Epitope Mapping Protocols Volume 66 || Production of Phage-Display Antibodies for Epitope Mapping

33

Production of Phage-Display Antibodies for Epitope iMapping

Jenny Walker and George Banting

1. Introduction The development of genetic engineering has enabled the production of anti

bodies in Escherichia coli (1,2). An essential requirement for a good antibody expression system is that an antibody fragment is folded and in a functional state so that the selection or purification procedure can make use of its antigen-binding properties. The application of filamentous phage-display technology to the expression of antibody fragments enables the rapid cloning of an immunological repertoire to produce large libraries that can be screened for appropriate antigen-binding specificities.

New expression systems have been developed, inspired by a method where diverse peptides are displayed on the surface of bacteriophage (3,4). In these systems, an antibody fragment is cloned into a phagemid vector next to a gene encoding a bacteriophage coat protein and subsequently expressed attached to the coat protein on the surface of the bacteriophage (5,6). Such surface expression allows the selection of specific antibodies based on their ability to bind to immobilized antigen. Those phage particles displaying a particular antibody fragment contain the gene encoding that fragment, thus creating a "genetic display package" (Fig. 1). This mimics the immune system where the rearranged gene encoding an antibody is found within the B-lymphocyte on whose surface the antibody is displayed.

Several variations of antibody phage-display vectors have been described (5—7). Each system involves polymerase chain reaction (PCR) amplification of the relevant regions of the antibody gene. PCR primers have been designed based on conserved nucleotide sequences of antibody genes extracted from the Kabat database (8). The oligonucleotide primers used have included rare

From Methods m Molecular Biology, vol 66' Epitope Mapping Protocols Edited by G E Morns Humana Press Inc , Totowa, NJ

391

392 Walker and Banting

Phagemfd Vector

r-1

n

Fig. 1. Cartoon representing a bacteriophage particle containing phagemid DNA that encodes for the Fab expressed on its surface. The Fab fragment is expressed attached to a gene 3 bacteriophage coat protein.

restriction sites in order to reduce the chances of digesting the imported immunoglobulin gene when restricting PCR products prior to cloning.

The most complex proteins to be heterologously expressed on the surface of filamentous phage are antibody Fab fragments (5). The cloning of these fragments is the basis of the system described in this chapter for the production of phage-display antibody fragments.

Antibody Fab fragments are composed of the complete light chain and the heavy-chain Fd region, which consists of the variable domain and the first constant region of the heavy chain. The heavy- and light-chain variable domains alone make up the antibody Fv fragment. The constant region domains present in Fab antibody fragments facilitate strong association between the heavy and light chains relative to their smaller Fv counterparts (9). The binding between the heavy and light constant domains is both covalent (with the formation of a disulfide bridge) and noncovalent in nature. The resultant additional stability in the structure overcomes the need for an artificial covalent link between the chains, such as the peptide linker in single-chain Fv fragments.

The phagemid vector pComb3H-SS is a modified version of pComb3, which was derived ft-om the phagemid pBluescript (5,10) (see Fig. 2). In this system, unique restriction sites are provided for the independent cloning of the heavy-chain variable domain, and first constant domain (Fd) sequences (Xhol and Spel) and light-chain sequences (Sad andXbal). The SS designation (see Fig. 2)

Phage-Display Antibodies 393

Nhel

M13on

Fig. 2. pComb3H-SS phagemid cloning vector. See text for details.

represents the presence of stuffer fragments in the antibody fragment cloning sites. On expression, both the heavy- and light-chain antibody fragments are targeted to the periplasmic space by appropriate leader peptides. The cDNA encoding the light chain is inserted immediately 3' of sequence encoding the outer membrane omp A leader peptide oiE. coli, whereas the cDNA encoding the Fd chain is inserted between sequences encoding the pel B leader peptide of Erwinia camtovora and the C-terminal domain of the phage coat protein g3p. The g3p portion of the resultant expressed fusion protein accumulates in the inner membrane of the E. coli, with the Fd region in the periplasmic space, where it can associate with a light chain to form a functional Fab fragment (Fig. 3). The expression of the heavy and light chain sequences is under the control of a lac promoter/operator sequence. The phagemid vector is propagated in XL 1-blue cells (11), thus allowing the induction of transcription by the addition of IPTG (12).

Inclusion of the bacteriophage intergenic region in the vector allows helper phage initiated transcription of single-stranded phagemid DNA. The helper phage infection leads to the expression of native g3p as well as phagemid-derived Fab-g3p. The resultant packaged phage carries native g3p, which is


Phagemid Veclot

g S f i - - - ; Fab

mm •mmm mmm mmm tmammmmmm

v_/

mmm

^F,ab Periplasm

'• —~'^..~..>-m.~~~~..... -IrmticMprnDHBa...—

OmpA Leader Sequence

L chain Antibody

pe!B Leader Sequence

H chain Antibody

g3p

Fig. 3. Cartoon representing tlie pathway for the assembly of an Fab antibody fragment in the bacteriophage expression system. The antibody heavy and light chains are directed to the periplasmic space by a leader sequence, which is subsequently cleaved. The heavy and light chains assemble in the periplasmic space to form a functional Fab fragment, which is secreted from the cell attached to a gene 3 bacteriophage coat protein.

necessary for infection, and the encoded Fab-g3p, which is displayed for selection. According to published reports, Fab fragments expressed with this system are monomeric in nahire, thereby facilitating the isolation of specific, high-affinity antibody clones (13.14).

The random combinatorial principles of the antibody library construction result in a complete scrambling of association between protein sequences encoded by the heavy- and light-chain genes, i.e., any heavy chain can associate with any light chain. As a result, the original heavy- and light-chain pair is


unlikely to be recovered, and high-affinity (>10^ moH) antigen-binding heavy and light chain combinations are likely to be rare. Two factors that improve their frequency in antibody libraries are (1) chain promiscuity—the ability of particular heavy chains to bind effectively antigen when combined with different light-chain partners (6), and (2) using RNA from an immune source in library construction (5,6). Affinity purification of phage antibodies by "panning" enables the selection of even rare antigen-binding antibody combinations fi-om a library. Antibody fragments can be displayed to antigen in the following ways:

1. Antigen adsorbed to a plastic surface (5,15). 2. Columns of antigen linked to a matrix (16). 3. Biotinylated antigen in solution, subsequendy captured on streptavidin-coated

magnetic beads (17). 4. Antigen expressed on the surface of an immobilized cell (18).

Nonbinders can be removed by washing and the bound phage can then be eluted at low pH (5), high pH (18), or by addition of excess antigen (6). Successive rounds of selection can be achieved by infecting bacteria with the enriched phage and panning the phage prepared from the culture. The enrichment resulting from repeated rounds of selection should be sufficient to isolate specific phage occurring only singly in the initial library (i.e., about 1 in 10'). Once phage-displayed antibody fragments have been selected against a specific antigenic target, they can be expressed without the g3p and can be screened for reactivity with antigen, usually by conventional enzyme-linked immunosorbent assay (ELISA).

In this chapter, detailed methods for the production and panning of Fab antibody libraries will be described. In addition to the obvious advantage of obtaining a number of epitope-specific antibodies from a single library, the smaller size of Fab fragments (relative to whole antibody molecules) produced with this system may facilitate greater accessibility than whole antibodies to the target antigen. Antibody library construction circumvents the laborious process of hybridoma production and allows new approaches to antibody selection and design. With antibody cloning, one has immediate access to the DNA encoding the Fab fragment of interest. The sequence encoding a Fab fragment that binds to an antigen with low affinity can be manipulated in order to encode a higher affinity antibody (19).

2. Materials 1. Mice immunized with the required immunogen (following protocols used for the

production of monoclonal or polyclonal antibodies [20J; see also Chapter 32). 2. Mechanical homogenizer, e.g., Polytron. 3. RNA extraction kit (Pharmacia, Cat. No. 27-9270-01). 4. mRNA purification kit (Pharmacia, Cat. No. 27-9258-01).


5. cDNA synthesis (Pharmacia, Cat. No. 27-9661-01) kits obtained from LKB Pharmacia.

6. The mouse immunoglobulin primers required for the amplification of heavy- and light-chain antibody fragments are given below (21).

Heavy-chain Fd 3'-primers (reverse): IGgl5'-AGGCTTACIAQIACAATCCCTGGGCACAAT-3' IGg2a5'-GTTCTGACIAQTGGGCACTCTGGGCTC-3'

Heavy-chain variable domain 5' primers (forward)' Hcl 5'-AGGTCCAGCTG£l£fiMiTCTGG -3' Hc2 5'-AGGTCCAGCTG£lCQAQTCAGG -3' Hc3 5'-AGGTCCAGCTT£l£QAGTCTGG-3' Hc4 5'-AGGTCCAGCTT£lCQAQTCAGG-3' Hc5 5'-AGGTCCAACTG£l£OAQTCTGG-3' Hc6 5'-AGGTCCAACTG£l£eAQTCAGG-3' Hc7 5'-AGGTCCAACTTCTCGAGTCTGG-3' Hc8 5'-AGGTCCAACTTCTCGAGTCAGG-3' Hc9 5'-AGGTIIAICTICI£QAaTC(TA)GG-3' (see Note 1)

Murine K light-chain 3'-primer (reverse): 5'-GCGCCGlCIAQAATTAACACTCATTCCTGTTGAA-3'

Murine light-chain variable-domain 5'-primers (forward): Lcl5'-CCAGTTCCQAQ£lQGTTGTGACTCAGGAATCT-3' Lc2 5'-CCAGTTCCOMiCICGTGTTGACGCAGCCGCCC-3' Lc3 5'-CCAGTTCCfiA(ieiCGTGCTCACCCAGTCTCCA-3' LC4 5'-CCAGTTCCQAQCIJCCAGATGACCCAGTCTCCA-3' Lc5 5'-CCAGATGTGAGCTCGTGATGACCCAGACTCCA-3' Lc6 5'-CCAGATGTQAfi£TQTCATGACCCAGTCTCCA-3' Lc7 5'-CCAGTTCCQAQClCGTGATGACACAGTCTCCA-3'

7. Tag Polymerase with supplied buffer and dNTP solution (5 mAf stock solution). 8. Preparative gels should be made with Genetic Technology Grade (GTG) agarose

available from Flowgen. Seakem or Nusieve GTG agarose is recommended. 9. Electrophoresis buffer (TAE): 0.2M Tris base, 0.0005M EDTA, and 0.12% ace

tic acid adjusted to pH 7.0. 10. DNA mol-wt standard markers. 11. Electroelution apparatus or see Note 2. 12. Restriction enzymes a l , Sad, Xhol, Spel, and Nhel (Boehringer Mannheim, to

be used with the supplied buffers). 13. Chromaspin 100 column (Clontech Laboratories Inc.). 14. The phagemid vector pComb3H-SS (5) can be obtained from: Carlos Barbas

III at The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037.

15. XLl-blue MRF cells (Strategene / ; / ; ) . 16. LB agar: 10 g/L bacto-tryptone, 5 g/L bacto-teast, 5 g/L NaCl, 16 g/L agar. 17. Carbenicillin (carb) made up into 100 mg/mL stock. 18. Super broth (SB): 30 g/L bacto-tryptone, 20 g/L bacto-yeast extract, lOg/LMOPS.


19. Preparative DNA kit with tip-500 columns (Qiagen). 20. T4 DNA ligase (Gibco BRL recommended, to be used with 4X buffer supplied). 21. 0,025-).im microdialysis membranes (Millipore). 22. SOC: 20 g/L bacto-tryptone, 5 g/L bacto-yeast extract, 0.5 g/L NaCl, 2.5 mAfKCl,

10 mM MgClj, and 20 mM glucose (filter-sterilized and added after autoclaving). 23. Tetracycline (tet); 10 mg/mL stock solution. 24. M13 helper phage M13K07 (Pharmacia Cat. No. 27-1525-01). Stocks of the

helper phage should be prepared and titered following standard procedures (15). 25. Kanamycin (Kan): 100 mg/mL stock solution. 26. PEG8000. 27. PBS: 0.9% NaCl, 25 mM sodium phosphate buffer, pH 7.0. 28. Blocking buffer: 3% BSA in PBS. 29. Microtiter plates; any high binding capacity plate suitable for ELISA can be used. 30. Relevant antigen diluted in coating buffer, e.g., O.IM bicarbonate, pH 8.6 (opti

mal coating buffer may vary with antigen). 31. Humidified incubator at 37°C. 32. PBS containing 0.5% Tween-20 solution (PEST). 33. Elution buffer: O.lA/HCl (adjusted with glycine to pH 2.2), 1 mg/mL BSA. 34. 2M Tris base neutrahzing buffer. 35. IPTG; 0. IM stock solution in HjO. 36. 10mMTris,pH8.0. 37. Appropriate enzyme-conjugated anti-Fab second antibody, e.g., alkaline phos-

phatase-conjugated rabbit antimouse Fab (Pierce or Sigma). 38. Appropriate developing substrate solutions, refer to ref 20 for details.

3. Methods

3.1. Obtaining cDNA from an Immunized Source

The preferred and reliable method is to use Pharmacia kits, although other appropriate methods could be adopted. RNA can be quickly and efficiently obtained from the spleen of an immunized mouse (see Note 3) using a Pharmacia RNA extraction kit, following the manufacturer's protocol. Precautions should be taken to minimize RNA digestion by ribonucleases present in the spleen cells (see Note 4).

1. Remove the spleen aseptically, directly transfer it to an aliquot (3 mL/spleen) of extraction buffer, and immediately homogenize.

2. Isolate polyadenylated mRNA on oligo(dT)-cellulose spun columns supplied in a Pharmacia mRNA Purification Kit, following the manufacturer's protocol.

3. Prepare first-strand cDNA from either total RNA or mRNA using a Pharmacia First-Strand cDNA Synthesis Kit, following the manufacturer's protocol. Prime the reactions with primers supplied with the kit, using either 5 |.ig of a A/brt-d(T)18 bifunctional primer or 0.04 jig pd(N)* in the reaction.


3.2. Preparation and Cloning of Fab Fragments

The procedures followed are summarized in Fig. 4.

1. Follow standard techniques for PCR using an annealing temperature of 52°C and 35 cycles. Each reaction should be performed with individual primer sets in reaction volumes of 50 ixL

2. Test for the presence of amplified antibody DNA by running an aliquot (7%) of each individual PCR reaction on a 1.5% agarose gel. The expected size of the amplified antibody fragments is 660 bp.

3 Pool the PCR products with common 3'-primers, and extract once with phenol and once with phenol:chlorofonn:isoamyl prior to precipitating the DNA using standard procedures.

4. Run the recovered DNA on a preparative 1.5% agarose gel. 5. Excise the appropriate (660 bp) DNA bands (other bands may be present and

should be ignored) from the gel, and extract the DNA by electroelution (see Note 2). 6. Quantify the purified DNA, and digest with the appropriate restriction enzymes

for 3 h at 37°C. The light chain should be incubated with Xbal and Sad and the heavy chain with^ol and Spel. The number of enzyme units and buffers to use are summarized m Table 1.

7. Precipitate the restricted DNA and resuspend the DNA in 50 |j,L sterile water. 8. Purify the insert for ligation by passage through a Chromaspin 100 column, fol

lowing the manufacturer's instructions.

3.3. Preparation of Vector DNA 1. Electroporate 1 |aL of a 1:100 dilution of supplied vector DNA into a 40-\iL

aliqout of electrocompetent XLl-blue cells (see Section 3.5.), plate out dilutions onto LB agar plates (100 |xg/mL carbenicillin), and incubate overnight at 37°C.

2. Pick a single colony, inoculate 10 mL SB (50 |xg/mL carb), and incubate in a shaking incubator for 8 h.

3. Use this culture to inoculate 1 L of SB/carb and incubate, shaking (at 250 rpm) at 37°C overnight.

4. The following day, prepare phagemid DNA using the Qiagen method with a tip-500 column.

5. In preparation for the ligation of the light chain into the vector (see Section 3.4.), digest the pComb3H-SS vector DNA with Xbal and Sad for 3 h at 37°C (using the number of enzyme units and required buffers as summarized in Table 1).

6. Precipitate the digested DNA prior to running it out on a 0.8% Seakem GTG gel with appropriate mol-wt markers.

7. Excise the appropriate linearized vector band, and extract the DNA from the gel chip by electroelution (see Note 3).

8. Quantitate the DNA (see Note 5). 9. After ligation of the light chain into the vector (see Section 3.4.), repeat the above

steps from 2, but restrict the DNA with^^ol and Spel for 3 h at 37°C (using the number of enzyme units and required buffers as summarized in Table 1), thus


Purify and extract iriRNA

Hybridoma calls Spleen cells

\ y' I AAAAA

Synthesise first strand cDNA

Amplify of cDNA of Heavy and Light chain antibody CHI and V domair\s by PCR

Clone Light Chain into pCombSH Vector

• 4 Competent Celts (XLl - Blue)

Clone Heavy Cham into Vector plus Light Cham

I- Competent Cells (XLl - Blue)

Package Phage • ^ ~ " Helper Phage

I Pan (x4) with Antigen

Make Soluble Fab

TestbyELISA

Fig. 4. Cartoon representing the preparation and cloning of Fab fragments into pComb3H-SS.

preparing the vector containing the light-chain library for ligating in the heavy chain (see Section 3.4.).

3.4. Ligation of PCR Fragments Into the Vector

The prepared antibody PCR fragments are ligated in turn (first light chain, followed by heavy chain) into the restricted, quantitated vector. The insert


Table 1 Equivalent Enzyme Amounts and Buffers for Vector and PCR Insert Digestion (Using Enzymes and Buffers Obtained from Boehringer Mannheim)

Enzyme

Spel Xhol Sad Mai

Buffer

H H A A

pComb3H-SS or pComb3H-H + L,

U/jag DNA

3 9 5 9

PCR inserts

U/i^g DNA

17 70 35 70

ligation efficiencies and the extent of background (vector alone) ligation should be tested on a small scale prior to library construction.

1. For the test ligation, ligate 250 ng vector with or without 50 ng insert for 2 h at room temperature. These ligations should be performed in a 20-|iL total volume with 1 jiL T4 ligase. Electroporate 1 iL of each ligation mix into 40-|aL aliquots of competent XL 1-blue cells (see Section 3.5.), and plate out aliquots of 1, 10, and 100 nL. The background ligation should be <20% of the ligation with insert.

2. For production of the library, incubate 1400 ng of vector with 450 ng of the prepared PCR product at room temperature overnight, in the presence of 10 |.iL T4 ligase and 40 iL 5X buffer in a total reaction volume of 200 \iL.

3. Stop the reaction by heating at 65°C for 10 mm, ethanol-precipitate, and resus-pend in 15 |LiL of water.

4. Microdialyze the DNA solution against water on a 0.025-^m Millipore membrane prior to transformation (this removes excess salt from the sample, which may interfere with electroporation).

3.5. Transformation of the Library Electrocompetent XL 1-blue cells should be prepared and tested with 1 fxL

0.01 |jg/mL control pUC DNA before library preparation. Cells should transform with an efficiency of at least 2 x 10 CFU/|j.g {see Note 6).

1. Add all 15 yiL of Hbrary DNA to two aUquots of 200 )iL electrocompetent cells. 2. Transfer to 0.2-cm chilled cuvets and electroporate by pulsing at 2.5 kV, 2 |aF

and 200 fi. 3. Flush the electroporation cuvets with a total volume of 5 mL SOC. 4. Immediately incubate the cells, shaking at 37°C for 1 h.

3.6. Recovery of the Library

Initially the antibody light-chain fragments are cloned into the vector to prepare the light-chain library. The vector DNA is recovered from this and pre-


pared for the insertion of the heavy-chain antibody fragments. The combined heavy- and light-chain (Fab) library is expressed, and recombinant phage-car-rying Fab fragments are recovered.

1. After 1 h of incubation at 37°C, add 10 mL prewarmed (37°C) SB containing 20 )j,g/mL carb and 10 jig/mL tet to the transformed cells.

2. Titer the library by further diluting 10, 1, and 0.1 | L of the culture into 100 |j,L SB and plating out onto LB (carb) plates. Leave the plates to grow in a 37°C oven overnight.

3. Incubate the 10-mL culture for a further 1 h at 37°C with shaking. 4. Increase the carbenicillin concentration to 50 jjg/mL, and incubate for another

hour with shaking at 37°C. 5. Transfer the 10-mL culture to 100 mL prewarmed (37°C) SB containing 50 |j,g/mL

carb and 10 fig/mL tet. 6. For recovery of the light-chain library, leave this culture incubating at 37°C while

shaking, overnight (go to step 9 below). 7. For recovery of the final Fab (heavy- and light-chain) library, immediately add

10'2 PFU of M13K07 helper phage to the culture, and then incubate at 37°C with shaking.

8. After 2 h, add 70 |xg/niL of Kan, and then leave the culture to grow in a shaking incubator at 37°C overnight.

9. After construction of both the light and combined Fab libraries, prepare phagemid DNA from the overnight culture, using the Qiagen method.

10. After construction of the Fab (light and heavy chain) library, the phage-contain-ing supernatant can be recovered and used for the selection of antigen-specific antibody fragments. Transfer the supernatant after pelleting the cells to a clean bottle, and precipitate the phage by adding 4% (w/v) PEG-800 and 3% (w/v) NaCl.

11. Place the bottle on a shaker for 5 min to dissolve the PEG and NaCl, and then incubate on ice for 30 min.

12. Pellet the precipitate by centrifugation at 10,000g in a JAIO rotor at 4°C for 20 min.

13. Discard the supernatant and allow the bottle to drain on paper towel for about 10 min to remove as much PEG solution as possible.

14. Resuspend the pellet in 2 mL PBS/1% BSA, and transfer to Eppendorf tubes prior to microcentriftigation for 10 min to pellet any residual cell debris. Recover the supematants to fresh Eppendorf tubes, and store at 4°C.

Always reamplify library phage preparations prior to use in panning if they had been stored for more than 24 h, because the attachment of the Fab fragment to the g3p phage coat protein is relatively unstable. Reamplification can be achieved by foUowmg the panning protocol firom step 6 in Section 3.7. below.

15. Titer the phage suspension by infecting 50-|xL volumes of XLl-blue cells (OD • 600 = 0-5) with 1 i L of 10~ , 10" , and 10^ dilutions of the phage suspension at room temperature for 15 min before plating out on LB carb plates and incubating overnight at 37°C.


3.7. Panning

Several rounds of panning of the Fab-carrying phage will be required to select for antigen-specific binding clones within a library.

1. Coat ELISA plate wells overnight at 4°C with 1-0.1 \xg antigen solution in coating buffer (see Note 7).

2. On the following day, empty the wells (the antigen may be retained for repeated use), wash them three times with water, fill them with 3% BSA in PBS blocking buffer, and incubate at 37°C in a humidified incubator.

3. After 1 h, replace the blocking buffer with 100 \iL fresh phage suspension, and incubate for a further 2 h at 37°C m the humidified incubator.

4. Remove the phage fi"om the wells (see Note 8), and wash by filling them with PBST, pipeting vigorously up and down, and leaving for 5 min before removal. In the first round of panning, wash the wells once in this fashion; in the second round, wash five times; and in the third and subsequent rounds, wash 10 times.

5. After washing the wells, elute the phage by adding 50 |J.L of elution buffer (0. \M HCl [adjusted with glycine to pH 2.2]/l mg/mL BSA) to each well for exactly 10 min of incubation at room temperature.

6. Pipet up and down vigorously, remove the eluate, and neutralize with 3 \xL neutralizing buffer.

7. Infect 2 mL (per well) log phase (OD A^QQ = 1) XL 1-blue cells with the eluted phage. Incubate the cells at room temperature for 15 mm to allow reinfection of the phage before proceeding with replication as described for the original Fab library in Section 3.6. (beginning with step 1, the addition of 10 mL prewarmed, antibiotic containing SB).

3.8. Conversion ofpCombSH-SS to Soiuble Fab-Producing Form

1. Prepare double-stranded DNA from the cellular pellet collected after the final round of panning, reinfection, and replication.

2. Digest 5 |ig DNA with Spel and Nhel with the appropriate buffer (see Table 1) for 3 h at 37°C. This removes the g3p component from the vector, and the compatible "sticky" ends of vector sequence can be religated.

3. Precipitate the DNA and resuspend the pellet in 50 pL sterile water. 4. Run the digested product on a 0.8% GTG agarose gel. 5. Excise the 4136-bp vector band, and electroelute the DNA. 6. Self-ligate 200 ng vector DNA in 20 |uL total volume for at least 2 h at room

temperature. 7. Transform 1 jiL of the ligation mix into a 40-)xL aliquot of electrocompetent

XL 1-blue cells, and plate out dilutions. 8. Pick single colonies and inoculate 10 mL SB containing 50 ng/mL carb, and

grow for 6 h at 37''C with shaking. 9. Streak or spot an LB carb plate with each culture prior to induction, because they

may not be viable on the next day.


10. Add IPTG to a final concentration of 1 mAf, and incubate overnight at 30°C {see Note 9),

11. Recover the cells from the overnight cultures by centrifiigation. Although both supematants and cell pellets will contain Fab fragments, a higher concentration will be obtained from the cell pellet.

12. Prepare periplasmic protein from the cells by freezing the pellets in a dry ice/ ethanol bath for 5 min. Once frozen, add 100 \LL 10 mM Tris, pH 8.0, to the pellets, and allow them to defrost at room temperature, with occasional gentle mixing. As soon as the pellets have defrosted and resuspended, transfer the tubes to ice. After 1 h, pellet the cell debris by centrifugation and transfer the supematants to fresh tubes ready to use in ELISA.

3.9. ELISA Analysis of Potential Antigen Recognizing Clones ELISA analysis should be performed following standard procedures (see

also Chapters 5, 6,16, and 24).

1. Coat wells of an ELISA plate with 25 yL of 4.0 jig/mL antigen diluted in coating buffer.

2. Block the plates with 3% (w/v) BSA in PBS 3. Wash the plates four times with 3% (w/v) BSA in PBS. 4. For the first antibody, use 50 |jL/well of Fab preparation directly (or diluted in

PBS/3% BSA). 5. Wash the plates four times with 3% (w/v) BSA in PBS. 6. For the second antibody/enzyme conjugate, use anti-Fab antibody. 7. Wash the plates four times with 3% (w/v) BSA in PBS. 8. Identify positive clones by developing the ELISA with appropriate substrate

solution (20).

3.10. Fab Protein Purification

The production of periplasmic proteins described in Section 3.8. can be scaled up. The cells should be induced with IPTG when their OD A^QQ is about 1.0. Add 100 \xM PMSF to the lO-mM Tris, pH 8.0, solution during the periplasmic preparation stage. After removal of the cell debris by centrifugation, the Fab protein can be purified from the supernatant. Refer to ref. 20 for a variety of appropriate methods for antibody purification. Final analysis of the Fab activity and purity prior to epitope mapping can be achieved by ELISA titration and protein gel analysis.

4. Notes 1. I represents inosine, a base that will base-pair with T, A, and C. 2. Electroelution is the preferred method for obtaining efficient recovery of good-

quality DNA (GeneClean will yield lower-quality inserts). If an electroelution apparatus is not available, the following protocol can be used: Transfer the gel chip to a length of preboiled dialysis tubing (e.g., Medicell size 5) with 400 fiL


0. IX TAE buffer. All the air bubbles should be removed before clipping the tubing at both ends. Rest the "package" just submerged in an electrophoresis tank containing 0.2X TAE. Electrophoresis was carried out at 200 V for 30-60 min. The buffer containing DNA was illuminated over a UV transilluminator and removed to an Eppendorf tube. Chill the solution on ice for 2 min, prior to spinning in a microfuge for 10 min, to remove any residual gel material. Remove the supernatant to a fresh tube. Extract first with an equal volume of phenol and then with an equal volume of phenol:chloroform:isoamyl alcohol (24:24:1). Precipitate the DNA.

3. The antibody response should be assessed by ELISA from a test bleed prior to recovery of the mouse spleens.

4. All RNA work must be performed wearing gloves and with sterilized RNase-free pipets and solutions.

5. The concentration of the DNA can be ascertained on a 0.7% agarose plate containing 0.7 jag/mL ethidium bromide. Dot 1 |LIL of the DNA to be assessed onto the plate along with 1 \iL of dilutions of plasmid DNA of known concentration. Examine the intensity of fluorescence of the DNA dots by illumination with UV, and estimate the concentration of the sample from the fluorecence it gives relative to the fluorecence observed from the plasmid DNA dilutions.

6. The preparation of highly electrocompetent cells is essential for the production of reasonably sized antibody libraries. Standard procedures should be followed to prepare the cells (22). It is imperative to keep the cells as cold as possible during the procedures. All solutions, pipets, and centrifugation rotors and bottles should be prechilled before use. Alternatively, ready-prepared electrocompetent XL 1-blue MRF' cells can be obtained from Strategene (200158).

7. Coating may also be performed at 37°C for 1 h. Other solid supports, such as Sepharose, may also be used for the immobilization of the antigen.

8. Bacteriophage should be removed to bleach solution to prevent it from contaminating future experiments.

9. Optimum induction times will vary. It may be necessary to do a time-course analysis of the Fab production.

References 1. Pluckthun, A. and Skerra, A. (1990) Expression of functional antibody Fv and

f Ah. Methods Enzymol. 178,497-515. 2. Pluckthun, A. (1992) Biotechnological aspects of antibody production in E. coll

Immunol. Rev. 130,151-188. 3. Smith, G. P. (1985) Filamentous fusion phage; novel expression vectors that

express cloned antigens on the virion surface. Science 228, 1315-1317. 4. Cesareni, G. (1992) Peptide display on filamentous phage capsids. FEBS Lett.

307,66-70. 5. Barbas, C. P., Ill, Kang, A. S., Lemer, R. A., and Benkovic, S. J. (1991) Assem

bly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc. Natl. Acad. Sci. USA 88, 7978-7982.


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Epitope Mapping Protocols Volume 66 || Production of Phage-Display Antibodies for Epitope Mapping