Chapter 11

Phage Display and Selections on Biotinylated Antigens Patrick Chames and Daniel Baty

11.1

Introduction

Phage antibody library selections on peptides or proteins are usually carried out using antigens directly coated on a plastic surface (e.g., Petri dishes, microtiter plate well, immunotubes). This straightforward method is easy to perform and has been shown to be very successful for a diverse set of antigens (for review (Winter et al. 1994). However, phage-antibody selections on some proteins, and especially on peptides are not always successful, which is often caused by immobilizationassociated features. The main problem observed for selection on peptides is the very poor coating efficiency of some peptides and the altered availability of epitopes on plastic-coated peptides. The direct coating of proteins on plastic is usually more efficient but can also be problematic, because the passive adsorption on plastic at pH 9.6 is a mechanism of protein denaturation. Under these conditions, 95% of adsorbed proteins are nonfunctional (Butler et al. 1992; Davies et al. 1994).This problem is not very important for a classical ELISA because mostly a small fraction of proteins having a native conformation is still detectable. However, this phenomenon can be very troublesome for phage antibody library selections, because phage antibodies binding to epitopes only present in denatured molecules may be selected. Several methods have been developed to increase peptide coating, including coupling to bigger proteins (Oshima and Atassi 1989), to amino acid linkers binding plastic (Loomans et al. 1998; Pyun et al. 1997), or to multiple antigen peptide (Tam and Zavala 1989). The most successful method had been the indirect coating of biotinylated antigens via streptavidin: biotinylation of the peptide and immobilization via streptavidin improves the sensitivity in ELISA (Ivanov et al. 1992) and allows more efficient selection of anti-peptide phage-antibodies (de Haard et al. 1999; Henderikx et al. 1998).

P. Chames (*) and D. Baty INSERM – U624, 163 avenue de Luminy, 13288 Marseille Cedex 09, France e-mail: [email protected]

R. Kontermann and S. Du¨bel (eds.), Antibody Engineering Vol. 1, DOI 10.1007/978-3-642-01144-3_11, # Springer-Verlag Berlin Heidelberg 2010

151

152

P. Chames and D. Baty

In the case of phage library selection against proteins, the indirect coating via streptavidin results in higher density coating, more uniform distribution of antigens on the well surface, and above all, 60–70% of active molecules (Butler et al. 1992; Davies et al. 1994). Most importantly, however, the use of biotinylated peptide or protein allows the use of paramagnetic streptavidin-coated microbeads to capture the biotinylated antigens with the phage bound to them. The interaction between the phage particle and the antigen therefore takes place in solution; antigen-bound phage is retrieved via a short incubation with the beads. This technique permits precise control of the antigen concentration and the time of exposure of the antigen to the phage-antibody library, two parameters that are very useful in affinity selection, for example, during affinity maturation protocols (Hawkins et al. 1992; Schier et al. 1996). This interaction between antigen and phage antibody in solution leaves a maximum of epitopes available for binding, and avoids the selection of scFv fragments with low affinity but with a high tendency to form dimers (Schier et al. 1996). The latter will be preferentially selected on antigen-coated surfaces because of their avid binding. This chapter contains protocols for chemical or enzymatic biotinylation, as well as phage library selection in solution and sensitive ELISA procedures for using indirectly-coated biotinylated antigen. The advantages and drawbacks of each method are discussed.

11.2

Biotinylation of Antigens

11.2.1 Biotinylation of Proteins/Peptides with NHS-ss-Biotin 11.2.1.1

Purpose

Chemical biotinylation is the most common way to obtain a biotinylated antigen. There are many commercially available reagents that can be used for biotinylation using a variety of chemistries. For most of our biotinylations, we prefer to use the chemical reagent NHS-SS-Biotin (Sulfo-Succinimidyl-2-(biotinamido)ethyl-1, 3-dithiopropionate MW ¼ 606.70). This molecule is a unique biotin analog with ˚ in length, capable of reacting with primary amine an extended spacer arm of 24.3 A groups (lysines and NH2 termini). The long chain reduces steric hindrances associated with binding of biotinylated molecules to avidin or streptavidin and should not interfere with the structure of the protein/peptide involved. The presence of the S–S linker in NHS-S-S-Biotin enables the use of a reducing agent (DTT, DTE, b-mercaptoethanol) to separate the antigen and all phageantibodies bound to it from the beads. This feature allows a more specific elution, which is very useful when undesired streptavidin binders are preferentially selected from a phage antibody repertoire. The following method is modified from (Hnatowich et al., 1987) and the Pierce instruction manual.

11

Phage Display and Selections on Biotinylated Antigens

11.2.1.2

153

Materials

– 50 mM Sodiumbicarbonate pH ¼ 8.5:4.2 g NaHCO3 adjust pH with 6 M NaOH to pH 8.5, adjust to 1 l with H2O, filter-sterilize, store at RT – NHS-SS-Biotin (Pierce 21331, Rockford, Illinois) – Peptide/Protein of interest – The best molar ratio of biotin to the protein has to be determined empirically. Try different molar ratios if possible. NHS-SS-Biotin MW ¼ 606.70 NHS-LCBiotin MW ¼ 556.58 – Alternatively, if no dialysis is used: Centricon 30 or Centricon 10 (Amicon, 4306, 4304; Millipore, Billerica, MA) – Dialysis tubing (tubing with MW-cut-off of 1,000–50,000 kD can be found at Cellu.Sep, Waterloo, Belgium) – 5* PBS pH ¼ 7.4: 43.8 g NaCl (750 mM), 7.1 g Na2HPO4 (40 mM), 1.08 g KH2PO4 (7.8 mM), adjust volume to 1 l. Before the test, add 800 ml H2O to 200 ml of 5* PBS and check the pH (7.4)

Precautions Avoid buffers containing amines (such as Tris or glycine) since these compete with peptide/protein the biotinylation reaction. Also, reducing agents should not be included in the conjugation step to prevent cleavage of the disulphide bond within NHS-SS-Biotin.

11.2.1.3

Procedure

1. Dissolve 1–10 mg/ml of the peptide/protein of interest in 50 mM NaHCO3 (pH ¼ 8.5). If the peptide/protein is already in another solvent, dialyse against 50 mM NaHCO3 (2–3 x for at least 4 h in 1 l) 2. Calculate the required amount of NHS-SS-Biotin taking into account a Molar ratio Biotin:Protein ¼ 5:1–20:1. Although this amount depends on the number of lysines present within the protein, usually a ratio of 5:1 works fine. When enough protein is available, it is advised to test different ratios of Protein: Biotin. Ideally 1–2 biotinylated residues per molecule are present. Overbiotinylation often results in nonfunctional protein (aggregation, etc.) 3. Dissolve the required amount of NHS-SS-Biotin in distilled water and immediately add this to your protein sample, or alternatively, when using larger amounts of protein, add NHS-SS-Biotin directly to the protein solution 4. Place the tube on ice for 2 h or 30 min at room temperature 5. Add 1 M Tris (pH ¼ 7.5) to a final concentration of 50 mM, and incubate 1 h on ice to block free biotin 6. To remove free biotin, dialyse against the biotinylated protein in PBS at 4 C (3 for at least 4 h in 1 L PBS) overnight or alternatively: do steps 7 to 9. For

154

7. 8. 9. 10. 11.

P. Chames and D. Baty

small peptides (