Single‐Domain Antibodies and Their Utility

Toya Nath Baral1, Roger MacKenzie2, Mehdi Arbabi Ghahroudi3

1 Human Health Therapeutics, Life Sciences Division, National Research Council Canada, Ottawa, Ontario, 2 University of Guelph, Guelph, Ontario, 3 Department of Biology, Carleton University, Ottawa, Ontario
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 2.17
DOI:  10.1002/0471142735.im0217s103
Online Posting Date:  November, 2013
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Abstract

Engineered monoclonal antibody fragments have gained market attention due to their versatility and tailor‐made potential and are now considered to be an important part of future immunobiotherapeutics. Single‐domain antibodies (sdAbs), also known as nanobodies, are derived from VHHs [variable domains (V) of heavy‐chain‐only antibodies (HCAb)] of camelid heavy‐chain antibodies. These nature‐made sdAbs are well suited for various 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. In this unit, we will present and discuss comprehensive and step‐by‐step protocols routinely practiced in our laboratory for isolating sdAbs from immunized llamas (or other members of the Camelidae family) against target antigens using phage‐display technology. Expression, purification, and characterization of the isolated sdAbs will then be described, followed by presentation of several examples of applications of sdAbs previously characterized in our laboratory and elsewhere. Curr. Protoc. Immunol. 103:2.17.1‐2.17.57. ©2013 by John Wiley & Sons, Inc.

Keywords: single‐domain antibody; heavy‐chain antibodies; VHH; antibody fragment libraries dimeric VHH; pentabodies; multivalency

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Immunization of Llamas
  • Alternate Protocol 1: Immunization of Llamas Using Short Immunization Protocol
  • Support Protocol 1: Enzyme Linked Immunosorbent Assay (ELISA)
  • Support Protocol 2: Llama Serum Fractionation to Prepare Pooled IgG2a, IgG2b, and IgG2c Isotypes
  • Basic Protocol 2: Construction of VHH Library
  • Support Protocol 3: Purification of DNA Fragments from Agarose Gels
  • Support Protocol 4: Preparation of Electrocompetent Cells
  • Alternate Protocol 2: Construction of Non‐Immune (Naïve) Library
  • Basic Protocol 3: Rescue of Phagemid Library and Panning
  • Basic Protocol 4: Monitoring the Progress of Biopanning by Phage ELISA
  • Basic Protocol 5: Subcloning of VHH Fragments in Bacterial Expression Vector
  • Basic Protocol 6: Expression of Single‐Domain Antibodies in Bacterial Systems
  • Basic Protocol 7: Purification of Single‐Domain Antibodies
  • Support Protocol 5: SDS‐Page and Western Blotting
  • Basic Protocol 8: GEL‐Filtration/Size‐Exclusion Chromatography
  • Basic Protocol 9: Increasing Functional Affinity and Potency of VHHs
  • Basic Protocol 10: Preparation of VHH Fc Fusion
  • Support Protocol 6: Pentamerization of VHHs
  • Basic Protocol 11: Characterization of Antigen‐Specific VHHs
  • Support Protocol 7: Surface Plasmon Resonance (SPR) and Epitope Mapping
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Immunization of Llamas

  Materials
  • Llama (Lama glama) (Cedarlane Laboratories)
  • Antigen (1 mg required)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Freund's complete and incomplete adjuvant (Sigma; also see unit 2.4)
  • 1‐ to 2‐ml syringes with 21‐G, 1‐ to 1.5‐in. long needles for llama immunization and 10‐ to 15‐ml Vacutainer for blood collection
  • Heparin‐coated tubes (Becton Dickinson)
  • Additional reagents and equipment for emulsifying antigen using Freund's adjuvant (unit 2.4), isolation of lymphocytes (unit 7.1), ELISA ( protocol 3), and fractionation of sera ( protocol 4)

Alternate Protocol 1: Immunization of Llamas Using Short Immunization Protocol

  Materials
  • Antigen at 5 to 10 µg/ml
  • 5 µg/ml BSA in PBS (filter sterilize and store at 4°C)
  • PBS (see recipe)
  • PBST: PBS (see recipe) containing 0.05% (v/v) Tween 20
  • Blocking buffer A: 3% (w/v) BSA in PBS‐T
  • Goat anti‐llama IgG and swine anti‐goat IgG labeled with horseradish peroxidase (HRP) (Cedarlane)
  • HRP substrate solutions for ELISA (GE Healthcare)
  • 1 M H 3PO 4
  • 96‐well MaxiSorp microtiter plates (VWR)
  • Microtiter plate reader (Biochrom)
  • Additional reagents and equipment for ELISA (unit 2.2)

Support Protocol 1: Enzyme Linked Immunosorbent Assay (ELISA)

  Materials
  • Sera prepared from blood collected on Day 57 and Day 71 of protocol 1 or day 42 of protocol 2
  • 20 mM NaPi buffer: 8.46 ml of 1 M NaH 2PO 4, 11.54 ml of 1 M Na 2HPO 4, diluted in 1 liter of distilled, deionized H 2O (pH 7.0)
  • 100 mM citrate buffer: 3.11 g citric acid, 1.53 g sodium citrate, pH 3.5, per 200 ml of distilled, deionized H 2O—adjust to pH 3.5 with 1 M NaOH, filter sterilize, and store at 4°C.
  • 1 M Tris·Cl, pH 8.8 ( appendix 2A)
  • 100 mM glycine buffer: 1.5 g glycine, pH 2.7, per 200 ml of distilled H 2O—adjust to pH 2.7 with 3 M HCl, filter sterilize, and store at 4°C.
  • 100 mM sodium acetate buffer: 2.7 g sodium acetate, pH 4.5, per 200 ml of distilled H 2O—adjust to pH 4.5 with acetic acid, filter sterilize, and store at 4°C.
  • Phosphate‐buffered saline (PBS; see recipe)
  • Dialysis tubing with MWCO 8000 (Biodesign Inc., http://biodesignofny.com
  • 1‐ml HiTrap Protein G HP and HiTrap Protein A HP columns (GE Healthcare)
  • ÄKTA FPLC purification system (GE Healthcare)
  • pH paper
  • Additional reagents and equipment for dialysis ( appendix 3H), SDS‐PAGE ( protocol 14; also see unit 8.4), and ELISA ( protocol 3)

Support Protocol 2: Llama Serum Fractionation to Prepare Pooled IgG2a, IgG2b, and IgG2c Isotypes

  Materials
  • Llama blood drawn on Day 71 of protocol 1 (or Day 42 of protocol 2)
  • RPMI 1640 medium (e.g., Life Technologies)
  • Lymphoprep Tube (Cedarlane)
  • Trizol (Life Technologies)
  • First‐Strand cDNA Synthesis kit (GE Healthcare)
  • 10 pmol/µl primers: CH2FORTA4, CH2B3‐F, MJ1, MJ2, MJ3, MJ7, MJ8, PN2, M13RP (see Table 2.17.2)—primers were purchased from Integrated DNA Technology (IDT)
  • 5 U/µl Taq DNA polymerase and 10× PCR buffer
  • dNTPs: 10 mM each of dTTP, dATP, dCTP and dGTP (New England Biolabs)
  • QIAquick Gel Extraction and QIAquick PCR purification kits (Qiagen)
  • pMED1 phagemid vector (Arbabi Ghahroudi et al., ; the vector is available from the authors upon request)
  • SfiI, XhoI, and PstI restriction endonucleases and their respective 10× buffers
  • LigaFast Rapid DNA Ligation System (Promega)
  • Electrocompetent TG1 E. coli cells (see protocol 7)
  • SOC medium (see recipe)
  • 2xYT medium (see recipe)
  • 2xYT‐Amp plates: 2×YT plates (see recipe) containing 100 µg/ml ampicillin2xYT‐Amp‐Glu medium: 2xYT medium (see recipe) containing 100 µg/ml ampicillin and 2% (w/v) glucose
  • 70% (v/v) glycerol, autoclave to sterilize
  • ND‐1000 spectrophotometer (Thermo Scientific) or similar instrument
  • Electroporation cuvettes (BioRad)
  • MicroPulser electroporator (BioRad) or equivalent electroporation device
  • 37°C shaking water bath or incubator
  • Refrigerated centrifuge
  • Additional reagents and equipment for measuring nucleic acid concentration ( appendix 3L and Gallagher and Desjardins, ), counting cells ( appendix 3A), agarose gel electrophoresis (see protocol 6 and unit 10.4), and DNA sequencing (unit 10.25)
Table 2.7.2   MaterialsPrimers Used in Working with Single‐Domain Antibodies

Primer no. Primer name Primer sequence
First PCR VH/VHH5′ end primers
1 MJ1 GCC CAG CCG GCC ATG GCC SMK GTG CAG CTG GTG GAK TCT GGG GGA
2 MJ2 GCC CAG CCG GCC ATG GCC CAG GTA AAG CTG GAG GAG TCT GGG GGA
3 MJ3 GCC CAG CCG GCC ATG GCC CAG GCT CAG GTA CAG CTG GTG GAG TCT
First PCR VH/VHH3′ end primers
4 CH2FORTA4 CGC CAT CAA GGT ACC AGT TGA
5 CH2B3‐F CH2B3‐F
Second PCR VH/VHH5′ end primers
6 MJ7 CAT GTG TAG ACT CGC GGC CCA GCC GGC CAT GGC C
Second PCR VH/VHH3′ end primers
7 MJ8 CAT GTG TAG ATT CCT GGC CGG CCT GGC CTG AGG AGA CGG TGA CCTGG
Subcloning VHH5′ end primers
8 BbsI1‐VHH TAT GAA GAC ACC AGG CCC AGG TAA AGC TGG AGG AGT CT
9 BbsI2‐VHH TAT GAA GAC ACC AGG CCC AGG TGC AGC TGG TGG AGT CT
Subcloning VHH3′ end primers
10 BamHI‐VHH TTG TTC GGA TCC TGA GGA GAC GGT GAC CTG
Colony‐PCR, sequencing5′ end primers
11 M13RP CAG GAA ACA GCT ATG AC
Colony‐PCR, sequencing 3′ end primers
12 M13FP GTA AAA CGA CGG CCA GT
13 PN2 CCC TCA TAG TTA GCG TAA CGA TCT
Primers for dimeric VHH
14 DIMBACK1 GGT GGC AGT GGC GGT GGA GGT GGC GGA GGC AGC GGA GGC GGT GGC AGT CAG GTG CAG CTG GAG GAG TCT
15 DIMFOR1 TCC ACC GCC ACT GCC ACC GCT ACC TCC GCC ACC AGA ACC TCC ACC GCC TGA GGA GAC GGT GAC CTG GGT
Primers for mouse Fc fusion
16 FC‐FW GGT GTA CAG TGT XXXXXX ( Xs are the nucleotides at the 5′ of the VHH gene)
17 FC‐RE AAT GGG CCC GCT GGG CTC AAG TTT TTT GTC CAC CGT CAT
18 pTT5‐mFC7 AGC TGT TGG GGT GAG TAC TCC
19 pTT5‐mFC8 GGA TGG TCC ACC CT CGA GGT TAG G
Primer for pentamerization
20 VT‐BbsI‐f TAA TAA GAA GAC ACC AGG CCG ATG TGCA GCT GCA GGC GTC TG
21 VT‐ApaI‐r ATT ATT ATG GGC CCT GAG GAG ACG GTG ACC TGG GTC

Basic Protocol 2: Construction of VHH Library

  Materials
  • Agarose
  • I× TAE buffer (prepare from 50× stock; see recipe)
  • DNA sample ( protocol 5)
  • Loading dye (unit 10.4)
  • Ethidium bromide staining solution (unit 10.4)
  • UV transilluminator (see unit 10.4)
  • Gel Extraction Kit (Qiagen)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 10.4) and measurement of DNA concentration ( appendix 3L)

Support Protocol 3: Purification of DNA Fragments from Agarose Gels

  Materials
  • TG1 cells (Stratagene)
  • LB medium (see recipe)
  • 10% (v/v) glycerol
  • 37°C shaking water bath or incubator
  • Spectrophotometer for measuring OD 600
  • 0.5‐ml microcentrifuge tubes, autoclaved

Support Protocol 4: Preparation of Electrocompetent Cells

  Additional Materials (also see protocol 5)
  • Blood (50 ml) from at least five non‐immunized llamas (or other camelid species if available)
  • Fd‐tet‐M vector (Zacher et al., ; Kumaran et al., )

Alternate Protocol 2: Construction of Non‐Immune (Naïve) Library

  Materials
  • VHH library glycerol stock ( protocol 5)
  • M13KO7 helper phage (New England Biolabs)
  • 2xYT‐Amp‐Glu medium: 2xYT medium (see recipe) containing 100 µg/ml ampicillin and 2% (w/v) glucose
  • 2xYT‐Amp‐Kan medium: 2xYT medium (see recipe) containing 100 µg/ml ampicillin and 50 µg/ml kanamycin
  • PEG/NaCl (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • Frozen stock of E. coli TG1 cells (Stratagene)
  • M9 minimal medium plate (see recipe)
  • 2xYT medium (see recipe)
  • 2xYT‐Amp plates: 2×YT plates (see recipe) containing 100 µg/ml ampicillin
  • Antigens (100 to 200 µg required)
  • Buffer B: PBS plus 1% (w/v) casein (autoclave to sterilize)
  • PBST: PBS (see recipe) containing 0.05% (v/v) Tween 20
  • 0.1 M triethylamine, prepared fresh daily
  • 1 M Tris·Cl, pH 7.4 ( appendix 2A)
  • Kanamycin: 50 mg/ml, filter sterilize and store at −20°C in 1‐ml aliquots
  • Spectrophotometer
  • 37°C shaking water bath or incubator
  • Refrigerated centrifuge
  • 0.2‐µm filter units
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • 37°C shaking water bath or incubator
  • 32°C incubator
  • 96‐well MaxiSorp microtiter plates (VWR)

Basic Protocol 3: Rescue of Phagemid Library and Panning

  Materials
  • 2xYT‐Amp‐Glu medium: 2xYT medium (see recipe) containing 100 µg/ml ampicillin and 2% (w/v) glucose
  • TG1 cells containing the VHH phagemid (previously screened from the titer plate by colony PCR and DNA sequencing; protocol 9)
  • M13KO7 helper phage (New England Biolabs)
  • 2xYT‐Amp‐Kan medium: 2xYT medium (see recipe) containing 100 µg/ml ampicillin and 50 µg/ml kanamycin
  • Antigens
  • Phosphate‐buffered saline (PBS; see recipe)
  • Buffer B: PBS (see recipe) plus 1% (w/v) casein (autoclave to sterilize)
  • PBST: PBS (see recipe) plus 0.05% (v/v) Tween 20
  • Anti‐M13 IgG conjugated to HRP (GE Healthcare)
  • HRP substrate solutions for ELISA (GE Healthcare)
  • 1 M H 3PO 4
  • 15‐ml conical centrifuge tubes
  • Spectrophotometer
  • 96‐well MaxiSorp microtiter plates (VWR)
  • Microtiter plate reader (Biochrom, http://www.biochrom.co.uk/)
  • Additional reagents and equipment for preparing phage ( protocol 9, steps 1 to 2)

Basic Protocol 4: Monitoring the Progress of Biopanning by Phage ELISA

  Materials
  • 10× PCR buffer (Hoffmann‐La Roche)
  • Expand High‐Fidelity Taq DNA polymerase (Hoffmann–La Roche)
  • dNTPs: 10 mM each of dTTP, dATP, dCTP and dGTP (New England Biolabs)
  • Primers: VHBbs1, VHBbs2, VHBam, M13RP, M13FP (see Table 2.17.2; purchase from Integrated DNA Technology)
  • Titer reference plates with positive colonies ( protocol 9, step 18)
  • QIAquick PCR Purification kit (Qiagen)
  • Restriction enzymes BbsI, BamHI (New England Biolabs)
  • QIAquick Gel Extraction kit (Qiagen)
  • pSJF2 expression vector (Baral and Arbabi Ghahroudi, ; cut with BbsI and BamHI before ligation)
  • LigaFast Rapid DNA Ligation System (Promega)
  • TG1 electroporation‐competent cells ( protocol 7)
  • SOC medium (see recipe)
  • LB‐Amp plates: Prepare LB agar (see recipe) and add filter‐sterilized ampicillin at a final concentration of 100 µg/ml when agar medium has cooled to ∼50°C, but before pouring plates
  • 5 U/µl regular Tag DNA polymerase (not “Expand”)
  • Thermal Cycler (GeneAmp PCR System 9700, Applied Biosystems) or similar instrument
  • Sterile toothpicks or 10‐µl (P‐10) pipet tip
  • Disposable electroporation cuvettes (BioRad)
  • MicroPulser electroporator (BioRad Laboratories) or similar instrument
  • 37°C shaking water bath or incubator
  • 32°C incubator
  • ND‐1000 spectrophotometer (Thermo Scientific) or similar instrument (Gallagher and Desjardins, )
  • Additional reagents and equipment for agarose gel electrophoresis (see protocol 6 and unit 10.4), DNA sequencing (unit 10.25), and microvolume determination of DNA concentration (Gallagher and Desjardins, )

Basic Protocol 5: Subcloning of VHH Fragments in Bacterial Expression Vector

  Materials
  • Plate with colonies of positive clones ( protocol 11)
  • B2YT‐Amp medium: B2YT medium (see recipe) containing 100 µg/ml filter‐sterilized ampicillin
  • 1 M isopropyl‐β‐D‐thio‐galactopyranoside (IPTG; Rose Scientific, http://www.rosesci.com/) stock solution in H 2O
  • LB‐Amp medium: LB medium (see recipe) containing 100 µg/ml filter‐sterilized ampicillin
  • M9 medium (see recipe) containing 100 µg/ml filter‐sterilized ampicillin
  • 10× induction medium (see recipe)
  • Wash solution: 10 mM Tris·Cl, pH 8.0 ( appendix 2A) containing 150 mM NaCl
  • Sucrose solution: 10 mM Tris·Cl, pH 8.0 ( appendix 2A) containing 1 mM EDTA and 25% (w/v) sucrose
  • Shock solution: 10 mM Tris·Cl, pH 8.0 ( appendix 2A) containing 0.5 mM MgCl 2, ice cold
  • Mouse anti‐His 6 antibody (GE Healthcare, cat. no. 27‐4710‐01)
  • Starting buffer (see recipe)
  • Lysis buffer (see recipe), ice cold
  • 100 mM phenylmethylsulfonyl fluoride (PMSF; Thermo Scientific)
  • 1 M dithiothreitol (DTT; Sigma‐Aldrich)
  • 3 mg/ml lysozyme (USB Corporation), freshly prepared
  • 15 U/µl DNase I (Sigma‐Aldrich) in 1 M MgCl 2
  • Incubator with rotary shaking
  • Beckman Coulter J2‐21M/E high‐speed centrifuge or equivalent
  • Sorvall high‐speed, swinging‐bucket bench‐top (RT6000B Refrigerated) centrifuge or equivalent
  • 250‐ml Erlenmeyer flask
  • Cell density meter (Biochrome Ltd., http://www.biochrom.co.uk/) or equivalent
  • Dialysis tubing with MWCO 8000 (Biodesign Inc., http://biodesignofny.com/)
  • 0.2 µm GP Express Plus membrane filtration system (Millipore)
  • Additional reagents and equipment for SDS‐PAGE ( protocol 14; also see unit 8.4) and western blotting (immunoblotting; protocol 14; also see unit 8.10)

Basic Protocol 6: Expression of Single‐Domain Antibodies in Bacterial Systems

  Materials
  • Pure Proteome Nickel Magnetic Beads (Millipore)
  • Lysis buffer (see recipe)
  • Cell lysate, dialyzed and filtered ( protocol 12)
  • Wash solution: 10 mM Tris·Cl, H 8.0 ( appendix 2A) containing 150 mM NaCl
  • Elution buffer (see recipe)
  • 5 mg/ml NiCl 2
  • Starting buffer (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Sodium azide (Thermo Scientific Pierce)
  • Pure Proteome Magnetic Stand (Millipore)
  • 10‐ml glass collection tubes (Fisher Scientific, cat. no. 14‐961‐26)
  • 5 ml HiTrap Chelating HP column (GE Healthcare)
  • ÄKTA FPLC purification system (GE Healthcare)
  • Dialysis tubing with MWCO 8000 (Biodesign Inc., http://biodesignofny.com 0.2 µm GP Express Plus Membrane filtration system (Millipore)
  • ND‐1000 spectrophotometer (Thermo Scientific; also see Gallagher and Desjardins, ) or a similar instrument
  • Additional reagents and equipment for SDS‐PAGE ( protocol 14; also see unit 8.4), dialysis ( appendix 3H), and microvolume spectrophotometry (Gallagher and Desjardins, )

Basic Protocol 7: Purification of Single‐Domain Antibodies

  Materials
  • 70% ethanol for cleaning glass plates of gel‐casting apparatus
  • 30% polyacrylamide (BioRad)
  • 1.5 M (pH 8.8) and 0.5 M (pH 6.8) Tris·Cl ( appendix 2A)
  • 10% (w/v) sodium dodecyl sulfate (SDS) in H 2O
  • 10% (w/v) ammonium persulfate (APS) in H 2O
  • TEMED (Life Technologies)
  • Isopropanol (ACP Chemicals, Inc., http://www.acpchem.com/)
  • 10× electrophoresis running buffer: 30.35 g Tris, 141.75 g glycine, and 10 g of SDS per liter of water
  • 4× SDS loading buffer (see recipe)
  • Coomassie blue dye: combine 50 ml isopropyl alcohol, 20 ml acetic acid, 50 mg of Coomassie blue dye, and 130 ml of water
  • Destaining solution: 10% (v/v) acetic acid, 30% (v/v) methanol, and 60% (v/v) water
  • 1× transfer buffer: 3.03 g Tris hydrochloride, 14.41 g glycine, 40 ml methanol, H 2O up to 1 liter
  • Phosphate‐buffered saline (PBS; see recipe)
  • PBST: PBS (see recipe) containing 0.1% (v/v) Tween 20
  • MPBS blocking buffer: 2% (w/v) skim milk in PBS
  • Mouse anti‐His 6 primary antibody (GE Healthcare)
  • Alkaline phosphatase–conjugated goat anti‐mouse secondary antibody (Jackson Immunoresearch)
  • AP‐conjugated substrate kit (BioRad)
  • Electrophoresis apparatus: e.g., Protean II 16‐cm cell (BioRad) or SE 600/400 16‐cm unit (Hoefer) with clamps, glass plates, casting stand, and buffer chambers
  • Spacers
  • Teflon comb with 1, 3, 5, 10, 15, or 20 teeth
  • 100°C water bath or heat block
  • Immobilon PVDF membrane (Millipore)
  • Whatman 3MM filter paper
  • Trans‐Blot SD (BioRad) transfer apparatus for semi‐dry western blotting (also see unit 8.4)
  • Additional reagents and equipment for electrophoresis (unit 8.4) and immunoblotting (western blotting; unit 8.10)

Support Protocol 5: SDS‐Page and Western Blotting

  Materials
  • Distilled, deionized H 2O, filtered and degassed
  • Phosphate‐buffered saline (PBS; see recipe), filtered and degassed
  • IMAC‐purified VHHs ( protocol 13)
  • Superdex 75 size‐ exclusion column (GE Healthcare)
  • ÄKTA FPLC purification system (GE Healthcare)

Basic Protocol 8: GEL‐Filtration/Size‐Exclusion Chromatography

  Materials
  • 5 ng/µl pSJF2‐VHH1 plasmid
  • 5 ng/µl pSJF2‐VHH2 plasmid
  • 10 pmol/µl Primers: M13RP, M13 FP, DIMBACK1, and DIMFOR1 (see Table 2.17.2)—primers were purchased from Integrated DNA Technology (IDT)
  • Restriction enzymes EcoRI and HindIII (New England Biolabs)
  • B2YT medium (see recipe)
  • Additional reagents and equipment for amplification of VHH genes ( protocol 11), expression of single‐domain antibodies in bacterial systems ( protocol 12), and DNA sequencing (unit 10.25)

Basic Protocol 9: Increasing Functional Affinity and Potency of VHHs

  Materials
  • Construct containing sdab gene ( protocol 11, step 8)
  • Primers: FC‐FW, FC‐RE, pTT5‐mFc7, and pTT5‐mFc8 (see Table 2.17.2)—the primers were purchased from Integrated DNA Technology (IDT).
  • Restriction enzymes: BsrGI and ApaI (New England Biolabs)
  • pTT5‐mFc vector (Zhang et al., )
  • QIAquick PCR purification kit (Qiagen)
  • 2xYT‐Amp plates: 2×YT plates (see recipe) containing 100 µg/ml ampicillin
  • Maxiprep plasmid extraction kit (Qiagen)
  • HEK‐293 cells (Dr. Yves Durocher, National Research Council Canada)
  • L FreeStyle F17 medium (Life Technologies): add 10 ml of 10% (100×) Pluronic F‐68 stock, 10 ml of 200 mM (100×) L‐glutamine stock, and 10 ml of 100× antibiotic/antimycotic stock per liter of Freestyle F17 medium just prior to use
  • Phosphate‐buffered saline (PBS; see recipe)
  • Polyethyleneimine (PEI; Polyplus, http://www.polyplus.com/)
  • Trypton N1 (TN1, Organotechnie S.A., cat. no. 19553; http://www.organotechnie.com/): make 20% stock solution in prewarmed Freestyle F17 culture medium just prior to use
  • Goat anti‐mouse HRP conjugate (Cedarlane)
  • MicroPulser electroporator (BioRad) or equivalent electroporation device
  • Disposable Erlenmeyer 125‐ml plastic shaker flasks (Corning, cat no. 43143)
  • 5‐ml HiTrap protein A HP column (GE Healthcare)
  • 0.22‐µm sterilization filter (e.g., Millipore)
  • 0.45 um Steritop filter unit with 500 ml filtrate capacity (Millipore)
  • Additional reagents for PCR (as described in protocol 11, step 1) and colony PCR (as described in protocol 11, step 7), counting cells and determining cell viability ( appendix 3A & ), purification of sdAb on a protein A column ( protocol 4), SDS‐PAGE ( protocol 14; also see unit 8.4), ELISA ( protocol 3), and SPR ( protocol 20)

Basic Protocol 10: Preparation of VHH Fc Fusion

  Materials
  • dNTPs: 10 mM dATP, dCTP, dGTP, dTTP (New England Biolabs)
  • 10× PCR buffer (Hoffmann‐La Roche)
  • Primers: VT‐BbsI‐f and VT‐ApaI‐r (see Table 2.17.2)——purchased from Integrated DNA Technology (IDT).
  • Plasmid template containing sdAb gene ( protocol 11, step 8)
  • Expand High‐Fidelity Taq DNA polymerase (Hoffmann–La Roche)
  • 500 ng/µl pVT2 vector (Zhang et al., )
  • Restriction enzymes BbsI and ApaI (New England Biolabs)
  • NEBuffer 4 (New England Biolabs)
  • QIAquick Gel Extraction Kit (Qiagen)
  • LigaFast Rapid DNA Ligation System (Promega)
  • Electrocompetent TG1 E. coli cells (see protocol 7)
  • LB medium (see recipe)
  • LB‐Amp plates: Prepare LB agar (see recipe) and add filter‐sterilized ampicillin at a final concentration of 100 µg/ml when agar medium has cooled to ∼50°C, but before pouring plates
  • Mouse anti‐VT antibody (National Research Council Canada)
  • Goat anti mouse–alkaline phosphatase (Jackson Immunoresearch)
  • Alkaline phosphatase substrate (BioRad Laboratories)
  • Thermal cycler
  • ND‐1000 spectrophotometer (Thermo Scientific; see Gallagher and Desjardins, )
  • 16°C water bath
  • MicroPulser electroporator (BioRad) or equivalent electroporation device
  • Water bath or incubator with rotary shaker
  • Additional reagents and equipment for PCR (unit 10.20), microvolume quantitation of DNA (Gallagher and Desjardins, ), agarose gel electrophoresis (unit 10.4), DNA sequencing (unit 10.25), and SDS‐PAGE and western blotting ( protocol 14)

Support Protocol 6: Pentamerization of VHHs

  Materials
  • Purified antigen (100 µg/ml stock)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Purified VHH solution ( protocol 13, step 4b)
  • PBST: PBS (see recipe) containing 0.05% Tween 20
  • Anti‐His 6 mouse monoclonal antibody (GE Healthcare, cat. no. 27‐4710‐01)
  • HRP substrate solutions for ELISA (GE Healthcare)
  • ELISA plates: 96‐well MaxiSorp microtiter plates (VWR)
  • Additional reagents and equipment for blocking ELISA plate (see Basic Protocols protocol 93 and protocol 104) and ELISA procedure ( protocol 3)

Basic Protocol 11: Characterization of Antigen‐Specific VHHs

  Materials
  • HBS‐E buffer: 10 mM HEPES (pH 7.4) containing 150 mM NaCl, and 3 mM EDTA (this buffer can be purchased from GE Healthcare; if it is not purchased from GE Healthcare, it should be thoroughly degassed before use)
  • Surfactant P20 (GE Healthcare)
  • IMAC‐purified VHH protein ( protocol 13, step 4b)
  • Protein assay kit
  • Amine coupling kit that contains N‐hydroxysuccinimide (NHS), N‐ethyl‐N′‐(3‐dimethylaminopropyl)carbodiimide hydrochloride (EDC), and ethanolamine (GE Healthcare)
  • Antigen (same antigen used for llama immunization in protocol 1)
  • Immobilization buffer: 10 mM sodium acetate buffer, pH 4.0 (GE Healthcare, cat. no. BR 100349)
  • sdAb‐Fc fusion ( protocol 17)
  • sdAb pentabodies ( protocol 18)
  • 10 mM phosphate buffer, pH 7.3: dissolve 1.4 g Na 2HPO 4 and 0.24 g KH 2PO 2 (pH should be 7.3) in 1 liter of distilled H 2O, filter‐sterilize, and store at room temperature.
  • Superdex 75 10/300 GL and Superdex 200 10/300 GL gel filtration columns (GE Healthcare; also see protocol 15)
  • BIACORE 3000 (GE Healthcare) or another surface plasmon resonance instrument with similar capabilities
  • CM5 sensor chips (GE Healthcare)
  • BIAevaluation software 4.1 (GE Healthcare)
  • 10‐mm quartz cuvette
  • A J‐850 CD spectrometer (JASCO)
  • GraphPad Prism software (http://www.graphpad.com/)
  • Additional reagents and equipment for size‐exclusion chromatography ( protocol 15) and determination of circular dichroism (CD) spectrum (Pain, )
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