Alpaca immune procedures and techniques

TIANJIN, China - Nov. 20, 2023 - PRLog -- At present, the acquisition of single-domain antibodies is generally achieved by immunizing alpaca and maturing the antibodies of the immune system itself in alpaca. B lymphocytes are separated, RNA is extracted, cDNA is obtained by reverse transcription, cDNA is used as the substrate for PCR amplification to obtain diversified nanoparticle antibody gene fragments, and then the diversified nanoparticle antibody gene fragments are connected to phage granules to construct phage library. Then, the most suitable antibody was selected from the alpaca antibody library by phage display screening technology and the nano-antibody was verified. The whole process mainly includes alpaca immunization, phage library construction, antibody screening, expression purification and verification.

Alpaca immune process

(1) Antigen Production: an alpaca can be immunized with 1-3 antigens at the same time, the total amount of antigen for each immunization is maintained between 1-2mg, the volume is below 2mL, and the antigen and adjuvant are 1:1 emulsified before immunization to form a uniform mixture, which is stored at 4℃.

(2) Immunizing alpacas: After recording a blank alpaca ear number, the immunization experiment was started. Immunizations are given every 2 weeks, at least 4 times.

(3) Blood collection: 50ml of blood was collected from the neck vein of alpaca at an interval of 5-7 days after the fourth immunization.

(4) Serum separation: Before each antigen immunization, blood was taken for immune evaluation, 5mL of blood was taken each time; On the same day, the blood was precooled by a 25℃ centrifuge and centrifuged at 400 xg for 30 minutes to separate and preserve the upper serum for subsequent antibody titer detection.

(5) Isolation of lymphocytes.

Immunization Techniques

(1) The selection of alpaca and the immune antigen are the key to the success of immunization. Choose healthy and strong, good mental state, moderate size of the alpaca is blank alpaca. The purity of the immune antigen and its correct conformation are crucial to the screening of suitable antibodies after immunizing alpacas for subsequent use, and the purity of the protein antigen is generally not less than 90%.

(2) lymphocyte separation: Timely cell separation can effectively prevent hemolysis after blood collection to achieve the best separation effect.

(3) The choice of immune cycle can affect the immune effect, according to experience, 1-2 weeks of immunization interval can make alpacas have a good immune response to most antigens.

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