1. Production method of nano antibody
The development of nano antibodies is different from the traditional monoclonal antibody Production method by hybridoma, which generally selects candidate nano antibodies by immunizing alpaca, constructing phage library and displaying phage, and then carries out the verification experiment of binding to antigen after expression and purification of nano antibodies.
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.
2. Production process of nano antibody
2.1 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. Each time, the alpaca was injected into the left and right sides near the neck lymph nodes, and each side was injected into 2 points, and about 0.4mL of emulsified antigen was injected into each point. Observation for half an hour after immunization confirmed that the alpaca was in good condition and had no discomfort symptoms. 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: 15mL of cell separation solution was first added to a 50mL centrifuge tube, and then 15mL of blood was slowly added. Add blood slowly and carefully to prevent mixing of blood and separation solution. Then the centrifuge was precooled to 25℃ and centrifuged at 400 xg for 30 minutes. The separation of blood in the centrifuge tube was observed. The upper level serum was stored in the new centrifuge tube at -80 ℃. Each tube was added with 10mL PBS buffer at room temperature and centrifuged at 400 xg at 25℃ for 20 min. The supernatant was removed, 5mL of PBS buffer at room temperature was added to each tube, and the number of cells was calculated using a blood cell counting plate, and then centrifuged at 25℃ and 400 xg for 20 min. The supernatant was removed, and the isolated lymphocytes were dissolved with RNAiso Plus according to the number of cells to obtain 107/mL cytolytic solution, which was stored at -80℃.
2.2 Phage construction process
(1) RNA extraction: Peripheral blood lymphocytes preserved with Trizol were dissolved on ice and transferred to a 1.5mL centrifuge tube, where 1/5 volume of chloroform was added to shake and mix. Stand at room temperature for 5 minutes at 4℃, centrifuge at 12000g for 15 minutes; Transfer the supernatant after centrifugation to a new centrifuge tube; Isopropyl alcohol of equal volume was added to the new centrifuge tube. Let stand at room temperature for 10 minutes and centrifuge at 4℃ 12000g for 10 minutes; Clean and precipitate with 75% ethanol, centrifuge at 4℃ 7500g for 5 minutes, discard the supernatant, precipitate and dry at room temperature, and dissolve in appropriate amount of RNA-free water.
(2) Reverse transcription cDNA: According to the instructions of the reverse transcription kit, the RNA obtained in the previous step was divided into two parts and reverse-transcribed into cDNA. The reverse transcription primers were Oligo T and random primers, respectively.
(3) Amplification of antibody fragments: Specific antibody fragments are amplified from reverse-transcribed cDNA, and PCR amplification is performed using Taq DNA Polymerase Hot Start enzyme.
(4) Two rounds of PCR reaction were performed, and the obtained PCR amplification products were recovered using DNA purification recycling kit according to the instructions.
(5) Cloning to phage plasmid: The diversified antibody gene sequence and phage vector amplified in the previous step are enzymically cut, respectively purified and linked. The connected products were recovered by DNA purification recycling kit according to the instructions, and dissolved in ultra-pure water.
(6) TG1 conversion: Place the cup on ice for pre-cooling, wait for 100ul of TG1 receptive cells to melt and add 100ng of the recovered connection products, transfer the mixed receptive cells and connection products to the pre-cooled cup, use the Bacteria transformation procedure preset by the tester for electric shock transformation, immediately add 1mL SOC culture medium into the cup. After at least 20 revolutions, the cells were resuscitated at 37℃ for 60 minutes and coated on a LB culture plate containing ampicillin resistance for overnight growth. The cells on the culture plate after overnight growth in the previous step were washed and scraped off with 2xYT medium and a coating rod, and then stored at -80℃ after adding 20% glycerin.
(7) Amplification and purification of phage library: After mixing the bacteria scraped from the previous step, about 10^9 bacteria were transferred to 100mL 2x YT culture medium pre-added with ampicillin antibiotics, and cultured at 220rpm at 37℃ until OD600nm reached 0.5. According to the ratio of helper phage: the number of bacterial cells was 20:1, the auxiliary phage was added and continued to be cultured at 37℃ for 30 minutes. The final concentration of kanamycin was 50ug/ml and cultured in shaking bed at 30℃ overnight. The bacteria cultured overnight were centrifuged at 13000rpm at 4℃ for 5 minutes, the supernatant was transferred to a new centrifuge tube, and 1/4 volume of pre-cooled 5x PEG8000/NaCl was added, and incubated on ice for 30 minutes. Centrifuge at 4℃ at 13000rpm for 10 min to remove the supernatant and add 1mL PBS buffer to dissolve the precipitation. 250ul 5X PEG8000/NaCl was added again and incubated on ice for 10 minutes. After centrifugation at 4℃ at 16000g for 15 minutes, the supernatant was removed and the precipitation was dissolved in 1mL PBS to obtain the phage bank.
(1) Coated immune tube: 50ug antigen was added to 2mL PBS and added to the immune tube for overnight incubation at 4℃.
(2) Isolation: The amplified and purified phage was added to 1mL 3% BSA and incubated for 2h at room temperature. At the same time, 2-3mL 3% BSA was added to the coated immune tubes and incubated at room temperature for 2h.
(3) Antigen and bacteriophage incubation: The sealed immune tube was washed 3 times with PBS containing 0.01% Tween for 5 minutes each time. The closed phage library was added to the closed immune tube, PBS was added until 2-3mL, and the phage library was incubated at room temperature for 1h.
(4) Cleaning: After incubating the antigen and phage, the immune tube containing 0.1% Tween was washed 20 times with PBS for 5 minutes each time.
(5) Elution: 1mL 100mM Trimethymime was added to the immune tube, incubated at room temperature for 10 minutes, 1M Tris-HCl was added to neutralize Trimethymime, and the last 1.5mL eluted phage was transferred to a new centrifuge tube. The eluted phage was amplified and purified according to the phage library, and then the screening process was repeated twice after amplification, and the amount of antigen covered by the immune tube was halved successively to obtain the eluted phage after 3 screening. The elution phage can be sequenced by NGS to obtain the candidate nanoantibody DNA sequence library.
(6) ELISA identification: At the same time, after gradient dilution of the bacteriophages obtained in the previous step, 100ul of each bacteriophage was added to TG1 bacterial solution with OD600nm of 0.5, cultured at 37℃ for 30 minutes, coated with 2x YT culture plate containing ambenomycin, and incubated at 37℃ overnight for the next day to obtain monoclonal colonies. At least 192 single colonies were randomly selected onto 96-well cell culture plates containing 2x YT culture solution and cultured overnight at 37℃ as seed bacteria plates, and the bacterial solution 2ul was re-added to the new 96-well plates (each well containing 200ul 2x YT fresh culture solution). 100ug/ml ammobenzyl) 5 hours later, the auxiliary phage was added into the culture hole, and the final concentration of 50ug/ml kanamycin was added at 37℃ for 30 minutes, and the culture was incubated overnight at 30℃. On the second day, the bacteria solution after overnight culture was centrifuged to obtain the supernatant containing phage.
The overnight coated antigen hole and the BSA coated control hole were sealed with 3% BSA, then the bacteriophage supernatant obtained in the previous step was added, and incubated at room temperature for 1h. After three times of cleaning with PBS containing 0.1% Twain, the phage antibody was added for incubation, and the light absorption value of each well was read at a wavelength of 450nm after color development with TMB. Colonies with large absorbency ratio between antigen-coated pores and corresponding control pores were selected for sequencing (two phage-Elisa analyses independently) to obtain the gene sequence of the nano antibody.
3. Committed step
3.1 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.
3.2 Tips for building phage library
(1) The capacity and diversity of phage library is one of the important criteria to measure the quality of the library, and the larger the capacity and the better the diversity of the library is an effective guarantee for the successful screening of nano antibodies;
(2) The key factors affecting the capacity and diversity of the library include: degradation of RNA during RNA extraction, template and primer during reverse transcription, selection of primer and amount of template during amplification, number of PCR amplification cycles, conversion efficiency of receptive bacteria, size of linking system, etc.
3.3 Antibody screening techniques
(1) The appropriate amount of antigen coating is related to the molecular weight of the antigen, hydrophobic and hydrophilic properties, structure, and also to the selection of coated buffer and coated medium. Reasonable coating is the basis for successful screening. If necessary, pre-experiments can be conducted to determine the conditions of coating or the method of antigen binding magnetic beads can be selected for screening.
(2) The titer of the phage was determined after elution. After 2-4 rounds of antigen encapsulation by panning, the enrichment degree of the phage should be within a reasonable range.
Fig. 1 Production process of nano antibody
KMD Bioscience has established a complete and mature phage antibody display technology platform. Based on the phage display technology platform, KMD Bioscience can provide major experimental links including antigen design, alpaca immunity, library construction and screening, and activity function verification, and provide highly specific and high-affinity alpaca VHH antibodies to scientists around the world. In addition, KMD Bioscience has rich experience in antibody engineering construction, and can provide three-dimensional antibody upstream and downstream services, including antibody humanization service, human scFv antibody library construction service, human Fab antibody library construction service, human antibody phage library Production service, phosphorylated antibody customization service, antibody affinity maturation service, etc.
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 Rossotti MA, Trempe F, van Faassen H, Hussack G, Arbabi-Ghahroudi M. Isolation and Characterization of Single-Domain Antibodies from Immune Phage Display Libraries. Methods Mol Biol. 2023;2702:107-147.