269
Journal of Immunological Methods, 136 (1991) 269-278 © 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 ADONIS 002217599100083Y
JIM05829
Characterisation of m o n o c l o n a l antibodies to h u m a n factor X / X a Initial observations with a quantitative ELISA procedure * Richard B. H o a d and Carolyn L. Geczy Heart Research Institute, Sydney, A ustralia (Received 23 July 1990, revised received 8 October 1990, accepted 22 October 1990)
Monoclonal antibodies to human plasma factor X (FX) and factor Xa (FXa) have been developed using several modifications of previously described techniques. These include the use of footpad immunisation with a suspension of free and nitrocellulose-bound antigen with subsequent fusion of popliteal lymph node cells. From a panel of 17 reactive hybridomas to FX, 3 were selected for further characterisation. An additional hybridoma reactive to FXa but not FX was also selected. Two monoclonal antibodies designated FX52 and FX64 were specific for FX with no reactivity to FXa, while antibody FXa24 was specific for FXa. Another FX/FXa95 reacted with both FX and FXa. All selected antibodies were of the IgG isotype and reacted both with native antigen and antigen transferred to nitrocellulose by Western blotting. Initial observations suggest that Mab FX52 may be used to quantitate FX levels in plasma. Key words." Monoclonal antibody; Factor X; Factor Xa; Immunization; Nitrocellulose; Popliteal lymph node cell fusion; ELISA
Introduction
Human factor X (FX), in common with other serine proteases, is highly conserved both within and between species (Jackson and Nemerson,
Correspondence to: R.B. Hoad, Heart Research Institute, 145 Missenden Road, Camperdown, Sydney, N.S.W. 2050, Australia. * This work was supported by" National Health and Medical Research Council (NH&MRC), Australia and America Diagnostica Inc., CT, U.S.A. Abbreviations: FX, human factor X; FXa, human factor Xa; DMSO, dimethyl sulphoxide; NPS, nitrocellulose particle suspension; CM, culture medium; ConA, concanavalin A; CAS, ConA-stimulated spleen cell-conditioned medium; H, hypoxanthine; T, thymidine; TBS, Tris-buffered saline; BSA, bovine serum albumin; HSA, human serum albumin; NB, nitrocellulose bound; SDS, sodium dodecyl sulphate; CR, coagulation assayed reference; CFA, complete Freund's adjuvant.
1980). It is a glycoprotein of apparent molecular weight 72 kDa (Mertens and Bertina, 1980) present in plasma at approximately 8/~g/ml (Epstein et al., 1984) and is situated at the critical interface of the intrinsic and extrinsic coagulation cascades. Human factor Xa (FXa) is derived by proteolytic cleavage of the heavy chain of FX to produce a 54 kDa product (designated aFXa) which can the be cleaved further by autolysis to flFXa (50 kDa) (Mertens and Bertina, 1980). Once formed, FXaa or fl complex with factor Va, phospholipid and calcium (prothrombinase complex) to cleave prothrombin to thrombin with subsequent cleavage of fibrinogen to fibrin and clot formation (Jackson and Nemerson, 1980). Two groups have generated monoclonal antibodies (Mabs) which react with both FX and FXa (Church and Mann, 1985; Doellgast, 1987; Church et al., 1988). However by modifying conventional procedures for antibody production we have suc-
270
ceeded in obtaining Mabs which react specifically with either FX or FXa.
Materials and methods
FX, FXa, and spectrozyme FXa chromogenic substrate were all gifts from American Diagnostica (CT).
Preparation of nitrocellulose particles and antigen binding Nitrocellulose particles were generated essentially as previously described (Abou-Zeid et al., 1987). Briefly a 4 cm 2 piece of nitrocellulose (0.1 /zm) (Schleicher and Schuell, Dassel, F.R.G.) was dissolved in 4 ml dimethyl sulphoxide (DMSO, Merck, Darmstadt, F.R.G.). The nitrocellulose was precipitated by adding it dropwise to 40 ml carbonate buffer (0.015 M NazCO 3, 0.03 M N a H C O 3, pH 9.6) with vortexing over 2 min. Large particles were allowed to settle by gravity for 1 rain and remaining particles in suspension were aspirated through a 21 gauge needle to generate smaller particles. Particles were sedimented by centrifugation at 500 x g for 10 rain and the pellet washed twice in carbonate buffer (50 ml/wash). A 0.5 ml packed vol of nitrocellulose particles was resuspended in 2 ml carbonate buffer and the suspension aspirated several times through a 25 gauge needle to further break up the particles. The nitrocellulose particle suspension (NPS) was stored at - 20 ° C prior to use. Nitrocellulose-bound FX (NB-FX) was prepared by adding 50 /~1 of the NPS to 50 /L1 solution of FX (28.5 /xg in 10 mM Tris-HCl/0.1 M NaC1, pH 7.5). The mixture was incubated at 3 7 ° C for 1 h, diluted with 0.9 ml NaC1 (0.9% w / v ) and stored at - 2 0 ° C prior to use. 25% of the FX added to the nitrocellulose particles bound to the particles. This was estimated by comparing the total amount of FX in the supernatant of samples with and without the addition of NPS. After 1 h incubation at 37°C, supernatants were collected and pooled for testing after centrifugation of the particles at 500 x g for 10 min and three subsequent washes with 0.01 M Tris-HC1 pH 7.9 to remove any weakly bound antigen. FX in serial dilutions of both fractions
was estimated by activation of FX to FXa with Russel's viper venom (Sigma, St. Louis, MO) and FXa detected using the Spectrozyme FXa chromogenic substrate according to the manufacturer's instructions.
Animals Female B A L B / c mice (5-6 weeks old) were initially obtained from Animal Resources Centre, Murdock, Australia and maintained in the Hospital Animal House Royal North Shore Hospital, Sydney.
Immunisation Three female B A L B / c mice were immunised in the rear footpads (under mild ether anaesthesia) with the solution of FX and NB-FX emulsified in an equal volume of complete Freund's adjuvant (CFA; Comonwealth Serum Laboratories, Parkville, Australia; 40 /~1 per footpad, equivalent to approximately 1.15 /~g FX per mouse). The mice were boosted in the same footpads with the solution of FX and NB-FX (without adjuvant) on days 9 and 12 after the initial injection (40 #1 per footpad, equivalent to approximately 2.3 /~g FX per mouse on each day).
Cell culture and fusion The culture medium (CM) used in the fusion studies was Iscove's (Gibco, Grand Island, NY) supplemented with 20% young calf serum (Bioclone, Marrickville, Australia) 2 mM glutamine (Flow Laboratories North Ryde, Australia) penicillin 100 U / m l , streptomycin sulphate 1 0 0 / t g / m l (both from Glaxo, Melbourne, Australia) and 5 x 10 --~ M 2-/3-mercaptoethanol (Sigma). All cultures were maintained at 3 7 ° C in a humidified atmosphere of 10% CO 2 in air. On day 1 prior to fusion, resident peritoneal macrophages were lavaged from B A L B / c mice, washed once in Iscove's medium and plated into 96 well microtitre plates (Nunc, Roskilde, Denmark) at a density of 104 cells in 150/xl CM per well. Concanavalin A (ConA) stimulated spleen cell conditioned medium (CAS) (Snick et al., 1986) was prepared by stimulating spleen cells from B A L B / c mice (107 cells/ml in CM) with 5 / z g / m l ConA Sepharose (Pharmacia, Sweden) for 24 h. The CAS was harvested by centrifugation at 300
271 X g for 10 rain, filter sterilised using a 0.22 /~m Milex-GV filter (Millipore, Bedford, MA) stored at 4 ° C and used within 2 weeks. 14 days after the initial immunisation, popliteal nodes from the three immunised mice were removed, pooled and single-cell suspensions prepared. Lymph node cells (LNCs) were fused with the non-secreting mouse myeloma P3-X63-Ag8.653 (ATCC CRL 1580) (Kearney et al., 1982) using standard methods (Galfr~ and Milstein, 1981). The fusion ratio was 4 × 10 v LNCs:2 X 10 7 myeloma cells. Post fusion, the equivalent of 105 LNCs in 150 /~1 CM were added to the wells of microtitre plates containing resident peritoneal macrophages. On day 1, selection of hybridomas was commenced by removal of 150 /~1 medium from each well and the addition of 150/~1 CM supplemented with 200 /~M hypoxanthine (H) 800 nM aminopterin and 32 /~M thymidine (T) (all from Sigma). On day 7, 150 /~1 from each well was removed and 150 ~I of CM (100/xM H, 16 b~M T and 4% CAS) added.
Expansion of hybridomas Supernatants from wells containing growing hybridomas were screened by an ELISA procedure to FX (see below) 12-14 days post fusion. Selected hybridomas were expanded until approximately 5 × 10 6 cells were obtained and the cells then frozen to - 7 0 ° C in CM supplemented with 10% DMSO. The original fusion plates were frozen to - 7 0 ° C on day 16 post fusion by the method of Wells and Price (1983). Hybridomas were cloned by limiting dilution. The original fusion plates were thawed 1 month post fusion and hybridomas screened for specific reactivity to FXa (but not FX) by an ELISA procedure (see below). The selected hybridoma FXa24 was identified and processed as described. Ascitic fluid was produced (Gillette, 1987) and Mabs purified using Protein A-Sepharose (Pharmacia) according to the manufacturer's instructions (binding buffer: 1.5 M glycine, 3 M NaC1, pH 8.9). Purified Mabs were concentrated and dialysed into 0.01 M Tris-HC1 + 0.9% NaC1 pH 7.9, using an Amicon concentrator (Amicon, Danvers, MA) with Diaflo YM10 membrane.
ELISA procedure for detection of hybridomas reactive with FX or FXa 100 btl vols of FX or FXa (5 ~ g / m l ) in Trisbuffered saline (TBS; 0.02 M Tris-HC1, 150 mM NaC1, pH 7.4) were added to polyvinyl chloride microtitre plates (Dynatech Laboratories, Virginia) Control wells contained 100 ~1 human serum albumin (HSA, fraction V; 5 /~g/ml; Calbiochem, San Diego, CA). Plates were incubated overnight at 4 o C and then washed four times in TBS using a Microplate washer ($8/12 Titerteck; Flow Labs). Unless specified, all subsequent washes in the ELISA procedure were with the same buffer. Nonspecific binding sites were blocked with 0.5% gelatin (BDH, Poole, England) in TBS (200 /~l/well) for 2 h at 37 ° C. The plates were washed and 100 ~1 aliquots of the supernatants (previously diluted 1 / 6 in Iscove's medium) added. Plates were incubated at 3 7 ° C for 90 min, followed by four washes. 100 /~1 vols of horseradish peroxidase-conjugated rabbit anti-mouse Ig (Dakopatts, Gostrup, Denmark) diluted 1/1000 in TBS + 1% bovine serum albumin (BSA, fraction V, Calbiochem) were added and plates incubated at 37 ° C for a further 1 h. The plates were washed, and peroxidase activity detected using 100/~l/well 2,2'-azino-di-3-ethylbenzthiazoline sulphonate (Boehringer Mannheim, F.R.G., 1 m g / m l ) in 0.1 M citrate buffer, 0.006% H202, pH 4.3. After 20 min at room temperature absorbance values at 414 nm (dual wave-length 414 nm, reference 690 nm) were read using a Multiscan plate reader (Titertek, Flow). After the initial screening, the assay was modified using streptavidin-biotin amplification so that less antigen was required per test well. 200 /~1 vols of FX or FXa (250 n g / m l or 500 n g / m l ) in TBS were added to Nunc-maxisorp plates for 2 h at 37°C prior to blocking nonspecific binding sites with 0.1% Tween 20 (Sigma) in TBS for 30 min at 37°C. FX or FXa was reconstituted according to the manufacturer's instructions and used within 1 h at 4 ° C. Control wells contained no antigen. Plates were washed and 200/~l/well of a biotinylated sheep anti-mouse Ig (species specific and (Fab')2 fragment; Amersham, North Ryde, Sydney) diluted 1/10,000 in TBS, 0.2% ovalbumin, 0.05% Tween were added and the plate incubated for a further hour. The plates were washed and 200 ~1 streptavidin peroxidase (Amersham)
272 (diluted 1/1000 in TBS 0.2% ovalbumin, 0.05% Tween) added to each well for 30 min at 37 o C. The plates were washed and peroxidase activity detected as described (dual wave-length 414 nm, reference 492 nm) except that 200 #1 vol. of substrate were used and the plates read after 10 min.
ELISA procedure for class determination and isotyping of Mabs One hundred/~1 goat anti-mouse Ig (either/t or y chain specific, Cappel, Organon, Teknika, North Ryde, Sydney, diluted 1/500 in carbonate buffer) were added to the wells of PVC plates and incubated overnight at 4 ° C. The plates were blocked with 1% BSA in TBS for 1 h at 37°C, washed and undiluted hybridoma supernatants added. Control myelomas were obtained from ICN Immunobiologicals, IL. After 1 h at 3 7 ° C the plates were washed and 100 ~1 of peroxidase-labelled rabbit anti-mouse Ig (Dakopatts; 1/1000 in TBS, 1% BSA) added. The plates were incubated at 3 7 ° C for 1 h and washed prior to detection of peroxidase activity as described. In assays where specific isotypes were required, procedures were processed up to the washing step after the capture of Mabs. 100/tl rabbit anti-mouse subclass-specific antibodies (diluted 1/500 in TBS, 1% BSA, Litton Bionetics, SC) were added and the plates incubated at 3 7 ° C for 1 h. The plates were then washed and 100 /~1 goat anti-rabbit-peroxidase (Bio-rad, Richmond, CA; 1/1000 in TBS, 1% BSA) added for 2 h at 37 °C. Finally, the plates were washed prior to detection of peroxidase activity as described.
diluted 1 / 5 in TBS and then 1 / 2 in 4% SDS containing 12% glycerol, 5 mM T r i s + 0 . 0 1 % bromophenol blue, pH 6.8 (sample buffer). FX and FXa (8 ~ g / m l ) in TBS were diluted 1 / 2 in sample buffer. Samples were boiled for 2 min and 25 ~1 loaded per lane. Electrophoresis of proteins was at 50 V through the stacking gel and 40 V for 20 h through the main gel. Proteins were electrotransferred to nitrocellulose (0.1 /zm; Schleicher and Schuell) (Towbin et al., 1979) for 2 h at 100 V using a Bio-Rad Transblot cell apparatus (BioRad). Amidoblack staining was carried out according to the method described by Harlow and Lane (1988). All washing steps were in 0.02 M Tris-HC1, 0.5 M NaC1, 0.05% Tween 20, pH 7.5 and consisted of three washes for 5 min with rocking. Membranes were blocked with 0.5% w / v fish gelatin (FG; Sigma) in 0.02 M Tris-HC1, 0.5 M NaCI, pH 7.5, for 2 h at room temperature, and probed with protein A-purified Mabs (1 ttg/ml in 0.02 M Tris-HC1, 0.5 M NaC1, pH 7.5, 0.05% Tween 20, 0.1% FG). Pure FX or FXa was probed for 1 h at room temperature. Plasma samples were probed overnight at 4 °C. Membranes were washed, incubated for 1 h at room temperature in rabbit anti-mouse IgG (Dakopatts) absorbed with human serum (1/400 in 0.02 M Tris-HC1, 0.5 M NaC1, 0.05% Tween 20, 0.1% FG, pH 7.5) washed and incubated with alkaline phosphatase-labelled swine anti-rabbit Ig (Dakopatts; diluted 1/800 in the same buffer) for 30 min. Membranes were washed and alkaline phosphatase activity detected as described by Harlow and Lane (1988).
Protein blotting
Quantitative ELISA procedure for estimating FX in plasma
Tricine-sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis was carried out by the method of Schagger (1987) using a gel (10% T, 3% C) with dimensions of 1.5 mm x 11 cm x 16 cm with a 4 cm stacking gel (4% T, 3% C; where T denotes the total % concentration of both acrylamide and bisacrylamide, C denotes the % concentration of crosslinker relative to the total concentration of T). Samples of normal citrated plasma and FX-deficient plasma (antigen deficient; Hoechst-Behring, Marburg, F.R.G.) were
200/~1 of purified Mab FX52 (5/~g/ml in TBS) were added to Nunc-maxisorp plates and incubated at 37 ° C for 2 h. Plates were washed prior to blocking nonspecific binding sites with 300 /~1 TBS + 1% ovalbumin for 1 h at 37°C. The plates were washed before adding 200 /.tl coagulation assayed reference (CR) plasma (Helena, Mount Waverly, Victoria) or factor-deficient plasma (Organon Technica, North Ryde, Sydney) diluted 1/100 in TBS, 0.05% Tween. In some assays pure FX (20 or 40 ng) was added to 200 /tl of the
273
diluted plasma. After incubation at 37 ° C for 2 h, the plates were washed and 200 /L1 peroxidaselabelled rabbit anti FX (Diagnostica Stago, Asnieres, France; 4 /~g/ml diluted in TBS 0.2% ovalbumin, 0.05% Tween) added. The plates were then incubated at 3 7 ° C for 2 h, washed and peroxidase activity detected as described after 30 min at room temperature.
positive (i.e., absorbance reading > 0.2 after 20 min with FX but < 0.05 with HSA). After isolation of reactive clones the original fusion plates were frozen prior to confluency on day 16 post fusion. Positive hybridomas were tested for reactivity to FX and FXa on nitrocellulose by dot blotting of the antigens before or after SDS treatment (i.e., boiling in the sample buffer used for SDS-PAGE), according to the methods described for Western blotting. The 17 hybridomas tested were all positive by dot blotting and those which were strongly positive were isotyped prior to subcloning. Of the 11 hybridomas tested, four were IgM, five were IgG and two contained both IgM and IgG isotypes. Three hybridomas FX52, FX64 and F X / F X a 95 were selected for subcloning. As a specific Mab to FXa was required, and
Results
A single fusion was carried out 14 days after initial immunisation with 4 × 107 LNC from three immunised mice. From day 12 post fusion, hybridomas (121 in total) were screened for reactivity to FX using the ELISA procedure and 17 were
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Fig. 1. Reactivity of Mabs to FX and FXa by anamplification EL1SA procedure. A : reactivity of Mab FX52 to FX (--©--) and Xa (~); B: Mab FX64 to FX (--©--) and Xa ( B - ) ; C: Mab F X / F X a 9 5 to FX (--©--) and Xa ({3-); and D: Mab FXa24 to FX (--©--) and Xa ( [] ). Assays were carried out with 50 ng antigen/well for graphs A and B, and 100 ng/well for graphs C and D. Tween blocking and initial Mab dilution in Tween was according to the materials and methods section. Background absorbance (no antigen) for all Mabs was < 0.08 for all dilutions tested. Mean of three values and standard deviations from the mean are given.
274 given the success of the fusion, we rethawed original fusion plates for further screening. H y b r i d o m a F X a 2 4 was selected by screening 28 s u p e r n a t a n t s for reactivity to F X a n d F X a by ELISA. The M a b was reactive with F X a (i.e., a b s o r b a n c e > 0.2 after 20 m i n but < 0.05 with F X or HSA), reacted with F X a but not F X after S D S - t r e a t m e n t when screened by dot b l o t t i n g a n d was of I g G isotype. The reactivity of the Mabs to F X a n d F X a was d e t e r m i n e d by amplification E L I S A procedures. Initially the blocking agent was 1% o v a l b u m i n for 1 at 37 ° C. Mabs were diluted in 0.2% o v a l b u m i n , 0.05% Tween. All Mabs, except FXa24, reacted well using these c o n d i t i o n s (data not shown). The procedure was modified by blocking with 0.1% T w e e n a n d dilution of the M a b in 0.05% T w e e n so that all could be effectively c o m p a r e d (Fig. 1). Both Mabs FX52 ( I g G 2 b ) a n d F X 6 4 (IgG1) (Figs. 1A a n d 1B) reacted strongly to F X but not to
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Fig. 2. lmmunoblotting of FX and FXa. Following SDS-PAGE and electrotransfer to nitrocellulose, FX and FXa were probed with Mabs. FX (lane A) and FXa (lane B) were probed with FX52. FX (lane C) and FXa (lane D) were probed with FX64. FX (lane E) and FXa (lane F) were probed with FX/FXa95. FX (lane G) and FXa (lane H) were probed with FXa24. Conditions for gel electrophoresis and immunoblotting are as described in the materials and methods section. Equivalent to 100 ng of pure FX or FXa were loaded onto the gel.
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Fig. 3. Immunoblotting of normal and FX deficient plasma samples. Following SDS-PAGE gel electrophoresis and electrotransfer to nitrocellulose, normal and FX-deficient plasma were probed with Mabs. Normal plasma (lane A) and FX-deficient plasma (lane B) were stained with amido black. FX-deficient plasma (lane C) and normal plasma (lane D) were probed with FX52, FX-deficient plasma (lane E) and normal plasma (lane F) were probed with FX64 according to the conditions outlined in the materials and methods section. The equivalent of 20 ng of FX in normal plasma was loaded onto the gel.
FXa. M a b F X / F X a 9 5 (IgG1) reacted with both F X and F X a (Fig. 1C) whereas M a b FXa24 (IgG1) was specific for F X a (Fig. 1D). Mabs were tested following Western blotting of pure F X or F X a (Fig. 2). They reacted with identical specificities to those observed using the amplification ELISA procedures. A n t i b o d i e s FX52, FX64 a n d F X / F X a 9 5 reacted with the heavy chain of F X by W e s t e r n b l o t t i n g of reduced F X (data not shown). R e d u c t i o n of F X a destroyed the antigenic d e t e r m i n a n t detected by M a b FXa24. The specificity of Mabs FX52 and FX64 for F X was d e t e r m i n e d by Western blotting of n o r m a l a n d F X - d e f i c i e n t plasmas. Fig. 3 shows that both reacted with a single protein of approximately 72 k D a in n o r m a l p l a s m a whereas the c o m p o n e n t was a b s e n t in F X - d e f i c i e n t plasma. The detection system used for blots showed cross reactivity to h u m a n i m m u n o g l o b u l i n in both n o r m a l and FXdeficient plasmas. This was a p p a r e n t l y higher for M a b FX52 since a longer i n c u b a t i o n with substrate was required to detect the FX antigen.
275 0.8
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Fig. 4 shows that Mab FX52 could be used in a quantitative ELISA assay to detect FX in plasma. CR plasma reacted strongly with Mab FX52 whereas the FX-deficient plasma produced only a background signal (the values obtained were significantly different by Student's t test; P = 0.037). The reactivity of FVIII deficient plasma was not significantly different from that of CR plasma ( P = 0.118). When FX was added back to FX-deficient plasma the signal was increased (Fig. 4B). Similarly, increased reactivity was observed when FX was added to CR or FVIII-deficient plasma. The amount of FX present in the CR plasma was estimated to be 17.5 ng/well, equivalent to 8.75 /~g/ml. This value was obtained by reading absorbance values for the CR off the FX standard curve prepared by adding FX (from 0 to 40 ng/well) to FX-deficient plasma (Fig. 4B). The manufacturers specified that the CR plasma contained 105% of the concentration of FX in normal plasma. Therefore, the level of FX in normal plasma was estimated to be 8.3/~g/ml by adjusting the CR plasma to the concentration of FX in normal plasma.
Discussion
Production of Mabs requires the selection of a suitable immunisation schedule which generates high numbers of specifically activated B cells prior to fusion. Since we were unable to produce Mabs to FX using conventional immunisation procedures including in vitro stimulation, we investigated the use of footpad immunisation and fusion of LNCs. This procedure is known to generate a broader range of Mabs, including autoantibodies (Caterson et al., 1983; Holmdahl et al., 1985; Raymond and Suh, 1986; Mirza et al., 1987; Orlik and Altaner, 1988). Mice were immunised in the rear footpad using a combination of pure FX and FX passively bound to nitrocellulose particles (approximate ratio of 3 : 1 respectively). Abou-Zeid (1987) demonstrated that antigen-bound nitrocellulose particles produced following SDS-PAGE and transfer of the antigens to nitrocellulose were effective stimulants of antigen-specific T cell responses in vitro. Accessory cells phagocytose the particles and subsequently process and present antigen in association
276
with the major histocompatability complex (reviewed by Lamb et al., 1988). There are other reports describing the use of antigen bound to nitrocellulose following SDS-PAGE and transfer for the production of both polyclonal and monoclonal antibodies (Knudsen, 1985; Chiles et al., 1987; Diano et al., 1987; Nilsson et al., 1987). Our method is the first to describe the use of nitrocellulose particles mixed with antigen and footpad immunisation of both free and bound antigen for antibody production. The production of nitrocellulose particles prior to antigen binding permits the selection of very small particles without loss of antigen. The possible denaturation of antigenic epitopes by SDS or the solubilisation of the nitrocellulose (DMSO, freeze thawing, etc.) are also avoided. Rear footpad immunisation of pure FX and FX passively bound to nitrocellulose particles using CFA for the initial injection and saline for subsequent injections was tolerated well by the mice. Minimal swelling and no abnormal behavioural changes or ulceration developed within the 14 day period. The procedure described permitted the use of very low concentrations of FX (approximately 5.6 /~g/mouse). A high ratio (5:9) of the strongly positive hybridomas secreting antibodies to FX, were of IgG isotype. Two other strong positives contained a mixture of IgM and IgG isotypes but the reactive isotype was not determined. A highly specific Mab FXa24, specific for the serine protease FXa, was also produced. It is interesting to note that all antibodies to FX detected by ELISA also reacted with FX following dot blotting and all selected hybridomas were positive after transfer of antigen by Western blotting. This may be due to the way the antigen was presented during immunisation (i.e., the conformation of antigen on the nitrocellulose). The procedure described here has also been effective in generating Mabs against recombinant hirudin (Hoad, Lackmann and Geczy, in preparation) and human a-thrombin. Both hirudin (Spinner et al., 1986) and human a-thrombin (Dawes et al., 1984) have been reported to be poorly immunogenic. Furthermore FX has the potential to be converted to a potent protease which may produce toxic side effects and influence its immunogenicity. Thus the low con-
centrations of antigen used may be more effectively tolerated and immobilisation may allow it to be released more slowly into the general circulation. In addition the combined use of pure antigen and nitrocellulose-bound antigen may stimulate the immune response more effectively. Although antigen-bound nitrocellulose particles effectively stimulate T cell responses, free antigen may be required to stimulate virgin B cells which specifically phagocytose antigen via surface-bound immunoglobulin (Harlow and Lane, 1988). By using this highly effective method to stimulate the immune response we assume that only low quantities of antigen would be required and the method may be particularly useful when only limited quantities are available or when the antigen is toxic. FX in plasma samples, detected by Mabs FX52 and FX64 after Western blotting, had a similar Rf value to pure FX (72 kDa). The amount of FX in the plasma loaded onto the gel was approximately 20 ng. Mabs FX52 and FX64 failed to react with other proteins in normal plasma or any protein in FX-deficient plasma when the same quantities of plasma were loaded although non-specific reactivity of the swine anti-rabbit Ig-alkaline phosphatase to human Ig was observed. Using the quantitative ELISA procedure to measure FX (with Mab FX52 as capture antibody) we estimated the level of FX in plasma to be 8.3/~g/ml which compares well with the quantity reported by Epstein et al. (1984) who estimated FX to be 7.74 _+ 1.81 /~g/ml in 31 normal patients using radioimmunoassay (12sI-labelled FX and polyclonal goat anti-FX). Mab FX64 also permitted the quantitation of FX in plasma (data not shown) using the methods described and may have an advantage over FX52 if F(ab')2 fragments are to be produced (IgG1 vs IgG2b; Parham, 1983). Mabs previously reported to react with both FX and FXa include aflFX-2b (Church and Mann, 1985; Church et al., 1988) and BG-X2 and BG-X4 (Doellgast, 1987). Mab a/~FX-2b to bovine FX cross reacted with human FX and reacted in a similar manner to F X / F X a 9 5 to detect FX and FXa a or fl following Western blotting (Fig. 2). a/3FX-2b and F X / F X a 9 5 reacted with the heavy chain of FX or FXa. Church and colleagues used Mab a/~FX-2b to affinity purify FX from plasma,
277 i s o l a t i n g a 72 k D a p r o t e i n as d e t e r m i n e d b y S D S PAGE. Mabs FX52 and FX64 reacted with a p l a s m a c o m p o n e n t of s i m i l a r m o l e c u l a r w e i g h t using Western blotting. BG-X2 and BG-X4 ( D o e l l g a s t , 1987) r e a c t i n g w i t h d i f f e r e n t e p i t o p e s o n F X / F X a , w e r e e f f e c t i v e in t h e c a p t u r e o f F X o r F X a f r o m s o l u t i o n a n d h a v e b e e n u s e d to develop further sensitive enzyme-linked coagulat i o n assays ( D o e l l g a s t a n d R o t h b e r g e r , 1985; D o e l l g a s t , 1987). We have produced Mabs with a greater s p e c i f i c i t y for F X or F X a t h a n the p r e v i o u s l y reported Mabs which react with both FX and FXa. Mabs FX52 and FX64 reacted with FX but not with FXa by ELISA and Western blotting (Figs. 1 a n d 2) a n d s p e c i f i c a l l y d e t e c t e d F X in p l a s m a f o l l o w i n g W e s t e r n b l o t t i n g (Fig. 3). T h e s e M a b s also e f f e c t i v e l y c a p t u r e d F X f r o m p l a s m a a n d c o u l d b e u s e d to q u a n t i t a t e F X (Fig. 4). Antibody FX/FXa95 reacted with both FX and F X a b y E L I S A a n d W e s t e r n b l o t t i n g (Figs. 1 a n d 2) a n d was a p p a r e n t l y s i m i l a r to p r e v i o u s l y rep o r t e d M a b s to F X / F X a . T h i s a n t i b o d y m a y b e u s e f u l as a c a p t u r e a n t i b o d y in q u a n t i t a t i v e E L I S A assays for F X a - a n t i t h r o m b i n c o m p l e x e s in p l a s m a (American Diagnostica, personal communication). M a b F X a 2 4 r e a c t e d s p e c i f i c a l l y to F X a b y E L I S A a n d W e s t e r n b l o t t i n g (Figs. 1 a n d 2) w i t h n o r e a c t i v i t y to F X o r r e d u c e d F X a . S p e c i f i c M a b s r e a c t i n g to F X a m a y b e useful r e s e a r c h tools for f u r t h e r i n v e s t i g a t i o n s of F X a a c t i v a t i o n a n d regul a t i o n of its p r o t e a s e activity.
References Abou-Zeid, C., Filley, E., Steele, J. and Rook, G.A.W. (1987) A simple new method for using antigens separated by polyacrylamide gel electrophoresis to stimulate lymphocytes in vitro after converting bands cut from Western blots into antigen-bearing particles. J. Immunol. Methods 98, 5. Caterson, B., Christners, J.E. and Baker, J.R. (1983) Identification of a monoclonal antibody that specifically recognizes corneal and skeletal keratan sulfate. J. Biol. Chem. 258, 8848. Chiles, T.C., O' Brien, T.W. and Kilberg, M.S. (1987) Production of monospecific antibodies to a low-abundance hepatic membrane protein using nitrocellulose immobilized protein as antigen. Anal. Biochem. 163, 138. Church, W.R. and Mann, K.G. (1985) A simple purification of human FX using high affinity monoclonal antibody immunoabsorbant. Thromb. Res. 38, 417.
Church, W.R., Messier, T.L., Tucker, M.M. and Mann, K.G. (1988) An inhibitory monoclonal antibody to FX that blocks prothrombin activation but not prothombinase enzyme activity. Blood 72, 1911. Dawes, J., James, K., Mickley, L.R., Pepper, D.S. and Prowse, C.V. (1984) Monoclonal antibodies directed against human a thrombin and the thrombin-antithrombin 111 complex. Thromb. Res. 36, 397. Diano, M., Bivic, A.L. and Hirn, M. (1987) A method for the production of highly specific polyclonal antibodies. Anal. Biochem. 166, 224. Doellgast, G.J. (1987) Enzyme-linked coagulation assay. Sensitive immunoassay for clotting factors II, VII and X. Anal. Biochem. 162, 102. Doellgast, G.J. and Rothberger, H. (1985) Enzyme-linked coagulation assay. Anal. Biochem. 152, 199. Epstein, D.J., Bergum, P.W., Bajaj, P. and Rapaport, S.I. (1984) Radioimmunoassay for protein C and Factor X. Plasma antigen levels in abnormal hemostatic states. Am. J. Clin. Pathol. 82, 537. Galfr~, G. and Milstein, C. (1981) Preparation of monoclonal antibodies strategies and procedures. Methods Enzymol. 73, 3. Gillette, R.W. (1987) Alternatives to pristane priming for ascites fluid and monoclonal antibody production. J. Immunol. Methods 99, 21. Harlow, E. and Lane, D. (Eds.) (1988) Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory, NY, 1988. Holmdahl, R., Moran, T. and Andersson, M. (1985) A rapid and efficient immunization protocol for production of monoclonal antibodies reactive with autoantigens. J. Immunol. Methods 83, 379. Jackson, C.M. and Nemerson, Y. (1980) Blood coagulation. Ann. Rev. Biochem. 49, 765. Kearney, J.K., Radbruch, A., Liesegang, B. and Rajewsky, K. (1982) A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid lines. J. Immunol. 128, 1548. Knudsen, K.A. (1985) Proteins transferred to nitrocellulose for use as immunogens. Anal. Biochem. 147, 285. Lamb, J.R., O'Hehir, R.E. and Young, D.B. (1988) The use of nitrocellulose immunoblots for the analysis of antigen recognition by T cells. J. Immunol Methods 110, 1. Mertens, K. and Bertina, R.M. (1980) Pathways in the activation of human coagulation Factor X. Biochem. J. 185, 647. Mirza, I.H., Wilkin, T.J., Cantarini, M. and Moore, M. (1987) A comparison of spleen and lymph node cells as fusion partners for the raising of monoclonal antibodies after different routes of immunisation. J. Immunol. Methods 105, 235. Nilsson, B.O., Svalander, P.C. and Larsson, A. (1987) Immunization of mice and rabbits by intrasplenic deposition of nanogram quantities of protein attached to Sepharose beads or nitrocellulose paper strips. J. Immunol. Methods 99, 67. Orlik, O. and Altaner, C. (1988) Modifications of hybridoma technology which improve the yield of monoclonal antibody producing cells. J. lmmunol. Methods 115, 55. Parham, P. (1983) On the fragmentation of monoclonal IgG1,
278 IgG2a, and IgG2b from BALB/c mice. J. Immunol. 131, 2895. Raymond, Y. and Suh, M. (1986) Lymph node primary immunization of mice for production of polyclonal and monoclonal antibodies J. Immunol. Methods 93, 103. Schagger, H. and Jagow, G.V. (1987) Tricine-sodium dodecylsulfate polyacrylamide gel electrophoresis for the separation of proteins in the range of 1 to 100 kDa. Anal. Biochem. 166, 368. Snick, V.J., "Cayphas, S., Vink, A., Uyttenhove, C., Coulie, P.G., Rubira, M.R. and Simpson, R.J. (1986) Purification and NHz-terminal amino sequence of a T-cell derived
lymphokine with growth factor activity for B-cell hybridomas. Proc. Natl. Acad. Sci. U.S.A. 83, 9679. Spinner, S., Stoffler, G. and Fink. E. (1986) Quantitative enzyme linked immunosorbent assay (ELISA) for hirudin. J. Immunol. Methods 87, 79. Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc. Acad. Sci., U.S.A. 76, 4350. Wells, D.E. and Price, P.J. (1983) Simple rapid methods for freezing hybridomas in 96 well microculture plates. J. Immunol. Methods 59, 49.
Journal of Immunological Methods, 136 (1991) 269-278 © 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 ADONIS 002217599100083Y
JIM05829
Characterisation of m o n o c l o n a l antibodies to h u m a n factor X / X a Initial observations with a quantitative ELISA procedure * Richard B. H o a d and Carolyn L. Geczy Heart Research Institute, Sydney, A ustralia (Received 23 July 1990, revised received 8 October 1990, accepted 22 October 1990)
Monoclonal antibodies to human plasma factor X (FX) and factor Xa (FXa) have been developed using several modifications of previously described techniques. These include the use of footpad immunisation with a suspension of free and nitrocellulose-bound antigen with subsequent fusion of popliteal lymph node cells. From a panel of 17 reactive hybridomas to FX, 3 were selected for further characterisation. An additional hybridoma reactive to FXa but not FX was also selected. Two monoclonal antibodies designated FX52 and FX64 were specific for FX with no reactivity to FXa, while antibody FXa24 was specific for FXa. Another FX/FXa95 reacted with both FX and FXa. All selected antibodies were of the IgG isotype and reacted both with native antigen and antigen transferred to nitrocellulose by Western blotting. Initial observations suggest that Mab FX52 may be used to quantitate FX levels in plasma. Key words." Monoclonal antibody; Factor X; Factor Xa; Immunization; Nitrocellulose; Popliteal lymph node cell fusion; ELISA
Introduction
Human factor X (FX), in common with other serine proteases, is highly conserved both within and between species (Jackson and Nemerson,
Correspondence to: R.B. Hoad, Heart Research Institute, 145 Missenden Road, Camperdown, Sydney, N.S.W. 2050, Australia. * This work was supported by" National Health and Medical Research Council (NH&MRC), Australia and America Diagnostica Inc., CT, U.S.A. Abbreviations: FX, human factor X; FXa, human factor Xa; DMSO, dimethyl sulphoxide; NPS, nitrocellulose particle suspension; CM, culture medium; ConA, concanavalin A; CAS, ConA-stimulated spleen cell-conditioned medium; H, hypoxanthine; T, thymidine; TBS, Tris-buffered saline; BSA, bovine serum albumin; HSA, human serum albumin; NB, nitrocellulose bound; SDS, sodium dodecyl sulphate; CR, coagulation assayed reference; CFA, complete Freund's adjuvant.
1980). It is a glycoprotein of apparent molecular weight 72 kDa (Mertens and Bertina, 1980) present in plasma at approximately 8/~g/ml (Epstein et al., 1984) and is situated at the critical interface of the intrinsic and extrinsic coagulation cascades. Human factor Xa (FXa) is derived by proteolytic cleavage of the heavy chain of FX to produce a 54 kDa product (designated aFXa) which can the be cleaved further by autolysis to flFXa (50 kDa) (Mertens and Bertina, 1980). Once formed, FXaa or fl complex with factor Va, phospholipid and calcium (prothrombinase complex) to cleave prothrombin to thrombin with subsequent cleavage of fibrinogen to fibrin and clot formation (Jackson and Nemerson, 1980). Two groups have generated monoclonal antibodies (Mabs) which react with both FX and FXa (Church and Mann, 1985; Doellgast, 1987; Church et al., 1988). However by modifying conventional procedures for antibody production we have suc-
270
ceeded in obtaining Mabs which react specifically with either FX or FXa.
Materials and methods
FX, FXa, and spectrozyme FXa chromogenic substrate were all gifts from American Diagnostica (CT).
Preparation of nitrocellulose particles and antigen binding Nitrocellulose particles were generated essentially as previously described (Abou-Zeid et al., 1987). Briefly a 4 cm 2 piece of nitrocellulose (0.1 /zm) (Schleicher and Schuell, Dassel, F.R.G.) was dissolved in 4 ml dimethyl sulphoxide (DMSO, Merck, Darmstadt, F.R.G.). The nitrocellulose was precipitated by adding it dropwise to 40 ml carbonate buffer (0.015 M NazCO 3, 0.03 M N a H C O 3, pH 9.6) with vortexing over 2 min. Large particles were allowed to settle by gravity for 1 rain and remaining particles in suspension were aspirated through a 21 gauge needle to generate smaller particles. Particles were sedimented by centrifugation at 500 x g for 10 rain and the pellet washed twice in carbonate buffer (50 ml/wash). A 0.5 ml packed vol of nitrocellulose particles was resuspended in 2 ml carbonate buffer and the suspension aspirated several times through a 25 gauge needle to further break up the particles. The nitrocellulose particle suspension (NPS) was stored at - 20 ° C prior to use. Nitrocellulose-bound FX (NB-FX) was prepared by adding 50 /~1 of the NPS to 50 /L1 solution of FX (28.5 /xg in 10 mM Tris-HCl/0.1 M NaC1, pH 7.5). The mixture was incubated at 3 7 ° C for 1 h, diluted with 0.9 ml NaC1 (0.9% w / v ) and stored at - 2 0 ° C prior to use. 25% of the FX added to the nitrocellulose particles bound to the particles. This was estimated by comparing the total amount of FX in the supernatant of samples with and without the addition of NPS. After 1 h incubation at 37°C, supernatants were collected and pooled for testing after centrifugation of the particles at 500 x g for 10 min and three subsequent washes with 0.01 M Tris-HC1 pH 7.9 to remove any weakly bound antigen. FX in serial dilutions of both fractions
was estimated by activation of FX to FXa with Russel's viper venom (Sigma, St. Louis, MO) and FXa detected using the Spectrozyme FXa chromogenic substrate according to the manufacturer's instructions.
Animals Female B A L B / c mice (5-6 weeks old) were initially obtained from Animal Resources Centre, Murdock, Australia and maintained in the Hospital Animal House Royal North Shore Hospital, Sydney.
Immunisation Three female B A L B / c mice were immunised in the rear footpads (under mild ether anaesthesia) with the solution of FX and NB-FX emulsified in an equal volume of complete Freund's adjuvant (CFA; Comonwealth Serum Laboratories, Parkville, Australia; 40 /~1 per footpad, equivalent to approximately 1.15 /~g FX per mouse). The mice were boosted in the same footpads with the solution of FX and NB-FX (without adjuvant) on days 9 and 12 after the initial injection (40 #1 per footpad, equivalent to approximately 2.3 /~g FX per mouse on each day).
Cell culture and fusion The culture medium (CM) used in the fusion studies was Iscove's (Gibco, Grand Island, NY) supplemented with 20% young calf serum (Bioclone, Marrickville, Australia) 2 mM glutamine (Flow Laboratories North Ryde, Australia) penicillin 100 U / m l , streptomycin sulphate 1 0 0 / t g / m l (both from Glaxo, Melbourne, Australia) and 5 x 10 --~ M 2-/3-mercaptoethanol (Sigma). All cultures were maintained at 3 7 ° C in a humidified atmosphere of 10% CO 2 in air. On day 1 prior to fusion, resident peritoneal macrophages were lavaged from B A L B / c mice, washed once in Iscove's medium and plated into 96 well microtitre plates (Nunc, Roskilde, Denmark) at a density of 104 cells in 150/xl CM per well. Concanavalin A (ConA) stimulated spleen cell conditioned medium (CAS) (Snick et al., 1986) was prepared by stimulating spleen cells from B A L B / c mice (107 cells/ml in CM) with 5 / z g / m l ConA Sepharose (Pharmacia, Sweden) for 24 h. The CAS was harvested by centrifugation at 300
271 X g for 10 rain, filter sterilised using a 0.22 /~m Milex-GV filter (Millipore, Bedford, MA) stored at 4 ° C and used within 2 weeks. 14 days after the initial immunisation, popliteal nodes from the three immunised mice were removed, pooled and single-cell suspensions prepared. Lymph node cells (LNCs) were fused with the non-secreting mouse myeloma P3-X63-Ag8.653 (ATCC CRL 1580) (Kearney et al., 1982) using standard methods (Galfr~ and Milstein, 1981). The fusion ratio was 4 × 10 v LNCs:2 X 10 7 myeloma cells. Post fusion, the equivalent of 105 LNCs in 150 /~1 CM were added to the wells of microtitre plates containing resident peritoneal macrophages. On day 1, selection of hybridomas was commenced by removal of 150 /~1 medium from each well and the addition of 150/~1 CM supplemented with 200 /~M hypoxanthine (H) 800 nM aminopterin and 32 /~M thymidine (T) (all from Sigma). On day 7, 150 /~1 from each well was removed and 150 ~I of CM (100/xM H, 16 b~M T and 4% CAS) added.
Expansion of hybridomas Supernatants from wells containing growing hybridomas were screened by an ELISA procedure to FX (see below) 12-14 days post fusion. Selected hybridomas were expanded until approximately 5 × 10 6 cells were obtained and the cells then frozen to - 7 0 ° C in CM supplemented with 10% DMSO. The original fusion plates were frozen to - 7 0 ° C on day 16 post fusion by the method of Wells and Price (1983). Hybridomas were cloned by limiting dilution. The original fusion plates were thawed 1 month post fusion and hybridomas screened for specific reactivity to FXa (but not FX) by an ELISA procedure (see below). The selected hybridoma FXa24 was identified and processed as described. Ascitic fluid was produced (Gillette, 1987) and Mabs purified using Protein A-Sepharose (Pharmacia) according to the manufacturer's instructions (binding buffer: 1.5 M glycine, 3 M NaC1, pH 8.9). Purified Mabs were concentrated and dialysed into 0.01 M Tris-HC1 + 0.9% NaC1 pH 7.9, using an Amicon concentrator (Amicon, Danvers, MA) with Diaflo YM10 membrane.
ELISA procedure for detection of hybridomas reactive with FX or FXa 100 btl vols of FX or FXa (5 ~ g / m l ) in Trisbuffered saline (TBS; 0.02 M Tris-HC1, 150 mM NaC1, pH 7.4) were added to polyvinyl chloride microtitre plates (Dynatech Laboratories, Virginia) Control wells contained 100 ~1 human serum albumin (HSA, fraction V; 5 /~g/ml; Calbiochem, San Diego, CA). Plates were incubated overnight at 4 o C and then washed four times in TBS using a Microplate washer ($8/12 Titerteck; Flow Labs). Unless specified, all subsequent washes in the ELISA procedure were with the same buffer. Nonspecific binding sites were blocked with 0.5% gelatin (BDH, Poole, England) in TBS (200 /~l/well) for 2 h at 37 ° C. The plates were washed and 100 ~1 aliquots of the supernatants (previously diluted 1 / 6 in Iscove's medium) added. Plates were incubated at 3 7 ° C for 90 min, followed by four washes. 100 /~1 vols of horseradish peroxidase-conjugated rabbit anti-mouse Ig (Dakopatts, Gostrup, Denmark) diluted 1/1000 in TBS + 1% bovine serum albumin (BSA, fraction V, Calbiochem) were added and plates incubated at 37 ° C for a further 1 h. The plates were washed, and peroxidase activity detected using 100/~l/well 2,2'-azino-di-3-ethylbenzthiazoline sulphonate (Boehringer Mannheim, F.R.G., 1 m g / m l ) in 0.1 M citrate buffer, 0.006% H202, pH 4.3. After 20 min at room temperature absorbance values at 414 nm (dual wave-length 414 nm, reference 690 nm) were read using a Multiscan plate reader (Titertek, Flow). After the initial screening, the assay was modified using streptavidin-biotin amplification so that less antigen was required per test well. 200 /~1 vols of FX or FXa (250 n g / m l or 500 n g / m l ) in TBS were added to Nunc-maxisorp plates for 2 h at 37°C prior to blocking nonspecific binding sites with 0.1% Tween 20 (Sigma) in TBS for 30 min at 37°C. FX or FXa was reconstituted according to the manufacturer's instructions and used within 1 h at 4 ° C. Control wells contained no antigen. Plates were washed and 200/~l/well of a biotinylated sheep anti-mouse Ig (species specific and (Fab')2 fragment; Amersham, North Ryde, Sydney) diluted 1/10,000 in TBS, 0.2% ovalbumin, 0.05% Tween were added and the plate incubated for a further hour. The plates were washed and 200 ~1 streptavidin peroxidase (Amersham)
272 (diluted 1/1000 in TBS 0.2% ovalbumin, 0.05% Tween) added to each well for 30 min at 37 o C. The plates were washed and peroxidase activity detected as described (dual wave-length 414 nm, reference 492 nm) except that 200 #1 vol. of substrate were used and the plates read after 10 min.
ELISA procedure for class determination and isotyping of Mabs One hundred/~1 goat anti-mouse Ig (either/t or y chain specific, Cappel, Organon, Teknika, North Ryde, Sydney, diluted 1/500 in carbonate buffer) were added to the wells of PVC plates and incubated overnight at 4 ° C. The plates were blocked with 1% BSA in TBS for 1 h at 37°C, washed and undiluted hybridoma supernatants added. Control myelomas were obtained from ICN Immunobiologicals, IL. After 1 h at 3 7 ° C the plates were washed and 100 ~1 of peroxidase-labelled rabbit anti-mouse Ig (Dakopatts; 1/1000 in TBS, 1% BSA) added. The plates were incubated at 3 7 ° C for 1 h and washed prior to detection of peroxidase activity as described. In assays where specific isotypes were required, procedures were processed up to the washing step after the capture of Mabs. 100/tl rabbit anti-mouse subclass-specific antibodies (diluted 1/500 in TBS, 1% BSA, Litton Bionetics, SC) were added and the plates incubated at 3 7 ° C for 1 h. The plates were then washed and 100 /~1 goat anti-rabbit-peroxidase (Bio-rad, Richmond, CA; 1/1000 in TBS, 1% BSA) added for 2 h at 37 °C. Finally, the plates were washed prior to detection of peroxidase activity as described.
diluted 1 / 5 in TBS and then 1 / 2 in 4% SDS containing 12% glycerol, 5 mM T r i s + 0 . 0 1 % bromophenol blue, pH 6.8 (sample buffer). FX and FXa (8 ~ g / m l ) in TBS were diluted 1 / 2 in sample buffer. Samples were boiled for 2 min and 25 ~1 loaded per lane. Electrophoresis of proteins was at 50 V through the stacking gel and 40 V for 20 h through the main gel. Proteins were electrotransferred to nitrocellulose (0.1 /zm; Schleicher and Schuell) (Towbin et al., 1979) for 2 h at 100 V using a Bio-Rad Transblot cell apparatus (BioRad). Amidoblack staining was carried out according to the method described by Harlow and Lane (1988). All washing steps were in 0.02 M Tris-HC1, 0.5 M NaC1, 0.05% Tween 20, pH 7.5 and consisted of three washes for 5 min with rocking. Membranes were blocked with 0.5% w / v fish gelatin (FG; Sigma) in 0.02 M Tris-HC1, 0.5 M NaCI, pH 7.5, for 2 h at room temperature, and probed with protein A-purified Mabs (1 ttg/ml in 0.02 M Tris-HC1, 0.5 M NaC1, pH 7.5, 0.05% Tween 20, 0.1% FG). Pure FX or FXa was probed for 1 h at room temperature. Plasma samples were probed overnight at 4 °C. Membranes were washed, incubated for 1 h at room temperature in rabbit anti-mouse IgG (Dakopatts) absorbed with human serum (1/400 in 0.02 M Tris-HC1, 0.5 M NaC1, 0.05% Tween 20, 0.1% FG, pH 7.5) washed and incubated with alkaline phosphatase-labelled swine anti-rabbit Ig (Dakopatts; diluted 1/800 in the same buffer) for 30 min. Membranes were washed and alkaline phosphatase activity detected as described by Harlow and Lane (1988).
Protein blotting
Quantitative ELISA procedure for estimating FX in plasma
Tricine-sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis was carried out by the method of Schagger (1987) using a gel (10% T, 3% C) with dimensions of 1.5 mm x 11 cm x 16 cm with a 4 cm stacking gel (4% T, 3% C; where T denotes the total % concentration of both acrylamide and bisacrylamide, C denotes the % concentration of crosslinker relative to the total concentration of T). Samples of normal citrated plasma and FX-deficient plasma (antigen deficient; Hoechst-Behring, Marburg, F.R.G.) were
200/~1 of purified Mab FX52 (5/~g/ml in TBS) were added to Nunc-maxisorp plates and incubated at 37 ° C for 2 h. Plates were washed prior to blocking nonspecific binding sites with 300 /~1 TBS + 1% ovalbumin for 1 h at 37°C. The plates were washed before adding 200 /.tl coagulation assayed reference (CR) plasma (Helena, Mount Waverly, Victoria) or factor-deficient plasma (Organon Technica, North Ryde, Sydney) diluted 1/100 in TBS, 0.05% Tween. In some assays pure FX (20 or 40 ng) was added to 200 /tl of the
273
diluted plasma. After incubation at 37 ° C for 2 h, the plates were washed and 200 /L1 peroxidaselabelled rabbit anti FX (Diagnostica Stago, Asnieres, France; 4 /~g/ml diluted in TBS 0.2% ovalbumin, 0.05% Tween) added. The plates were then incubated at 3 7 ° C for 2 h, washed and peroxidase activity detected as described after 30 min at room temperature.
positive (i.e., absorbance reading > 0.2 after 20 min with FX but < 0.05 with HSA). After isolation of reactive clones the original fusion plates were frozen prior to confluency on day 16 post fusion. Positive hybridomas were tested for reactivity to FX and FXa on nitrocellulose by dot blotting of the antigens before or after SDS treatment (i.e., boiling in the sample buffer used for SDS-PAGE), according to the methods described for Western blotting. The 17 hybridomas tested were all positive by dot blotting and those which were strongly positive were isotyped prior to subcloning. Of the 11 hybridomas tested, four were IgM, five were IgG and two contained both IgM and IgG isotypes. Three hybridomas FX52, FX64 and F X / F X a 95 were selected for subcloning. As a specific Mab to FXa was required, and
Results
A single fusion was carried out 14 days after initial immunisation with 4 × 107 LNC from three immunised mice. From day 12 post fusion, hybridomas (121 in total) were screened for reactivity to FX using the ELISA procedure and 17 were
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m
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'
ioo . . . . . . . .
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50
Fig. 1. Reactivity of Mabs to FX and FXa by anamplification EL1SA procedure. A : reactivity of Mab FX52 to FX (--©--) and Xa (~); B: Mab FX64 to FX (--©--) and Xa ( B - ) ; C: Mab F X / F X a 9 5 to FX (--©--) and Xa ({3-); and D: Mab FXa24 to FX (--©--) and Xa ( [] ). Assays were carried out with 50 ng antigen/well for graphs A and B, and 100 ng/well for graphs C and D. Tween blocking and initial Mab dilution in Tween was according to the materials and methods section. Background absorbance (no antigen) for all Mabs was < 0.08 for all dilutions tested. Mean of three values and standard deviations from the mean are given.
274 given the success of the fusion, we rethawed original fusion plates for further screening. H y b r i d o m a F X a 2 4 was selected by screening 28 s u p e r n a t a n t s for reactivity to F X a n d F X a by ELISA. The M a b was reactive with F X a (i.e., a b s o r b a n c e > 0.2 after 20 m i n but < 0.05 with F X or HSA), reacted with F X a but not F X after S D S - t r e a t m e n t when screened by dot b l o t t i n g a n d was of I g G isotype. The reactivity of the Mabs to F X a n d F X a was d e t e r m i n e d by amplification E L I S A procedures. Initially the blocking agent was 1% o v a l b u m i n for 1 at 37 ° C. Mabs were diluted in 0.2% o v a l b u m i n , 0.05% Tween. All Mabs, except FXa24, reacted well using these c o n d i t i o n s (data not shown). The procedure was modified by blocking with 0.1% T w e e n a n d dilution of the M a b in 0.05% T w e e n so that all could be effectively c o m p a r e d (Fig. 1). Both Mabs FX52 ( I g G 2 b ) a n d F X 6 4 (IgG1) (Figs. 1A a n d 1B) reacted strongly to F X but not to
94 kD
FX
67 kD
FXa - FXa~
43 kD
30 kD
A
B
C
D
E
F
GH
Fig. 2. lmmunoblotting of FX and FXa. Following SDS-PAGE and electrotransfer to nitrocellulose, FX and FXa were probed with Mabs. FX (lane A) and FXa (lane B) were probed with FX52. FX (lane C) and FXa (lane D) were probed with FX64. FX (lane E) and FXa (lane F) were probed with FX/FXa95. FX (lane G) and FXa (lane H) were probed with FXa24. Conditions for gel electrophoresis and immunoblotting are as described in the materials and methods section. Equivalent to 100 ng of pure FX or FXa were loaded onto the gel.
94 kD
67 kD 43 kD 30 kD
A
B
C
D
E
F
Fig. 3. Immunoblotting of normal and FX deficient plasma samples. Following SDS-PAGE gel electrophoresis and electrotransfer to nitrocellulose, normal and FX-deficient plasma were probed with Mabs. Normal plasma (lane A) and FX-deficient plasma (lane B) were stained with amido black. FX-deficient plasma (lane C) and normal plasma (lane D) were probed with FX52, FX-deficient plasma (lane E) and normal plasma (lane F) were probed with FX64 according to the conditions outlined in the materials and methods section. The equivalent of 20 ng of FX in normal plasma was loaded onto the gel.
FXa. M a b F X / F X a 9 5 (IgG1) reacted with both F X and F X a (Fig. 1C) whereas M a b FXa24 (IgG1) was specific for F X a (Fig. 1D). Mabs were tested following Western blotting of pure F X or F X a (Fig. 2). They reacted with identical specificities to those observed using the amplification ELISA procedures. A n t i b o d i e s FX52, FX64 a n d F X / F X a 9 5 reacted with the heavy chain of F X by W e s t e r n b l o t t i n g of reduced F X (data not shown). R e d u c t i o n of F X a destroyed the antigenic d e t e r m i n a n t detected by M a b FXa24. The specificity of Mabs FX52 and FX64 for F X was d e t e r m i n e d by Western blotting of n o r m a l a n d F X - d e f i c i e n t plasmas. Fig. 3 shows that both reacted with a single protein of approximately 72 k D a in n o r m a l p l a s m a whereas the c o m p o n e n t was a b s e n t in F X - d e f i c i e n t plasma. The detection system used for blots showed cross reactivity to h u m a n i m m u n o g l o b u l i n in both n o r m a l and FXdeficient plasmas. This was a p p a r e n t l y higher for M a b FX52 since a longer i n c u b a t i o n with substrate was required to detect the FX antigen.
275 0.8
A
B
"i
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O
T
~-~ 0.4
// ///
,~ 0~
/
¢~ 0.2
/ / / / / / / / /
~
//
3 I~
///
/ / / / / / ,.-// / / / / / / ,..- / /
0 o.1
O.4
/
0.2
o.1
1o5%
o.o
o
plasma
plasma
+ FX
lo
2'o
ng
of
~'o
~o
FX
Fig. 4. Quantitative ELISA procedure for FX in plasma. A: absorbance readings of FX-deficient plasma (11), CR plasma ([]), FVIlI-deficient plasma (D), in the absence (left histogram) or presence of 20 ng added FX (right histogram). B: absorbance readings of different concentrations of FX added to FX-deficient plasma. Concentration of FX in CR plasma was estimated from the standard curve. Both assays were carried out as described in the materials and methods section. Means of three values and standard deviations from the mean are shown.
Fig. 4 shows that Mab FX52 could be used in a quantitative ELISA assay to detect FX in plasma. CR plasma reacted strongly with Mab FX52 whereas the FX-deficient plasma produced only a background signal (the values obtained were significantly different by Student's t test; P = 0.037). The reactivity of FVIII deficient plasma was not significantly different from that of CR plasma ( P = 0.118). When FX was added back to FX-deficient plasma the signal was increased (Fig. 4B). Similarly, increased reactivity was observed when FX was added to CR or FVIII-deficient plasma. The amount of FX present in the CR plasma was estimated to be 17.5 ng/well, equivalent to 8.75 /~g/ml. This value was obtained by reading absorbance values for the CR off the FX standard curve prepared by adding FX (from 0 to 40 ng/well) to FX-deficient plasma (Fig. 4B). The manufacturers specified that the CR plasma contained 105% of the concentration of FX in normal plasma. Therefore, the level of FX in normal plasma was estimated to be 8.3/~g/ml by adjusting the CR plasma to the concentration of FX in normal plasma.
Discussion
Production of Mabs requires the selection of a suitable immunisation schedule which generates high numbers of specifically activated B cells prior to fusion. Since we were unable to produce Mabs to FX using conventional immunisation procedures including in vitro stimulation, we investigated the use of footpad immunisation and fusion of LNCs. This procedure is known to generate a broader range of Mabs, including autoantibodies (Caterson et al., 1983; Holmdahl et al., 1985; Raymond and Suh, 1986; Mirza et al., 1987; Orlik and Altaner, 1988). Mice were immunised in the rear footpad using a combination of pure FX and FX passively bound to nitrocellulose particles (approximate ratio of 3 : 1 respectively). Abou-Zeid (1987) demonstrated that antigen-bound nitrocellulose particles produced following SDS-PAGE and transfer of the antigens to nitrocellulose were effective stimulants of antigen-specific T cell responses in vitro. Accessory cells phagocytose the particles and subsequently process and present antigen in association
276
with the major histocompatability complex (reviewed by Lamb et al., 1988). There are other reports describing the use of antigen bound to nitrocellulose following SDS-PAGE and transfer for the production of both polyclonal and monoclonal antibodies (Knudsen, 1985; Chiles et al., 1987; Diano et al., 1987; Nilsson et al., 1987). Our method is the first to describe the use of nitrocellulose particles mixed with antigen and footpad immunisation of both free and bound antigen for antibody production. The production of nitrocellulose particles prior to antigen binding permits the selection of very small particles without loss of antigen. The possible denaturation of antigenic epitopes by SDS or the solubilisation of the nitrocellulose (DMSO, freeze thawing, etc.) are also avoided. Rear footpad immunisation of pure FX and FX passively bound to nitrocellulose particles using CFA for the initial injection and saline for subsequent injections was tolerated well by the mice. Minimal swelling and no abnormal behavioural changes or ulceration developed within the 14 day period. The procedure described permitted the use of very low concentrations of FX (approximately 5.6 /~g/mouse). A high ratio (5:9) of the strongly positive hybridomas secreting antibodies to FX, were of IgG isotype. Two other strong positives contained a mixture of IgM and IgG isotypes but the reactive isotype was not determined. A highly specific Mab FXa24, specific for the serine protease FXa, was also produced. It is interesting to note that all antibodies to FX detected by ELISA also reacted with FX following dot blotting and all selected hybridomas were positive after transfer of antigen by Western blotting. This may be due to the way the antigen was presented during immunisation (i.e., the conformation of antigen on the nitrocellulose). The procedure described here has also been effective in generating Mabs against recombinant hirudin (Hoad, Lackmann and Geczy, in preparation) and human a-thrombin. Both hirudin (Spinner et al., 1986) and human a-thrombin (Dawes et al., 1984) have been reported to be poorly immunogenic. Furthermore FX has the potential to be converted to a potent protease which may produce toxic side effects and influence its immunogenicity. Thus the low con-
centrations of antigen used may be more effectively tolerated and immobilisation may allow it to be released more slowly into the general circulation. In addition the combined use of pure antigen and nitrocellulose-bound antigen may stimulate the immune response more effectively. Although antigen-bound nitrocellulose particles effectively stimulate T cell responses, free antigen may be required to stimulate virgin B cells which specifically phagocytose antigen via surface-bound immunoglobulin (Harlow and Lane, 1988). By using this highly effective method to stimulate the immune response we assume that only low quantities of antigen would be required and the method may be particularly useful when only limited quantities are available or when the antigen is toxic. FX in plasma samples, detected by Mabs FX52 and FX64 after Western blotting, had a similar Rf value to pure FX (72 kDa). The amount of FX in the plasma loaded onto the gel was approximately 20 ng. Mabs FX52 and FX64 failed to react with other proteins in normal plasma or any protein in FX-deficient plasma when the same quantities of plasma were loaded although non-specific reactivity of the swine anti-rabbit Ig-alkaline phosphatase to human Ig was observed. Using the quantitative ELISA procedure to measure FX (with Mab FX52 as capture antibody) we estimated the level of FX in plasma to be 8.3/~g/ml which compares well with the quantity reported by Epstein et al. (1984) who estimated FX to be 7.74 _+ 1.81 /~g/ml in 31 normal patients using radioimmunoassay (12sI-labelled FX and polyclonal goat anti-FX). Mab FX64 also permitted the quantitation of FX in plasma (data not shown) using the methods described and may have an advantage over FX52 if F(ab')2 fragments are to be produced (IgG1 vs IgG2b; Parham, 1983). Mabs previously reported to react with both FX and FXa include aflFX-2b (Church and Mann, 1985; Church et al., 1988) and BG-X2 and BG-X4 (Doellgast, 1987). Mab a/~FX-2b to bovine FX cross reacted with human FX and reacted in a similar manner to F X / F X a 9 5 to detect FX and FXa a or fl following Western blotting (Fig. 2). a/3FX-2b and F X / F X a 9 5 reacted with the heavy chain of FX or FXa. Church and colleagues used Mab a/~FX-2b to affinity purify FX from plasma,
277 i s o l a t i n g a 72 k D a p r o t e i n as d e t e r m i n e d b y S D S PAGE. Mabs FX52 and FX64 reacted with a p l a s m a c o m p o n e n t of s i m i l a r m o l e c u l a r w e i g h t using Western blotting. BG-X2 and BG-X4 ( D o e l l g a s t , 1987) r e a c t i n g w i t h d i f f e r e n t e p i t o p e s o n F X / F X a , w e r e e f f e c t i v e in t h e c a p t u r e o f F X o r F X a f r o m s o l u t i o n a n d h a v e b e e n u s e d to develop further sensitive enzyme-linked coagulat i o n assays ( D o e l l g a s t a n d R o t h b e r g e r , 1985; D o e l l g a s t , 1987). We have produced Mabs with a greater s p e c i f i c i t y for F X or F X a t h a n the p r e v i o u s l y reported Mabs which react with both FX and FXa. Mabs FX52 and FX64 reacted with FX but not with FXa by ELISA and Western blotting (Figs. 1 a n d 2) a n d s p e c i f i c a l l y d e t e c t e d F X in p l a s m a f o l l o w i n g W e s t e r n b l o t t i n g (Fig. 3). T h e s e M a b s also e f f e c t i v e l y c a p t u r e d F X f r o m p l a s m a a n d c o u l d b e u s e d to q u a n t i t a t e F X (Fig. 4). Antibody FX/FXa95 reacted with both FX and F X a b y E L I S A a n d W e s t e r n b l o t t i n g (Figs. 1 a n d 2) a n d was a p p a r e n t l y s i m i l a r to p r e v i o u s l y rep o r t e d M a b s to F X / F X a . T h i s a n t i b o d y m a y b e u s e f u l as a c a p t u r e a n t i b o d y in q u a n t i t a t i v e E L I S A assays for F X a - a n t i t h r o m b i n c o m p l e x e s in p l a s m a (American Diagnostica, personal communication). M a b F X a 2 4 r e a c t e d s p e c i f i c a l l y to F X a b y E L I S A a n d W e s t e r n b l o t t i n g (Figs. 1 a n d 2) w i t h n o r e a c t i v i t y to F X o r r e d u c e d F X a . S p e c i f i c M a b s r e a c t i n g to F X a m a y b e useful r e s e a r c h tools for f u r t h e r i n v e s t i g a t i o n s of F X a a c t i v a t i o n a n d regul a t i o n of its p r o t e a s e activity.
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Church, W.R., Messier, T.L., Tucker, M.M. and Mann, K.G. (1988) An inhibitory monoclonal antibody to FX that blocks prothrombin activation but not prothombinase enzyme activity. Blood 72, 1911. Dawes, J., James, K., Mickley, L.R., Pepper, D.S. and Prowse, C.V. (1984) Monoclonal antibodies directed against human a thrombin and the thrombin-antithrombin 111 complex. Thromb. Res. 36, 397. Diano, M., Bivic, A.L. and Hirn, M. (1987) A method for the production of highly specific polyclonal antibodies. Anal. Biochem. 166, 224. Doellgast, G.J. (1987) Enzyme-linked coagulation assay. Sensitive immunoassay for clotting factors II, VII and X. Anal. Biochem. 162, 102. Doellgast, G.J. and Rothberger, H. (1985) Enzyme-linked coagulation assay. Anal. Biochem. 152, 199. Epstein, D.J., Bergum, P.W., Bajaj, P. and Rapaport, S.I. (1984) Radioimmunoassay for protein C and Factor X. Plasma antigen levels in abnormal hemostatic states. Am. J. Clin. Pathol. 82, 537. Galfr~, G. and Milstein, C. (1981) Preparation of monoclonal antibodies strategies and procedures. Methods Enzymol. 73, 3. Gillette, R.W. (1987) Alternatives to pristane priming for ascites fluid and monoclonal antibody production. J. Immunol. Methods 99, 21. Harlow, E. and Lane, D. (Eds.) (1988) Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory, NY, 1988. Holmdahl, R., Moran, T. and Andersson, M. (1985) A rapid and efficient immunization protocol for production of monoclonal antibodies reactive with autoantigens. J. Immunol. Methods 83, 379. Jackson, C.M. and Nemerson, Y. (1980) Blood coagulation. Ann. Rev. Biochem. 49, 765. Kearney, J.K., Radbruch, A., Liesegang, B. and Rajewsky, K. (1982) A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid lines. J. Immunol. 128, 1548. Knudsen, K.A. (1985) Proteins transferred to nitrocellulose for use as immunogens. Anal. Biochem. 147, 285. Lamb, J.R., O'Hehir, R.E. and Young, D.B. (1988) The use of nitrocellulose immunoblots for the analysis of antigen recognition by T cells. J. Immunol Methods 110, 1. Mertens, K. and Bertina, R.M. (1980) Pathways in the activation of human coagulation Factor X. Biochem. J. 185, 647. Mirza, I.H., Wilkin, T.J., Cantarini, M. and Moore, M. (1987) A comparison of spleen and lymph node cells as fusion partners for the raising of monoclonal antibodies after different routes of immunisation. J. Immunol. Methods 105, 235. Nilsson, B.O., Svalander, P.C. and Larsson, A. (1987) Immunization of mice and rabbits by intrasplenic deposition of nanogram quantities of protein attached to Sepharose beads or nitrocellulose paper strips. J. Immunol. Methods 99, 67. Orlik, O. and Altaner, C. (1988) Modifications of hybridoma technology which improve the yield of monoclonal antibody producing cells. J. lmmunol. Methods 115, 55. Parham, P. (1983) On the fragmentation of monoclonal IgG1,
278 IgG2a, and IgG2b from BALB/c mice. J. Immunol. 131, 2895. Raymond, Y. and Suh, M. (1986) Lymph node primary immunization of mice for production of polyclonal and monoclonal antibodies J. Immunol. Methods 93, 103. Schagger, H. and Jagow, G.V. (1987) Tricine-sodium dodecylsulfate polyacrylamide gel electrophoresis for the separation of proteins in the range of 1 to 100 kDa. Anal. Biochem. 166, 368. Snick, V.J., "Cayphas, S., Vink, A., Uyttenhove, C., Coulie, P.G., Rubira, M.R. and Simpson, R.J. (1986) Purification and NHz-terminal amino sequence of a T-cell derived
lymphokine with growth factor activity for B-cell hybridomas. Proc. Natl. Acad. Sci. U.S.A. 83, 9679. Spinner, S., Stoffler, G. and Fink. E. (1986) Quantitative enzyme linked immunosorbent assay (ELISA) for hirudin. J. Immunol. Methods 87, 79. Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc. Acad. Sci., U.S.A. 76, 4350. Wells, D.E. and Price, P.J. (1983) Simple rapid methods for freezing hybridomas in 96 well microculture plates. J. Immunol. Methods 59, 49.
