I. Preparation Methods of Nanobodies

Nanobodies, also known as single-domain antibodies, differ from traditional monoclonal antibodies produced via hybridoma technology. They are typically developed by immunizing alpacas, constructing a phage library, and screening for nanobodies through phage display. The selected nanobodies are then expressed, purified, and validated for antigen binding.

Single-domain antibodies are generally obtained by immunizing alpacas. After the alpaca’s immune system naturally matures the antibodies, B lymphocytes are isolated, RNA is extracted, and cDNA is obtained via reverse transcription. The cDNA serves as a template for PCR amplification to generate diverse nanobody gene fragments. These fragments are then ligated into phagemid vectors to construct a phage library. Suitable antibodies are identified through phage display screening, followed by nanobody validation. The entire process mainly includes alpaca immunization, phage library construction, antibody screening, expression and purification, and validation.

II. Nanobody Preparation Process

2.1 Alpaca Immunization Process

(1) Antigen Preparation: A single alpaca can be immunized with 1–3 antigens simultaneously. The total antigen dose per immunization should be maintained between 1–2 mg in a volume of less than 2 mL. Before immunization, the antigen is emulsified 1:1 with an adjuvant to form a homogeneous mixture and stored at 4°C.

(2) Alpaca Immunization: After recording the blank alpaca’s ear tag number, the immunization experiment begins. The emulsion is injected at two sites on each side (left and right) near the cervical lymph nodes, with approximately 0.4 mL per injection site (total ~1.6 mL per immunization). The alpaca is observed for 30 minutes post-injection to confirm no adverse reactions. Immunizations are performed every two weeks, with at least four immunizations in total.

(3) Blood Collection: Five to seven days after the fourth immunization, 50 mL of blood is collected from the alpaca’s jugular vein.

(4) Serum Separation: Before each antigen immunization, 5 mL of blood is collected for immune response evaluation. On the same day, the blood is centrifuged at 400 ×g for 30 minutes in a pre-chilled (25°C) centrifuge. The upper serum layer is separated and stored for subsequent antibody titer detection.

(5) Isolation of Lymphocytes: First, add 15 mL of cell separation solution to a 50 mL centrifuge tube, then slowly layer 15 mL of blood on top. Exercise caution during the addition to prevent mixing of the blood and separation solution. Pre-cool the centrifuge to 25°C and centrifuge at 400 xg for 30 minutes. Transfer the upper serum layer to a new centrifuge tube and store at -80°C. Using a pipette, carefully aspirate the intermediate cloudy layer containing immune cells into a new 50 mL centrifuge tube.Add 10 mL of PBS buffer (pre-equilibrated to room temperature) to each tube, and centrifuge at 25°C, 400 xg for 20 minutes. Discard the supernatant, then add 5 mL of room-temperature PBS buffer to each tube, gently resuspend the cells, and count the cells using a hemocytometer. Centrifuge again at 25°C, 400 xg for 20 minutes.After discarding the supernatant, lyse the isolated lymphocytes with RNAiso Plus to achieve a cell lysate concentration of 10⁷/mL, and store at -80°C.

2.2 Construction of the Phage Display Library：

(1) RNA Extraction: Thaw the peripheral blood lymphocytes preserved in Trizol on ice and transfer them to a 1.5 mL centrifuge tube. Add 1/5 volume of chloroform, vortex thoroughly, and let stand at room temperature for 5 minutes. Centrifuge at 12,000g for 15 minutes at 4°C. Transfer the supernatant to a new centrifuge tube. Add an equal volume of isopropanol to the supernatant. Let stand at room temperature for 10 minutes, then centrifuge at 12,000g for 10 minutes at 4°C. Wash the precipitate with 75% ethanol, centrifuge at 7,500g for 5 minutes at 4°C, and discard the supernatant. Air-dry the precipitate at room temperature and dissolve it in an appropriate amount of RNase-free water.

(2) cDNA Reverse Transcription: According to the instructions of the reverse transcription kit, divide the obtained RNA into two equal parts for reverse transcription into cDNA, using Oligo T and random primers as reverse transcription primers, respectively.

(3) Antibody Fragment Amplification: Amplify specific antibody fragments from the reverse-transcribed cDNA using Taq DNA Polymerase Hot Start enzyme for PCR amplification.

(4) Purification of PCR Products: Purify and recover the PCR amplification products obtained from the two rounds of PCR reactions using a DNA purification and recovery kit.

(5) Cloning into Phagemid Vector: Digest the amplified diverse antibody gene sequences and the phagemid vector with restriction enzymes, purify them separately, and perform a ligation reaction. Recover the ligation products using a DNA purification and recovery kit and dissolve them in ultrapure water.

(6) Transformation of TG1: Pre-chill the electroporation cuvette on ice.  Once the 100 μL of TG1 competent cells have thawed, add 100 ng of the recovered ligation product and transfer the mixture into the pre-chilled electroporation cuvette.  Perform electroporation using the preset Bacteria transformation program on the electroporation device.  Immediately after electroporation, add 1 mL of SOC medium to the cuvette.  Carry out at least 20 electroporations, then recover the cells at 37°C for 60 minutes before plating them on LB agar plates containing ampicillin for overnight growth.  After overnight incubation, scrape the cells from the plates using 2xYT medium and a spreader, then resuspend in 20% glycerol and store at -80°C.

(7) Amplification and Purification of Phage Library: Mix the scraped bacterial cells thoroughly and transfer approximately 10^9 bacteria into 100 mL of 2x YT medium pre-supplemented with ampicillin.  Culture at 37°C with 220 rpm shaking until OD600 reaches 0.5.  Add helper phage at a ratio of 20:1 (phage:bacterial cells) and incubate at 37°C for 30 minutes.  Then, add kanamycin and continue incubation overnight at 30°C with shaking.  Centrifuge the overnight culture at 13,000 rpm for 5 minutes at 4°C, transfer the supernatant to a new tube, and add 1/4 volume of pre-chilled 5x PEG8000/NaCl.  Incubate on ice for 30 minutes.  Centrifuge at 13,000 rpm for 10 minutes at 4°C, discard the supernatant, and resuspend the pellet in 1 mL PBS buffer.  Add 250 μL of 5X PEG8000/NaCl again, incubate on ice for 10 minutes, then centrifuge at 16,000 × g for 15 minutes at 4°C.  Discard the supernatant and dissolve the pellet in 1 mL PBS to obtain the phage library.

2.3 Antibody Screening and Identification Procedure：

(1) Coating the Immunotube: Add 50 μg of antigen to 2 mL of PBS and transfer the mixture into the immunotube. Incubate overnight at 4°C.

(2) Blocking: Add an appropriate amount of amplified and purified phage to 1 mL of 3% BSA and incubate with rotation at room temperature for 2 hours. Meanwhile, add 2–3 mL of 3% BSA to the coated immunotube and incubate with rotation at room temperature for 2 hours.

(3) Antigen and Phage Incubation: Wash the blocked immunotube three times (5 minutes each) with PBS containing 0.01% Tween. Then, add the blocked phage library to the blocked immunotube and adjust the volume to 2–3 mL with PBS. Incubate with rotation at room temperature for 1 hour.

(4) Washing: Wash the immunotube (after antigen-phage incubation) 20 times (5 minutes each) with PBS containing 0.1% Tween.

(5) Elution: Add 1 mL of 100 mM Trimethylamine to the immunotube and incubate at room temperature for 10 minutes. Neutralize the Trimethylamine by adding 1 M Tris-HCl, then transfer the final 1.5 mL of eluted phage to a new centrifuge tube. Amplify and purify the eluted phage using the phage library amplification and purification protocol. Repeat the screening process twice more, successively halving the amount of coated antigen in the immunotube each time, to obtain the eluted phage after three rounds of screening. The eluted phage can be subjected to NGS sequencing to obtain a DNA sequence library of candidate nanobodies that bind to the antigen.

(6) ELISA Identification: Meanwhile, dilute the phage obtained from the previous screening step in a gradient, take 100 μL of each dilution and add it to TG1 bacterial culture with an OD600 of 0.5. Incubate at 37°C for 30 minutes, then plate on 2x YT agar plates containing ampicillin and incubate overnight at 37°C to obtain single colonies. Randomly pick at least 192 single colonies into a 96-well cell culture plate containing 2x YT medium with ampicillin. After overnight incubation at 37°C, use this as a seed culture plate. Transfer 2 μL of the bacterial culture to a new 96-well plate (each well containing 200 μL of fresh 2x YT medium with 100 μg/mL ampicillin). After 5 hours, add helper phage to each well and incubate at 37°C for 30 minutes. Then add kanamycin to a final concentration of 50 μg/mL and incubate overnight at 30°C. Centrifuge the culture and collect the supernatant containing the phage.

After overnight coating of antigen wells and BSA-coated control wells, block with 3% BSA and add the phage supernatant obtained in the previous step. Incubate at room temperature for 1 hour. Wash three times with PBS containing 0.1% Tween, then incubate with phage antibodies. After color development with TMB, measure the absorbance at 450 nm for each well. Select colonies with a high ratio of absorbance in antigen-coated wells compared to control wells for sequencing (performing two independent phage-ELISA analyses) to obtain the gene sequences of the nanobodies.

III. Key Steps

3.1 Immunization Techniques：

(1) The selection of alpacas and the immunizing antigen are key to successful immunization. Choose healthy, non-immunized alpacas. The purity and correct conformation of the antigen are crucial for obtaining suitable antibodies after immunization and for subsequent applications. Generally, the purity of the protein antigen should be no less than 90%.

(2) Lymphocyte isolation: Timely cell separation can effectively prevent hemolysis after blood collection, ensuring optimal isolation results.

(3) The immunization cycle affects immune response: A 1-2 week immunization interval allows alpacas to develop a strong immune response to most antigens.

3.2 Techniques for Constructing a Phage Display Library：

(1) The library size and diversity of a phage display library are important criteria for evaluating its quality.  A large library size and high diversity are essential for successfully screening nanobodies.

(2) Key factors affecting library size and diversity include: RNA degradation during RNA extraction, the template and primers used in reverse transcription, the selection of primers and the amount of template during amplification, the number of PCR cycles, the transformation efficiency of competent bacteria, and the size of the ligation system.

3.3 Antibody Screening Techniques：

(1) The appropriate amount of antigen coating depends on factors such as the molecular weight, hydrophobicity/hydrophilicity, and structure of the antigen, as well as the choice of coating buffer and solid phase. Proper coating is the foundation for successful screening. If necessary, preliminary experiments can be conducted to optimize coating conditions, or antigen-coupled magnetic beads can be used as an alternative screening method.

(2) After elution, the titer of the phage should be measured. Following 2-4 rounds of antigen-coated panning, the enrichment level of the phage should fall within a reasonable range.

KMD Bioscience has established a comprehensive and well-developed phage antibody display technology platform. Based on this platform, KMD Bioscience offers key experimental services, including antigen design, alpaca immunization, library construction and screening, and functional activity validation, providing highly specific and high-affinity alpaca VHH antibodies.Additionally, KMD Bioscience possesses extensive expertise in antibody engineering and delivers end-to-end upstream and downstream antibody services. These include antibody humanization, human scFv antibody library construction, human Fab antibody library construction, human antibody phage library preparation, customized phosphospecific antibody development, and antibody affinity maturation. These services are designed to meet diverse research needs for clients.

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