Journal of Immunological Methods, 132 (1990) 227-237 Elsevier
227
JIM 05680
A sandwich type enzyme-linked immunosorbent assay for proliferating cell nuclear antigen (PCNA)/cyclin using monoclonal antibodies * Yoshinari Takasaki 1, Makoto Ohgaki 2,3, Akira K o d a m a 1,4, Kenji Ogata 5, Hiroshi Hashimoto t, Toshikazu Shirai 2 and Shun-Ichi Hirose 1 1 Division of Rheumatology, Department of Medicine, Juntendo University School of Medicine, Tokyo, Japan, 2 Second Department of Pathology, Juntendo University School of Medicine, Tokyo, Japan, 3 Mitsubishi Kasei Corporation, Research Center, Kanagawa, Japan, 4 Biomedical Laboratories, Saitama, Japan, and 5 Hoechst Japan, Research Center, Saitama, Japan (Received 14 November 1989, revised received 15 May 1990, accepted 17 May 1990)
Three hybridomas producing monoclonal antibodies to proliferating cell nuclear antigen (PCNA)/cyclin were newly derived. The specificity of established monoclonal antibodies to PCNA, TOB7, TO17 and TO30, was compared with that of the previously reported PCNA-specific monoclonal antibodies, 19A2 and 19F4. TOB7, TO17 and TO30 reacted with purified PCNA in an enzyme-linked immunosorbent assay (ELISA). A 34 kDa PCNA polypeptide was noted by immunoblotting, the same polypeptide as recognized by 19A2 and 19F4. Epitopes recognized by all those monoclonal antibodies to PCNA were analyzed by competitive inhibition tests using ELISA. The results of those experiments suggested that the epitope recognized by TO17 and TO30 was almost identical to that of 19A2 and closely related to that of 19F4, but different from that of TOB7. Based on those results, a sandwich type ELISA for detection of PCNA was developed using TO17 and TOB7. This system was applied to measure the concentration of PCNA in normal peripheral blood lymphocytes (PBL) before and after stimulation by phytohemagglutinin (PHA), and in tissue extracts from rabbit kidney and thymus (RKE and RTE, respectively). Before mitogenic stimulation, the PCNA concentration in PBL extracts was less than 6 ng/ml (cell concentration 2.5 x 107/ml), but at 48 h after PHA stimulation, when more than 70% of cells were in S phase, its concentration became 1280 ng/ml. RTE which contained many proliferative lymphocytes had much higher concentration of PCNA than RKE. Those results suggested that sandwich type ELISA using TO17 and TOB7 was useful as the quantitative assay to detect blast-transformation and proliferation of cells in vivo. Key words: Proliferating cell nuclear antigen; Cyclin; Monoclonal antibody; Cell profiferation; Antinuclear antibody; ELISA
Correspondence to: Y. Takasaki, Division of Rhenmatology, Department of Medicine, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-Ku, Tokyo, 113 Japan. * Supported by a research grant from the Ministry of Education of Japan (Grant no. 01480220) and a 1988 research grant from the Mixed Connective Tissue Disease Research Committee, the Ministry of Welfare of Japan. Abbreviations: PCNA, proliferating cell nuclear antigen; ELISA, enzyme-linked immunosorbent assay; DID, double immunodiffusion; CIE, counterlmmnnoelectrophoresis; CTE, calf thymus extract; HPLC, high performance fiquid chromatography; T-PBS, 0.1~ Tween 20/phosphate-buffered saline; HRP, horseradish peroxidase; ABTS, 2,2'-azino-di-(3-ethylbenzthiazolin-6-sulfonicacid); SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; BrdU, bromodeoxy-uridine; 3H-TdR, tritiated thymidine. 0022-1759/90//$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
228 Introduction
Antibodies to proliferating cell nuclear antigen (PCNA) were detected in some patients with systemic lupus erythematosus (SLE) and reacted with a nuclear antigen expressed predominantly by proliferating cells such as cultured cells and mitogentransformed cells (Miyachi et al., 1978). PCNA has been identified as a nuclear protein with a molecular weight (MW) of about 33 kDa and isoelectric point of 4.8 (Takasaki et al., 1984a; Ogata et al., 1985). These observations led to an insight that PCNA was identical to cyclin, a nuclear protein associated with cell proliferation and identified by two-dimensional (2D) gel electrophoresis (Bravo et al., 1980); the identity of these two proteins was recently demonstrated (Mathews et al., 1984). Subsequent studies have revealed that the expression of PCNA/cyclin increased in the late G1 phase of the cell-cycle immediately preceding DNA synthesis (Bravo et al., 1980; Takasaki et al., 1981; Bravo, 1984; Bravo and MacDonald-Bravo, 1984). The molecular cloning of cDNA coding for PCNA/cyclin allowed detection of cell cycle-specific expression of PCNA mRNA (Almendral et al., 1987; Matsumoto et al., 1987). These observations implied an association of PCNA/cyclin with DNA replication. Recent findings suggest that PCNA/cyclin is an auxiliary protein of DNA polymerase delta (Bravo et al., 1987; Prellch et al., 1987). These characteristics of PCNA enabled autoantibodies to PCNA from lupus patients to be used as a reagent for detecting blast cells. This was used to detect the crisis phase of patients with chronic myeloid leukemia (Takasaki et al., 1984b). However, sera from lupus patients often contain other autoantibodies and it is quite difficult to obtain sufficient m o u n t s of monospecific sera for PCNA because of its low incidence in lupus patients. To solve these problems, monoclonal antibodies to PCNA were generated and two mouse monoclonal PCNA-specific antibodies, 19A2 and 19F4, established (Ogata et al., 1987). Using these monoclonal antibodies as probes, detection of proliferating cells by immunofluorescence microscopy and flow cytometry was recently reported (Kurki et al., 1988). In addition, it was shown that these monoclonal antibodies were useful for labeling
immunocytochemically the nuclei of proliferating cells in mouse lung tissue (Thaete et al., 1989). These results suggest that monoclonal antibodies to PCNA are useful tools to study the cell proliferation and blast transformation. However, it was impossible to make a sandwich type enzyme-linked immunosorbent assay (ELISA) for detection of PCNA because these two monoclonal antibodies recognized closely related epitopes. Here we report the generation of three hybridomas producing monoclonal antibodies to PCNA and establishment of specific quantitative assay for PCNA using two of these monoclonal antibodies.
Materials and methods
Sera Anti-PCNA serum obtained from a lupus patient (AK) was used to purify PCNA and test the specificity of anti-PCNA monoclonal antibodies. Serum AK showed a single precipitin line which was identical to anti-PCNA standard serum (PT) (kindly given to us by Eng M. Tan, M.D., Scripps Clinic and Research Foundation, La Jolla, CA, U.S.A.) in a standard double immunodiffusion system ( D I D ) and by counterimmunoelectrophoresis (CIE) (Takasaki et al., 1981). This serum was used to monitor the antigenic activity of PCNA in DID and CIE during the antigen purification procedure, and as the source of antiPCNA IgG to make a affinity chromatography column. Antigen purification Calf thymus extract (CTE) was prepared as previously reported (Sharp et al., 1972) and used as the source material containing PCNA. PCNA in CTE was firstly purified by 40-65% ammonium sulfate fractionation and Sephadex G-200 (Pharmacia, Uppsala, Sweden) gel filtration as previously reported (Takasaki et al., 1984a). An IgG fraction of serum AK prepared by 33% ammonium sulfate fractionation and DE52 (Whatman Lab. Products, Clifton, NJ, U.S.A.) ion exchange chromatography coupled to CNBr activated Sepharose 4B (Pharmacia) (Douvas et al., 1979), and anti-PCNA affinity chromatography were performed to further purify PCNA.
229 Anti-PCNA gels were packed into Bio-Rad econo-colunm (Bio-Rad, Richmond, CA), and PCNA containing fractions were passed over the column at 5 ml/h. After washing with more than three column bed volume of phosphate-buffered saline (PBS, 0.15 M NaC1, 0.01 M phosphate buffer, pH 7.4) and 1 M NaC1, 0.01 M phosphate buffer, pH 7.4, the bound material was eluted with 3 M NaSCN. The recovered material was dialyzed against PBS. As a final purification procedure, high performance liquid chromatography (HPLC) gel filtration using a TSK gel G3000 SW (7.5 × 600 mm, Toyo Soda, Japan) was performed. The immunoaffinity purified PCNA was eluted with PBS at 1 ml/min flow rate. PCNA active fractions were collected and used as the purified PCNA.
Monoclonal antibodies to PCNA Hybridomas producing monoclonal antibodies to PCNA were generated by the method of Oi and Herzenberg (1980) with some modification, and their specificities were compared with those of other two monoclonal antibodies, 19A2 (IgM, K) and 19F4 (IgG1, x), which were previously established (Ogata et al., 1987). Female BALB/c mice (4 weeks old) were immunized intraperitoneally with 10 #g of the purified PCNA in Freund's complete adjuvant. 2 weeks later, each mouse received an intravenous injection of 10 #g of PCNA. An intravenous injection was repeated three times at 2 week intervals. One mouse whose serum had the highest antibody activity against PCNA was killed and a spleen cell suspension was prepared 3 days after the final immunization. Spleen cells were fused with mouse myeloma cells (P3X63Ag8.653) (Kearney et al., 1979) by 50% polyethylene glycol 1500 (BDH Chemical, England). After hybridization, hypoxanthin aminopterin thymidine selection was continued for 16 days. Anti-PCNA activity in mouse sera and supernatants of cultured medium were tested by ELISA using the purified PCNA as described below. Hybridoma producing immunoglobulins against PCNA were tested by this ELISA and cloned by limited dilution. Established hybridomas (TOB7, IgG1, K; TO17, IgM, ~¢;TO30, IgM, K) were transplanted into pristane primed BALB/c n u / n u mice to obtain ascites fluid. The
monoclonal antibodies were purified as previously reported (Ogata et al., 1987). 19A2 and 19F4 were purified in the same manner.
Biotinylation of monoclonal antibodies Monoclonal antibodies were biotinylated by the method of Guesdon et al. (1979) with some modifications. 1 ml of 0.1 M NaHCO 3 containing 1 mg of antibody was mixed with 60 gl of dimethylsulfoxide (DMSO) containing 0.06 mg of N-hydroxysuccinimide biotin (Sigma, St. Louis, MO, U.S.A.). The reaction mixture was incubated at room temperature for 4 h and then dialyzed at 4 °C for 24 h against PBS with three changes. ELISA Two types of ELISA were performed. One was for the detection of anti-PCNA activities in culture supernatants of hybridomas and the other was for inhibition tests to analyze epitopes of monoclonal antibodies. 50 gl of purified PCNA (5.0 gg/ml) in 0.05 M carbonate buffer, pH 8.5, was added to the wells of Nunc Immunoplates II (Nunc, Den Mark) and incubated overnight at 4 o C. Flicking off the coating solution, the plates were washed three times with 0.1% Tween 20/PBS (T-PBS). After the plates were drained, 250 #1 of 1% bovine serum albumin (BSA) (Sigma) T-PBS was added to wells and incubated overnight at 4°C. After washing as before, 100 gl of mouse anti-PCNA sera diluted 1/500 by T-PBS or culture supernatants were added for 2 h at room temperature. After washing, 100 #1 of alkaline phosphatase-labeled anti-mouse ~/-globulin (KPL, Gaithersburg, MD) in T-PBS diluted 1/1000 were added to the wells and incubated for 2 h at room temperature. Washed as before, 250 gl of the substrate solution (1 mg p - n i t r o p h e n y l p h o s p h a t e / m l , diethanolamine buffer, pH 9.8) was added and optical density (OD) at 405 nm was measured. ELISA was also used in inhibition tests to analyze epitope specificity of monoclonal antibodies as reported (Ogata et al., 1987). A PCNA coated plate was prepared as described above, blocking antibody in the form of 100 gl of serially diluted monoclonal antibodies (TOB7, TO17, TO30, 19A2, 19F4 and control antibody (anti-Sm mouse monoclonal antibody (Cappel, West Ches-
230 ter, PA)) were added to the wells and incubated for 2 h at room temperature. After washing with T-PBS, biotinylated 19A2 (1 #g/ml), 19F4 (2 gg/ml), TOB7 (2 #g/ml), TO17 (1 gg/ml) and TO30 (1/~g/ml) were added, and incubated for 90 rain at room temperature. The wells were washed and 100 gl of horseradish peroxidase (HRP)-conjugated streptavidin (BRL, Bethesda, MD) diluted 1/2000 was added and incubated for 1 h. After washing, 250 gl of substrate solution (1 mg/ml 2,2'-azino-di-(3-ethylb enzthiazolin-6-sulfonic acid), Sigma (ABTS) and 0.005% H202 in 0.1 M Mcllvain's buffer, pH 4.6) was added and absorbance at 405 nm was measured after 1 h incubation at room temperature. Inhibition was calculated as follows: inhibition(%) = [10--D--~ 5 O D 4 °sw lsleo f ~with ~ y °blocking f antibody ]] x 100
Sandwich assay of PCNA using monoclonal antibodies Based on the results of inhibition tests, TO17 and TOB7 were selected for sandwich assay to detect PCNA. Various concentrations of TO17, 5, 10, 20 and 40/~g/ml, were prepared and 50 gl of those solutions in carbonate buffer, pH 8.5, were added into wells of Nunc Immunoplates II. The coating of antibodies was performed at 4 °C overnight, and wells were washed with T-PBS. After post-coating with 1% BSA T-PBS, 100 gl of purified PCNA from CTE diluted from 0.002 to 6 g g / m l was added into wells and incubated for 2 h at room temperature. After washing wells five times, 100 gl of biotinylated TOB7 adjusted to 5 g g / m l was added and incubated for 2 h at room temperature. The wells were washed five times and developed as the same as the inhibition tests using HRP-conjugated streptavidin and ABTS. Based on the results obtained by the experiments described above, the concentration of TO17 was defined as 20 gg/ml and amounts of PCNA in extracts from tissue and mitogen stimulated lymphocytes were measured.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PA GE) SDS-PAGE was performed to analyze the protein profile of the purified PCNA. The slab gel
consisted of 10% acrylamide and 0.1% SDS in Tris-HC1, pH 8.8, with 15 mm of stacking gel containing 5% acrylamide and 0.1% SDS in TrisHCI, pH 6.8. The gel was electrophoresed at 1.5 m A / c m at 4 ° C. Samples were dissolved in the sample buffer (3% SDS, 5% 2-mercaptoethanol, 55 mM Tris HC1, pH 6.8, 10% glycerol, bromphenol blue) and boiled for 3 minutes. After electrophoresis, gels were stained by Coomassie Blue R250.
Immunoblotting The purified PCNA was transferred electrophoretically to a nitrocellulose filter (Bio-Rad) as previously reported (Takasaki et al., 1984). For immunochemical detection of PCNA on the nitrocellulose filter, the filter strip was first treated with 3% BSA in T-PBS for 24 h in order to reduce subsequent background staining. It was then incubated for 2 h with anti-PCNA monoclonal antibodies diluted in T-PBS (1 gg/ml) or anti-PCNA serum AK in a 1/200 dilution of T-PBS. Anti-Sm monoclonal antibody (1 gg/ml) was also reacted as control. After washing with T-PBS, HRP-conjugated goat anti-mouse y-globulin (Cappel) diluted 1/1000 with T-PBS or HRP-conjugated goat anti-human IgG (Cappel) diluted 1/2000 was added and incubated for 2 h. After final washing, the bound conjugate was detected by incubation with substrate solution (250 gg of 3,3-diaminobenzidine-4 HC1/ml, 0.5 gl of 30% H202//ml, 0.05 M Tris-HC1 buffer, pH 7.6). The strips to which the marker proteins and purified PCNA had been transferred were stained with 0.1% amido black 10B in 7% acetic acid.
Preparation of phytohemagglutinin-stimulated lymphocytes Normal human peripheral blood lymphocytes (PBL) were prepared as described (B/Syum, 1968), and stimulated by 30 g g / m l phytohemagglutinin (PHA, Wellcome Reagents, Greenville, N.C.) as previously reported (Takasaki et al., 1981). The DNA synthesis in those cells after stimulation was detected by immunofluorescence using antibromodeoxy-uridine (BrdU) monoclonal antibodies (Becton Dickinson Monoclonal Center, Mountain View, CA) by the procedure recommended by supplier. The mitotic index was also defined by staining with Schiff's solution.
231
PCNA extraction from PBL and tissue PCNA in PBL before and after PHA stimulation was extracted as described below and its concentration was measured by sandwich type ELISA using TOB7 and TO17. After cells were harvested, they were washed with PBS twice and resuspended in 50 mM Tris-HC1, pH 7.4, with 150 mM NaC1, 5 mM EDTA, 0.5% Nonidet P40 and 2 mM phenylmethylsulfonylfluoride at the concentration of 2.5 × 107/ml. Cell suspension was vortexed for 5 rain at room temperature and centrifuged at 8000 x g. Supernatant was collected and used to measure the PCNA concentration. Rabbit thymus and kidney extract were also prepared as previously reported (Takasaki, et al., 1981) and the PCNA concentration in those extracts was measured by the same method.
Other methods Protein concentration was measured by the method reported by Lowry et al. (1951).
Results
Purification of PCNA Protein profile of PCNA purified by ammonium sulfate fractionation, Sephadex G-200 gel filtration and anti-PCNA affinity chromatography was analyzed by SDS-PAGE (Fig. 1). The purified PCNA showed a 34 kDa single band. This purified antigen was immunized into mice and used as the antigen source in ELISA and immunoblotting.
Reactivity and specificity of TOB7, TO17 and TO30 Reactivities of TOB7, TO17 and TO30 were first tested by ELISA as shown on Fig. 2. Those monoclonal antibodies strongly reacted with the purified PCNA, while control mouse monoclonal antibody to Sm was negative. Reactivities of those monoclonal antibodies to PCNA were further analyzed by immunoblotting (Fig. 3). TOB7 (lane 3), TO17 (lane 4) and TO30 (lane 5) reacted with 34 kDa PCNA polypeptide the same as 19F4 (lane 7) and anti-PCNA standard serum AK (lane 6). On the other hand, control antibody, anti-Sm monoclonal antibody (lane 8), did not react with it.
Fig. 1. Sodium dodecyl sulfate-polyacrylamidegel electrophoresis of the purified PCNA from calf thymus extract. Protein profiles of the purified PCNA (lane B) and molecular weightmarkers(laneA). The immunofluorescence staining of normal human PBL before and after mitogenic stimulation was performed to analyze the specificity of those monoclonal antibodies to PCNA. As shown on Fig. 4, TOB7 did not react with nuclei of normal PBL (Fig. 4A), but strongly reacted with nuclei of lymphocytes stimulated by PHA showing the speckled pattern (Fig. 4B). TO17 and TO30 behaved similarly to TOB7 and specifically reacted with PHA-stimulated blast-trasformed lymphocytes. Those results indicated that three monoclonal antibodies, TOB7, TO17 and TO30 were antibodies against PCNA.
Epitope analysis of monoclonal antibodies ELISA was used to analyze the epitope specificities of the monoclonal antibodies in relation to each other (Figs. 5A-5E). When serial dilutions of TOB7 (A), TO17 (B), TO30 (C), 19A2 (D) and 19F4 (E) were added as inhibitors, binding of biotinylated TOB7 was not blocked by
232 TABLE I PCNA CONCENTRATION IN HUMAN PBL BEFORE AND AFTER PHA STIMULATION AND IN RABBIT TISSUE EXTRACT Rabbit tissue extract
Human PBL stimulated by PHA
Concentration of PCNA (ng/ml) % of cells in DNA synthesis Mitotic index (%)
0h
22 h
48 h
Thymus
Kidney
227
JIM 05680
A sandwich type enzyme-linked immunosorbent assay for proliferating cell nuclear antigen (PCNA)/cyclin using monoclonal antibodies * Yoshinari Takasaki 1, Makoto Ohgaki 2,3, Akira K o d a m a 1,4, Kenji Ogata 5, Hiroshi Hashimoto t, Toshikazu Shirai 2 and Shun-Ichi Hirose 1 1 Division of Rheumatology, Department of Medicine, Juntendo University School of Medicine, Tokyo, Japan, 2 Second Department of Pathology, Juntendo University School of Medicine, Tokyo, Japan, 3 Mitsubishi Kasei Corporation, Research Center, Kanagawa, Japan, 4 Biomedical Laboratories, Saitama, Japan, and 5 Hoechst Japan, Research Center, Saitama, Japan (Received 14 November 1989, revised received 15 May 1990, accepted 17 May 1990)
Three hybridomas producing monoclonal antibodies to proliferating cell nuclear antigen (PCNA)/cyclin were newly derived. The specificity of established monoclonal antibodies to PCNA, TOB7, TO17 and TO30, was compared with that of the previously reported PCNA-specific monoclonal antibodies, 19A2 and 19F4. TOB7, TO17 and TO30 reacted with purified PCNA in an enzyme-linked immunosorbent assay (ELISA). A 34 kDa PCNA polypeptide was noted by immunoblotting, the same polypeptide as recognized by 19A2 and 19F4. Epitopes recognized by all those monoclonal antibodies to PCNA were analyzed by competitive inhibition tests using ELISA. The results of those experiments suggested that the epitope recognized by TO17 and TO30 was almost identical to that of 19A2 and closely related to that of 19F4, but different from that of TOB7. Based on those results, a sandwich type ELISA for detection of PCNA was developed using TO17 and TOB7. This system was applied to measure the concentration of PCNA in normal peripheral blood lymphocytes (PBL) before and after stimulation by phytohemagglutinin (PHA), and in tissue extracts from rabbit kidney and thymus (RKE and RTE, respectively). Before mitogenic stimulation, the PCNA concentration in PBL extracts was less than 6 ng/ml (cell concentration 2.5 x 107/ml), but at 48 h after PHA stimulation, when more than 70% of cells were in S phase, its concentration became 1280 ng/ml. RTE which contained many proliferative lymphocytes had much higher concentration of PCNA than RKE. Those results suggested that sandwich type ELISA using TO17 and TOB7 was useful as the quantitative assay to detect blast-transformation and proliferation of cells in vivo. Key words: Proliferating cell nuclear antigen; Cyclin; Monoclonal antibody; Cell profiferation; Antinuclear antibody; ELISA
Correspondence to: Y. Takasaki, Division of Rhenmatology, Department of Medicine, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-Ku, Tokyo, 113 Japan. * Supported by a research grant from the Ministry of Education of Japan (Grant no. 01480220) and a 1988 research grant from the Mixed Connective Tissue Disease Research Committee, the Ministry of Welfare of Japan. Abbreviations: PCNA, proliferating cell nuclear antigen; ELISA, enzyme-linked immunosorbent assay; DID, double immunodiffusion; CIE, counterlmmnnoelectrophoresis; CTE, calf thymus extract; HPLC, high performance fiquid chromatography; T-PBS, 0.1~ Tween 20/phosphate-buffered saline; HRP, horseradish peroxidase; ABTS, 2,2'-azino-di-(3-ethylbenzthiazolin-6-sulfonicacid); SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; BrdU, bromodeoxy-uridine; 3H-TdR, tritiated thymidine. 0022-1759/90//$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
228 Introduction
Antibodies to proliferating cell nuclear antigen (PCNA) were detected in some patients with systemic lupus erythematosus (SLE) and reacted with a nuclear antigen expressed predominantly by proliferating cells such as cultured cells and mitogentransformed cells (Miyachi et al., 1978). PCNA has been identified as a nuclear protein with a molecular weight (MW) of about 33 kDa and isoelectric point of 4.8 (Takasaki et al., 1984a; Ogata et al., 1985). These observations led to an insight that PCNA was identical to cyclin, a nuclear protein associated with cell proliferation and identified by two-dimensional (2D) gel electrophoresis (Bravo et al., 1980); the identity of these two proteins was recently demonstrated (Mathews et al., 1984). Subsequent studies have revealed that the expression of PCNA/cyclin increased in the late G1 phase of the cell-cycle immediately preceding DNA synthesis (Bravo et al., 1980; Takasaki et al., 1981; Bravo, 1984; Bravo and MacDonald-Bravo, 1984). The molecular cloning of cDNA coding for PCNA/cyclin allowed detection of cell cycle-specific expression of PCNA mRNA (Almendral et al., 1987; Matsumoto et al., 1987). These observations implied an association of PCNA/cyclin with DNA replication. Recent findings suggest that PCNA/cyclin is an auxiliary protein of DNA polymerase delta (Bravo et al., 1987; Prellch et al., 1987). These characteristics of PCNA enabled autoantibodies to PCNA from lupus patients to be used as a reagent for detecting blast cells. This was used to detect the crisis phase of patients with chronic myeloid leukemia (Takasaki et al., 1984b). However, sera from lupus patients often contain other autoantibodies and it is quite difficult to obtain sufficient m o u n t s of monospecific sera for PCNA because of its low incidence in lupus patients. To solve these problems, monoclonal antibodies to PCNA were generated and two mouse monoclonal PCNA-specific antibodies, 19A2 and 19F4, established (Ogata et al., 1987). Using these monoclonal antibodies as probes, detection of proliferating cells by immunofluorescence microscopy and flow cytometry was recently reported (Kurki et al., 1988). In addition, it was shown that these monoclonal antibodies were useful for labeling
immunocytochemically the nuclei of proliferating cells in mouse lung tissue (Thaete et al., 1989). These results suggest that monoclonal antibodies to PCNA are useful tools to study the cell proliferation and blast transformation. However, it was impossible to make a sandwich type enzyme-linked immunosorbent assay (ELISA) for detection of PCNA because these two monoclonal antibodies recognized closely related epitopes. Here we report the generation of three hybridomas producing monoclonal antibodies to PCNA and establishment of specific quantitative assay for PCNA using two of these monoclonal antibodies.
Materials and methods
Sera Anti-PCNA serum obtained from a lupus patient (AK) was used to purify PCNA and test the specificity of anti-PCNA monoclonal antibodies. Serum AK showed a single precipitin line which was identical to anti-PCNA standard serum (PT) (kindly given to us by Eng M. Tan, M.D., Scripps Clinic and Research Foundation, La Jolla, CA, U.S.A.) in a standard double immunodiffusion system ( D I D ) and by counterimmunoelectrophoresis (CIE) (Takasaki et al., 1981). This serum was used to monitor the antigenic activity of PCNA in DID and CIE during the antigen purification procedure, and as the source of antiPCNA IgG to make a affinity chromatography column. Antigen purification Calf thymus extract (CTE) was prepared as previously reported (Sharp et al., 1972) and used as the source material containing PCNA. PCNA in CTE was firstly purified by 40-65% ammonium sulfate fractionation and Sephadex G-200 (Pharmacia, Uppsala, Sweden) gel filtration as previously reported (Takasaki et al., 1984a). An IgG fraction of serum AK prepared by 33% ammonium sulfate fractionation and DE52 (Whatman Lab. Products, Clifton, NJ, U.S.A.) ion exchange chromatography coupled to CNBr activated Sepharose 4B (Pharmacia) (Douvas et al., 1979), and anti-PCNA affinity chromatography were performed to further purify PCNA.
229 Anti-PCNA gels were packed into Bio-Rad econo-colunm (Bio-Rad, Richmond, CA), and PCNA containing fractions were passed over the column at 5 ml/h. After washing with more than three column bed volume of phosphate-buffered saline (PBS, 0.15 M NaC1, 0.01 M phosphate buffer, pH 7.4) and 1 M NaC1, 0.01 M phosphate buffer, pH 7.4, the bound material was eluted with 3 M NaSCN. The recovered material was dialyzed against PBS. As a final purification procedure, high performance liquid chromatography (HPLC) gel filtration using a TSK gel G3000 SW (7.5 × 600 mm, Toyo Soda, Japan) was performed. The immunoaffinity purified PCNA was eluted with PBS at 1 ml/min flow rate. PCNA active fractions were collected and used as the purified PCNA.
Monoclonal antibodies to PCNA Hybridomas producing monoclonal antibodies to PCNA were generated by the method of Oi and Herzenberg (1980) with some modification, and their specificities were compared with those of other two monoclonal antibodies, 19A2 (IgM, K) and 19F4 (IgG1, x), which were previously established (Ogata et al., 1987). Female BALB/c mice (4 weeks old) were immunized intraperitoneally with 10 #g of the purified PCNA in Freund's complete adjuvant. 2 weeks later, each mouse received an intravenous injection of 10 #g of PCNA. An intravenous injection was repeated three times at 2 week intervals. One mouse whose serum had the highest antibody activity against PCNA was killed and a spleen cell suspension was prepared 3 days after the final immunization. Spleen cells were fused with mouse myeloma cells (P3X63Ag8.653) (Kearney et al., 1979) by 50% polyethylene glycol 1500 (BDH Chemical, England). After hybridization, hypoxanthin aminopterin thymidine selection was continued for 16 days. Anti-PCNA activity in mouse sera and supernatants of cultured medium were tested by ELISA using the purified PCNA as described below. Hybridoma producing immunoglobulins against PCNA were tested by this ELISA and cloned by limited dilution. Established hybridomas (TOB7, IgG1, K; TO17, IgM, ~¢;TO30, IgM, K) were transplanted into pristane primed BALB/c n u / n u mice to obtain ascites fluid. The
monoclonal antibodies were purified as previously reported (Ogata et al., 1987). 19A2 and 19F4 were purified in the same manner.
Biotinylation of monoclonal antibodies Monoclonal antibodies were biotinylated by the method of Guesdon et al. (1979) with some modifications. 1 ml of 0.1 M NaHCO 3 containing 1 mg of antibody was mixed with 60 gl of dimethylsulfoxide (DMSO) containing 0.06 mg of N-hydroxysuccinimide biotin (Sigma, St. Louis, MO, U.S.A.). The reaction mixture was incubated at room temperature for 4 h and then dialyzed at 4 °C for 24 h against PBS with three changes. ELISA Two types of ELISA were performed. One was for the detection of anti-PCNA activities in culture supernatants of hybridomas and the other was for inhibition tests to analyze epitopes of monoclonal antibodies. 50 gl of purified PCNA (5.0 gg/ml) in 0.05 M carbonate buffer, pH 8.5, was added to the wells of Nunc Immunoplates II (Nunc, Den Mark) and incubated overnight at 4 o C. Flicking off the coating solution, the plates were washed three times with 0.1% Tween 20/PBS (T-PBS). After the plates were drained, 250 #1 of 1% bovine serum albumin (BSA) (Sigma) T-PBS was added to wells and incubated overnight at 4°C. After washing as before, 100 gl of mouse anti-PCNA sera diluted 1/500 by T-PBS or culture supernatants were added for 2 h at room temperature. After washing, 100 #1 of alkaline phosphatase-labeled anti-mouse ~/-globulin (KPL, Gaithersburg, MD) in T-PBS diluted 1/1000 were added to the wells and incubated for 2 h at room temperature. Washed as before, 250 gl of the substrate solution (1 mg p - n i t r o p h e n y l p h o s p h a t e / m l , diethanolamine buffer, pH 9.8) was added and optical density (OD) at 405 nm was measured. ELISA was also used in inhibition tests to analyze epitope specificity of monoclonal antibodies as reported (Ogata et al., 1987). A PCNA coated plate was prepared as described above, blocking antibody in the form of 100 gl of serially diluted monoclonal antibodies (TOB7, TO17, TO30, 19A2, 19F4 and control antibody (anti-Sm mouse monoclonal antibody (Cappel, West Ches-
230 ter, PA)) were added to the wells and incubated for 2 h at room temperature. After washing with T-PBS, biotinylated 19A2 (1 #g/ml), 19F4 (2 gg/ml), TOB7 (2 #g/ml), TO17 (1 gg/ml) and TO30 (1/~g/ml) were added, and incubated for 90 rain at room temperature. The wells were washed and 100 gl of horseradish peroxidase (HRP)-conjugated streptavidin (BRL, Bethesda, MD) diluted 1/2000 was added and incubated for 1 h. After washing, 250 gl of substrate solution (1 mg/ml 2,2'-azino-di-(3-ethylb enzthiazolin-6-sulfonic acid), Sigma (ABTS) and 0.005% H202 in 0.1 M Mcllvain's buffer, pH 4.6) was added and absorbance at 405 nm was measured after 1 h incubation at room temperature. Inhibition was calculated as follows: inhibition(%) = [10--D--~ 5 O D 4 °sw lsleo f ~with ~ y °blocking f antibody ]] x 100
Sandwich assay of PCNA using monoclonal antibodies Based on the results of inhibition tests, TO17 and TOB7 were selected for sandwich assay to detect PCNA. Various concentrations of TO17, 5, 10, 20 and 40/~g/ml, were prepared and 50 gl of those solutions in carbonate buffer, pH 8.5, were added into wells of Nunc Immunoplates II. The coating of antibodies was performed at 4 °C overnight, and wells were washed with T-PBS. After post-coating with 1% BSA T-PBS, 100 gl of purified PCNA from CTE diluted from 0.002 to 6 g g / m l was added into wells and incubated for 2 h at room temperature. After washing wells five times, 100 gl of biotinylated TOB7 adjusted to 5 g g / m l was added and incubated for 2 h at room temperature. The wells were washed five times and developed as the same as the inhibition tests using HRP-conjugated streptavidin and ABTS. Based on the results obtained by the experiments described above, the concentration of TO17 was defined as 20 gg/ml and amounts of PCNA in extracts from tissue and mitogen stimulated lymphocytes were measured.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PA GE) SDS-PAGE was performed to analyze the protein profile of the purified PCNA. The slab gel
consisted of 10% acrylamide and 0.1% SDS in Tris-HC1, pH 8.8, with 15 mm of stacking gel containing 5% acrylamide and 0.1% SDS in TrisHCI, pH 6.8. The gel was electrophoresed at 1.5 m A / c m at 4 ° C. Samples were dissolved in the sample buffer (3% SDS, 5% 2-mercaptoethanol, 55 mM Tris HC1, pH 6.8, 10% glycerol, bromphenol blue) and boiled for 3 minutes. After electrophoresis, gels were stained by Coomassie Blue R250.
Immunoblotting The purified PCNA was transferred electrophoretically to a nitrocellulose filter (Bio-Rad) as previously reported (Takasaki et al., 1984). For immunochemical detection of PCNA on the nitrocellulose filter, the filter strip was first treated with 3% BSA in T-PBS for 24 h in order to reduce subsequent background staining. It was then incubated for 2 h with anti-PCNA monoclonal antibodies diluted in T-PBS (1 gg/ml) or anti-PCNA serum AK in a 1/200 dilution of T-PBS. Anti-Sm monoclonal antibody (1 gg/ml) was also reacted as control. After washing with T-PBS, HRP-conjugated goat anti-mouse y-globulin (Cappel) diluted 1/1000 with T-PBS or HRP-conjugated goat anti-human IgG (Cappel) diluted 1/2000 was added and incubated for 2 h. After final washing, the bound conjugate was detected by incubation with substrate solution (250 gg of 3,3-diaminobenzidine-4 HC1/ml, 0.5 gl of 30% H202//ml, 0.05 M Tris-HC1 buffer, pH 7.6). The strips to which the marker proteins and purified PCNA had been transferred were stained with 0.1% amido black 10B in 7% acetic acid.
Preparation of phytohemagglutinin-stimulated lymphocytes Normal human peripheral blood lymphocytes (PBL) were prepared as described (B/Syum, 1968), and stimulated by 30 g g / m l phytohemagglutinin (PHA, Wellcome Reagents, Greenville, N.C.) as previously reported (Takasaki et al., 1981). The DNA synthesis in those cells after stimulation was detected by immunofluorescence using antibromodeoxy-uridine (BrdU) monoclonal antibodies (Becton Dickinson Monoclonal Center, Mountain View, CA) by the procedure recommended by supplier. The mitotic index was also defined by staining with Schiff's solution.
231
PCNA extraction from PBL and tissue PCNA in PBL before and after PHA stimulation was extracted as described below and its concentration was measured by sandwich type ELISA using TOB7 and TO17. After cells were harvested, they were washed with PBS twice and resuspended in 50 mM Tris-HC1, pH 7.4, with 150 mM NaC1, 5 mM EDTA, 0.5% Nonidet P40 and 2 mM phenylmethylsulfonylfluoride at the concentration of 2.5 × 107/ml. Cell suspension was vortexed for 5 rain at room temperature and centrifuged at 8000 x g. Supernatant was collected and used to measure the PCNA concentration. Rabbit thymus and kidney extract were also prepared as previously reported (Takasaki, et al., 1981) and the PCNA concentration in those extracts was measured by the same method.
Other methods Protein concentration was measured by the method reported by Lowry et al. (1951).
Results
Purification of PCNA Protein profile of PCNA purified by ammonium sulfate fractionation, Sephadex G-200 gel filtration and anti-PCNA affinity chromatography was analyzed by SDS-PAGE (Fig. 1). The purified PCNA showed a 34 kDa single band. This purified antigen was immunized into mice and used as the antigen source in ELISA and immunoblotting.
Reactivity and specificity of TOB7, TO17 and TO30 Reactivities of TOB7, TO17 and TO30 were first tested by ELISA as shown on Fig. 2. Those monoclonal antibodies strongly reacted with the purified PCNA, while control mouse monoclonal antibody to Sm was negative. Reactivities of those monoclonal antibodies to PCNA were further analyzed by immunoblotting (Fig. 3). TOB7 (lane 3), TO17 (lane 4) and TO30 (lane 5) reacted with 34 kDa PCNA polypeptide the same as 19F4 (lane 7) and anti-PCNA standard serum AK (lane 6). On the other hand, control antibody, anti-Sm monoclonal antibody (lane 8), did not react with it.
Fig. 1. Sodium dodecyl sulfate-polyacrylamidegel electrophoresis of the purified PCNA from calf thymus extract. Protein profiles of the purified PCNA (lane B) and molecular weightmarkers(laneA). The immunofluorescence staining of normal human PBL before and after mitogenic stimulation was performed to analyze the specificity of those monoclonal antibodies to PCNA. As shown on Fig. 4, TOB7 did not react with nuclei of normal PBL (Fig. 4A), but strongly reacted with nuclei of lymphocytes stimulated by PHA showing the speckled pattern (Fig. 4B). TO17 and TO30 behaved similarly to TOB7 and specifically reacted with PHA-stimulated blast-trasformed lymphocytes. Those results indicated that three monoclonal antibodies, TOB7, TO17 and TO30 were antibodies against PCNA.
Epitope analysis of monoclonal antibodies ELISA was used to analyze the epitope specificities of the monoclonal antibodies in relation to each other (Figs. 5A-5E). When serial dilutions of TOB7 (A), TO17 (B), TO30 (C), 19A2 (D) and 19F4 (E) were added as inhibitors, binding of biotinylated TOB7 was not blocked by
232 TABLE I PCNA CONCENTRATION IN HUMAN PBL BEFORE AND AFTER PHA STIMULATION AND IN RABBIT TISSUE EXTRACT Rabbit tissue extract
Human PBL stimulated by PHA
Concentration of PCNA (ng/ml) % of cells in DNA synthesis Mitotic index (%)
0h
22 h
48 h
Thymus
Kidney
