An immuno-precipitation assay for determining specific interactions between antibodies and phage selected from random peptide expression libraries

Journal of Immunological Methods 264 (2002) 163 – 171 www.elsevier.com/locate/jim

Recombinant Technology

An immuno-precipitation assay for determining specific interactions between antibodies and phage selected from random peptide expression libraries T.A.M.A. Al-bukhari, P. Tighe, I. Todd* Division of Molecular and Clinical Immunology, School of Clinical Laboratory Sciences, University of Nottingham, A Floor West Block, Queen’s Medical Centre, Nottingham NG7 2UH, UK Received 30 May 2001; received in revised form 24 October 2001; accepted 27 November 2001

Abstract Libraries of random peptides displayed by bacteriophage can be screened to select phage expressing peptides that specifically bind antibodies, so that the peptide sequence motifs expressed by the phage can help to define the epitopes of the antibodies. It is often desirable to screen antibody-selected phage for binding of the selecting antibody in an immunoassay in order to verify the specificity of the interaction. Enzyme-linked immunosorbent assays (ELISAs) are commonly used for this purpose. However, for many antibodies, the best techniques for measuring specific, high affinity interactions are immunoprecipitation assays. Immuno-precipitation was therefore investigated as a means of measuring interactions between antibodies and phage clones selected from random peptide display libraries. Three mouse monoclonal antibodies specific for glutamic acid decarboxylase were used to select peptides as 9-mers on T7 phage, linear 12-mers on pIII of M13 phage, or constrained 15-mers on pVIII of M13 phage. Following the cloning and sequencing of selected phage, mixtures of antibody and phage were incubated in solution and the immune complexes were precipitated with Protein G bound to Sepharose beads. In order to detect and quantitate the phage that had formed immune complexes and been precipitated, advantage was taken of the biological properties of the phage by inducing infection of Escherichia coli by the precipitated phage. The aim was to quantitate the phage precipitated by determining the number of plaques produced, which would therefore be proportional to the degree of interaction between the phage and the antibody in solution. The results presented here indicate that this method of measuring monoclonal antibody interactions with phage selected for expression of peptides recognised by the monoclonal antibody is highly specific and sensitive. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Immuno-precipitation; Monoclonal antibody; Phage peptide display

Abbreviations: BSA, bovine serum albumin; C-mAb, monoclonal antibody specific for the C-terminus of GAD; ELISA, enzyme-linked immunosorbent assay; GAD65/GAD67, 65 kDa/67 kDa isoforms of glutamic acid decarboxylase; KLH, keyhole limpet haemocyanin; mAb, monoclonal antibody; N-mAb, monoclonal antibody specific for the N-terminus of GAD; OD, optical density; PCR, polymerase chain reaction; PEG, polyethylene glycol; pfu, plaque forming unit; TBS, Tris-buffered saline; TBS – T, Tris-buffered saline containing 0.1% Tween-20. * Corresponding author. Tel.: +44-115-970-9128; fax: +44-115-970-9125. E-mail address: [email protected] (I. Todd). 0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 ( 0 2 ) 0 0 0 8 0 - 7

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1. Introduction A range of applications have been introduced for libraries of random peptides displayed by bacteriophage (Cortese et al., 1995). These include the selection of phage expressing peptides that specifically bind antibodies, so that the peptide sequence motifs expressed by the phage can help to define the epitopes of the antibodies (Cortese et al., 1995). It is often desirable to screen antibody-selected phage for binding of the selecting antibody in an immunoassay in order to verify the specificity of the interaction. This can be performed by immunoblotting on nitrocellulose membranes, but this does not give quantitative data for precise comparisons between different phage clones. Enzyme-linked immuno-sorbent assays (ELISAs) are also used to measure antibody– phage interactions: these can give very useful, quantitative data, but can also be problematic with certain phage and/or antibody combinations. For example, there may be problems in optimising binding of phage to the wells of the ELISA plate. There may also be non-specific interactions between the different reagents (e.g., binding of the anti-immunoglobulin enzyme conjugate to the phage or bacterial contaminants, or to the antiphage antibody if a capture system has to be used). For many antibodies, the best techniques for measuring specific, high affinity interactions are immunoprecipitation assays, in which immune complexes are precipitated following binding between antibodies and antigens in solution (in contrast to the solid-phase assays mentioned above). We therefore decided to investigate immuno-precipitation as a means of measuring interactions between antibodies and phage clones selected from random peptide display libraries. The technique employed was simply to incubate a mixture of antibody and phage in solution and then precipitate the immunoglobulins, including the antibody – phage immune complexes, with Protein G bound to Sepharose beads. An issue was obviously how to detect and quantitate the phage that had formed immune complexes and been precipitated. A standard method in immuno-precipitation assays is to use radio-labelled antigens, so that the amount of radioactivity in the precipitate is proportional to the amount of antigen bound. However, in the current instance, radio-labelling each individual phage clone would have greatly complicated the assay, and using

radioactivity was undesirable on safety grounds. We therefore investigated whether we could take advantage of the biological properties of the phage by inducing infection of Escherichia coli by the precipitated phage. The idea was to quantitate the phage precipitated by determining the number of plaques produced, which would therefore be proportional to the degree of interaction between the phage and the antibody in solution.

2. Materials and methods 2.1. Monoclonal antibodies GAD-6, a murine IgG2a monoclonal antibody (mAb) specific for the 65 kDa isoform of glutamic acid decarboxylase (GAD65) (Chang and Gottlieb, 1988), was purchased as purified lyophilised antibody (Roche Diagnostics, Lewes, UK). GAD-6 was produced in ascites from the hybridoma and purified by ammonium sulphate precipitation and anionexchange chromatography; it was > 90% pure as determined by anion-exchange HPLC. The following antibodies were purchased from Affiniti Research Products (Exeter, UK) as immunoglobulin preparations, partially purified from exhausted hybridoma culture supernatants, dissolved in phosphate-buffered saline containing 0.01 M sodium azide: N-mAb (GC3208, clone 11) is a murine IgG1 mAb that specifically recognises resi-dues 4 – 17 of GAD65 (Ziegler et al., 1994); C-mAb (GC3108, clone 111) is a murine IgG1 mAb that recognises the C-terminus of GAD65 and GAD67 (residues 572– 585 of GAD65) (Ziegler et al., 1996). The isotype-matched negative control antibodies used in ELISAs (Section 2.5) were a mouse IgG2a anti-keyhole limpet haemocyanin (KLH) mAb, and mouse IgG1 and IgG2a myeloma proteins (Sigma, Poole, UK). 2.2. Preparation of the T7 constrained 9-mer random peptide display library A T7 phage library was constructed that was designed to express 9-mer peptides that were constrained by a disulphide bridge between cysteine residues immediately adjacent to both ends of the 9-

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mer. The peptides were expressed near the C-terminus of the T7 gene X surface coat protein (415 copies per phage). The random peptides of the T7 library were encoded by double stranded DNA inserts assembled from synthetic degenerate oligonucleotides and cloned into gene X of the vector (T7select415-1) (Bioscience, Cambridge, UK) at HindIII and EcoRI restriction sites. The vector DNA and insert DNA were ligated with T4 DNA ligase, and assembled into phage using T7Select packaging extract (BioScience, Cambridge, UK). The phage were amplified in E. coli BL21. 2.3. Screening the T7 constrained 9-mer random peptide display library GAD-6 mAb (10 Ag/ml), or 10 Al of N-mAb or CmAb in 1 ml 0.05 M sodium carbonate/sodium bicarbonate buffer pH 9.6, was coated onto Nunc immuno-tubes that were subsequently blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS). The constrained 9-mer T7 phage random peptide display library [about 1  1010 plaque forming units (pfu) per tube] was added to antibody-coated tubes and incubated at 4 jC for 20 –30 min. The tubes were washed extensively in TBS –0.1% Tween-20 (TBS – T) and then 1 ml of a mid-log phase E. coli BL 21 culture was added and incubated at room temperature for 5 min for infection by the bound phage (termed the ‘eluate’). The eluate phage were amplified and subjected to three further rounds of selection, as above. The phage from the fourth round of selection were plated on LB agar at 100 – 200 plaques per dish. A nitrocellulose membrane (0.45 AM pore size) (Millipore, UK) was placed onto the plate and incubated for 30 min at room temperature. The membrane was then blocked with 5% BSA/TBS. GAD-6 (10 Ag/ml), or NmAb or C-mAb (1:1000 dilution) were added to the membrane and incubated for 1 –2 h. The membrane was washed and alkaline phosphatase-conjugated sheep anti-mouse IgG (whole molecule) (Sigma) (1:1000 dilution in 2.5% BSA/TBS –T; pre-absorbed with phage/E. coli lysate-treated nitrocellulose membrane for 30 min at room temperature) was then added to the membrane and incubated at room temperature for 1 h. The membrane was washed and 5-bromo-4-

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chloro-3-indolyl phosphate/nitro-blue tetrazolium substrate (Sigma) with 5 mM levamisole was added to the membrane. Following the appearance of blue spots, the membrane was washed with TBS, then with water and dried. Antibody-specific phage clones, which developed as blue spots on the membrane, were selected from the original plate and each of them mixed with 1 ml of mid-log phase E. coli BL 21 culture for amplification. The lysed cultures were then centrifuged to obtain the supernatants containing the phage. Each clone of specific phage was amplified by polymerase chain reaction (PCR). The PCR products were then added to BigDye Terminator (Perkin-Elmer Applied Biosystems) and T7 sequencing primer, and run on a sequencing reaction program. The samples underwent cycle sequencing in an ABI PRISM 310 Genetic Analyser (Perkin-Elmer). 2.4. Screening the M13 random peptide display libraries The M13 pIII linear 12-mer library (referred to here as pIII phage) was obtained commercially (New England Biolabs, UK). The unconstrained 12-mer peptides were expressed near the N-terminus of the M13 gene III surface coat protein (three to five copies per phage). The M13 pVIII (5C4C4) phage library (referred to here as pVIII phage) was kindly provided by Dr. George Smith (Missouri, USA). The constrained 15-mer peptides contained an internal disulphide bridge between cysteines at positions 6 and 11: they were expressed near the N-terminus of a proportion of the 2500 copies of the M13 gene VIII surface coat protein. GAD-6, N-mAb or C-mAb was coated onto Nunc immunotubes essentially as described in Section 2.3. The tubes were blocked with BSA solution and washed. The M13 phage were added to the antibody-coated immunotubes (about 2  1011 pfu per tube for the pIII phage library or 1  1013 pfu per tube for the pVIII phage library) in TBS –T and incubated at 4 jC for 30 min. The tubes were washed extensively and 1 ml of elution buffer (0.2 M glycine – HCl pH2.2, 0.1% BSA) was added and incubated at room temperature for 10 min. The eluate was then neutralised with 1 M Tris –HCl pH9.1. The pIII phage eluate was amplified in E. coli 2537 and the pVIII phage

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eluate was amplified in E. coli K91 BluKan. The enriched phage were subjected to further rounds of selection, as above: two rounds for the pIII phage, and four or five for the pVIII phage. Selected M13 phage clones were screened on immunoblots for specific reactivity with the selecting antibodies, essentially as described for the T7 phage clones in Section 2.3. The pIII phage clones were selected from the plates used for immunoblotting, as was done with the T7 phage (Section 2.3). For the pVIII phage, by contrast, clones were selected and amplified prior to being transferred to nitrocellulose membranes for immunoblotting. Selected clones were sequenced in an ABI PRISM 310 Genetic Analyser (Perkin-Elmer). 2.5. ELISAs for the detection of antibody binding to phage The T7 purified phage clones were coated directly onto wells (100 Al/well) of maxisorp ELISA plates (Life Technologies, UK) in carbonate/bicarbonate coating buffer pH 9.7. Plates were incubated at 4 jC overnight. Wells were washed three times (200 Al/ well) with TBS – T. The wells were then blocked with 3% BSA/TBS (120 Al/well) at room temperature for 1 h. Blank wells, which were not coated with phage, were also blocked with 3% BSA/TBS, as above. A capture ELISA system was employed with the M13 pIII and pVIII phage clones rather than direct coating because M13 phage (unlike T7 phage) did not bind well to the plastic and tended to give high nonspecific immunoglobulin binding. Rabbit anti-fd IgG (Sigma) (diluted 1:100 in coating buffer) was coated onto wells (100 Al/well) of maxisorp ELISA plates on a shaker. The plates were incubated at 4 jC overnight. Wells were washed three times (200 Al/well) in TBS – T, and blocked with 3% BSA/TBS (120 Al/well) at room temperature for 1 h. The selected phage, unselected phage or helper M13 phage as a negative control (diluted 1:20 in 1% BSA/TBS – T) were applied (100 Al/well) to test wells, while 1% BSA/ TBS – T was applied to blank wells, and shaken at room temperature for 2 h. The wells were then washed. GAD-6 (1 Ag/ml, in 1% BSA/TBS – T), N-mAb or C-mAb (diluted 1:1000, in 1% BSA/TBS –T), or a heavy chain isotype-matched negative control anti-

body (1 Ag/ml in 1% BSA/TBS – T), described in Section 2.1, was applied (100 Al/well) in duplicate to T7 or M13 phage-coated and blank wells and shaken at room temperature for 2 h. Following three washes, sheep anti-mouse IgG (whole molecule) alkaline phosphatase conjugate (diluted 1:1000 in 1% BSA/TBS –T) (Sigma) was added to all wells (100 Al/well) and shaken at room temperature for 1 h. Wells were washed three times in TBS – T and substrate [1 Ag/ml p-nitrophenyl-phosphate substrate (Sigma) in diethanolamine buffer, 25 mM MgCl2, 15 mM NaN3, pH 9.8] was added to all wells (100 Al/well) and incubated at room temperature for 15 min for pVIII phage, for 30 min for pIII phage, and overnight at 37 jC with the T7 phage. Plates were read at optical density (OD) 405 nm on a microtitre plate reader (Molecular Devices). The mean OD of the antigencoated wells was corrected by subtracting the mean OD of the equivalent blank wells. 2.6. Antibody – phage immunoprecipitation and detection One microlitre of N-mAb or C-mAb, or 1 Ag of GAD-6 (1 Ag/Al), was added to 50 Al of TBS – T in microcentrifuge tubes. Five microlitres of each specific or non-specific amplified clone of the three different phage peptide libraries (T7 C9C, M13 pIII 12-mers or M13 pVIII 5C4C4) was added to the appropriate microcentrifuge tubes, mixed and incubated on a rotator for 4 h at 4 jC. Also, 5 Al of each amplified phage clone was added to 50 Al of TBS – T in another microcentrifuge tube as a negative control. Protein G Sepharose 4B (Sigma) was prepared for use by mixing 1 ml Protein G Sepharose 4B slurry with 30 ml of TBS –T, centrifuging at 2800 g for 5 min at 4 jC, and re-suspending the pellet in 10 ml of TBS –T. One hundred microlitres of the Protein G Sepharose were added to each microcentrifuge tube and incubated on a rotator for 1 h at 4 jC. The tubes were washed by adding 750 Al of TBS –T and centrifuged at 4 jC and 2800 g for 5 min. The supernatant was removed and the pellets were saved for the next wash. Ten washes were applied. The pellets were re-suspended in 100 Al of TBS – T. One microlitre of each suspension was serially diluted in LB broth and the phage at each dilution were infected into E. coli and plated as follows.

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(i) T7 phage were added to mid-log phase E. coli BL 21 culture and then mixed with 3 ml of top agarose (0.7% agarose and 0.1% MgCl2
6H2O in LB broth), which were pre-warmed to 45 jC, in a Pijuo tube. The mixture was poured on to an LB agar Petri dish and incubated at 37 jC for 3 h. (ii) M13 pIII phage were added to mid-log phase E. coli ER 2537 culture and then mixed and incubated at room temperature for 1 –5 min (to allow the M13 pIII phage to infect the E. coli ER 2537). This culture was then mixed with 3 ml of top agarose, which were prewarmed to 45 jC, in a Pijuo tube. The mixture was poured onto an LB agar Petri dish and incubated at 37 jC overnight. (iii) M13 pVIII phage were added to late-log phase E. coli K91BluKan culture, mixed and incubated at room temperature for 5 min (to allow the M13 pVIII phage to infect the E. coli ER 2537). This culture was then mixed with LB broth containing 0.2 Ag/ml of tetracycline and incubated on a shaker vigorously at 37 jC for 35 min. Two hundred microlitres from that mixture were poured onto LB agar Petri dishes containing 40 Ag/ml of tetracycline and 100 Ag/ml of kanamycin and incubated at 37 jC overnight. In each case, the phage plaques on the plates were counted and expressed as pfu per microlitre of the 100 Al of resuspended pellet used for the serial dilutions described above.

3. Results and discussion 3.1. Selection of phage and sequencing of the peptides expressed The three phage random peptide display libraries (T7 C9C constrained 9-mer, M13 pIII linear 12-mer, M13 pVIII 5C4C4 constrained 15-mer) underwent several rounds of selection for phage expressing peptides bound by one of the three mouse mAbs (GAD-6, N-mAb, C-mAb), as described in Section 2. The number of pfu obtained following each round of selection increased consecutively, indicating that the immunopanning rounds were successful. A number of the selected phage clones were picked for DNA sequencing of the expressed peptides on the basis of showing strong and specific staining with the selecting mAb in an immunoblot assay (Sections

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2.3 and 2.4). The sequences of the peptides expressed by the phage clones, selected for the present study on the basis of representing a range of peptide sequences selected from the different phage libraries, are shown in Table 1 using the single letter amino acid code (except for the T7 clones selected with NmAb or C-mAb, and the clone Ct/III-1, for which peptide sequences were not obtained). Similarities between peptides selected from a library by a particular mAb, in terms of identical amino acids and conservative substitutions, are indicated by bold type. These represent motifs that are probably important for the epitopic recognition of the peptides by the mAbs. The upper panel of Table 1 shows the sequences of the peptides expressed by phage clones selected with GAD-6 mAb from the T7 and M13 pVIII libraries. The T7 clone G6/T7-2 expressed a different peptide sequence from clones G6/T7-1 and -3, but this was similar to another 12 selected clones not used here.

Table 1 Sequences of peptides expressed by phage clones selected by monoclonal antibodies from random peptide phage expression libraries Phage clonea

Sequenceb

G6/T7-2 G6/T7-1 G6/T7-3 G6/VIII-1,2,3

C K I A Kc C Q P M D Q A C D Lc C Q P M E Q A C D Lc A R RW D C D G H M C WA Q I

N/III-21 N/III-22 N/III-24 N/VIII-1 N/VIII-2 N/VIII-3

LCSTVHCCPGSST DPGRSNWSMSFD S P S FPLW SF SYL AINTICSTPLCWNEA AT NR PCST PMC MGS Y ASHDSCSTPMCSTPR

C/III-2 C/III-3 C/III-4

SFLQTEIDNMGR W D LT Y E L D R LW T GFLIWEVDTLSP

a

Each clone is designated by: selecting mAb/Phage libraryClone number. G6 = GAD6; N = N-mAb; C = C-mAb; T7 = T7 9mer library; III = M13 pIII 12-mer library; VIII = M13 pVIII 5C4C4 15-mer library. b Sequences represented by the single letter amino acid code. Amino acids that are identical, or show conservative substitutions, between peptides selected from the same library with the same antibody are shown in bold type. c Indicates a stop codon terminating the peptide sequence.

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Although this T7 library had been designed to express 9-mer peptides constrained by a disulphide bridge formed between cysteine residues at either end of the sequence, the inserts all contained a stop codon prior to the position of the second cysteine (indicated in Table 1). The three clones selected with GAD-6 from the M13 pVIII library all expressed the same peptide sequence as each other, and as another 16 clones selected in the same experiment. The sequences of the peptides expressed by the M13 pIII and pVIII phage selected with the N-mAb are shown in the middle panel of Table 1. The pIII peptide sequences show similarities to each other, and so do the pVIII peptide sequences. The sequences of the peptides expressed by the M13 pIII phage selected with the C-mAb are shown in the lower panel of Table 1: these show homologies with each other. The strong homologies between the peptides expressed by phage clones selected from a particular library with a particular mAb are consistent with the specificity of the interactions between the mAb and the selected peptides. 3.2. Measurements of mAb/phage interactions by ELISA ELISAs were performed, as described in Section 2.5, to measure binding between the phage clones listed in Table 1 and the mAb used to select them from the phage libraries. The specificities of these interactions were assessed by comparison with the negative controls given by interactions of the selecting mAbs with wild type phage (i.e., not expressing a peptide insert), and the binding to the selected phage of irrelevant negative control mAbs that were of the same heavy chain isotype as the selecting mAbs. The results, shown in Fig. 1, are expressed on a linear scale as optical densities (ODs) in the ELISAs and indicate that each selecting mAb interacted specifically with the phage clones it had selected from the libraries. The clearest results were given with the M13 pVIII phage (Fig. 1d and e), where ratios of up to >60 were obtained for the OD of specific mAb binding divided by the OD of non-specific mAb binding. This was primarily due to the very low binding of the nonspecific mAbs in these ELISAs. However, the specific/non-specific mAb binding ratios were lower in the ELISAs with the T7 and M13 pIII phage, mainly

being less than 10 (Fig. 1a,b and c). This was primarily due to higher binding of the non-specific mAbs in these ELISAs compared with the ELISAs with the pVIII phage. It is also notable that the ELISAs with the M13 pVIII phage required the shortest time of incubation for enzyme substrate conversion (see Section 2.5). 3.3. Measurements of mAb/phage interactions by immuno-precipitation and plaque formation Phage clones were immuno-precipitated with the mAbs used for their selection, as described in Section 2.6. The specificities of these interactions were assessed, for most phage clones, by comparison with the immuno-precipitation of the phage by the mAbs used for the selection of other phage. For example, N-mAb and/or C-mAb served as negative controls for GAD-6, and so on. The phage/mAb immune complexes precipitated with Protein G Sepharose were serially diluted, and the phage enumerated as plaques following infection of E. coli and plating. The results, shown in Fig. 2, are expressed on a logarithmic scale as numbers of pfu precipitated per microlitre of a 100-Al suspension of the precipitate. They indicate highly specific precipitation of phage by the mAb used for their selection, compared with the irrelevant mAb(s). For the majority of phage clones, the ratios of pfu precipitated following specific mAb binding divided by the pfu precipitated following non-specific mAb binding were >100, or even >1000 (which is why logarithmic scales are used in Fig. 2, rather than linear scales as for the ELISA results in Fig. 1). The lowest ratios for specific versus non-specific mAb binding were given by the T7 phage clones, but even in this case the highest ratio was >450 (clone N/T7-2) and the lowest ratio was 9 (clone C/T7-2). Very few phage were precipitated in the absence of any mAb, the numbers ranging from 5/Al to 120/Al. The immuno-precipitation method facilitated strong recognition of phage clones that gave only weak signals by ELISA. This is illustrated by clone C/III1, which gave a ratio for specific versus non-specific mAb binding of only about 2 in ELISA (Fig. 1c), but a ratio of 100 by immuno-precipitation (Fig. 2b). Similarly, clone N/III-21 gave a ratio of < 5 by ELISA (Fig. 1b), but a ratio of >1000 by immuno-precipitation (Fig. 2b).

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Fig. 1. ELISAs to measure the interactions of mAbs with phage clones selected from random peptide phage expression libraries. The ELISAs were performed as described in Section 2.5. The designation of clones is as indicated in footnote to Table 1. (a) T7 9-mer clones selected with GAD-6 mAb; (b) M13 pIII linear 12-mer clones selected with N-mAb; (c) M13 pIII linear 12-mer clones selected with C-mAb; (d) M13 pVIII constrained 15-mer clones selected with GAD-6; (e) M13 pVIII constrained 15-mer clones selected with N-mAb. The negative control antibodies used are indicated on the figures. WT = wild type phage.

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Fig. 2. Enumeration of plaque forming units of phage clones precipitated by the mAbs indicated, as described in Section 2.6. The designation of clones is as indicated in footnote to Table 1. (a) T7 9-mer clones; (b) M13 pIII linear 12-mer clones; (c) M13 pVIII constrained 15-mer clones. A circle indicates that the interaction of the particular phage clone and the mAb (designated by the shading) was not assessed.

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These results indicate that this method of measuring mAb interactions with phage selected for expression of peptides recognised by the mAb is highly specific and sensitive. We suggest that the specificity results from the fact that the mAb/phage interactions take place in solution, which tend to be more stringent for high affinity binding than do solid-phase interactions. The sensitivity may be due to the fact every phage particle can generate a plaque and so contributes to the quantitative readout. The advantage of using plaque formation as the readout of phage immuno-precipitation is that it employs standard techniques already available for phage amplification and quantification in the laboratory. Furthermore, it requires no special manipulation (i.e., labelling) of the phage for their enumeration in the immunoprecipitate, and avoids the use of radioactivity.

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References Chang, Y., Gottlieb, D., 1988. Characterization of the proteins purified with monoclonal antibodies to glutamic acid decarboxylase. J. Neurosci. 8, 2123. Cortese, R., Monaci, P., Nicosia, A., Luzzago, A., Felici, F., Galfre, G., Pessi, A., Tramontano, A., Sollazzo, M., 1995. Identification of biologically active peptides using random libraries displayed on phage. Curr. Opin. Biotechnol. 6, 73. Ziegler, B., Augstein, P., Luhder, F., Northemann, W., Hamann, J., Schlosser, M., Kloting, I., Michaelis, D., Ziegler, M., 1994. Monoclonal antibodies specific to the glutamic acid decarboxylase 65 kDa isoform derived from a non-obese diabetic (NOD) mouse. Diabetes Res. 25, 47. Ziegler, B., Augstein, P., Schroder, D., Mauch, L., Hahmann, J., Schlosser, M., Ziegler, M., 1996. Glutamate decarboxylase (GAD) is not detectable on the surface of rat islet cells examined by cytofluorometry and complement-dependent antibody-mediated cytotoxicity of monoclonal GAD antibodies. Horm. Metab. Res. 28, 11.