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Monoclonal antibodies against a minor and the major coat proteins of filamentous phage M13: their application in phage display
ELSEVIER
JOURNALOF WIUMMGICAL METHODS Journal
of Immunological Methods 179 (1995) 165-175
Monoclonal antibodies against a minor and the major coat proteins of filamentous phage M13: their application in phage display D. Bhardwaj, S.S. Singh, S. Abrol, V.K. Chaudhary
*
Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi-110021, India
Received 26 July 1994; revised 13 October 1994; accepted 19 October 1994
Abstract We have produced monoclonal antibodies (MAbs) which react with a minor and the major coat proteins of filamentous phage Ml3 and have characterised them by combining the techniques of enzyme-linked immunosorbent assay (ELISA) and Western blotting. These coat proteins are the minor coat protein, gIIIp, the product of gene III and the major coat protein, gVIIIp, the product of gene VIII. Both gIIIp and gVIIIp are important in the context of
‘phage display’ of foreign peptides/proteins as fusions to these proteins. The anti-gIIIp MAbs were able to detect native gIIIp as well as the fusion proteins comprising foreign proteins and gIIIp in ELISA and on Western blot, indicating their utility for studying the expression of foreign proteins in phage display. Similarly anti-gVIIIp MAbs detected gVIIIp both in ELISA and on Western blot. In an ‘affinity capture phage ELISA’, phages that were captured by virtue of the interaction between the foreign protein (ligand fused to the gIIIp and displayed on the phage surface) and the immobilised counterpart (receptor), could be easily detected using anti-gVIIIp MAbs. Considering the potential of ‘phage display’ technology in protein engineering, these antibodies should find wide applications. Keywords:
ELISA, affinity capture phage; Filamentous
phage M13; Fusion protein;
Monoclonal
antibody; Phage
display
1. Introduction Abbreviations: aa, amino acid(s); ABTS, 2,2’-azino-bis(3’ethylbenzthiazoline&sulfonic acid); BSA, bovine serum albumin; cfu, colony-forming unit(s); ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence activated cell sorter; gIIIp, gene III encoded minor coat protein of bacteriophage M13; gVIIIp, gene VIII encoded major coat protein of bacteriophage M13; HRP, horseradish peroxidase; IgG. immunoglobulin G; pelB signal sequence, signal sequence of the protein pectate lyase from Erwinia carotovora; MAb, monoclonal antibody; OD, optical density; PAGE, polyacrylamide gel electrophoresis; lac p/o, promoter and operator sequences of the lac operon; SDS, sodium dodecyl sulfate 0022-1759/95/$09.50 0 1995 Elsevier SSDI 0022-1759(94)00280-O
Science
B.V. All rights
The filamentous phages (fl, M13, and fd) are single-stranded DNA phages which infect only male specific E. coli strains that contain F’ factor (Marvin and Hohn, 1969). The genome of Ml3 (and fd) is a closed circular DNA molecule of 6.4
* Corresponding author. Tel.: 91-11-678876, 91-11-6885270 and 91-1 l-6886427. reserved
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of Immunological Methods I79 (1995) 165-I 75
kb, 90% of which codes for the ten genes essential for the completion of life cycle of the phage (Van Wezenbeek et al., 1980). The phage particles have an appearance of a long flexible tube (Berkowitz and Day, 1976) with a 1.5-2 nm thick shell and a diameter of about 6.5 nm (Glucksman et al., 1992; Makowski, 1993) and have a molecular weight of approximately 1.2-1.4 X 10’ Da (Parmley and Smith, 1988). The major coat protein, gVIIIp (product of the gene VIII) is present in about 2700 copies per phage particle. Each phage particle also contains four minor proteins, the products of genes III, VI, VII and IX present in 3-5 copies each. Recently, vectors based on filamentous phages have been developed for displaying the proteins and peptides fused to phage coat proteins on the phage surface and are being used as genetic tools for selecting high affinity variants of engineered proteins/peptides. Their applications include mapping of epitopes for monoclonal antibodies, identification of peptide mimics for non-peptide ligands and for selection of proteins with improved properties (Barbas, 1993; Scott and Craig, 1994; Wells and Lowman, 1992). These studies have resulted in the selection of variants of human growth hormone with 400-fold higher affinity towards its receptor (Lowman and Wells, 19931, identification of peptides that bind to concanavalin A (Scott et al., 19921, identification of a peptide that mimics the binding of carbohydrates to its monoclonal antibody (Hoess et al., 1993) and in selecting human antibodies of desired specificity (Lerner et al., 1992; Winter et al., 1994). In a majority of these vectors, the DNA encoding the desired peptide or protein is cloned at the 5’ end of gene III which encodes a 42 kDa protein, gIIIp. In some vectors, the fusions have been made at the 5’ end of gene VIII (Greenwood et al., 1991; Kang et al., 1991; Markland et al., 19911, which encodes a 5600 Da protein, gVIIIp. Upon expression, the peptide/protein is displayed on the surface of the phage particle as a fusion with one of the coat proteins while its corresponding DNA remains encapsulated in the same phage particle. The interaction between the displayed peptide/pro-
bilized partner followed by elution of the bound phages with acidic (Parmley and Smith, 1988) or alkaline (Marks et al., 1991) buffers. The number of viable phages in eluates is estimated by a time consuming process in terms of colony/plaque forming units after infecting a suitable strain of E. coli. The expression of foreign proteins fused with the phage coat proteins could be readily studied if MAbs against the coat proteins were available. The interaction between the peptide/protein displayed on the phage surface fused to one of the coat proteins with its receptor immobilised on the surface of a microtitre plate could be detected in an ELISA as a quick estimate of the binding of phages, with MAbs against the other coat proteins. In this report, we describe the production, characterisation and utility of a panel of MAbs against gIIIp and gVIIIp of filamentous phage M13. These antibodies were able to detect the respective phage proteins both in ELISA as well as in Western blot analysis. The anti-gIIIp MAbs were also able to detect gIIIp fusion proteins on Western blots. The anti-gVIIIp MAbs were useful in detecting the phage particles displaying a ligand fused to the gIIIp when captured by the immobilised receptor coated on the microtitre plates.
tein and its binding studied by incubating
PAGE) and gelatin were mond, CA, USA). Bovine
partner (receptor) is often the phages with the immo-
2. Materials and methods 2.1. Materials Bacterial media components were obtained from DIFCO Laboratories (Detroit, MI, USA) or Gibco BRL (Gaithersburg, MD, USA). Antibiotics were either from United States Biochemicals Corp. (Cleveland, OH, USA), Sigma Chemical Co. (St. Louis, MO, USA) or Gibco BRL. Freund’s adjuvants, polyethylene glycol (PEG) 1300-1600 and 50 x HAT supplement were from Sigma Chemical Co. Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum and prestained molecular weight standards were purchased from Gibco BRL. Reagents for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSfrom Bio-Rad serum albumin
(Rich(BSA,
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of Immunological Methods 179 (1995) 165-I 75
fraction V) was purchased from Boehringer-Mannheim (Indianapolis, IN, USA). HRP (horseradish peroxidase) conjugated secondary antibodies used for ELISA and Western blot were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). All other reagents and chemicals used were of analytical grade. PVDF membranes (Immobilon) were purchased from Millipore (Bedford, MA, USA). Plasticware used in cell culture was from either Costar (Cambridge, MA, USA) or Corning (Corning, NY, USA). Bacteriophages M13mp19 and M13K07 were obtained from Gibco BRL and Stratagene (La Jolla, CA, USA) respectively. E. coli strain XL-1 blue and plasmid pBluescript KS + were obtained from Stratagene. Inbred mice of BALB/c strain were procured from National Institute of Nutrition, Hyderabad (India) and maintained at the departmental animal house facility. 2.2. Production of phage particles The phages M13mp19 were propagated in E. blue, and purified according to the method of Lin et al. (1980).
coli XL-l
2.3. Production and screening of hybridomas Highly purified phage particles were used for immunizing the Balb/c mice. Primary immunization was done by intraperitoneal injections of 200 pg of total phage protein in Freund’s complete adjuvant in 4-6-week-old mice weighing 20-25 g, followed by a secondary immunisation after 21 days with 100 pg of total phage protein using Freund’s incomplete adjuvant. The animals were test bled 21 days after the secondary immunisation and their sera were titrated for the presence of anti-phage antibodies by ELISA. The positive animals were given an intravenous booster of 100 pug phage protein in saline. Seven days later, the animal was sacrificed and the spleen was removed under aseptic conditions. A single cell suspension of the splenocytes was prepared using a frosted glass homogeniser. The cells were washed twice with serum-free DMEM. Cells of myeloma line, Sp2/0 were also washed with serum-free DMEM and mixed with the spleno-
167
cytes in one to one ratio followed by centrifugation. The mixed cell pellet was then suspended in 0.5 ml of 50% solution of PEG 1300-1600 (prepared in serum-free DMEM containing penicillin and streptomycin at a concentration of 100 U/ml and 100 pg/ml respectively) over a period of 1 min by constant hand mixing. Stirring was continued for an additional minute followed by addition of 1 ml of serum-free medium. Over the next 2 min, 9 ml of serum-free medium was added while keeping the cells under constant and gentle stirring. The cells were pelleted and suspended to a final concentration of 2 X lo6 splenocytes per ml in 40 ml medium containing antibiotics and supplemented with 10% heat inactivated fetal calf serum and HAT (hypoxanthine, 1 X lo-” M; aminopterin, 4 X 10-h M and thymidine, 1.6 x 10e4 Ml. The cell suspension (40 ml) was mixed with normal spleen cells (40 ml, 2 x 10h cells per ml) and dispensed in four 96 well plates (200 ~1 per well). Plates were kept in a humidified incubator at 37°C with 5% CO,. After 14 days, the supernatants from wells bearing colonies were tested by ELISA and Western blot analyses (described later). The cells from antibody positive wells were taken for cloning by limiting cell dilution method. Ascitic fluids were produced in pristane primed BALB/c mice by intraperitoneal injections of 1 X lo6 actively growing hybridoma cells that were washed with serum-free DMEM. 2.4. Screening of the hybridomas The hybridomas were screened by ELISA and Western blot analyses at every stage of cloning and subcloning. Since purified phage proteins were not available, ELISA using phage coated microtitre plates was performed to identify the wells which contained cells secreting anti-phage antibody. Western blot analysis using total phage proteins was employed to determine the specificity of the antibodies in terms of their reactivity with individual phage proteins. 2.5. Enzyme-linked immunosorbent assay Phage particles equivalent to 100 ng protein (1 pg/ml) were coated on the surface of polystyrene
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of Immunological Methods 179 (1995) 165-l 75
microtitre plates (Corning) in 50 mM bicarbonate buffer pH 9.2, for 1 h at 37°C. After washing with PBST (20 mM phosphate buffer pH 7.4, 150 mM NaCl containing 0.05% Tween 20), the unoccupied reactive sites were blocked with 2% non-fat dry milk solution in PBST at 37°C for 1 h followed by washing. The serum of the immunized mice or antibody containing culture supernatant were diluted in PBST and added to the coated plates and incubated at 37°C for 1 h. After washing the wells with PBST, 100 ~1 of HRP-conjugated goat anti-mouse IgG (diluted in PBST containing 2% non-fat dry milk) was added and incubated at 37°C for 1 h. After washing the wells with PBST, the reaction was revealed by incubating with 100 ~1 of 1 mg/ml solution of ABTS (2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) in citrate-phosphate buffer (76 mM citric acid and 218 mM disodium hydrogen phosphate, pH 4.5) containing 1 pi/ml of 30% H,O,. After incubating at 37°C for 10 min the reaction was stopped by adding 100 ~1 of 10% SDS and recorded at 405 nm using an ELISA reader (BioTek Instruments, Winooski, VT, USA). 2.6. Affinity capture phage ELISA In this assay anti-gVIIIp MAbs were employed to detect the phages displaying a ligand and captured by immobilised receptor on microtitre plates. For this purpose the wells of polystyrene ELISA strips were coated with 100 ~1 solution of normal human IgG (10 pg/ml in bicarbonate buffer) at 37°C for 1 h followed by washing with PBST for three times. After blocking the unoccupied reactive sites with 3% gelatin solution in PBST at 37°C for 1 h, phages displaying the B domain of protein A, an IgG binding protein from S. aureus, were added in the IgG coated wells in different concentrations (Kushwaha et al., 1994). The strips were incubated at 37°C on a vibrating platform to ensure constant mixing of the contents of the wells for 1 h. After four washings with PBST, the captured phages were eluted using 0.1 N HCl (pH adjusted to 2.2 with solid glycine) containing BSA at 1 mg/ml (Parmley and Smith, 1988) in three different sets of ELISA strips. One set was eluted once, another
was eluted twice and the third set was eluted three times. The eluates from each set were pooled and were used to determine the number of eluted phages by infecting the E. coli XL-l blue cells and counting colonies and plaques (Smith and Scott, 1994). One set was kept as a control from which the phages were not eluted. The number of phage particles which still remained bound to the plate before and after elutions were probed and quantitated with anti-gVIIIp MAbs (from clone 2911) and HRP-conjugated goat anti-mouse IgG, as described earlier. 2.7. Western blot analysis The supernatants positive for anti-phage antibodies in ELISA were further screened by Western blot analysis to determine their specificity against Ml3 phage coat proteins. 1 pug of total phage protein in 200 ~1 of 1 X Laemmli buffer was loaded on a preparative 0.1% SDS/12.5% polyacrylamide gel and electrophoresis was performed as described (Laemmli, 1970). Following electrophoresis, the proteins were electroblotted (Towbin et al., 1979) onto PVDF membranes which were subsequently blocked with 2% non-fat dry milk solution in PBST. The membrane was cut into 7-9 mm wide strips, which were incubated with culture supernatants (diluted l/50 in PBST) followed by washing and incubation with HRP-conjugated goat anti-mouse IgG. All the incubations were carried out at ambient temperature (25-27°C). The binding of anti-phage antibodies to phage proteins was revealed by incubating the strips with 1 mg/ml solution of 3,3’-diaminobenzidine (DAB) in PBS, containing 1 pi/ml of 30% H,O,. 2.8. Production of gIIIp fusion proteins and gIIIp deletion derivatives for Western blot analysis
Various fusion proteins consisting of foreign proteins fused at the N-terminus of gIIIp were expressed in E. coli and the total cell lysate was used for their detection by anti-gIIIp MAbs on Western blot. The fusion proteins, CD4-gIIIp consisting of the first 176 amino acids of human CD4, EB-gIIIp consisting of a single IgG-binding
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of Immunological Methods 179 (1995) 165-175
domain of staphylococcal protein A and antiTat-gIIIp consisting of a single chain antibody which binds to the ~5.5 subunit of the human IL2 were expressed using phagemids receptor, pVCCD43426 (Abrol et al., 1994), pVCEB3426 (Kushwaha et al., 1994) and pVC73426 (our unpublished results), respectively. These vectors express aa +3 to +406 of mature gIIIp with the ligand fused at +3 codon. The gene fusion is preceded by pelB signal sequence and the expression of these proteins is under the control of lac p/o. Briefly, the overnight grown cultures of XL-l blue cells carrying these phagemids were diluted ten-fold in LB medium containing glucose cl%), ampicillin (100 pg/ml) and tetracycline (10 pgg/ml) and grown at 37°C to OD,,, of 0.3-0.4. The expression of fusion proteins was induced by diluting the cultures ten-fold in glucose-free medium and incubating for 2 h. The cells were harvested by centrifugation and the cell pellet was boiled in 1 X Laemmli buffer (Laemmli, 1970) for 3-5 min, electrophoresed and transferred onto PVDF membranes. The membranes were probed with anti-gIIIp MAbs (l/500 dilution of culture supernatant of clone 30421) followed by incubation with HRP-conjugated goat anti-mouse IgG as described above. To detect the fusion proteins displayed on the surface of phage particles, CD41923 phages displaying the first 176 amino acids of human CD4 fused to the gIIIp of Ml3 phages (Abrol et al., 1994) were electrophoresed on SDS-polyacrylamide gel and transferred onto PVDF membranes. The presence of fusion proteins was probed using culture supernatant from clone 30421 as described above. To identify the epitope recognised by anti-glIIp MAbs, deletion derivatives of gIIIp were expressed in E. coli using phagemids pVC326, pVC1326 and pVC2326 which encode aa +3 to +406, aa +56 to +406 and aa + 198 to +406 of gIIIp respectively. At the N-terminus, these proteins carry a 10 aa peptide tag, c-myc recognised by MAb 9ElO (Evan et al. 1985 and Munro and Pelham, 1987) followed by a pentapeptide linker (SGGGG). The proteins were expressed and used for Western blot analysis as described above for other gIIIp fusion proteins.
169
3. Results 3.1. identification of the hybridoma clones producing antibodies against glllp and gP7IZp
Following 14 days of fusion, the supernatant from wells with colonies were selected for screening. Initially, the supernatants were tested for the presence of anti-phage antibodies using phage particles coated on the microtitre plates and a total of 44 wells were identified with varying extents of reactivity (data not shown). Further analysis was done by Western blotting, using total phage proteins separated by SDS-PAGE, which identified three clones producing anti-gIIIp and another 20 producing anti-gVIIIp antibodies. A few of the antibody-producing clones were further purified by limiting dilution and supernatants were analysed by ELISA and by Western blots (data not shown). Finally the culture supernatants or ascitic fluid from two clones of each type of MAb were used for further characterisation of the antibodies. 3.2. Anti-glllp antibodies can detect both native gIIIp and its fusion proteins The ability of anti-gIIIp MAbs to detect the gIIIp in ELISA was checked by coating phage particles directly on microtitre plates. In this assay, 1.0 ng of gIIIp (contributed by 3 X lo9 phage particles) was detectable using l/100 dilution of culture supernatants from two different clones, 30421 and 30122 (Fig. 1). Similar results were obtained with l/50000 dilution of ascitic fluid produced by these two clones (data not shown). End point titration showed that l/1000 dilution of culture supernatant and l/100000 dilution of the ascitic fluid produced color intensity which was 20 times higher than the background. Since purified gIIIp was not available, detectability of gIIIp on Western blots using the monoclonal antibodies was established by separating Ml3 phage proteins on SDS-polyacrylamide gel and transferring on to PVDF membranes. As shown in Fig. 2, as little as 0.1 ng of gIIIp could be detected which was contributed by 3 x 10’ phage
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of Immunological
particles (lane 1). Anti-gIIIp MAbs were used to detect the gIIIp fusion proteins when expressed in E. coli cells and also when displayed on the surface of phage particles. The anti-gIIIp MAbs from clone 30421 detected gIIIp on Western blots in the form of various fusion proteins, when expressed in E. coli and separated by SDS-PAGE (Fig. 3). The culture supernatant from anti-gIIIp clone 30122 also showed the same results (data not shown). In this experiment it was difficult to estimate the relative reactivity of the anti-gIIIp MAbs towards the fusion proteins in comparison to native gIIIp because the purified fusion proteins were not available. However, the reactivity of anti-gIIIp MAbs to the fusion proteins was further evaluated by using a purified phage preparation of CD41923, that displays all the copies of gIIIp as fusion protein with the human CD4 (Abrol et al., 1994). The reactivity of anti-gIIIp MAb with the CD4-gIIIp fusion protein (Fig. 4, lane 3) is similar to the native gIIIp (Fig. 4, lane 4) thereby indicating that the anti-gIIIp MAbs are equally reactive to gIIIp when it is present as fusion protein and these MAbs might
I
2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10” lQf0 Phages per well
log I
0.35
.“‘.‘I
“““‘I
“““‘I
3.5
35.0
350
glllp (ng per well) Fig. 1. ELISA using anti-gIIIp MAbs. The indicated number of phages were coated on the surface of microtitre plates as described in Materials and Methods. l/100 dilutions of culture supernatants of hybridoma clones 30122 (0) and 30421 (a 1 were used to detect the gIIIp. The calculated amount of gIIIp per well is also shown.
Methods 179 (1995) 165-175
kDa
97 \
Std
1
2 3 4 5
29 1814-
Fig. 2. Western blot analysis using anti-gIIIp MAbs. Ml3 phage particles (produced by M13mp19) were boiled in IX Laemmli buffer, electrophoresed on 15% polyacrylamide gel, transferred onto the PVDF membranes and probed with anti-gIIIp MAb (clone 30421) followed by HRP-conjugated goat anti-mouse IgG. Lane 1, 3 X 10’ phages (0.11 ng gIIIp); lane 2, 9 X lo8 phages (0.33 ng gIIIp); lane 3, 2.7 X lo9 phages (0.977 ng gIIIp); lane 4, 8.3 X lo9 phages (2.94 ng gIIIp); lane 5, 2.5 x 10” phages (8.8 ng gIIIp). The sizes of the molecular weight markers is shown in kDa. Std, prestained molecular weight standards (Gibco BRL).
be recognising an epitope distal to the site of fusion which is +3 aa of mature gIIIp. To identify the epitope recognised by anti-gIIIp MAbs, various truncated forms of gIIIp with the c-myc tag at the N-terminus were expressed in E. coli. The proteins in the total cell pellets were separated by SDS-PAGE for Western blot analysis. The Western blot in Fig. 5 shows that fusion proteins carrying full length gIIIp or both the deletion derivatives were reactive to 9ElO which recognises the c-myc tag in the expressed protein (panel A). However, anti-gIIIp MAbs from clone 30421 were reactive to full length gIIIp and the truncated derivative which contained aa t-56 to + 406 of gIIIp (panel B, lanes 1 and 2) but not to the derivative consisting of aa + 198 to + 406 of gIIIp (panel B, lane 3). Similar observations were made using MAbs from the clone 30122 (data not shown). These results show that the anti-gIIIp MAbs described here recognised an epitope between aa + 55 and + 197 of the mature gIIIp which is distal to the site of fusion and therefore the binding of these antibodies is not affected by
D. Bhardwaj et al. /Journal
of Immunological Methods 179 (1995) 165-I 75
the presence of ligands such as CD4 or anti-Tat (sFv) fused to gIIIp at its N-terminus.
12
171
34
3.3. Anti-gVIIIp monoclonal antibodies can be used in Western blot analysis and phage ELISA
The culture supernatants containing anti-gVIIIp MAbs from two clones 713 and 2911 were used to detect the phages that were directly coated on the surface of microtitre plate. As shown in Fig. 6, 1.1 X 10s phage particles which correspond to 2.56 ng of gVIIIp produced a signal which was approximately 25 times more than the background using l/100 dilution of the culture supernatant. End point titration demonstrated that l/500 dilution of culture supernatant or l/64000 dilution of the ascitic fluid could be used in phage ELISA. The specificity of these MAbs was further established in Western blot analysis using phage proteins separated by SDSPAGE which showed a dose dependent increase
kDa
g7\ 6%
-
1
Std
.(
2 3 4
,.,_<_* ;
‘, ,..
43 29
_
m-
Fig. 4. Western blot analysis of phages displaying CD4-gIIIp fusion protein. CD41923 phages (Abrol et al., 1994) were boiled in 1 X Laemmli’s sample buffer for 3-5 mitt, electrophoresed on 0.1% SDS/12.5% polyacrylamide gel and transferred to PVDF membrane, and probed with anti-gIIIp MAbs (clone 30421) followed by HRP-conjugated goat antimouse IgG. Lane 1. 1.4~ 10’ phages (equivalent to 0.074 ng fusion protein); lane 2, 4.2~ 10s phages (0.222 ng fusion protein); lane 3, 1.4~ lo9 phages (0.74 ng fusion protein); lane 4, 7.5~ 10” M13mp19 phages (2.64 ng gIIIp) per IO ~1 sample. Arrows indicate the position of CD4-gIIIp fusion protein and gIIIp respectively. Number of phage particles as described above have been corrected for the loss of infectivity due to the presence of CD4 at the N-terminus of gIIIp in comparison to the parent vector MlJmpl923, which does not have CD4 (Abrol et al., 1994).
^,
.
in binding to gVIIIp (N 6 kDa) without any reaction to any other phage or E. coli protein (Fig. 7). However, the reactivity of anti-gVIIIp MAbs to gVIIIp on Western blot was weak as reaction was visible only when phages equivalent to 25 ng of gVIIIp were loaded. 3.4. Affinity capture phage ELBA
Fig. 3. Western blot analysis of gIIIp fusion proteins. Various gIIIp fusion proteins were expressed in XL-l blue cells as described in the materials and methods section. Total cell pellet from 500 ~1 culture of each was boiled in 150 ~1 of 1 x Laemmli buffer and 10 PI sample was electrophoresed on 0.1% SDS/lS% polyacrylamide gel, transferred onto PVDF membrane and probed with anti-gIIIp MAb (clone 304211 as described. Lane 1, XL-1 blue cells carrying pBluescript KS: lane 2, anti-Tat gIIIp fusion protein ( - 87 kDa1 expressed by pVC73426; lane 3, EB-gIIIp fusion protein ( - 70 kDa) expressed by pVCEB3426, and lane 4, CD4-gIIIp fusion protein ( - 82 kDa) expressed by pVCCD43426. Std, prestained molecular weight standards (Gibco BRL).
The anti-gVIIIp MAbs were also used to detect phages which were captured by virtue of interaction between a ligand, fused to gIIIp and displayed on the phage surface, with its receptor immobilised on the microtitre plates (Fig. 8A). In this assay anti-gVIIIp MAbs were able to detect phages displaying the B domain of staphylococcal protein A captured in an IgG coated well when 3 x 10’ phages were added per well (Fig. 8B). These studies were further extended to deter-
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1 2
kDa
3
4
of Immunological
Methods 179 (1995) 165-175
1234
200 -
9s; 43 29 18
Fig. 5. Western blot analysis of gIIIp deletion derivatives. gIIIp derivatives were expressed in XL-l blue cells as described in the materials and methods section. The total cell pellets were processed as described in legends to Fig. 3 followed by electrophoresis on 0.1% SDS/lO% polyacrylamide gel. The Western blot analysis was performed using MAb 9E10, l/l000 dilution of the ascitic fluid (panel Al and MAb against gIIIp from clone 30421, l/1000 dilution of the ascitic fluid (panel B). Lane 1, gIIIp fusion protein encoded by pVC326 ( - 60 kDa1; lane 2, gIIIp fusion protein encoded by pVC1326 ( - 54 kDa1; lane 3, gIIIp fusion protein encoded by pVC2326 ( - 30 kDa); lane 4, 7.5 X lo9 M13mp19 phages. The values in parantheses are the observed molecular weights of the gIIIp fusion protein derivatives. Position of molecular weight standards is shown in kDa.
mine as to what fraction of the added phages was captured by immobilised human IgG. For this purpose the captured phages were eluted as described in the materials and methods section. After each elution, the wells were incubated with anti-gVIIIp MAbs to quantitate the remaining phages by ELBA. The number of phages that remained bound to the IgG coated plates even after elution was estimated by measuring the intensity of color produced in these wells in comparison to that produced in the wells in which elution was not done. ELISA results revealed that only 2, 1.5 and less than 1% phages remained bound after one, two and three rounds of elution respectively (data shown only for one round of elution). This simple ELISA using antigVIIIp MAb could therefore be used to check the binding of phages as well as to evaluate the efficiency of elution of bound phages. The number of eluted phages encapsulating the phagemids
Phages per well
I 0.23
‘...“‘I
“““‘I 23.0 2.3 gVlllp (ng per well)
““,“l 230
Fig. 6. ELISA using anti-gVIIIp MAbs. The indicated phage numbers were coated on the surface of microtitre plates and l/100 dilutions of culture supernatants of clones 713 (0) and 2911 (0) were used to detect the gVIIIp as described in the materials and methods section. The calculated amount of gVIIIp per well is also shown.
was estimated by infecting E. coli cells and counting colonies (colony forming units), which revealed that only 1.5-2.0% of the added phage
kDa
12345
Fig. 7. Western blot analysis using anti-gVIIIp MAbs. Different numbers of Ml3 phage particles (produced hy M13mp19) and XL-1 blue cells were boiled in 1 X Laemmli buffer, electrophoresed on 0.1% SDS/12.5% polyacrylamide gel and transferred onto the PVDF membrane. The membrane was probed with l/100 dilution of culture supernatant from clone 2911. Lanes 1, XL-1 blue cells; lane 2, 3.6~ lOa phages (8.5 ng gVIIIp); lane 3, 1.1~ lo9 phages (25 ng gVIIIp); lane 4, 3.3 x lo9 phages (77 ng gVIIIp); lane 5, 1 X 10” phages (232 ng gVIIIp1. The sizes of the low range prestained molecular weight markers (Gibco BRL) are shown in kDa.
D. Bhardwaj et al. /Journal
of Immunological Methods 179 (1995) 165-I 75
173
immobilised receptor as well as on the number of fusion proteins per phage particle. These results clearly show the utility of anti-gVIIIp MAbs in detecting bound phages in a simple ELISA format and also show that one round of elution is sufficient to dissociate 98% of the bound phages.
(4
Affinity Capture Phage ELBA
4. Discussion
0.03 107
““‘7 108 Phages
“““3
‘10s
10’0
added per well
Fig. 8. Affinity capture phage ELISA. A: cartoon depicting the ‘affinity capture phage ELISA’ showing the interaction of phages displaying the B domain of staphylococcal protein A with human IgG coated on the well surface of a microtitre plate. B: microtitre plate wells were coated with 1 pg human IgG and incubated with indicated number of phages displaying B domain of staphylococcal protein A as fusion with gIIIp. The bound phages were detected using anti-gVIIIp MAbs to) as described in the materials and methods section. The captured phages were eluted once as described and phages that remained bound after elution were detected by ELISA using anti-gVIIIp MAbs (A). The number of eluted phages measured as colony forming units (cfu) after infecting XL-l blue cells is shown in the inset.
were able to bind the immobilised human IgG under the conditions employed (Fig. 8B inset). This low frequency of capture is due to the fact that phages displaying the B domain of staphylococcal protein A were produced using phagemid system in which only 10% of the phage population displays one or more fusion protein per phage particle (Bass et al., 1990; Kushwaha et al., 1994). However, in this assay the phage number required for positive signal would depend upon the affinity between the displayed ligand and the
Monoclonal antibodies have been widely used for detecting proteins in a variety of immunoassays such as ELISA and Western blot. Employing the techniques of ELISA and Western blotting, we have selected monoclonal antibodies against two coat proteins of Ml3 phage although the purified proteins were not available, either for immunization or for detection. These MAbs were found to be useful in studying the expression of fusions with phage coat proteins in E. cd, and the production of Ml3 phage particles that display foreign proteins fused at the N-terminus of gIIIp. Earlier, several groups have used polyclonal antisera raised against the whole Ml3 phage or the purified gIIIp for this purpose (Goldsmith and Konigsberg, 1977; Stengele et al., 1990). Since the gIIIp is present in only 3-5 copies per phage particle, it is difficult to purify and therefore production of a good quality polyclonal anti-gIIIp antiserum becomes a formidable task. Moreover, polyclonal anti-Ml3 antibodies may not be very suitable in experiments where phage preparations are contaminated with E. co& proteins (Kushwaha et al., 19941. The epitope mapping studies for anti-gIIIp MAbs from clones 30421 and 30122 revealed that these MAbs recognised an epitope between aa + 56 to + 197, therefore, these should be useful with phage display vectors which use full length gIIIp (McCafferty et al. 1990; Hoogenboom et al. 1991; Abrol et al. 1994; Kushwaha et al. 1994). Our results in the present study showed that anti-gVIIIp MAbs were very useful in detecting the binding of phages, that display ligands fused to gIIIp and were captured by immobilised receptor in an ‘affinity capture phage ELISA’. This assay has been successfully used to study: the interaction of phages displaying IgG-binding do-
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mains of staphyloccocal protein A with various subclasses of human IgG (Fig. 8 and Kushwaha et al., 19941, the interaction between the human CD4 borne on phages with gp120, the HIV envelope glycoprotein (Abrol et al., 1994), and the binding of the phages displaying a single chain antibody to its antigen on the surface of cancer cell line (our unpublished results). Although, polyclonal anti-Ml3 antisera could also be used for this purpose (McCafferty et al., 1991), presence of E. coli proteins in the phage preparations may give higher background. Using antigVIIIp MAbs, we have also been able to detect the binding of phages bearing a variety of ligands to their respective immobilised receptor using the total bacterial culture containing ligand bearing phages without any interference by E. coli cells (our unpublished results). Smith and Scott (1994) have described an ELISA in which phages are directly immobilised in the wells of microtitre plates followed by detection of ligand borne on the phage surface fused to gIIIp. This assay requires purified phages as impurities might interfere with adsorption to the plastic or give a high background signal. Purification of phages is tedious, therefore immobilised anti-gVIIIp MAbs should also find utility in capturing phages to assay for presence of ligand fused to gIIIp and displayed on the phage surface. Recently, Nissim et al. (1994) have developed phages bearing antibodies to a variety of proteins and used them as immunochemical reagent to detect proteins on Western blot or on the surface of the intact cells. Anti-gVIIIp MAbs should be useful in this assay as they should give lower background. Use of phages as immuno-chemical agents may have advantage due to the amplification of signal because of a large number of epitopes for anti-gVIIIp MAbs. The anti-gIIIp and anti-gVIIIp MAbs should also find usefulness in studying binding of phages to intact mammalian cells as detected by immunoflourescence or by FACS. In conclusion, the anti-gIIIp MAbs should be very useful in studying the expression of foreign proteins displayed on the surface of phages fused to gIIIp. The anti-gVIIIp MAbs should find wide application in studying the parameters of interac-
tion between the displayed protein (fused to the gIIIp) and its counterpart which is immobilised on the microtitre plates using affinity capture phage ELISA.
Acknowledgements
D.B. and S.A. are recipients of Research Fellowships from the Council of Scientific and Industrial Research, Government of India. Thanks are due to Dr. K. Kannan for providing help in immunisations. We are thankful to Drs. P.K. Ghosh, J.K. Batra, Maria Gallo, Donna Roscoe and Dinkar Sahal for critically reading the manuscript and helpful suggestions, and to Mr. R. Chawla and Mr. S. Simon for typing the manuscript. This work was partly funded by the Department of Biotechnology and Department of Science and Technology, Government of India.
References Abrol, S., Sampath, A.. Arora, K. and Chaudhaty, V.K. (1994) Construction and characterization of Ml3 bacteriophages displaying gp120 binding domains of human CD4. Ind. J. Biochem. Biophys. 31, 302. Barbas III, C.F. (1993) Recent advances in phage display. Curr. Opin. Biotech. 4, 526. Bass, S., Green, R. and Wells, J.A. (1990) Hormone phage: an enrichment method for variant proteins with altered binding proteins. Proteins 8, 309. Berkowitz, S. and Day, L. (1976) Mass, length, composition and structure of the filamentous bacteria1 virus fd. J. Mol. Biol. 102, 531. Evan, G.I., Lewis, G.K., Ramsay, G. and Bishop, M. (1985) Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol. Cell. Biol. 5, 3610. Glucksman, M.J., Bhattacharjee, S. and Makowski, L. (1992) Three dimensional structure of a cloning vector: X-ray diffraction studies of filamentous bacteriophage Ml3 at 7 A resolution. J. Mol. Biol. 226, 455. Goldsmith, M.E. and Konigsberg, W.H. (1977) Adsorption protein of the bacteriophage fd: isolation, molecular properties, and location in the virus. Biochemistry 16, 2686. Greenwood, J., Willis, A.E. and Perham, R.N. (1991) Multiple display of foreign peptides on a filamentous bacteriophage: peptides from Plasmodium falcipanrm circumsporozoite protein as antigens. J. Mol. Biol. 220, 821. Hoess, R., Brinkmann, U., Handel, T. and Pastan, I. (1993)
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Identification of a peptide which binds to the carbohydrate-specific monoclonal antibody B3. Gene 128, 43. Hoogenboom, H.R., Griffiths, A.D., Johnson, KS., Chiswell, D.J., Hudson, P., and Winter, G. (1991) Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucl. Acids Res. 19. 4133. Kang, AS., Barbas, C.F., Janda. K.D., Benkovic, S.J. and Lerner, R.A. (1991) Linkage of recognition and replication functions by assembling combinatorial antibody Fab libraries along phage surfaces. Proc. Natl. Acad. Sci. USA 88, 4363. Kushwaha. A., Chowdhury, P.S., Arora, K., Abrol, S. and Chaudhary, V.K. (1994) Construction and characterization of Ml3 bacteriophages displaying functional IgG-binding domains of staphylococcal protein A. Gene, in press. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227, 680. Lerner. R.A., Kang, A.S., Bain. J.D., Burton, D.R. and Barbas III, C.F. (1992) Antibodies without immunization. Science 258. 1313. Lin. T.-C., Webste, R.E. and Konigsberg, W. (1980) Isolation and characterization of the C and D proteins coded by gene IX and gene VI in the filamentous bacteriophage fl and fd. J. Biol. Chem. 255, 10331. Lowman. H.B. and Wells, J.A. (1993) Affinity maturation of human growth hormone by monovalent phage display. J. Mol. Biol. 234, 564. Makowski, L. (1993) Structural constraints on the display of foreign peptides on filamentous bacteriophages. Gene 128, 5. Markland, W.. Roberts, B.L., Saxena, M.J., Guterman, S.K. and Ladner, R.C. (1991) Design, construction and function of a multicopy display vector using fusions to the major coat protein of bacteriophage M13. Gene 109, 13. Marks, J.D., Hoogenboom, H.R., Bonnert, T.P., McCafferty, J., Griffiths, A.D. and Winter, G. (1991) By-passing immunization: human antibodies from V-gene libraries displayed on phage. J. Mol. Biol. 222, 581. Marvin, D. and Hohn, B. (1969) Filamentous bacterial viruses. Bacterial. Rev. 33, 172. McCafferty, J., Griffiths, A.D., Winter, G. and Chiswell, D.J.
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(1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348, 552. McCafferty, J., Jackson, R.H. and Chiswell, D.J. (1991) Phage enzymes: expression and affinity chromatography of functional alkaline phosphatase on the surface of bacteriophage. Prot. Eng. 4, 955. Munro, S. and Pelham, H.R.B. (1987) A C-terminal signal prevents secretion of luminal ER proteins. Cell 48, 899. Nissim, A., Hoogenboom, H.R., Tomlinson, I.M., Flynn, G., Midgley, C., Lane, D. and Winter, G. (1994) Antibody fragments from a ‘single pot’ phage display library as immunochemical reagents. EMBO J. 13, 692. Parmley, SF. and Smith, G.P. (1988) Antibody-selectable filamentous fd phage vectors: affinity purification of target genes. Gene 73, 305. Scott, J.K. and Craig, L. (1994) Random peptide libraries. Curr. Opin. Biotech. 5, 40. Scott, J.K., Loganathan, D., Easley, R.B., Gong, X. and Goldstein, I.J. (1992) A family of concanavalin A-binding peptides from a hexapeptide epitope library. Proc. Natl. Acad. Sci. USA 89, 5398. Smith, G.P. and Scott, J.K. (1994) Libraries of peptides and proteins displayed on filamentous phage. Methods Enzymol. 217, 228. Stengele, I., Brass, P., Garces, X., Giray, J. and Rasched, 1. (1990) Dissection of functional domains in phage fd adsorption protein: discrimination between attachment and penetration sites. J. Mol. Biol. 212, 143. Towbin, H., Staehelin, T. and Gordon, I. (1979) Electrophoretic transfer of protein from the polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc. Natl. Acad. Sci. USA 76, 4350. Van Wezenbeek, P.M.G.F., Hulsebos, T.J.M. and Schoenmakers, J.G.G. (19801 Nucleotide sequence of the filamentous bacteriophage Ml3 genome: comparison with phage fd. Gene 11, 129. Wells, J.A. and Lowman, H.B. (1992) Rapid evolution of peptide and protein binding properties in vitro. Curr. Opin. Biotech. 3, 3.55. Winter, G., Griffiths, A.D., Hawkins, R.E. and Hoogenboom. H.R. (19941 Making antibodies by phage display technology. Ann. Rev. Immunol. 12. 433.
JOURNALOF WIUMMGICAL METHODS Journal
of Immunological Methods 179 (1995) 165-175
Monoclonal antibodies against a minor and the major coat proteins of filamentous phage M13: their application in phage display D. Bhardwaj, S.S. Singh, S. Abrol, V.K. Chaudhary
*
Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi-110021, India
Received 26 July 1994; revised 13 October 1994; accepted 19 October 1994
Abstract We have produced monoclonal antibodies (MAbs) which react with a minor and the major coat proteins of filamentous phage Ml3 and have characterised them by combining the techniques of enzyme-linked immunosorbent assay (ELISA) and Western blotting. These coat proteins are the minor coat protein, gIIIp, the product of gene III and the major coat protein, gVIIIp, the product of gene VIII. Both gIIIp and gVIIIp are important in the context of
‘phage display’ of foreign peptides/proteins as fusions to these proteins. The anti-gIIIp MAbs were able to detect native gIIIp as well as the fusion proteins comprising foreign proteins and gIIIp in ELISA and on Western blot, indicating their utility for studying the expression of foreign proteins in phage display. Similarly anti-gVIIIp MAbs detected gVIIIp both in ELISA and on Western blot. In an ‘affinity capture phage ELISA’, phages that were captured by virtue of the interaction between the foreign protein (ligand fused to the gIIIp and displayed on the phage surface) and the immobilised counterpart (receptor), could be easily detected using anti-gVIIIp MAbs. Considering the potential of ‘phage display’ technology in protein engineering, these antibodies should find wide applications. Keywords:
ELISA, affinity capture phage; Filamentous
phage M13; Fusion protein;
Monoclonal
antibody; Phage
display
1. Introduction Abbreviations: aa, amino acid(s); ABTS, 2,2’-azino-bis(3’ethylbenzthiazoline&sulfonic acid); BSA, bovine serum albumin; cfu, colony-forming unit(s); ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence activated cell sorter; gIIIp, gene III encoded minor coat protein of bacteriophage M13; gVIIIp, gene VIII encoded major coat protein of bacteriophage M13; HRP, horseradish peroxidase; IgG. immunoglobulin G; pelB signal sequence, signal sequence of the protein pectate lyase from Erwinia carotovora; MAb, monoclonal antibody; OD, optical density; PAGE, polyacrylamide gel electrophoresis; lac p/o, promoter and operator sequences of the lac operon; SDS, sodium dodecyl sulfate 0022-1759/95/$09.50 0 1995 Elsevier SSDI 0022-1759(94)00280-O
Science
B.V. All rights
The filamentous phages (fl, M13, and fd) are single-stranded DNA phages which infect only male specific E. coli strains that contain F’ factor (Marvin and Hohn, 1969). The genome of Ml3 (and fd) is a closed circular DNA molecule of 6.4
* Corresponding author. Tel.: 91-11-678876, 91-11-6885270 and 91-1 l-6886427. reserved
ext. 406; Fax:
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kb, 90% of which codes for the ten genes essential for the completion of life cycle of the phage (Van Wezenbeek et al., 1980). The phage particles have an appearance of a long flexible tube (Berkowitz and Day, 1976) with a 1.5-2 nm thick shell and a diameter of about 6.5 nm (Glucksman et al., 1992; Makowski, 1993) and have a molecular weight of approximately 1.2-1.4 X 10’ Da (Parmley and Smith, 1988). The major coat protein, gVIIIp (product of the gene VIII) is present in about 2700 copies per phage particle. Each phage particle also contains four minor proteins, the products of genes III, VI, VII and IX present in 3-5 copies each. Recently, vectors based on filamentous phages have been developed for displaying the proteins and peptides fused to phage coat proteins on the phage surface and are being used as genetic tools for selecting high affinity variants of engineered proteins/peptides. Their applications include mapping of epitopes for monoclonal antibodies, identification of peptide mimics for non-peptide ligands and for selection of proteins with improved properties (Barbas, 1993; Scott and Craig, 1994; Wells and Lowman, 1992). These studies have resulted in the selection of variants of human growth hormone with 400-fold higher affinity towards its receptor (Lowman and Wells, 19931, identification of peptides that bind to concanavalin A (Scott et al., 19921, identification of a peptide that mimics the binding of carbohydrates to its monoclonal antibody (Hoess et al., 1993) and in selecting human antibodies of desired specificity (Lerner et al., 1992; Winter et al., 1994). In a majority of these vectors, the DNA encoding the desired peptide or protein is cloned at the 5’ end of gene III which encodes a 42 kDa protein, gIIIp. In some vectors, the fusions have been made at the 5’ end of gene VIII (Greenwood et al., 1991; Kang et al., 1991; Markland et al., 19911, which encodes a 5600 Da protein, gVIIIp. Upon expression, the peptide/protein is displayed on the surface of the phage particle as a fusion with one of the coat proteins while its corresponding DNA remains encapsulated in the same phage particle. The interaction between the displayed peptide/pro-
bilized partner followed by elution of the bound phages with acidic (Parmley and Smith, 1988) or alkaline (Marks et al., 1991) buffers. The number of viable phages in eluates is estimated by a time consuming process in terms of colony/plaque forming units after infecting a suitable strain of E. coli. The expression of foreign proteins fused with the phage coat proteins could be readily studied if MAbs against the coat proteins were available. The interaction between the peptide/protein displayed on the phage surface fused to one of the coat proteins with its receptor immobilised on the surface of a microtitre plate could be detected in an ELISA as a quick estimate of the binding of phages, with MAbs against the other coat proteins. In this report, we describe the production, characterisation and utility of a panel of MAbs against gIIIp and gVIIIp of filamentous phage M13. These antibodies were able to detect the respective phage proteins both in ELISA as well as in Western blot analysis. The anti-gIIIp MAbs were also able to detect gIIIp fusion proteins on Western blots. The anti-gVIIIp MAbs were useful in detecting the phage particles displaying a ligand fused to the gIIIp when captured by the immobilised receptor coated on the microtitre plates.
tein and its binding studied by incubating
PAGE) and gelatin were mond, CA, USA). Bovine
partner (receptor) is often the phages with the immo-
2. Materials and methods 2.1. Materials Bacterial media components were obtained from DIFCO Laboratories (Detroit, MI, USA) or Gibco BRL (Gaithersburg, MD, USA). Antibiotics were either from United States Biochemicals Corp. (Cleveland, OH, USA), Sigma Chemical Co. (St. Louis, MO, USA) or Gibco BRL. Freund’s adjuvants, polyethylene glycol (PEG) 1300-1600 and 50 x HAT supplement were from Sigma Chemical Co. Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum and prestained molecular weight standards were purchased from Gibco BRL. Reagents for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSfrom Bio-Rad serum albumin
(Rich(BSA,
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of Immunological Methods 179 (1995) 165-I 75
fraction V) was purchased from Boehringer-Mannheim (Indianapolis, IN, USA). HRP (horseradish peroxidase) conjugated secondary antibodies used for ELISA and Western blot were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). All other reagents and chemicals used were of analytical grade. PVDF membranes (Immobilon) were purchased from Millipore (Bedford, MA, USA). Plasticware used in cell culture was from either Costar (Cambridge, MA, USA) or Corning (Corning, NY, USA). Bacteriophages M13mp19 and M13K07 were obtained from Gibco BRL and Stratagene (La Jolla, CA, USA) respectively. E. coli strain XL-1 blue and plasmid pBluescript KS + were obtained from Stratagene. Inbred mice of BALB/c strain were procured from National Institute of Nutrition, Hyderabad (India) and maintained at the departmental animal house facility. 2.2. Production of phage particles The phages M13mp19 were propagated in E. blue, and purified according to the method of Lin et al. (1980).
coli XL-l
2.3. Production and screening of hybridomas Highly purified phage particles were used for immunizing the Balb/c mice. Primary immunization was done by intraperitoneal injections of 200 pg of total phage protein in Freund’s complete adjuvant in 4-6-week-old mice weighing 20-25 g, followed by a secondary immunisation after 21 days with 100 pg of total phage protein using Freund’s incomplete adjuvant. The animals were test bled 21 days after the secondary immunisation and their sera were titrated for the presence of anti-phage antibodies by ELISA. The positive animals were given an intravenous booster of 100 pug phage protein in saline. Seven days later, the animal was sacrificed and the spleen was removed under aseptic conditions. A single cell suspension of the splenocytes was prepared using a frosted glass homogeniser. The cells were washed twice with serum-free DMEM. Cells of myeloma line, Sp2/0 were also washed with serum-free DMEM and mixed with the spleno-
167
cytes in one to one ratio followed by centrifugation. The mixed cell pellet was then suspended in 0.5 ml of 50% solution of PEG 1300-1600 (prepared in serum-free DMEM containing penicillin and streptomycin at a concentration of 100 U/ml and 100 pg/ml respectively) over a period of 1 min by constant hand mixing. Stirring was continued for an additional minute followed by addition of 1 ml of serum-free medium. Over the next 2 min, 9 ml of serum-free medium was added while keeping the cells under constant and gentle stirring. The cells were pelleted and suspended to a final concentration of 2 X lo6 splenocytes per ml in 40 ml medium containing antibiotics and supplemented with 10% heat inactivated fetal calf serum and HAT (hypoxanthine, 1 X lo-” M; aminopterin, 4 X 10-h M and thymidine, 1.6 x 10e4 Ml. The cell suspension (40 ml) was mixed with normal spleen cells (40 ml, 2 x 10h cells per ml) and dispensed in four 96 well plates (200 ~1 per well). Plates were kept in a humidified incubator at 37°C with 5% CO,. After 14 days, the supernatants from wells bearing colonies were tested by ELISA and Western blot analyses (described later). The cells from antibody positive wells were taken for cloning by limiting cell dilution method. Ascitic fluids were produced in pristane primed BALB/c mice by intraperitoneal injections of 1 X lo6 actively growing hybridoma cells that were washed with serum-free DMEM. 2.4. Screening of the hybridomas The hybridomas were screened by ELISA and Western blot analyses at every stage of cloning and subcloning. Since purified phage proteins were not available, ELISA using phage coated microtitre plates was performed to identify the wells which contained cells secreting anti-phage antibody. Western blot analysis using total phage proteins was employed to determine the specificity of the antibodies in terms of their reactivity with individual phage proteins. 2.5. Enzyme-linked immunosorbent assay Phage particles equivalent to 100 ng protein (1 pg/ml) were coated on the surface of polystyrene
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of Immunological Methods 179 (1995) 165-l 75
microtitre plates (Corning) in 50 mM bicarbonate buffer pH 9.2, for 1 h at 37°C. After washing with PBST (20 mM phosphate buffer pH 7.4, 150 mM NaCl containing 0.05% Tween 20), the unoccupied reactive sites were blocked with 2% non-fat dry milk solution in PBST at 37°C for 1 h followed by washing. The serum of the immunized mice or antibody containing culture supernatant were diluted in PBST and added to the coated plates and incubated at 37°C for 1 h. After washing the wells with PBST, 100 ~1 of HRP-conjugated goat anti-mouse IgG (diluted in PBST containing 2% non-fat dry milk) was added and incubated at 37°C for 1 h. After washing the wells with PBST, the reaction was revealed by incubating with 100 ~1 of 1 mg/ml solution of ABTS (2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) in citrate-phosphate buffer (76 mM citric acid and 218 mM disodium hydrogen phosphate, pH 4.5) containing 1 pi/ml of 30% H,O,. After incubating at 37°C for 10 min the reaction was stopped by adding 100 ~1 of 10% SDS and recorded at 405 nm using an ELISA reader (BioTek Instruments, Winooski, VT, USA). 2.6. Affinity capture phage ELISA In this assay anti-gVIIIp MAbs were employed to detect the phages displaying a ligand and captured by immobilised receptor on microtitre plates. For this purpose the wells of polystyrene ELISA strips were coated with 100 ~1 solution of normal human IgG (10 pg/ml in bicarbonate buffer) at 37°C for 1 h followed by washing with PBST for three times. After blocking the unoccupied reactive sites with 3% gelatin solution in PBST at 37°C for 1 h, phages displaying the B domain of protein A, an IgG binding protein from S. aureus, were added in the IgG coated wells in different concentrations (Kushwaha et al., 1994). The strips were incubated at 37°C on a vibrating platform to ensure constant mixing of the contents of the wells for 1 h. After four washings with PBST, the captured phages were eluted using 0.1 N HCl (pH adjusted to 2.2 with solid glycine) containing BSA at 1 mg/ml (Parmley and Smith, 1988) in three different sets of ELISA strips. One set was eluted once, another
was eluted twice and the third set was eluted three times. The eluates from each set were pooled and were used to determine the number of eluted phages by infecting the E. coli XL-l blue cells and counting colonies and plaques (Smith and Scott, 1994). One set was kept as a control from which the phages were not eluted. The number of phage particles which still remained bound to the plate before and after elutions were probed and quantitated with anti-gVIIIp MAbs (from clone 2911) and HRP-conjugated goat anti-mouse IgG, as described earlier. 2.7. Western blot analysis The supernatants positive for anti-phage antibodies in ELISA were further screened by Western blot analysis to determine their specificity against Ml3 phage coat proteins. 1 pug of total phage protein in 200 ~1 of 1 X Laemmli buffer was loaded on a preparative 0.1% SDS/12.5% polyacrylamide gel and electrophoresis was performed as described (Laemmli, 1970). Following electrophoresis, the proteins were electroblotted (Towbin et al., 1979) onto PVDF membranes which were subsequently blocked with 2% non-fat dry milk solution in PBST. The membrane was cut into 7-9 mm wide strips, which were incubated with culture supernatants (diluted l/50 in PBST) followed by washing and incubation with HRP-conjugated goat anti-mouse IgG. All the incubations were carried out at ambient temperature (25-27°C). The binding of anti-phage antibodies to phage proteins was revealed by incubating the strips with 1 mg/ml solution of 3,3’-diaminobenzidine (DAB) in PBS, containing 1 pi/ml of 30% H,O,. 2.8. Production of gIIIp fusion proteins and gIIIp deletion derivatives for Western blot analysis
Various fusion proteins consisting of foreign proteins fused at the N-terminus of gIIIp were expressed in E. coli and the total cell lysate was used for their detection by anti-gIIIp MAbs on Western blot. The fusion proteins, CD4-gIIIp consisting of the first 176 amino acids of human CD4, EB-gIIIp consisting of a single IgG-binding
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of Immunological Methods 179 (1995) 165-175
domain of staphylococcal protein A and antiTat-gIIIp consisting of a single chain antibody which binds to the ~5.5 subunit of the human IL2 were expressed using phagemids receptor, pVCCD43426 (Abrol et al., 1994), pVCEB3426 (Kushwaha et al., 1994) and pVC73426 (our unpublished results), respectively. These vectors express aa +3 to +406 of mature gIIIp with the ligand fused at +3 codon. The gene fusion is preceded by pelB signal sequence and the expression of these proteins is under the control of lac p/o. Briefly, the overnight grown cultures of XL-l blue cells carrying these phagemids were diluted ten-fold in LB medium containing glucose cl%), ampicillin (100 pg/ml) and tetracycline (10 pgg/ml) and grown at 37°C to OD,,, of 0.3-0.4. The expression of fusion proteins was induced by diluting the cultures ten-fold in glucose-free medium and incubating for 2 h. The cells were harvested by centrifugation and the cell pellet was boiled in 1 X Laemmli buffer (Laemmli, 1970) for 3-5 min, electrophoresed and transferred onto PVDF membranes. The membranes were probed with anti-gIIIp MAbs (l/500 dilution of culture supernatant of clone 30421) followed by incubation with HRP-conjugated goat anti-mouse IgG as described above. To detect the fusion proteins displayed on the surface of phage particles, CD41923 phages displaying the first 176 amino acids of human CD4 fused to the gIIIp of Ml3 phages (Abrol et al., 1994) were electrophoresed on SDS-polyacrylamide gel and transferred onto PVDF membranes. The presence of fusion proteins was probed using culture supernatant from clone 30421 as described above. To identify the epitope recognised by anti-glIIp MAbs, deletion derivatives of gIIIp were expressed in E. coli using phagemids pVC326, pVC1326 and pVC2326 which encode aa +3 to +406, aa +56 to +406 and aa + 198 to +406 of gIIIp respectively. At the N-terminus, these proteins carry a 10 aa peptide tag, c-myc recognised by MAb 9ElO (Evan et al. 1985 and Munro and Pelham, 1987) followed by a pentapeptide linker (SGGGG). The proteins were expressed and used for Western blot analysis as described above for other gIIIp fusion proteins.
169
3. Results 3.1. identification of the hybridoma clones producing antibodies against glllp and gP7IZp
Following 14 days of fusion, the supernatant from wells with colonies were selected for screening. Initially, the supernatants were tested for the presence of anti-phage antibodies using phage particles coated on the microtitre plates and a total of 44 wells were identified with varying extents of reactivity (data not shown). Further analysis was done by Western blotting, using total phage proteins separated by SDS-PAGE, which identified three clones producing anti-gIIIp and another 20 producing anti-gVIIIp antibodies. A few of the antibody-producing clones were further purified by limiting dilution and supernatants were analysed by ELISA and by Western blots (data not shown). Finally the culture supernatants or ascitic fluid from two clones of each type of MAb were used for further characterisation of the antibodies. 3.2. Anti-glllp antibodies can detect both native gIIIp and its fusion proteins The ability of anti-gIIIp MAbs to detect the gIIIp in ELISA was checked by coating phage particles directly on microtitre plates. In this assay, 1.0 ng of gIIIp (contributed by 3 X lo9 phage particles) was detectable using l/100 dilution of culture supernatants from two different clones, 30421 and 30122 (Fig. 1). Similar results were obtained with l/50000 dilution of ascitic fluid produced by these two clones (data not shown). End point titration showed that l/1000 dilution of culture supernatant and l/100000 dilution of the ascitic fluid produced color intensity which was 20 times higher than the background. Since purified gIIIp was not available, detectability of gIIIp on Western blots using the monoclonal antibodies was established by separating Ml3 phage proteins on SDS-polyacrylamide gel and transferring on to PVDF membranes. As shown in Fig. 2, as little as 0.1 ng of gIIIp could be detected which was contributed by 3 x 10’ phage
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of Immunological
particles (lane 1). Anti-gIIIp MAbs were used to detect the gIIIp fusion proteins when expressed in E. coli cells and also when displayed on the surface of phage particles. The anti-gIIIp MAbs from clone 30421 detected gIIIp on Western blots in the form of various fusion proteins, when expressed in E. coli and separated by SDS-PAGE (Fig. 3). The culture supernatant from anti-gIIIp clone 30122 also showed the same results (data not shown). In this experiment it was difficult to estimate the relative reactivity of the anti-gIIIp MAbs towards the fusion proteins in comparison to native gIIIp because the purified fusion proteins were not available. However, the reactivity of anti-gIIIp MAbs to the fusion proteins was further evaluated by using a purified phage preparation of CD41923, that displays all the copies of gIIIp as fusion protein with the human CD4 (Abrol et al., 1994). The reactivity of anti-gIIIp MAb with the CD4-gIIIp fusion protein (Fig. 4, lane 3) is similar to the native gIIIp (Fig. 4, lane 4) thereby indicating that the anti-gIIIp MAbs are equally reactive to gIIIp when it is present as fusion protein and these MAbs might
I
2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10” lQf0 Phages per well
log I
0.35
.“‘.‘I
“““‘I
“““‘I
3.5
35.0
350
glllp (ng per well) Fig. 1. ELISA using anti-gIIIp MAbs. The indicated number of phages were coated on the surface of microtitre plates as described in Materials and Methods. l/100 dilutions of culture supernatants of hybridoma clones 30122 (0) and 30421 (a 1 were used to detect the gIIIp. The calculated amount of gIIIp per well is also shown.
Methods 179 (1995) 165-175
kDa
97 \
Std
1
2 3 4 5
29 1814-
Fig. 2. Western blot analysis using anti-gIIIp MAbs. Ml3 phage particles (produced by M13mp19) were boiled in IX Laemmli buffer, electrophoresed on 15% polyacrylamide gel, transferred onto the PVDF membranes and probed with anti-gIIIp MAb (clone 30421) followed by HRP-conjugated goat anti-mouse IgG. Lane 1, 3 X 10’ phages (0.11 ng gIIIp); lane 2, 9 X lo8 phages (0.33 ng gIIIp); lane 3, 2.7 X lo9 phages (0.977 ng gIIIp); lane 4, 8.3 X lo9 phages (2.94 ng gIIIp); lane 5, 2.5 x 10” phages (8.8 ng gIIIp). The sizes of the molecular weight markers is shown in kDa. Std, prestained molecular weight standards (Gibco BRL).
be recognising an epitope distal to the site of fusion which is +3 aa of mature gIIIp. To identify the epitope recognised by anti-gIIIp MAbs, various truncated forms of gIIIp with the c-myc tag at the N-terminus were expressed in E. coli. The proteins in the total cell pellets were separated by SDS-PAGE for Western blot analysis. The Western blot in Fig. 5 shows that fusion proteins carrying full length gIIIp or both the deletion derivatives were reactive to 9ElO which recognises the c-myc tag in the expressed protein (panel A). However, anti-gIIIp MAbs from clone 30421 were reactive to full length gIIIp and the truncated derivative which contained aa t-56 to + 406 of gIIIp (panel B, lanes 1 and 2) but not to the derivative consisting of aa + 198 to + 406 of gIIIp (panel B, lane 3). Similar observations were made using MAbs from the clone 30122 (data not shown). These results show that the anti-gIIIp MAbs described here recognised an epitope between aa + 55 and + 197 of the mature gIIIp which is distal to the site of fusion and therefore the binding of these antibodies is not affected by
D. Bhardwaj et al. /Journal
of Immunological Methods 179 (1995) 165-I 75
the presence of ligands such as CD4 or anti-Tat (sFv) fused to gIIIp at its N-terminus.
12
171
34
3.3. Anti-gVIIIp monoclonal antibodies can be used in Western blot analysis and phage ELISA
The culture supernatants containing anti-gVIIIp MAbs from two clones 713 and 2911 were used to detect the phages that were directly coated on the surface of microtitre plate. As shown in Fig. 6, 1.1 X 10s phage particles which correspond to 2.56 ng of gVIIIp produced a signal which was approximately 25 times more than the background using l/100 dilution of the culture supernatant. End point titration demonstrated that l/500 dilution of culture supernatant or l/64000 dilution of the ascitic fluid could be used in phage ELISA. The specificity of these MAbs was further established in Western blot analysis using phage proteins separated by SDSPAGE which showed a dose dependent increase
kDa
g7\ 6%
-
1
Std
.(
2 3 4
,.,_<_* ;
‘, ,..
43 29
_
m-
Fig. 4. Western blot analysis of phages displaying CD4-gIIIp fusion protein. CD41923 phages (Abrol et al., 1994) were boiled in 1 X Laemmli’s sample buffer for 3-5 mitt, electrophoresed on 0.1% SDS/12.5% polyacrylamide gel and transferred to PVDF membrane, and probed with anti-gIIIp MAbs (clone 30421) followed by HRP-conjugated goat antimouse IgG. Lane 1. 1.4~ 10’ phages (equivalent to 0.074 ng fusion protein); lane 2, 4.2~ 10s phages (0.222 ng fusion protein); lane 3, 1.4~ lo9 phages (0.74 ng fusion protein); lane 4, 7.5~ 10” M13mp19 phages (2.64 ng gIIIp) per IO ~1 sample. Arrows indicate the position of CD4-gIIIp fusion protein and gIIIp respectively. Number of phage particles as described above have been corrected for the loss of infectivity due to the presence of CD4 at the N-terminus of gIIIp in comparison to the parent vector MlJmpl923, which does not have CD4 (Abrol et al., 1994).
^,
.
in binding to gVIIIp (N 6 kDa) without any reaction to any other phage or E. coli protein (Fig. 7). However, the reactivity of anti-gVIIIp MAbs to gVIIIp on Western blot was weak as reaction was visible only when phages equivalent to 25 ng of gVIIIp were loaded. 3.4. Affinity capture phage ELBA
Fig. 3. Western blot analysis of gIIIp fusion proteins. Various gIIIp fusion proteins were expressed in XL-l blue cells as described in the materials and methods section. Total cell pellet from 500 ~1 culture of each was boiled in 150 ~1 of 1 x Laemmli buffer and 10 PI sample was electrophoresed on 0.1% SDS/lS% polyacrylamide gel, transferred onto PVDF membrane and probed with anti-gIIIp MAb (clone 304211 as described. Lane 1, XL-1 blue cells carrying pBluescript KS: lane 2, anti-Tat gIIIp fusion protein ( - 87 kDa1 expressed by pVC73426; lane 3, EB-gIIIp fusion protein ( - 70 kDa) expressed by pVCEB3426, and lane 4, CD4-gIIIp fusion protein ( - 82 kDa) expressed by pVCCD43426. Std, prestained molecular weight standards (Gibco BRL).
The anti-gVIIIp MAbs were also used to detect phages which were captured by virtue of interaction between a ligand, fused to gIIIp and displayed on the phage surface, with its receptor immobilised on the microtitre plates (Fig. 8A). In this assay anti-gVIIIp MAbs were able to detect phages displaying the B domain of staphylococcal protein A captured in an IgG coated well when 3 x 10’ phages were added per well (Fig. 8B). These studies were further extended to deter-
172
D. Bhardwaj et al. /Journal
1 2
kDa
3
4
of Immunological
Methods 179 (1995) 165-175
1234
200 -
9s; 43 29 18
Fig. 5. Western blot analysis of gIIIp deletion derivatives. gIIIp derivatives were expressed in XL-l blue cells as described in the materials and methods section. The total cell pellets were processed as described in legends to Fig. 3 followed by electrophoresis on 0.1% SDS/lO% polyacrylamide gel. The Western blot analysis was performed using MAb 9E10, l/l000 dilution of the ascitic fluid (panel Al and MAb against gIIIp from clone 30421, l/1000 dilution of the ascitic fluid (panel B). Lane 1, gIIIp fusion protein encoded by pVC326 ( - 60 kDa1; lane 2, gIIIp fusion protein encoded by pVC1326 ( - 54 kDa1; lane 3, gIIIp fusion protein encoded by pVC2326 ( - 30 kDa); lane 4, 7.5 X lo9 M13mp19 phages. The values in parantheses are the observed molecular weights of the gIIIp fusion protein derivatives. Position of molecular weight standards is shown in kDa.
mine as to what fraction of the added phages was captured by immobilised human IgG. For this purpose the captured phages were eluted as described in the materials and methods section. After each elution, the wells were incubated with anti-gVIIIp MAbs to quantitate the remaining phages by ELBA. The number of phages that remained bound to the IgG coated plates even after elution was estimated by measuring the intensity of color produced in these wells in comparison to that produced in the wells in which elution was not done. ELISA results revealed that only 2, 1.5 and less than 1% phages remained bound after one, two and three rounds of elution respectively (data shown only for one round of elution). This simple ELISA using antigVIIIp MAb could therefore be used to check the binding of phages as well as to evaluate the efficiency of elution of bound phages. The number of eluted phages encapsulating the phagemids
Phages per well
I 0.23
‘...“‘I
“““‘I 23.0 2.3 gVlllp (ng per well)
““,“l 230
Fig. 6. ELISA using anti-gVIIIp MAbs. The indicated phage numbers were coated on the surface of microtitre plates and l/100 dilutions of culture supernatants of clones 713 (0) and 2911 (0) were used to detect the gVIIIp as described in the materials and methods section. The calculated amount of gVIIIp per well is also shown.
was estimated by infecting E. coli cells and counting colonies (colony forming units), which revealed that only 1.5-2.0% of the added phage
kDa
12345
Fig. 7. Western blot analysis using anti-gVIIIp MAbs. Different numbers of Ml3 phage particles (produced hy M13mp19) and XL-1 blue cells were boiled in 1 X Laemmli buffer, electrophoresed on 0.1% SDS/12.5% polyacrylamide gel and transferred onto the PVDF membrane. The membrane was probed with l/100 dilution of culture supernatant from clone 2911. Lanes 1, XL-1 blue cells; lane 2, 3.6~ lOa phages (8.5 ng gVIIIp); lane 3, 1.1~ lo9 phages (25 ng gVIIIp); lane 4, 3.3 x lo9 phages (77 ng gVIIIp); lane 5, 1 X 10” phages (232 ng gVIIIp1. The sizes of the low range prestained molecular weight markers (Gibco BRL) are shown in kDa.
D. Bhardwaj et al. /Journal
of Immunological Methods 179 (1995) 165-I 75
173
immobilised receptor as well as on the number of fusion proteins per phage particle. These results clearly show the utility of anti-gVIIIp MAbs in detecting bound phages in a simple ELISA format and also show that one round of elution is sufficient to dissociate 98% of the bound phages.
(4
Affinity Capture Phage ELBA
4. Discussion
0.03 107
““‘7 108 Phages
“““3
‘10s
10’0
added per well
Fig. 8. Affinity capture phage ELISA. A: cartoon depicting the ‘affinity capture phage ELISA’ showing the interaction of phages displaying the B domain of staphylococcal protein A with human IgG coated on the well surface of a microtitre plate. B: microtitre plate wells were coated with 1 pg human IgG and incubated with indicated number of phages displaying B domain of staphylococcal protein A as fusion with gIIIp. The bound phages were detected using anti-gVIIIp MAbs to) as described in the materials and methods section. The captured phages were eluted once as described and phages that remained bound after elution were detected by ELISA using anti-gVIIIp MAbs (A). The number of eluted phages measured as colony forming units (cfu) after infecting XL-l blue cells is shown in the inset.
were able to bind the immobilised human IgG under the conditions employed (Fig. 8B inset). This low frequency of capture is due to the fact that phages displaying the B domain of staphylococcal protein A were produced using phagemid system in which only 10% of the phage population displays one or more fusion protein per phage particle (Bass et al., 1990; Kushwaha et al., 1994). However, in this assay the phage number required for positive signal would depend upon the affinity between the displayed ligand and the
Monoclonal antibodies have been widely used for detecting proteins in a variety of immunoassays such as ELISA and Western blot. Employing the techniques of ELISA and Western blotting, we have selected monoclonal antibodies against two coat proteins of Ml3 phage although the purified proteins were not available, either for immunization or for detection. These MAbs were found to be useful in studying the expression of fusions with phage coat proteins in E. cd, and the production of Ml3 phage particles that display foreign proteins fused at the N-terminus of gIIIp. Earlier, several groups have used polyclonal antisera raised against the whole Ml3 phage or the purified gIIIp for this purpose (Goldsmith and Konigsberg, 1977; Stengele et al., 1990). Since the gIIIp is present in only 3-5 copies per phage particle, it is difficult to purify and therefore production of a good quality polyclonal anti-gIIIp antiserum becomes a formidable task. Moreover, polyclonal anti-Ml3 antibodies may not be very suitable in experiments where phage preparations are contaminated with E. co& proteins (Kushwaha et al., 19941. The epitope mapping studies for anti-gIIIp MAbs from clones 30421 and 30122 revealed that these MAbs recognised an epitope between aa + 56 to + 197, therefore, these should be useful with phage display vectors which use full length gIIIp (McCafferty et al. 1990; Hoogenboom et al. 1991; Abrol et al. 1994; Kushwaha et al. 1994). Our results in the present study showed that anti-gVIIIp MAbs were very useful in detecting the binding of phages, that display ligands fused to gIIIp and were captured by immobilised receptor in an ‘affinity capture phage ELISA’. This assay has been successfully used to study: the interaction of phages displaying IgG-binding do-
174
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of Immunological Methods I79 (1995) 165-l 75
mains of staphyloccocal protein A with various subclasses of human IgG (Fig. 8 and Kushwaha et al., 19941, the interaction between the human CD4 borne on phages with gp120, the HIV envelope glycoprotein (Abrol et al., 1994), and the binding of the phages displaying a single chain antibody to its antigen on the surface of cancer cell line (our unpublished results). Although, polyclonal anti-Ml3 antisera could also be used for this purpose (McCafferty et al., 1991), presence of E. coli proteins in the phage preparations may give higher background. Using antigVIIIp MAbs, we have also been able to detect the binding of phages bearing a variety of ligands to their respective immobilised receptor using the total bacterial culture containing ligand bearing phages without any interference by E. coli cells (our unpublished results). Smith and Scott (1994) have described an ELISA in which phages are directly immobilised in the wells of microtitre plates followed by detection of ligand borne on the phage surface fused to gIIIp. This assay requires purified phages as impurities might interfere with adsorption to the plastic or give a high background signal. Purification of phages is tedious, therefore immobilised anti-gVIIIp MAbs should also find utility in capturing phages to assay for presence of ligand fused to gIIIp and displayed on the phage surface. Recently, Nissim et al. (1994) have developed phages bearing antibodies to a variety of proteins and used them as immunochemical reagent to detect proteins on Western blot or on the surface of the intact cells. Anti-gVIIIp MAbs should be useful in this assay as they should give lower background. Use of phages as immuno-chemical agents may have advantage due to the amplification of signal because of a large number of epitopes for anti-gVIIIp MAbs. The anti-gIIIp and anti-gVIIIp MAbs should also find usefulness in studying binding of phages to intact mammalian cells as detected by immunoflourescence or by FACS. In conclusion, the anti-gIIIp MAbs should be very useful in studying the expression of foreign proteins displayed on the surface of phages fused to gIIIp. The anti-gVIIIp MAbs should find wide application in studying the parameters of interac-
tion between the displayed protein (fused to the gIIIp) and its counterpart which is immobilised on the microtitre plates using affinity capture phage ELISA.
Acknowledgements
D.B. and S.A. are recipients of Research Fellowships from the Council of Scientific and Industrial Research, Government of India. Thanks are due to Dr. K. Kannan for providing help in immunisations. We are thankful to Drs. P.K. Ghosh, J.K. Batra, Maria Gallo, Donna Roscoe and Dinkar Sahal for critically reading the manuscript and helpful suggestions, and to Mr. R. Chawla and Mr. S. Simon for typing the manuscript. This work was partly funded by the Department of Biotechnology and Department of Science and Technology, Government of India.
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