Urologische Abteilung, Marienkrankenhaus Hamburg', Institut fur Rechtsmedizin der Universitat Hamburg2, Urologische Universitatsklinik Hamburg3, Pharrnakologisches Institut, Abteilung Pharmakologie fur Pharmazeuten, Universitatskrankenhaus Eppendorf4/Germany

Monoclonal antibody to the prostate specific antigen

Monoklonaler Antikorper gegen das Prostataspezifische Antigen F. Donn', T. Bruns', L. von Meyerrinck4, C. Augustin2, M. Schulz2,H. Klosterhalfen3 and A. Schmoldt* Key wards. Monoclonal antibody, p34 antigen, prostate specific antigen, tumor marker, prostate cancer.

Summary. Somatic cell hybrids were made from mouse myeloma cells and spleen cells derived from BALB/c mice immunized with homogenized epithelial fractions of BPH. The screening by immunoperoxidase staining on human prostate and non-prostate tissue resulted in one monoclonal antibody identifying a prostate specific antigen. Upon SDSPAGE and Western blot this antigen exhibited a single band at the position of 34 kDa molecular weight. The immunoreactivity of the prostate antigen was found to be localized excluxsively in the epithelial lining of ducts and secretions of normal prostate, BPH and prostate cancer. Anti-p34 antibody reacted with an antigenic determinant on the PSA molecule and inhibited the binding of Anti-PSA antibody to PSA by about SO to 90% in the RIA. Zusammenfassung. Die Lymphozyten-Hybridisierung erfolgte zwischen Myelomzellen und Milzzellen imrnunisierter BALB/c-Mause. Zur Immunisierung wurde eine homogenisierte Epithelfraktion aus BPH-Geweben verwandt. Das Screening-Verfahren erfolgte immunhistologisch von Prostata und nicht-prostatischen Organen. Mit dieser Technik konnte ein monoklonaler Antikorper identifiziert werden, der ein Prostata-spezifisches Antigen erkennt. Das Antigen hat ein Molekulargewicht von 34 kDa und ist in den Driisenepithelien der normalen Prostata, der BPH und des Prostatakarzinoms lokalisiert. Der Antikorper reagiert mit antigenen Determinanten des PSA-Molekiils und inhibiert zu SO bis 90 % die Bindung des Anti-PSA-Antikorpers an PSA.

Introduction

PSA was first purified by Wang et al. (1979). The ammonium sulfate precipitate obtained from benign hyperplastic prostatic tissues was put through a series

of isolation steps including anion exchange chromatography on a DEAE-column, gel filtration on Sephadex G-100 and Sephadex G-75 columns. A simplified purification procedure has been described in 1982 (Wang et al.) eliminating

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Sephadex G-75 chromatography and replacing the tedious preparative gel electrophoresis by a simple pH gradient elution of the DEAE-column. The availability of the hybridoma technology (Kohler & Milstein, 1975) has revolutionized the field of detecting antigens in immunochemistry. A specific antibody can now be obtained without first purifying the antigen to homogeneity. This report describes the generating of a monoclonal antibody to a prostate specific antigen which is immunologically related to PSA. Material and methods Material Normal prostate glands were collected at necropsy from six young adults who had no history of prostate disease. Tissues of BPH, prostate cancer and lymph node metastases were collected at operation from prostatectomy and total radical prostatectomy and immediately frozen in liquid nitrogen. Non-prostatic tissues were obtained from urinary bladder, kidney, testis, thyroid and parotid gland, pancreas, adrenal gland, stomach, adrenocortical adenoma, colon, pituitary gland, bladder cancer, renal cell cancer, breast cancer, cancer of the testis and melanoma. Purified PSA was generously supplied by Dr Chu (Roswell Park Memorial Institute, Buffalo, New York/USA). Pooled seminal plasma was obtained from healthy human donors undergoing investigation for involuntary infertility at the Fertility Laboratory at the University of Hamburg. Spermatozoa and other particulate matter were removed from the seminal plasma by centrifugation at 800 x g for 15 minutes and then at 12000 x g for 20 minutes and resuspended in destilled water and dialysed at 4" against two changes of 2 1distilled water to remove any salts that might be present.

45

Serum samples for PSA measurements were obtained from ten patients with prostate cancer and bone metastases. Tumor staging was based on clinical examination, rectal biopsy of the prostate and bone scan. Diagnostic lymphadenectomy was not performed. Serum samples were separated from the blood within 2-3 h of collection and stored at -20" C until analysis. Immunization Pooled BPH tissues obtained from prostatectomy were separated into stromal and epithelial fractions by the method of Krieg et al. (1981). Based on morphological appearance, the purity of both fractions is virtually 100%. The epithelial fraction consists of strips and sheets of epithelial cells with well- preserved architecture. For immunization, a volume of the epithelial fraction containing 100 g of protein was inoculated into 3-week-old BALB/c mice on days 1, 28, and 56. Three days later, spleens were removed for cell fusion. Hybridoma technology Somatic cell hybrids were prepared by the method of Kohler & Milstein (1975). Briefly, 1 x lo8spleen cells were mixed with 1 x lo7 murine myeloma cells P3-x63 Ag8 6.5.3. (Kearney et al., 1979). The mixed cells were washed once in RPMI 1640 (Gibco Europe, Karlsruhe/FRG) without FCS. The pellet was suspended in 0.3 ml 50% PEG (Merck, Darmstadt/FRG) and centrifuged for 4 minutes at 400 x g (37"). The pellet was resuspended in RPMI 1640, centrifuged, and resuspended in about 120 ml HAT-medium (RPMI 1640 submitted with 100 U/ml penicillin- streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 20% FCS (Gibco Europe, Karlsruhe/ FRG), 3.9 mg/l thymidine, and 20 mgfl hypoxanthine (Calbiochem. Corp., La Jolla, CA/USA) and 0.152 mg/l aminopterin (Sigma, DeisenhofedFRG) and distributed in 12 microtiter plates (Greiner GmbH, NurtingedFRG). All hybridoma

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F. Donn et al.

supernatant fluids were screened for prostate specificity after 2 weeks by immunoperoxidase staining. Cloning of the hybridoma cell line was carried out by limited dilution.

Tissue section immunoperoxidase staining Cryostat tissue sections (6 pm thick) were performed as described by Naiem et al. (1982). After freezing at -80". sections were subjected to lyophilkation for 2 hours and then fixed in acetone and chloroform for 10 minutes each. Tissue sections were rinsed in TBS and then incubated with undiluted hybridoma culture supernatants for 30 minutes at room temperature. Sections were soaked in TBS 3 times for 10 minutes and incubated for 30 minutes at room temperature with 5% peroxidase conjugated rabbit-anti-mouse antiserum (Dakopatts, C0penhagedDenmar.k) diluted in 50% human serum PBS. After washing ( 3 times with TBS) sections were incubated for 30 minutes at room temperature with 5% peroxidase conjugated goat-anti-rabbit antiserum (Medac, Hamburg/FRG). After further washing (3 times, TBS), sections were incubated for 10 minutes at room temperature with substrate buffer (0.05 M Tris, dissolved in aqua dest. pH 7.6, diaminobenzidine [Rudolf KG, Kiel/FRG] 0.6 g/l and 5 pl of an 80% H,02 solution added just before use).

Hybridoma sub-iso typing We used the hybridoma sub-isotyping kit from Behringwerke (Marburg/FRG) as outlined in the manufacturer's guidelines. Culture supernatants were tested undiluted, whereas ascites fluids were diluted 1:500 and 1:1,000, respectively.

Indirect immunofluorescence Human erythrocytes (5 x lo5) of known blood group specificity were washed twice

in PBS and incubated with mAb 700 for 30 minutes at 4". After washing three times with RPMI 1640 containing 10% FCS, we applied to the erythrocytes 0.1 ml goatanti-mouse IgG FITC (fluorescein isothiocyanate, Medac, Hamburg/FRG) conjugated 1:lO fur 30 minutes at 44 After three washing steps, samples were examined with reflected-light fluorescence technique in a Zeiss fluorescence microscope^ SDS-PAGE Samples of the appropriate antigen were run on each well of a conventional vertical slab gel electrophoresis chamber (MiniProtean 11, Bio-Rad, Munich/FRG) on 6 x 8 cm gels of 0.75 mm gauge, 10% separation gels, and 3% stacking gels according to the guidelines of the manufacturer with 100 V for 5 minutes and 200 V for 45 minutes as outlined by Laemmli (1970). Samples were pretreated by addition of P-mercaptoethanol and SDS to final concentration of 5% and 1%, respectively, and placed into a boiling water bath for 10 minutes. Prestained gel electrophoresis standards of molecular weight were from Gibco-BRL /Karlsruhe/FRG) and were pretreated equally. Gels were removed from the apparatus when the bromophenolblue- stained front has migrated close to the bottom. If not stated otherwise, all electrophoresis chemicals were from BioRad (Munich/FRG).

Western blot analysis The removed gels were placed onto layers of filter paper on a semidry blotter (Sartorius, GottingedFRG), soaked in the appropriate buffer as recommended by the manufacturer. Transfer was performed to nitrocellulose BA 85 (Schleicher and Schiill, DasseVFRG) for 1hour at room temperature with 0.8 mA/

Monoclonal antibody

cm current, typically about 40 mA. Gels were removed and stained by the procedure outlined below: Washing three times for 10 minutes in TBS to remove traces of transfer buffer, 1 hour blocking of excess binding sites of nitrocellulose in 2% BSATTBS, overnight incubation in the appropriate antibody solution, washing three times for 10 minutes in TTBS, secondary antibody reaction with a goatanti-mouse alkaline phosphatase-conjugate specific for IgM and IgG (Jackson Immunoresearch Laboratories Inc., Avondale, PA/USA, obtained through Dianova, Hamburg/FRG) in a 20,000fold dilution in TTBS. Staining was obtained following three last washings for 10 minutes in TBS, p H 9.4, containing 0.4 M nitrobluetetrazolium (Sigma, DeisenhofedFRG) and 0.38 M of the toluidene salt of 5-bromo-4-chloro-indolylphosphate (Biomol, Ilvesheim/FRG) according to Leary et al. (1983).

Dot-blot analysis Samples (500 ng) of epithelial tissue fractions of BPH in 10 pl volume were spotted onto 0.8 x 6 cm strips of nitrocellulose BA 85. Each strip was placed into a well of an eight-well reservoir (Elab, OY/Finland) and washed with a series of buffers under gentle shaking on a horizontal shaker: 10 minutes washings, three times, in TBS, 1 hour blocking of excess binding sites of the nitrocellulose in 2% BSA (A7906, Sigma, DeisenhofedFRG) in TTBS, overnight incubation in 3 ml of anti-p34 antigen, washings three times for 10 minutes in TTBS, secondary antibody reaction with a goat-anti mouse alkaline phosphatase-conjugate specific for IgM and IgG in a 20,000-fold dilution in TTBS for 2 hours. Staining followed the three last washings for 10 minutes in TBS, pH 9.4, with 0.4 M nitrobluetetrazolium and 0.38 M of the toluidene salt of 5-bromo-4-chloro-indolylphosphate according to Leary et al. (1983).

47

Purification of monoclonal antibodies Female BALB/C mice were injected with 500 pl 2.6.10.14-tetramethylpentadecan (Merck, Darmstadt/FRG) on day one. On day ten 5 x lo5 - 1 x lo6 hybridoma cells in 200 1 of 0.9% NaCl were injected following the UKCCCR guidelines for the welfare of animals in experimental neoplasia (Workman et al., 1988). After 8-12 days the mice developed ascites tumors. The mice were punctured after light ether anesthesia, and ascites fluid was collected into heparinised cryotubes. The purification of the antibody was performed by anion-exchange chromatography on Q-Sepharose fast flow (Pharmacia, Freiburg/FRG) on a 1.6 x 40 cm column which was equilibrated with 50 mM Tris/HCI, pH 8.5 at 4" C. The antibody solution was centrifuged at 1000 x g and the remaining supernatant was dialysed against the equilibration buffer for 24 hours at 4" C. Any precipitated material was removed by additional centrifugation at 10 000 x g. Samples of 5 ml (equivalent to 250 mg of protein) were applied to the column. The column was washed twice with equilibration buffer. A linear gradient of 0-500 mM NaCl in 375 ml equilibration buffer was applied to the column and the antibody eluted. The fraction size was 4 ml, flow-rate was 1.1 ml/min, the adsorbance at 280 nm was recorded protein. Antibody containing fractions were collected, pooled, and concentrated by ultrafiltration (UM- 10 membrane, Berghof, TiibingedFRG). The concentrated antibody was stabilized with Na-azide (0.02% w/v) and stored at 4" C. The column was purified with 10 column volumes of 0.9 % NaCl for further use with the same antibody or the column material was washed on a filter with an acidic-alkali washing procedure as recommended by the manufacturer to avoid cross-contamination of antibodies. All liquid chromatography equipment

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F. Donn et al.

was from LKB-Pharmacia (Freiburg/ FRG).

PSA -RIA Serum PSA was measured by the PSARIA from Hybritech (San Diego, CA/ USA) according to the manufacturer’s instructions. In this RIA an anti-PSA antibody was used as primary and a 125J labelled anti-PSA antibody directed against a distinctly different antigenic site on the PSA molecule as secondary antibody. Radioactivity was measured in a LKB gamma-counter after the unbound labelled antibody was removed.

Results Analysis of hybridomas Two hundred monoclonal antibodies were screened on prostate specificity on 2500 tissue specimens by immunoperoxidase staining. This procedure resulted in one monoclonal antibody mAb 700, identifying a prostate specific antigen. The antibody is an IgG-1 subtype and not

Figure 1. Immunohistological localization of a prostate antigen. Human BPH tissue section (6 pm thick) fixed with formalin-methanol-acetone and stained using mAb 700. Original magnification= x 150.

directed against the blood group antigens ABO, Rhesus, Lewis, Kell & Lutheran studied by indirect immunofluorescence. The immunoreactivity of the prostate antigen was found to be localized exclusively in the epithelial lining of ducts and secretions (Fig. 1). Specific staining was not seen for other cell components of the prostate, including the stromal and vascular elements. No immunoreactivity could be demonstrated in non-prostatic tissues (Table 1).

I Table 1. List of nonprostatic tissues in which no p34 antigen could be demonstrated

I

Target tissue Normal urinary bladder Normal kidney Normal testis Renal cell cancer Bladder cancer Cancer of the testis Normal thyroid gland Normal parotid gland Breast cancer Normal pancreas Adrenal gland NNR Adenoma Normal stomach Lymphnode, metastatic from breast cancer Melanoma Pituitary gland Colon

positive / No. tested

o o o o o o o o o o o o o o o o o

/ / / / / / / / / / / / / / / / /

4 2 2 14 3 2 1 1 4 2 1 3 2 3 1 1 1

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49

Figure 2. Immunological localization of PSA. Human BPH tissue section (6 pm thick) fixed with formalin-methanol-acetone and stained using antip34 antibody. Original magnification= x 150.

The immunohistochemical localization of PSA in the same cell population in BPH could be clearly demonstrated (Fig. 2). On the strength of the immunohistological results we assumed that mAb 700 demonstrated similar staining patterns in prostate tissue as anti-PSA antibodies did.

Molecular nature of the prostate antigen The molecular weight of the prostate antigen was studied by SDS-PAGE and Western blot in homogenized BPH samples using culture supernatant of mAb 700. We found a single band in the 30 kDa region with a molecular mass of about 34 kDa, which we have designated as p34 antigen (Fig. 3). We found the same single band with a molecular mass of about 34 kDa when using the anti-PSA antibody in control (Fig. 3). The apparent size of the p34 antigen was unaltered when the electrophoresis was carried out under nonreducing conditions.

Purification of mAb 700 Purification of the antibody to satisfying homogeneity was achieved with anion-

Figure 3. Molecular analysis of the prostate specific antigen identified by mAb 700. SDS-PAGE and Western blot from BPH samples showing the immunoprecipitation of the antigen by mAb 700 in the 30 kDa region with a molecular mass of about 34 kDa (lane 1). Lane 2 shows the molecular weight standards with molecular weights as indicated.

exchange chromatography. Elution was performed with the equilibration buffer for 10 more fractions which was equivalent to more than one column volume to remove any unbound protein. Further elution was achieved with 500 ml of a linear 0-500 mM NaCl gradient made up in the equilibration buffer. The fractions (Fig. 4) 25-28 (peak 1) and 32-40 (peak 2) contained IgG-1 antibodies as detected by hybridoma subisotyping. The dot-blot positive fractions (Fig. 5) were pooled, concentrated on a UM 10 ultrafilter and stored under 0.05% sodium azide preservation at 4". The antibody yield with this procedure was usually about 70- 80% as determined by total protein concentration. This procedure yielded a 80- to 150fold purification of the antibody. The purity of the final preparation was confirmed by SDS-PAGE and Western blot. Peak 1 and peak 2 revealed single protein bands with molecular weights of 34 kDa (Fig. 6). The homogeneity of peak 1 and

F. Donn et al.

50

1 .o

0.0

,

4 0

20

40 fraction

60

W

Figure 4. Purification of mAb 700 by anion exchange chromatography. Anion exchange chromatogram showing dot-blot positive fractions between 25-30 (peak 1) and between 33-38 (peak 2). Solid line: protein, traced at 280 nm; dashed line: linear NaCl gradient 0-500 mM (= 375 ml).

peak 2 was 90% as judged by densitometry.

Quantitation of p34 antigen We used a semiquantitative method to assay p34 antigen in histologic sections, diluting the anti-p34 antibody in the immunoperoxidase assay. The staining was intensive, when antibody solutions were diluted 1:250 and 1:500, respectively.

Pattern of p34 antigen in normal, hyperplastic and neoplastic cells Sections of normal or hyperplastic and neoplastic glands were stained with each run as a positive control. The histologic type and the degree of the differentiation of each tissue was confirmed by a pathologist and assessed by reversing sections stained with hematoxylin-eosin. Omission of the primary antibody and substitution of the primary antibody with either normal mouse IgG or buffer solution constituted the antibody control. 6 normal prostates, 24 tissues of BPH, 13 primary prostate cancers, and 3 metastatic lesions were studied for p34 antigen and for PSA as control (Table 2). All cases of normal prostate showed intensive staining (Fig. 7a) for p34 anti-

Figure 5. Immunoblot of p34 antigen from BPH samples. p34 antigen was identified by anti-p34 antibody peak 1 (slot no.13) and peak 2 (slot no. 27).

1

2

3

i

*-

20,4

Figure 6. Purity analysis of purified anti-p34 antibody. SDS-PAGE and Western blot analysis from a BPH sample revealing a single band at 34 kDa for anti-p34 antibody peak 1 (lane 1) and peak 2 (lane 2). Lane 3 shows molecular weight standards.

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Table 2. P34 antigen and PSA immunoreactivity in prostate tissue examined by immunoperoxidase staining technique Tissue source Normal prostate Benign hyperplastic gland Prostate cancer Metastatic lesion

Number of specimens

Number of p34 antigen positive

Number of PSA positive

6

6

6

24 13 3

24 13 3

24 13 3

gen. A granular intraplasmatic reaction was found. This reaction was diffuse, often with a more intensive staining of the luminal part of the cytoplasm. Specific staining could not be detected for other cell components of the prostate including the stromal and vascular elements. All twenty-four tissues of BPH showed the identical localization of p34 antigen.

p34 immunoreactivity was found in the epithelial lining of acini, ducts and secretions. Although the staining was throughout the cytoplasm, the most intensive reactions were noted within the apical portions of each cell (Fig. 7b). Prostate cancer tissues showed positive intraplasmatic reactions for p34 antigen in many neoplastic cells (Fig. 7c). A ten-

a

b

Figure 7. Immunohistological localization of p34 antigen a. in normal prostate. Human normal prostate section (6 pm thick) fixed with formalin-methanol- acetone and stained using anti-p34 antibody. Original magnification= x 250. b. in BPH Human BPH tissue section (6 pm thick) fixed with formalin-methanol-acetone and stained using antip34 antibody. Original magnification= x 250. c. in prostate cancer Human prostate cancer section (6 pm thick) fixed with formalin-methanol- acetone and stained using anti-p34 antibody. Original magnification= x 180.

C

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F. Donn et al.

dency to less extensive staining in less differentiated tumors was seen. In individual tumors, there was a tendency for less intensive staining in lower differentiated areas. The metastatic lesions of prostate cancers demonstrated the same variability in staining intensity as the primary tumors. Peak 2 demonstrated essentially the same variability in staining intensity as peak 1 in tissues of normal prostate, BPH, prostate cancer and metastases of the primary tumor. The localization of PSA to the same epithelial cell population could be demonstrated in normal prostates (Fig. 8a), in BPH tissues (Fig. 2), and in prostate cancer (Fig. 8b).

Identification of p34 antigen in seminal plasma As the most intensive staining reaction of p34 antigen was seen throughout the cytoplasm of the epithelial cells we suggest that the p34 antigen is synthesized by the epithelial cells and secreted into the lumen of the prostate glands. This assumption was confirmed investigating the

1

2

3

- - -

4

tc-

Figure 8. Immunological localization of PSA a. in normal prostate Human normal prostate section (6 pm thick) fixed with formalin-methanol- acetone and stained using anti-PSA antibody. Original magnification= x 400. b. in prostate cancer Human prostate cancer section (6 pm thick) fixed with formalin-methanol- acetone and stained using anti-PSA antibody. Original magnification= x 180.

30

Figure 9. Identification of p34 antigen in seminal plasma SDS-PAGE and Western blot from seminal plasma showing the immunoprecipitation of p34-antigen by anti-p34 antibody peak 1 (lane l), for peak 2 (lane 2) and of PSA by anti-PSA antibody (lane 3). Lane 4 shows molecular weight standards.

presence of p34 antigen in the seminal plasma. Samples (500 ng) were run on a gel electrophoresis by SDS-PAGE and Western blot. As shown in Fig. 9 the p34 antigen was identified in seminal plasma by the anti-p34 antibody of peak 1 and 2.

Identification of PSA by anti-p34 antibody We expected that the anti-p34 antibody reacts with antigenic determinants on the

Monoclonal antibody

PSA molecule. We tested the p34-antibody by SDS-PAGE and Western blot. Samples of PSA (500 ng) in 10 pl volume were run on each well of the conventional vertical slab gel electrophoresis chamber as described before. Peak 1 and 2 exhibited single bands on the gel which were

1

2

3

b-

30

Figure 10. Identification of PSA by anti- p34 antibody SDS-PAGE and Western blot from PSA showing the immunoprecipitation of a single band at 34 kDa for anti-p34 antibody peak 1 (lane l), for peak 2 (lane 2) and for anti-PSA antibody (lane 3). Lane 4 shows molecular weight standards.

I

Table 3. Inhibition of

Case

1 2 3 4 5 6 7 8 9 10

lZSIlabelled

located at the position of 34 kDa molecular weight (Fig. 8). When we used the anti-PSA antibody as control the same single band was seen at the position of 34 kDa molecular weight (Fig. 10).

Identification of PSA in serum by anti-p34 antibody We measured PSA levels in pairs in serum of 10 patients with metastatic prostate cancer. The values were listed up in Table 3 and 4. Based on the same double monoclonal antibody assay principle we measured the same samples in a second experiment after having used anti-p34 antibody as secondary antibody. PSA levels were now reduced by about 80 to 90% of the original values by anti-p34 antibody peak 1 (Table 3) and anti-p34 antibody peak 2 (Table 4) indicating the competitive inhibition to serum PSA.

4

-

53

Discussion Antibodies to other prostate antigens generated by hybridoma technology have

anti-PSA antibody by anti-p34 antibody peak 1

Protocol

Age

Experiment I PSA-RIA

Experiment I1 PSA-RIA anti-p34 antibody as secondary antibody

890 872 450 643 712 312 577 845 345 632

76 72 69 74 78 68 72 75 74 71

23 1 278 694 459 112 108 170 450 225 275

39 56 119 77 10.4 1.o 10.8 65 38 50

The upper limit of normal is 2.5 ng/ml. In experiment I PSA levels were measured in 10 patients with metastatic prostate cancer by PSA-RIA using IZ5Ilabelled anti-PSA antibody as secondary antibody. In experiment I1 PSA levels were measured after having used anti-p34 antibody peak 1 as secondary antibody and inhibited the binding of 1251labelled anti-PSA antibody to PSA.

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F. Donn et al.

Table 4. Inhibition of 1251 labelled anti-PSA antibody by anti-p34 antibody peak 2

1 2 3 4 5 6 7 8 9 10

Protocol

Age

Experiment I PSA-RIA

890 872 450 643 712 312 577 845 345 632

76 72 69 74 78 68 72 75 74 71

23 1 278 694 459 112 108 170 450 225 275

Experiment I1 PSA-RIA anti-p34 antibody as secondary antibody 30.7 42.3 94.5 53.5 10.4 9.7 11.2 55 34 48

The upper limit of normal is 2.5 ng/ml. In experiment I PSA levels were measured in 10 patients with metastatic prostate cancer by PSA-RIA using lZsIlabelled anti-PSA antibody as secondary antibody. In experiment I1 PSA levels were measured after having used anti-p34 antibody peak 2 as secondary antibody and inhibited the binding of IZ5Ilabelled anti-PSA antibody to PSA.

been reported by Fraenkel et al. (1982b), Ware et al. (1982) and Webb et al. (1983), but none has yet been shown to bind to PSA so far. Monoclonal antibodies to PSA have previously been reported by Fraenkel et al. (1982a) using, however, purified PSA for immunization. Three antibodies defined two non-cross-blocking unique determinants on PSA, each present as one site per molecule. All of them reacted with PSA in a solid-phase radioimmunoassay and immunoprecipitated Iz5J-labelledPSA. This investigation is the first one in which a prostate specific antibody was known to originate from the epithelial cells of BPH by hybridoma technology without first purifying the antigen. From our results it follows that the p34 antigen is immunologically related to PSA. The primary structure of PSA shows a high degree of sequence homology with other serine proteases of the kallikrein family. Evidence coming up from Watt et al. (1986) and Lilja et al. (1988) that PSA is a single polypeptide chain containing 240 amino acid residues and having a calculated Mr of 26,496. A N-linked car-

bohydrate side chain is predicted at asparagine-45, and 0-linked carbohydrate side chain are possibly attached to serine69, threonine-70, and serine-7 1. Thus, we aim to determine the amino acid sequence of the p34 antigen so far and compare the primary structure with that of PSA and the other serine proteases of the kallikrein family. Acknowledgements We thank Professor G. Aumuller from the Department of Anatomy and Cell Biology, Philipps-University, Marburg/ FRG, for advice, suggestions, and immunoperoxidase staining of paraffin-embedded sections.

References Fraenkel, A.E., R. V. Rouse, M. C. Wang, T. M. Chu & L. A. Herzenberg (1982a): Monoclonal antibodies to a human prostate antigen. Cancer Res. 42: 3714-3718. Fraenkel, A.E., R. V. Rouse & L. A. Herzenberg (1982b): Human prostate specific and shared dif-

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ferentiation antigens defined by monoclonal antibodies. Proc. Natl. Acad. Sci. USA 19: 903-907. Kearney, J.F., A. Radbruch, B. Liesegang & K. Rajewsky (1979): A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J. Immunol. 123: 1548-1550. Kohler, G. & C. Milstein (1975): Continuous cultures of fused cells secreting antibody of predefined specificity. Nature (London) 256: 495- 497. Krieg, M., G. Klotzl, J . Kaufmann & K. D. Voigt (1981): Stroma of human benign prostatic hyperplasia: preferential tissue for androgen metabolism and oestrogen binding. Acta Endocrinol. 96: 422-432. Laemmli, U.K. (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 221: 680-685. Leary, J., J . Brigati & D. C. Ward (1983): Rapid and sensitive colorimetric method for visualizing biotin-labelled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots. Proc. Natl. Acad. Sci. USA 80: 4045-4049. Lilja, H. & P.-A. Abrahamsson (1988): Three predominant proteins secreted by the human prostate gland. The Prostate 12: 29-38. Naiem, M., J. Gerdes, Z. Abdulaziz, C. A. Sunderland, M. J. Allington & D. Y. Mason (1982): The value of immunohistological screening in the production of monoclonal antibodies. J. Immunol. Meth. 50: 145-160. Wang, M.C., L. A. Valenzuela, G. P. Murphy & T. M. Chu (1979): Purification of a human prostate specific antigen. Invest. Urol. L7 159-163. Wang, M.C., L. A. Valenzuela, G. P. Murphy & T. M. Chu (1982): Simplified purification procedure for human prostate antigen. Oncology 39: 1-5. Ware, J.L., D. F. Paulson, S. F. Parks & K. S. Webb (1982): Production of monoclonal antibody Pro 3 recognizing a human prostatic carcinoma antigen. Cancer Res. 42: 1215-1222.

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Watt, K.W.K., P.-J. Lee, T. M. Timkulu, W.-P. Chan & R. Loor (1986): Human prostate-specific antigen: Structural and functional similarity with serine proteases. Proc. Natl. Acad. Sci. USA 83: 3 166-3170. Webb, K.S., J. L. Ware, S. F. Parks, W. H. Briner & D. F. Paulson (1983): Monoclonal antibodies to different epitopes on a prostate tumor-associated antigen. Cancer Immunol. Immunother. 14: 155166. Workman, P., A. Balmain, J. A. Hickman, N. J. McNally, R. Raymond, C. Rowlatt, T. C. Stephens & J. Wallace (1988): UKCCCR guidelines for the welfare of animals in experimental neoplasia. Br. J. Cancer 58: 109-113. Abbreviations used:

BPH BSA DEAE FCS HAT mAb PAGE PBS PEG PSA RPMI RIA SDS TRIS TBS TTBS -

benign prostatic hypertrophy; bovine serum albumin; diethylaminoethyl; fetal calf serum; hypoxanthine-aminopterin-thymidine; monoclonal antibody; polyacrylamide gel electrophoresis; phosphate-buffered saline; polyethylene glycol; prostate specific antigen; Roswell Park Memorial Institute; radioimmunoassay; sodium dodecyl sulfate; Tris (hydroxymethyl) aminomethane; Tris-buffered saline; Tris-buffered saline 0.05% Tween 20.

+

Address: Dr F. Donn, Department of Urology, Marienhospital, Alfredstr. 9, D-2000 Hamburg 76, Germany.

Monoclonal antibody to the prostate specific antigen.

Somatic cell hybrids were made from mouse myeloma cells and spleen cells derived from BALB/c mice immunized with homogenized epithelial fractions of B...
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