From the brief overview of Nanobody production in the previous article, we understand the general process. Next, we will spend two articles introducing the specific production process. First, we will introduce the previous part of Nanobody production from alpaca immunization to library construction.

Alpaca immune process

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℃.

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.

Blood collection:

50ml of blood was collected from the neck vein of alpaca at an interval of 5-7 days after the fourth immunization.

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.

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℃.

Phage construction process

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.

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.

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.

Two rounds of PCR reaction were performed, and the obtained PCR amplification products were recovered using DNA purification recycling kit according to the instructions.

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.

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.

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 library.

In the next article, we will talk about the second half of nanobody production, which is library screening and antibody identification.

Fig.1 Alpaca immune and Phage construction process

KMD Bioscience provides healthy adult alpacas (28-36 months old) for immunization, and high-quality libraries and monoclonal antibody cell lines can be obtained in about 16-20 weeks, and complete experimental process records and immune dynamic videos can be delivered. According to the antigen type of customers, KMD Bioscience will make specific immune antigen generation plans and immunization plans. KMD Bioscience displayed VHH antibody (Nanobody) with pIII protein of M13 phage, prepared TG1 transduced competent cells, constructed phage vector, and measured phage storage capacity. (Learn more about antibody discovery services)

This article serves as a reference material for enthusiasts in scientific research. It does not substitute for professional knowledge or hands-on experimental procedures which require more detailed and professional information. In case of any content infringement, kindly reach out to the author for immediate deletion of the contentious material.

Reference

Muyldermans S. Nanobodies: natural single-domain antibodies. Annu Rev Biochem. 2013;82:775-97.