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Single Domain Antibody Library Construction Protocol

Single-domain antibodies (sdAbs) are engineered from VHHs [variable domains (V) of heavy-chain-only antibodies (HCAb)] of camelid antibodies. Single-domain antibodies lack both the light chain polypeptide and the CH1 domain and only comprise an Fc region (CH2 and CH3 domains) and the VH region. These nature-made sdAbs have several advantages for biotechnological applications due to their favorable characteristics such as small size, ease of genetic manipulation, high affinity and solubility, overall stability, resistance to harsh conditions (e.g., low pH, high temperature), and low immunogenicity. Most importantly, sdAbs have the feature of penetrating into cavities and recognizing hidden epitopes normally inaccessible to conventional antibodies, mainly due to their protruding CDR3/H3 loops.

Although single-domain antibodies later were also identified in particular cartilaginous fish (sharks, skates, and rays), most research on the biotechnological application of single domain antibodies was done using camelids because of their ease of handling, including immunization. However, shark antibody production service can still be provided by Creative Biolabs. There are two ways in a diverse single domain antibody (sdAb) library: 1. by amplification of VHH genes from isolated lymphocytes of naïve or immunized members of Camelidae; 2. by introducing diversity into a VHH scaffold synthetically. Comprehensive overview of sdAb generation was previously introduced for more researchers who interested biopharmaceutical field in Creative Biolabs.

pMED1 Vector Fig.1 pMED1 Vector

Here we only detail basic protocol of immunized llama single domain antibody library construction by the first way in above. This approach avoids the introduction of a peptide linker used in the protocol of scFv library construction which might create additional problems (e.g. reduced affinity, aggregation or proteolytic cleavage).

The llama goes through a 71-day schedule, during which they are boosted and bled to determine their specific antibody titer. Once the antibody titer up, the peripheral blood lymphocytes also called PBLs are isolated, total RNA are isolated from them and cDNAs are prepared. The VHH genes are then amplified from cDNA using proprietary PCR primers that cover the genetic diversity in this complex region. Next the PCR product is cloned into a phagemid vector (Fig. 1) creating the VHH antibody library.

Materials

  1. 3 mg of each antigen.
  2. Complete and incomplete Freund’s adjuvant.
  3. Llama.
  4. Phosphate-buffered saline (PBS). TRIzol reagent), Chloroform.
  5. Isopropyl alcohol, 75% ethanol.
  6. Kit for the preparation of mRNA (e.g., an oligo (dT)-purification system).
  7. Kit for the synthesis of cDNA (e.g., “First-strand cDNA synthesis kit”).
  8. Ligation kit, 5 x ligation buffer (500 mM Tris-HCl, 25 mM MgCl2, pH7.4).
  9. DEPC-treated H2O.
  10. PCR reagents: Taq DNA polymerase with 10 x Taq DNA polymerase buffer, a stock of deoxyribonucleoside triphosphates (dNTPs). PCR H2O [ACS] reagent.
  11. pMED1 phagemid vector (Fig. 1).
  12. T4 DNA ligase (5U/mL) and 10× ligation buffer.
  13. QIAquick PCR Purification kit.
  14. Electrocompetent TG1 E. coli cells.
  15. Qiaquick gel extraction kit.
  16. Primers: First PCR primers, second PCR primers.
  17. SOC medium. 2xYT-Amp plates: containing 100 μg/ml ampicillin. 2xYT-Amp-Glu medium: 2xYT medium containing 100 μg/ml ampicillin and 2% (w/v) glucose. RPMI medium.

Methods

Methods, which are not described step-by-step, should be carried out according to the instructions from manufacturers/suppliers of the kits and reagents, or by using methods described by Toya Nath Baral, or according to in-house protocols.

1. Immunization of a llama: As immunization plans, llama immunization schedules (see Table 1) are provided here and have worked well in authors’ laboratory.

  1. a.On Day 1, conduct a pre-immune bleed (10 to 15 ml) and then immunize the animal with 100 to 200 μg of each antigen. Mix the antigen solution well with 1 ml of complete Freunds adjuvant (CFA) to make a total immunization volume of 2 ml.
  2. b.On Days 22, 36, and 50, immunize with 100 μg of the same antigens mixed well with incomplete Freund’s adjuvant (IFA). On Day 64, immunize with 100 μg of each antigen with no adjuvant.
    Note: The proteinaceous antigens can be contaminated with other proteins (no proteases or toxic compound), but save an aliquot of the protein of highest purity for phage enrichment by panning and identification of VHH clones. However, make sure that the proteins are compatible (e.g., do not mix proteases with other proteinaceous antigens as the latter might be degraded upon storage of the mixture). Each antigen (mostly a recombinant protein) is purified and concentrated to 1 mg/mL in H2O or a “protein-friendly” buffer (glycine, Tris, Hepes, MOPS, avoid phosphate when using Gerbu adjuvant). Immunization with cells or a total proteome is feasible as well; however, it might be more difficult to select later on the sdAbs against your specific target expressed within that proteome/cell. The immune response will depend on the immunogenicity of the protein. Unfolded/unstructured proteins and small antigens such as haptens or synthetic oligopeptides elicit a poor titer even when coupled to carrier proteins (for haptens and oligopeptides).
  3. c.Collect blood (10 to 15 ml) on Days 29, 43, 57 as test bleed, and collect 50 ml of blood on Day 71 as production bleed.
  4. d.Isolate the lymphocytes from blood collected on Day 71 and use for phage display library construction.
  5. e.Following immunizations, analyze llama to monitor antigen-specific heavy-chain antibody responses by ELISA (The enzyme-linked immunosorbent assay is a routine and widely practiced assay for analyzing the specific interaction between antigens and antibodies in a relatively short period of time.).
    Note: It is important to have a pre-immune bleed on Day 1, which will be used as a non-immunized control for a subsequent ELISA. Short immunization protocol can be adapted and has the advantage of saving immunization time, if antigen is soluble protein.

Table 1 Immunization Schedules

Time Activity
Day 1 Pre-immune bleed
Day 1 Immunization (CFA as an adjuvant)
Day 22 Boost no.1 (IFA as an adjuvant)
Day 29 Test bleed 1
Day 36 Boost no.2 (IFA as an adjuvant)
Day 43 Test bleed 2
Day 50 Boost no.3 (IFA as an adjuvant) and test bleed 2
Day 57 Test bleed 3
Day 64 Boost no.4 (no adjuvant)
Day 71 Production bleed

2. Construction of single domain antibody library

2.1 Preparation of Peripheral Blood Lymphocytes (PBL), extraction of total RNA, synthesis of cDNA.

  1. a. Take 10 ml of llama blood drawn on Day 71, dilute 1:1 in RPMI medium. Centrifuge at room temperature for 10 min at. 1,000 × g. Collect the peripheral blood lymphocytes.
  2. b. Isolate total RNA by using the Trizol reagent according to the manufacturer’s instructions. Measure the RNA concentration and purity.
  3. c. cDNA is prepared using total RNA as template and commercially available reagents. Synthesize cDNA by using the First-Strand cDNA Synthesis kit, according to the manufacturer’s instructions.
    Note: A yield around 106 PBLs per mL of llama blood is usually obtained. It may sometimes be necessary to optimize the amount of input RNA, but generally 3 to 5 μg total RNA per cDNA synthesis reaction results in a good yield of DNA by RT-PCR.

2.2 PCR amplification of VHH repertoire and cloning of VHH fragments into a phagemid vector (pMED1 vector) for library construction.

In these steps we will amplify the VHH sequences from the cDNA pool. The cDNA is used as template in PCR with a combination (firs PCR primers) of IgG-specific 5’-end primers (VH/VHH framework 1 region) and 3’-end primers (constant region 2; CH2). This PCR will amplify both VH and VHH from IgG1, IgG2, and IgG3 genes. The first PCR normally yields a DNA band of about 800 to 900 bp corresponding to VH-CH1-Hinge and almost the first half of the CH2 region (called VH-CH2) of conventional antibodies, and another DNA band of about 550 to 650 bp corresponding to the VHH-Hinge and almost the first half of the CH2 region (called VHH-CH2) of heavy-chain antibodies The VHH region of heavy-chain antibodies, which is a pool from IgG2 and IgG3 genes, is amplified in a second PCR using VHH-specific primers (second PCR primers) anchored with SfiI restriction sites. The VHH fragments are cloned into the pMED1 phagemid vector between the two SfiI restriction sites, located at the 3’-end of the pelB leader sequence and 5’-end of the gene III coding sequence. This allows VHHs to be expressed as fusions to gene III, transported to the E. coli periplasmic space, and displayed on the surface of phage when it is super-infected by a suitable helper phage such as M13KO7. In principle, any other phagemid vector with similar features such as pHEN1 or pCANTAB 5E can be used.

  1. a. Perform PCR reaction system and thermal cycling program appropriately. Detailed parameters can be referred to author’s documentation.
  2. b. Gel-purify the VHH bands from a 1% agarose gel using the QIAquick Gel Extraction kit. Pool the DNA and measure the concentration.
  3. c. Re-amplify the purified product in a second PCR under the same conditions as above, using second PCR primers.
  4. d. Desalt the PCR products with the QIAquick PCR Purification kit and determine the DNA concentration.
  5. e.Digest the PCR products and pMED1 phagemid vector with SfiI in different reaction systems. Purify digested DNA and identified vector fragment respectively by QIAquick PCR Purification kit and measure their concentration.
  6. f.Ligate the SfiI-digested VHH DNA with SfiI-digested pMED1 vector using ligation kit.
    Note: With a ligation of this magnitude, library sizes of around 108 should be obtained. When larger-size libraries are required, as in the case of synthetic and naïve libraries, it is advisable to first identify the ligation conditions that will give the biggest library size. This can be done by performing small-scale ligations with different total DNA and molar ratios of insert to vector. Moreover, the scale of the ligation and the number of transformations needs to be significantly increased. In the case of naïve libraries, use of blood taken from several animals will increase the number of antibody-displaying B cells and the library diversity.
  7. g.Transform electrocompetent TG1 cells with the purified ligated material obtained in above step. Spread 100 μl of the diluted incubating cells on 2xYT-Amp plates and incubate overnight at 32°C.
  8. h.Centrifuge the amplified cultured cells 20 min at 5000 × g, 4°C. Discard the supernatant and resuspend the cells in 3 to 5 ml of 2xYT-Amp-Glu medium. Calculate the cell density (number of cells/ml) in the stock solution.
    Note: By putting the transformed cells on large plates, they will grow out into colonies. By measuring the OD of the cell suspension obtained by scraping the colonies and collecting the cells within these colonies, we can calculate the average amplifcation of the library. The library amplification factor equals the total number of colonies scraped from the plates divided by the total number of cells within the glycerol stock). This number (library amplification factor) corresponds to the average number of times each individual transformed cell is represented in the library.

It is not always feasible to construct an immune library, especially if the target antigen is nonimmunogenic, scarce, or unavailable. Alternatively, nonimmune or naïve libraries can be constructed by Creative Biolabs.

Reference:
  1. Reference: Toya Nath Baral, Roger MacKenzie, and Mehdi Arbabi Ghahroudi. Single-Domain Antibodies and Their UNIT 2.17 Utility. Current Protocols in Immunology. 2013 Nov 18.

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