 seconds, and cooled in an ice bath for 3 minutes. Add 600μl of LB medium to the centrifuge tube and incubate with shaking at 37°C for 60 minutes. Take 100 μl of the supernatant, spread it on the LB-A plate with a triangular spreader, and incubate it upside down at 37°C overnight.
[0063] 2.2 Induced expression of Nanobodies
[0064]...
Example Embodiment
[0067] Example 3. Determination of the affinity activity of anti-PCT Nanobody and antigen
[0068] 3.1 Chip antigen coupling
[0069] Prepare the PCT antigen with different pH sodium acetate buffers (pH 5.5, pH 5.0, pH 4.5, pH 4.0) into a working solution of 20 μg / mL, and prepare a 50 mM NaOH regeneration solution, using Biacore T100 protein interaction analysis system instrument The template method in Analyzes the electrostatic binding between antigens of different pH conditions and the surface of the chip (GE), and takes the signal increase of 5 times RL as the standard, selects the most suitable and most neutral pH system and according to needs Adjust the antigen concentration as a condition for coupling. The chip is coupled according to the template method that comes with the instrument: one channel selects the blank coupling mode, the second channel selects the Target coupling mode, and the target is set to the designed theoretical coupling amount. The coupling process takes...
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