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cyclin using monoclonal antibodies
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
<6 0 0
<6 2.2 0.02
1280 72.8 5.1
352 -
<6
Key: PBL, Peripheral blood lymphocyte; Cell concentration of PBL: 2.5 × 107/ml; Protein concentration: RTE 2.5 mg/ml, RKE 46 mg/ml.
19A2, TO17 and TO30 and only partially blocked by 19F4. On the other hand, binding of 19A2, TO17 and TO30 were markedly blocked by each other and moderately blocked by 19F4 and TOB7. Binding of 19F4 was blocked more than 80% by high concentrations of all other monoclonal antibodies except TOB7.
Sandwich type ELISA for detection of PCNA According to the results of epitope analysis of monoclonal antibodies, sandwich type ELISA for
detection of PCNA was performed using biotinylated TOB7 and TO17. Fig. 6 shows the results of a sandwich assay to determine the concentration of TO17 coated on a well of Immunoplate. When serial dilutions of purified PCNA were reacted and their binding to TO17 detected by biotinylated TOB7, absorbance of 405 nm increased in parallel with the concentration of TO17, except that there was no significant difference between 20 /xg/ml and 40 /xg/ml. The concentration of biotinylated TOB7 in this system was also studied. If
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Fig. 2. ELISA of monoclonal antibodies against PCNA. Monoclonal antibodies (TOB7, zx; TO17, n; TO30, ©) bound to PCNA on an ELISA plate were detected by alkaline phosphatase-labeled anti-mouse 7-globulin antibody. The undiluted protein concentration of TOB7 was 2 mg/ml, and that of TO17 and TO30 was 1 mg/ml. Anti-Sm specific monoclonal antibody (&) was used as control.
Fig. 3. Immunoblotting of monoclonal antibodies. Purified PCNA from calf thymus was detected by human anti-PCNA antibody from lupus patients and by monoclonal antibodies. Lanes: 1, molecular weight markers stained by amido black; 2, purified PCNA stained by amido black; 3, TOB7; 4, TO17; 5, TO30; 6, human anti-PCNA serum (AK); 7, 19F4; 8, anti-Sm monoclonal antibody.
233
Fig. 4. Immunofluorescencestaining of normal peripheral blood lymphocytes before and after mitogenic stimulation using TOB7. Before PHA stimulation (A), TOB7 did not react with lymphocyte nuclei, but cells harvested at 48 hours after PHA stimulation showed strong nuclear speckled staining (B). ( × 1200).
its concentration was more than 2 /~g/ml, P C N A was still reactive at a dilution of 0.012 # g / m l and there was no significant differences in the range to detect P C N A (data were not shown). Based on those results, it was determined that the concentration of TO17 and biotinylated TOB7 were 2 0 / x g / m l and 5 / x g / m l , respectively. Using this system, the concentration of P C N A in normal h u m a n PBL during mitogenic stimulation and the tissue extracts, R T E and RKE, were studied (Fig. 7, Table I). Before mitogenic stimulation (Fig. 7, C), P C N A concentration was less than 6 n g / m l (cell concentration, 2.5 × 107/ml). 22 h after P H A stimulation, only 2.2% of cells were in S phase of cell cycle (Fig. 7, D), and its P C N A concentration was still less than 6 n g / m l . But 72.8% of the cells harvested at 48 h after
mitogenic stimulation were in S phase and the P C N A concentration from those cells (Fig. 7, A) were estimated 1280 n g / m l . The concentrations of P C N A in R K E and R T E were also measured. P C N A concentration of R T E (Fig. 7, B) which contained stimulated lymphocytes was 352 n g / m l but that of R K E (Fig. 7, E ) was less than 6 ng/ml.
Discussion Recently, Ogata et al. (1987) described two mouse monoclonal PCNA-specific antibodies, 19A2 and 19F4. These antibodies offered a convenient tool for the detection of cell proliferation and were used to demonstrate the expression of
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PCNA in various human cell lines and blast-transformed cells by immunofluorescence microscopy and flow cytometry (Kurki et al., 1988). These antibodies were also used to analyze the proliferative grade in fixed, embedded human tumor tissues (Garcia et al., 1989) and to estimate the index of cell proliferation in mouse lung tissue (Thaete et al., 1989). One of our monoclonal antibodies, TOB7, was applied to detect activated peripheral blood lymphocytes in lupus patients, and it was demonstrated that the index of P C N A expression
in lymphocytes was useful for estimating the activity of the disease (Murashima et al., 1990). TOB7 was also used to study the correlation between expression of PCNA and the proliferative state of human keratinocytes (Miyagawa et al., 1989). These results suggested that monoclonal antibody to PCNA could be a reliable probe for the monitoring cell proliferation. However, the expression of PCNA in cells and tissues was shown only as an index in those reports, and could not be quantiffed. Sandwich type ELISA to detect PCNA was
235
performed when 19A2 and 19F4 were first reported (Ogata et al., 1987), but it was impossible to make the system detect PCNA using those two monoclonal antibodies because they recognized closely related epitopes. At the same time, it was shown that the epitopes which were recognized by 19A2 and 19F4 were different from those of certain human autoantibodies to PCNA. Sandwich type ELISA using the combination of mouse monoclonal antibodies and human autoantibodies to PCNA could quantify the amounts of PCNA (Ogata et al., 1987). The problem with this System was that anti-PCNA sera often contained antibodies to other nuclear antigens and it was difficult to obtain sufficient amounts of monospecific sera. Therefore, it was necessary to establish new hybridomas which produced monoclonal antibodies which recognize a different epitope from that of 19A2 and 19F4. The reaction of our monoclonal antibodies to PCNA, TO17 and TO30, was
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Fig. 7. Detection of PCNA in lymphocytes before and after mitogenic stimuration and in rabbit tissue extracts by sandwich type ELISA using TOB7 and TO17. PCNA was detected in lymphocyte* harvested at 48 h after PHA stimulation (A) but not in ceils before (C) or harvested 22 h after PHA stimulation (D) (cell concentration 2.5x107/ml). PCNA of RTE (protein concentration 2.5 mg/ml) (B) was also detected in much higher concentration than that of RKE (protein concentration 46 mg/ml) (E). The estimated concentrations of PCNA in those preparations were shown on Table I.
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Fig. 6. Comparison of sandwich type ELISA for detection of PCNA at various dilutions of TO17 coated onto plate*. Serial dilutions of PCNA were reacted with wells of plates coated with four different concentrations of TO17, 5 (O), 10 (,x), 20 ([2) and 40 /~g/ml (o), and reactions were detected using biotinylated TOB7 (5/~g/ml).
strongly inhibited by 19A2 in EL1SA, but not by TOB7. These results suggested that it would be possible to make a sandwich type ELISA assay to detect PCNA using TOB7 and either 19A2, TO17 or TO30. As shown above, our system using TOB7 and TO17 could detect the increased amount of PCNA after mitogenic stimulation. Total PCNA in cells harvested at 48 h were at least 200 times higher than in cells before mitogenic stimulation. The concentration of PCNA in tissue extracts was also tested using two different organs, rabbit thymus and kidney. Anti-PCNA antibodies react
236 with m a n y lymphocytes in rabbit thymus but not with kidney tubular or glomerular cells (Miyachi et al., 1978). Our system showed clearly a great difference in the P C N A concentration between R T E and RKE. Sandwich type E L I S A using monoclonal antibodies to P C N A could thus be used to quantitatively analyze the cell proliferation and activation. The levels of cyclin ( P C N A ) in normal and transformed h u m a n cultured cells was estimated by labelling with [35S]methionine in 2D gel electrophoresis (Celis et al., 1984). In that report, levels of cyclin were expressed as the percentage of labeled cyclin to total label. Cyclin was not detectable in normal h u m a n peripheral blood lymphocytes or mouse kidney, but 0.64% of the total label corresponded to cyclin in cultured cells such as Molt-4. Those results revealed the association of cyclin to cell proliferation semi-quantitatively but not the exact amounts of cyclin in tissues and cells. Therefore, our procedure using TOB7 and TO17 is the first specific quantitative system for detecting P C N A . A l t h o u g h the results of a sandwich type E L I S A using TOB7 with TO30 or 19A2 were not shown in this paper, they were almost the same as those obtained using TOB7 and TO17. A n o t h e r system using TOB7 and 19F4 was tested because it was suggested that the epitope recognized b y 19F4 was different f o r m that of TOB7 but the sensitivity was not high enough to detect P C N A in extracts of ceils and: tissue. Cell proliferation is generally studied by measuring the rate of D N A synthesis. Tritiated thymidine ( 3 H - T d R ) and BrdU incorporation have been usually used to detect the rate of D N A synthesis. It has been reported that P C N A labeling in individual ceils resembles that obtained by 3 H - T d R autoradiography (Takasaki et al., 1981) and monoclonal BrdU-specific antibodies (Kurki, et al., 1988). The a m o u n t of P C N A detected b y our system was also related to the labeling index obtained using monoclonal antibodies to BrdU. These results suggest that the quantitative assay for P C N A expression using TOB7 and TO17 can be used as a m e t h o d to estimate cell proliferation and transformation instead of BrdU or 3 H - T d R incorporation. The sensitivity of our system might be lower than that of BrdU or 3 H - T d R incorporation, but we believe that it has the advantage for
studying cell activation or proliferation in vivo where D N A synthesis is perturbed b y drugs (Kurki et al., 1987).
Acknowledgements The authors thank Ms. Misako Suzuki and Ms. Chie K o d a m a for excellent assistance and Dr. Eng M. T a n for helpful suggestions.
References Almendral, J.M., Huebsch, D., Blundell, P.A., MacDonaldBravo, H. and Bravo, R. (1987) Cloning and sequence of the human nuclear protein cyclin: homology with DNAbinding proteins. Proc. Natl. Acad. Sci. U.S.A. 84, 1575. B/Syum, A. (1968) Separation of leukocytes from blood and bone mallow. Scand. J. Clin. Lab. Invest. 21, 97. Bravo, R. (1984) Coordinated synthesis of the nuclear protein cyclin and DNA in serum-stimulated quiescent 3T3 ceils. FEBS Lett. 169, 185. Bravo, R. and Celis, J.E. (1980) Search for differencial polypeptide synthesis throughout the cell cycle of HeLa ceils. J. Cell Biol. 84, 795. Bravo, R. and MacDonald-Bravo, H. (1984) Induction of the nuclear protein "cyclin" in quiescent mouse 3T3 cells stimulated by serum and growth factors. Correlation with DNA synthesis. EMBO J. 3, 3177. Bravo, R., Frank, R., Blundell, P.A. and MacDonald-Bravo, H. (1987) Cyclin/PCNA is the auxiliary protein of DNA polymerase-& Nature 326, 515. Celis, J.E., Bravo, R., Larsen, P.M. and Fey, S. (1984) Cyclin: a nuclear protein whose level correlates directly with the proliferative stage of normal as well as transformed cells. Leukemia Res. 8, 143. Douvas, A.S., Stumph, W.E., Reyes, P. and Tan, E.M. (1979) Isolation and Chracterization of nuclear ribonucleoprotein complexes using human-nuclear ribonucleoprotein antibodies. J. Biol. Chem. 254, 3608. Garcia, R.L., Coltrera, M.D. and Gown, A.M. (1989) Analysis of prohferative grade using anti-PCNA/cyclin monoclonal antibodies in fixed, embedded tissues. Am. J. Pathol. 34, 733. Guesdon, J.L., Ternynck, T. and Avrameas, S. (1979) The use of avidin-biotin: interaction in immunoenzymatic techniques. J. Histochem. Cytochem. 27, 1131. Kearney, J.F., Radbruch, A., Liesegang, B. and Rajewsky, K. (1979) A new mouse myeloma cell line which has lost immunoglobulin expression but permits the constraction of antibody in secreting hybrid cell lines. J. Immunol. 123, 1548. Kurki, P., Ogata, K. and Tan, E.M. (1988) Monoclonal antibodies to proliferating cell nuclear antigen (PCNA)/cyclin as probes for proliferating cells by immunofluorescence
237 microscopy and flow cytometry. J. Immunol. Methods 109, 49. Lowry, O.H., Rosebrough, A.L., Faxr, A.L and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem, 193, 265. Mathews, M.B., Bernstein, R.M., Franza, Jr., R.B. and Garrels, J.I. (1984) Identity of proliferating cell nuclear antigen and cyclin. Nature 309, 374. Matsumoto, K., Moriuchi, T., Koji, T. and Nakane P.K. (1987) Molecular cloning of cDNA coding for rat proliferating cell nuclear antigen (PCNA)/cyclin. EMBO J. 6, 637. Miyachi, K., Fritzler, M.J. and Tan, E.M. (1978) Autoantibody to a nuclear antigen in proliferating cells. J. Immunol. 121, 2228. Miyagawa, S., Okada, N., Takasald, Y., Iida, T., Kitano, Y., Yoshikawa, K., Sakamoto, K. and Steiberg, M.L. (1989) Expression of proliferating cell nuclear antigen/cyclin in human keratinocytes. J. Invest. DermatoL 93, 678. Murashima, A., Takasaki, Y., Ohgaki, M., Hashimoto, H., Shirai, T. and Hirose, S. (1990) Activated peripheral blood mononucleax cells detected by murine monoclonal antibodies to proliferating cell nuclear antigen in active lupus patients. J. Clin. Immunol. 10, 28. Ogata, K., Ogata, Y., Nakamura, R.M. and Tan, E.M. (1985) Purification and N-terminal amino acid sequence of proliferating cell nuclear antigen (PCNA)/cyclinand development of ELISA for anti-PCNA antibodies. J. Immunol. 135, 2623. Ogata, K., Kurki, P., Cells, J.E., Nakamura, R.M. and Tan, E.M. (1987) Monoclonal antibodies to a nuclear protein
(PCNA/cyclin) associated with DNA replication. Exp. Cell Res. 168, 475. Oi, V. and Herzenberg, L.A. (1980) Selected methods in cellulax immunology. Mishell, B.B. and Shigii, E.S. (Eds.), W.H. Freeman, San Francisco, CA, p. 351. Plerich, G., Tan, C.-K., Marshak, D.R., Mathews, M.B., So, A.G., Downy, K.M. and Stillman, B. (1987) Functional identity of proliferating cell nuclear antigen and a DNA polymerase-8 auxiliary protein. Nature 326, 517. Sharp, G.C., Irvin, W.S., Tan, E.M., Gould, R.G. and Holman, H.R. (1972) Mixed connective tissue disease: an apparantly distinct rheumatic diseases syndrome associated with a specific antibody to extractable nuclear antigen (ENA). Am. J. Med. 52, 148. Takasaki, Y., Deng, J.S. and Tan, E.M. (1981) A nuclear antigen associated with cell proliferation and blast transformarion. Its distribution in synchronized cells. J. Exp. Med. 154, 1899. Takasaki, Y., Fishwild, D. and Tan, E.M. (1984a) Characterization of proliferating cell nuclear antigen recognized by autoantibodies in lupus sera. J. Exp. Med. 159, 981. Takasald, Y., Robinson, W.A. and Tan, E.M. (1984b) Proliferaring cell nuclear antigen in blast crisis cells of patients with chronic myeroid leukemia. J. Natl. Cancer Inst. 73, 655. Thaete, L.G., Atmen, D.J. and Malkinson, A.M. (1989) Proliferating cell nuclear antigen (PCNA/Cyclin) immunocytochemistry as a labeling index in mouse lung tissue. Cell Tissue Res. 256, 167.
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
<6 0 0
<6 2.2 0.02
1280 72.8 5.1
352 -
<6
Key: PBL, Peripheral blood lymphocyte; Cell concentration of PBL: 2.5 × 107/ml; Protein concentration: RTE 2.5 mg/ml, RKE 46 mg/ml.
19A2, TO17 and TO30 and only partially blocked by 19F4. On the other hand, binding of 19A2, TO17 and TO30 were markedly blocked by each other and moderately blocked by 19F4 and TOB7. Binding of 19F4 was blocked more than 80% by high concentrations of all other monoclonal antibodies except TOB7.
Sandwich type ELISA for detection of PCNA According to the results of epitope analysis of monoclonal antibodies, sandwich type ELISA for
detection of PCNA was performed using biotinylated TOB7 and TO17. Fig. 6 shows the results of a sandwich assay to determine the concentration of TO17 coated on a well of Immunoplate. When serial dilutions of purified PCNA were reacted and their binding to TO17 detected by biotinylated TOB7, absorbance of 405 nm increased in parallel with the concentration of TO17, except that there was no significant difference between 20 /xg/ml and 40 /xg/ml. The concentration of biotinylated TOB7 in this system was also studied. If
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Fig. 3. Immunoblotting of monoclonal antibodies. Purified PCNA from calf thymus was detected by human anti-PCNA antibody from lupus patients and by monoclonal antibodies. Lanes: 1, molecular weight markers stained by amido black; 2, purified PCNA stained by amido black; 3, TOB7; 4, TO17; 5, TO30; 6, human anti-PCNA serum (AK); 7, 19F4; 8, anti-Sm monoclonal antibody.
233
Fig. 4. Immunofluorescencestaining of normal peripheral blood lymphocytes before and after mitogenic stimulation using TOB7. Before PHA stimulation (A), TOB7 did not react with lymphocyte nuclei, but cells harvested at 48 hours after PHA stimulation showed strong nuclear speckled staining (B). ( × 1200).
its concentration was more than 2 /~g/ml, P C N A was still reactive at a dilution of 0.012 # g / m l and there was no significant differences in the range to detect P C N A (data were not shown). Based on those results, it was determined that the concentration of TO17 and biotinylated TOB7 were 2 0 / x g / m l and 5 / x g / m l , respectively. Using this system, the concentration of P C N A in normal h u m a n PBL during mitogenic stimulation and the tissue extracts, R T E and RKE, were studied (Fig. 7, Table I). Before mitogenic stimulation (Fig. 7, C), P C N A concentration was less than 6 n g / m l (cell concentration, 2.5 × 107/ml). 22 h after P H A stimulation, only 2.2% of cells were in S phase of cell cycle (Fig. 7, D), and its P C N A concentration was still less than 6 n g / m l . But 72.8% of the cells harvested at 48 h after
mitogenic stimulation were in S phase and the P C N A concentration from those cells (Fig. 7, A) were estimated 1280 n g / m l . The concentrations of P C N A in R K E and R T E were also measured. P C N A concentration of R T E (Fig. 7, B) which contained stimulated lymphocytes was 352 n g / m l but that of R K E (Fig. 7, E ) was less than 6 ng/ml.
Discussion Recently, Ogata et al. (1987) described two mouse monoclonal PCNA-specific antibodies, 19A2 and 19F4. These antibodies offered a convenient tool for the detection of cell proliferation and were used to demonstrate the expression of
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Fig. 5. Inhibition tests with monoclonal antibodies against P C N A . Serial concentrations of T O B 7 ( A ) , T O 1 7 ( B ) , T O 3 0 (C), 19A2 ( D ) and 19F4 ( E ) were used to inhibit the binding of biotinylated T O B 7 (A), T O 1 7 (n), T O 3 0 (O), 19A2 (II) and 19F4 (e) to P C N A coated on ELISA plates.
PCNA in various human cell lines and blast-transformed cells by immunofluorescence microscopy and flow cytometry (Kurki et al., 1988). These antibodies were also used to analyze the proliferative grade in fixed, embedded human tumor tissues (Garcia et al., 1989) and to estimate the index of cell proliferation in mouse lung tissue (Thaete et al., 1989). One of our monoclonal antibodies, TOB7, was applied to detect activated peripheral blood lymphocytes in lupus patients, and it was demonstrated that the index of P C N A expression
in lymphocytes was useful for estimating the activity of the disease (Murashima et al., 1990). TOB7 was also used to study the correlation between expression of PCNA and the proliferative state of human keratinocytes (Miyagawa et al., 1989). These results suggested that monoclonal antibody to PCNA could be a reliable probe for the monitoring cell proliferation. However, the expression of PCNA in cells and tissues was shown only as an index in those reports, and could not be quantiffed. Sandwich type ELISA to detect PCNA was
235
performed when 19A2 and 19F4 were first reported (Ogata et al., 1987), but it was impossible to make the system detect PCNA using those two monoclonal antibodies because they recognized closely related epitopes. At the same time, it was shown that the epitopes which were recognized by 19A2 and 19F4 were different from those of certain human autoantibodies to PCNA. Sandwich type ELISA using the combination of mouse monoclonal antibodies and human autoantibodies to PCNA could quantify the amounts of PCNA (Ogata et al., 1987). The problem with this System was that anti-PCNA sera often contained antibodies to other nuclear antigens and it was difficult to obtain sufficient amounts of monospecific sera. Therefore, it was necessary to establish new hybridomas which produced monoclonal antibodies which recognize a different epitope from that of 19A2 and 19F4. The reaction of our monoclonal antibodies to PCNA, TO17 and TO30, was
A
t-
O
O C t~ _O
B
1.0
0.~=
~'~'
. 0.75
2.0
,
,
0.188 0.047 0.012 0.003
0.370
0.094 0.023 0.006 0.002
Concentration of PCNA (/~g/m~)
Fig. 7. Detection of PCNA in lymphocytes before and after mitogenic stimuration and in rabbit tissue extracts by sandwich type ELISA using TOB7 and TO17. PCNA was detected in lymphocyte* harvested at 48 h after PHA stimulation (A) but not in ceils before (C) or harvested 22 h after PHA stimulation (D) (cell concentration 2.5x107/ml). PCNA of RTE (protein concentration 2.5 mg/ml) (B) was also detected in much higher concentration than that of RKE (protein concentration 46 mg/ml) (E). The estimated concentrations of PCNA in those preparations were shown on Table I.
1.5
o <
0.5
~ ,'.50.'75'
0.188 ' ' 0.047r 0.012 0.003 0.375 0.094 0,023 0.006 0.002
Concentration of PCNA (/lg/rnJ~)
Fig. 6. Comparison of sandwich type ELISA for detection of PCNA at various dilutions of TO17 coated onto plate*. Serial dilutions of PCNA were reacted with wells of plates coated with four different concentrations of TO17, 5 (O), 10 (,x), 20 ([2) and 40 /~g/ml (o), and reactions were detected using biotinylated TOB7 (5/~g/ml).
strongly inhibited by 19A2 in EL1SA, but not by TOB7. These results suggested that it would be possible to make a sandwich type ELISA assay to detect PCNA using TOB7 and either 19A2, TO17 or TO30. As shown above, our system using TOB7 and TO17 could detect the increased amount of PCNA after mitogenic stimulation. Total PCNA in cells harvested at 48 h were at least 200 times higher than in cells before mitogenic stimulation. The concentration of PCNA in tissue extracts was also tested using two different organs, rabbit thymus and kidney. Anti-PCNA antibodies react
236 with m a n y lymphocytes in rabbit thymus but not with kidney tubular or glomerular cells (Miyachi et al., 1978). Our system showed clearly a great difference in the P C N A concentration between R T E and RKE. Sandwich type E L I S A using monoclonal antibodies to P C N A could thus be used to quantitatively analyze the cell proliferation and activation. The levels of cyclin ( P C N A ) in normal and transformed h u m a n cultured cells was estimated by labelling with [35S]methionine in 2D gel electrophoresis (Celis et al., 1984). In that report, levels of cyclin were expressed as the percentage of labeled cyclin to total label. Cyclin was not detectable in normal h u m a n peripheral blood lymphocytes or mouse kidney, but 0.64% of the total label corresponded to cyclin in cultured cells such as Molt-4. Those results revealed the association of cyclin to cell proliferation semi-quantitatively but not the exact amounts of cyclin in tissues and cells. Therefore, our procedure using TOB7 and TO17 is the first specific quantitative system for detecting P C N A . A l t h o u g h the results of a sandwich type E L I S A using TOB7 with TO30 or 19A2 were not shown in this paper, they were almost the same as those obtained using TOB7 and TO17. A n o t h e r system using TOB7 and 19F4 was tested because it was suggested that the epitope recognized b y 19F4 was different f o r m that of TOB7 but the sensitivity was not high enough to detect P C N A in extracts of ceils and: tissue. Cell proliferation is generally studied by measuring the rate of D N A synthesis. Tritiated thymidine ( 3 H - T d R ) and BrdU incorporation have been usually used to detect the rate of D N A synthesis. It has been reported that P C N A labeling in individual ceils resembles that obtained by 3 H - T d R autoradiography (Takasaki et al., 1981) and monoclonal BrdU-specific antibodies (Kurki, et al., 1988). The a m o u n t of P C N A detected b y our system was also related to the labeling index obtained using monoclonal antibodies to BrdU. These results suggest that the quantitative assay for P C N A expression using TOB7 and TO17 can be used as a m e t h o d to estimate cell proliferation and transformation instead of BrdU or 3 H - T d R incorporation. The sensitivity of our system might be lower than that of BrdU or 3 H - T d R incorporation, but we believe that it has the advantage for
studying cell activation or proliferation in vivo where D N A synthesis is perturbed b y drugs (Kurki et al., 1987).
Acknowledgements The authors thank Ms. Misako Suzuki and Ms. Chie K o d a m a for excellent assistance and Dr. Eng M. T a n for helpful suggestions.
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