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Antibodies to Human Prostatic Acid Phosphatase*, †
Vol. 21, No.4, April1970 Printed in U.S.A.
FERTILITY AND STERILITY
Copyright © 1970 by The Williams & Wilkins Co.
ANTIBODIES TO HUMAN PROSTATIC ACID PHOSPHATASE*· LINDA PFEIFFER, R. J. ABLIN, M. J. GONDER,
AND
t
W. A. SOANES
Division of Immunology, Millard Fillmore Hospital Research Institute, Buffalo, New York
The immunologic and immunochemical and any extraneous material. Weighed properties of normal and pathologic human glands were then cut into small pieces with prostatic fluid have been the subject of a scalpel and extracted with saline (0.15 M numerous investigations. 2 • 3• 9 • 12 • 13 Re- N aCl). Details of this preparation appear cently, it has been demonstrated that acid elsewhere. 1 The protein concentration phosphatase, one of the predominant and of saline extracts of human prostatic tismost characteristic enzymes of the pros- sue (HPE) ranged from 10 to 25 mg. of tate gland, may be detected by the appli- protein/ml., as determined by the biuret cation of specific immunochemical stain- method of Gornall, Bardawill, and David.6 ing to the gel precipitation patterns ob- Extracts of other human tissues and fluids tained between human prostatic fluid and were prepared by the same methods derabbit antiserum to human prostatic scribed for the preparation of prostatic fluid. 10• 11 In addition, prostatic acid phos- extracts. Preparation of Antisera. Procedures for phatase was demonstrated in electrophoretic patterns of prostatic fluid in the re- inoculation were as described in a previous gion corresponding to the serum a 2 - and paper. 1 Briefly, HPE at a concentration of 10 mg. of proteinjml. was emulsified with ,B-globulins. 10 In the light of these observations, the an equal volume of complete Freund's possible production of antibodies to hu- adjuvant (Difco Laboratories, Detroit, man prostatic tissue and the subsequent Mich.). Approximatel y 1 ml. of this emulutilization of the adjunct specificity of sion was injected intradermally into rabantigen-antib ody interaction and enzy- bits in multiple sites. Trial bleedings were matic analysis in the study of the normal obtained prior to each inoculation. Immunodiffus ion. Double diffusion gel human prostate gland were explored. We herein report our initial observations in our precipitation was carried out according series of immunologic studies of the hu- to the method of Ouchterlony. 8 Difco Noble agar (Difco Laboratories, Detroit, Mich.) man prostate gland. was used at 1%, with 1:10,000 Merthiolate MATERIAL AND METHODS added as a preservative. Wells were 6 mm. Preparation of Extracts. Human pros- in diameter, 3 mm. deep, and separated tate glands from a variety of clinical cases edge to edge by 3 mm. After permitting other than diseases of the prostate were precipitation to proceed for 48 hr., plates removed at autopsy some hours after were washed in a saline (0.15 M NaCl) death and stored at -20° C. until use. bath at 4 o C. for 5 days, stained for the Individual prostate glands were partially presence of acid phosphatase by using a thawed, decapsulated, and trimmed of fat modification of the Gomori histochemical * Supported by a grant from the John A. procedure,5 and photographed. Hartford Foundation, Inc. Staining. Specific staining for the prest Address requests for reprints to: R. J. Ahlin, ence of prostatic acid phosphatase was Division of Immunology, Millard Fillmore Hospicarried out through the modification 7 of tal, Buffalo, N.Y. 14209. 344
April1970
PROSTATIC ACID PHOSPHATASE
the Gomori histochemical procedure and through inhibition of staining by L-tartaric acid/ utilizing the following reagents: (1) buffer: 0.05 M acetic acid-sodium acetate, pH 5.0, with lead nitrate incorporated at a concentration of 1.33 gm./L.; (2) substrate: 3% (w/v) sodium glycerophosphate, freshly prepared prior to usage; (3) rinse: 1% (w/v) acetic acid; (4) stain: 2% (w/v) ammonium sulfide; and (5) 0.01 M L-tartaric acid. Previously washed gel diffusion plates were: (1) incubated in the buffer-substrate mixture (10: 1 v /v) at 37° C. for approximately 30 min.; (2) rinsed in distilled
anti -HPE
HPE
HPE
water, 1% acetic acid, and distilled water; and (3) stained for approximately 1-2 min. with 2% ammonium sulfide. Identification of the stained precipitates as prostatic acid phosphatase was accomplished by incorporating L-tartaric acid at a final concentration of 0.01 M in the buffer-substrate mixture. Staining was then carried out as described above. RESULTS
Rabbit antisera to HPE revealed three to six bands of preq!,pJ.t_ate, depending upon the particular extract preparation and trial bleeding used. A representative gel pre-
NHS
~-0
anti-HPE
345
1
NHS
2
. FIG. 1. Gel diffusion precipitation pattern obtained between saline extract of normal human prostatic tissue (HPE No.3), rabbit antiserum to HPE (R531j), and normal human serum (NHS). FIG. 2. Gel diffusion precipitation reaction between HPE No.3, rabbit anti-HPE serum R531j, and NHS, as seen following staining of precipitin bands for acid phosphatase.
Vol. 21
PFEIFFER ET AL.
346
anti-HPE
NHS
HPE Fw. 3. Same reactants as in Figs. 1 and 2, illustrating inhibition of staining of precipitin bands by L-tartaric acid, which demonstrates the presence of antibodies to prostatic acid phosphatase.
cipitation pattern obtained between rabbit anti-HPE serum R531j and HPE No. 3 is shown in Fig. 1. It may be seen in Fig. 2 that two of the four bands of precipitate stained for acid phosphatase. One of these bands was a dense broad band located on or adjacent to the periphery of the antibody-contain ing well. The second band, of equal density but considerably thinner in appearance, was on the periphery of the antigen-conta ining well. Both of these bands of precipitate were brown in the original agar pattern. As shown in Fig. 3, the phosphatase staining of both of these bands was inhibited by the addition of 0.01 M L-tartaric acid to the buffer-substrate mixture. Block titrations of rabbit anti-HPE and HPE revealed end-points in the range of 1:8-1:16 and 1:8-1:16, respectively. In one series of experiments, HPE still gave a positive reaction at a dilution of 1: 1024 with undiluted antiserum. Normal human serum exhibited two to three bands of precipitate when tested with rabbit anti-HPE sera (Fig. 1). However, none of these bands of precipitate stained for acid phosphatase, even if the time of incubation with substrate was extended to 1 hr. (Fig. 2). Absorption of antiHPE with 30 mg. of lyophilized normal
human serum was sufficient to remove the reaction with normal human serum but did not remove the two bands of precipitate staining for acid phosphatase. Preparations of other human tissues (heart, liver, lung, kidney, and seminal vesicle) and human fluids (prostatic fluid and seminal plasma) yielded precipitation reactions with rabbit anti-HPE sera but, with the exception of prostatic fluid, they did not develop bands of precipitate staining for acid phosphatase. Prostatic fluid yielded one and occasionally two bands of precipitation. Preliminary gel diffusion precipitation studies employing several sera of patients with benign and malignant tumors of the prostate have yielded precipitation reactions accompanied by staining for prostatic acid phosphatase. However, the relevance of these reactions remains to be determined. DISCUSSION
The possible utilization of a modification7 of the Gomori histochemical procedure for the demonstration of acid phosphatase activity in precipitates obtained by the reaction of saline extracts of human prostatic tissue and rabbit antiserum to human prostatic tissue has been presented.
April1970
PROSTATIC ACID PHOSPHATASE
As the staining was inhibited by L-tartaric acid, which is specific for prostatic acid phosphatase, but not for plasma or erythrocytic phosphatase, the data indicate that specific staining of this prostatic enzyme has been demonstrated. Similar testing of other human tissue and fluid preparations (heart, liver, lung, kidney, seminal vesicle, prostatic fluid, and seminal plasma) for phosphatase activity were negative, with the exception of prostatic fluid. Such findings appear to be in agreement with previous studies.l 0 Presently, gel diffusion precipitation reactions obtained with sera of patients with benign and malignant tumors of the prostate, in addition to the isolation of prostatic acid phosphatase from such cases for the purpose of preparing specific antisera, are m progress. SUMMARY
The adjunct specificity of antigen-antibody interaction, as observed in gel diffusion precipitation and enzymatic analysis, has been employed in the study of rabbit antisera to saline extracts of normal human prostatic tissue for the demonstration of antibodies to human prostatic acid phosphatase. Preliminary gel diffusion precipitation studies employing sera of patients with benign and malignant tumors of the prostate also yield precipitation reactions accompanied by staining for prostatic acid phosphatase. Acknowledgments. The authors gratefully acknowledge the helpful assistance of Miss Eva Jane Brandt and Mr. Paul Bronson. REFERENCES 1. ABLIN, R. J., WITEBSKY, E., GONDER, M. J., AND SoANES, W. A. The serologic specificity
347
of extracts of normal human prostatic tissue. I. In preparation. 2. BARNES, G. W., SHULMAN, s., GONDER, M. J., AND SoANES, W. A. Further studies of the immunological properties of human prostatic fluid. J Lab Clin Med 66:741, 1965. 3. BARNEs, G. W., SoANEs, W. A., GoNDER, M. J., MAMROD, L., AND SHULMAN, S. Immunologic properties of human prostatic fluid. J Lab Clin Med 61:578, 1963. 4. FISHMAN, W. H., AND LERNER, F. Method for estimating serum acid phosphatase of prostatic origin. J Biol Chem 200:89, 1953. 5. GoMORI, G. Microtechnical demonstration of phosphatase in tissue sections. Proc Soc Exp Biol Med 42:23, 1939. 6. GoRNALL, A., BARDAWILL, C., AND DAviD, M. Determination of serum proteins by means of the biuret reaction. J Biol Chem 177:751, 1949. 7. MAcMANUS, J. F., AND MowDY, R. Staining Methods: Hi8tological and Hi8tochemical. Roeber, New York, 1960, p. 163. 8. OucHTERLONY, 0. In vitro method for testing the toxin-producing capacity of diphtheria bacteria. Acta Path Microbial Scand 25:186, 1948. 9. SHuLMAN, S., BRoNsoN, P., GoNDER, M. J., AND SoANEs, W. A. Ultracentrifugal constituents of human and canine prostatic fluid. Nature (London) 197:58, 1963. 10. SHULMAN, S., MAMROD, L., GoNDER, M. J., AND SoANEs, W. A. Measurement of prostatic acid phosphatase by antibody reactions in gel diffusion. J Immun 93:474, 1964. 11. SHULMAN, s., MAMROD, L., LANG, R. W., GoNDER, M. J., AND SoANES, W. A. Measurement of prostatic acid phosphatase by gel diffusion methods. J Reprod Fertil 10:55, 1965. 12. SOANES, w. A., GABRIELLI, E., AND FELCH, K. Biochemical classification of prostatitis: Based on electrophoretic study of prostatic fluid. J Ural 85:621, 1961. 13. SoANEs, W. A., SHULMAN, S., MAMROD, L., BARNES, G. W., AND GoNDER, M. J. Electrophoretic analysis of prostatic fluid. Invest Urol1 :269, 1963.
FERTILITY AND STERILITY
Copyright © 1970 by The Williams & Wilkins Co.
ANTIBODIES TO HUMAN PROSTATIC ACID PHOSPHATASE*· LINDA PFEIFFER, R. J. ABLIN, M. J. GONDER,
AND
t
W. A. SOANES
Division of Immunology, Millard Fillmore Hospital Research Institute, Buffalo, New York
The immunologic and immunochemical and any extraneous material. Weighed properties of normal and pathologic human glands were then cut into small pieces with prostatic fluid have been the subject of a scalpel and extracted with saline (0.15 M numerous investigations. 2 • 3• 9 • 12 • 13 Re- N aCl). Details of this preparation appear cently, it has been demonstrated that acid elsewhere. 1 The protein concentration phosphatase, one of the predominant and of saline extracts of human prostatic tismost characteristic enzymes of the pros- sue (HPE) ranged from 10 to 25 mg. of tate gland, may be detected by the appli- protein/ml., as determined by the biuret cation of specific immunochemical stain- method of Gornall, Bardawill, and David.6 ing to the gel precipitation patterns ob- Extracts of other human tissues and fluids tained between human prostatic fluid and were prepared by the same methods derabbit antiserum to human prostatic scribed for the preparation of prostatic fluid. 10• 11 In addition, prostatic acid phos- extracts. Preparation of Antisera. Procedures for phatase was demonstrated in electrophoretic patterns of prostatic fluid in the re- inoculation were as described in a previous gion corresponding to the serum a 2 - and paper. 1 Briefly, HPE at a concentration of 10 mg. of proteinjml. was emulsified with ,B-globulins. 10 In the light of these observations, the an equal volume of complete Freund's possible production of antibodies to hu- adjuvant (Difco Laboratories, Detroit, man prostatic tissue and the subsequent Mich.). Approximatel y 1 ml. of this emulutilization of the adjunct specificity of sion was injected intradermally into rabantigen-antib ody interaction and enzy- bits in multiple sites. Trial bleedings were matic analysis in the study of the normal obtained prior to each inoculation. Immunodiffus ion. Double diffusion gel human prostate gland were explored. We herein report our initial observations in our precipitation was carried out according series of immunologic studies of the hu- to the method of Ouchterlony. 8 Difco Noble agar (Difco Laboratories, Detroit, Mich.) man prostate gland. was used at 1%, with 1:10,000 Merthiolate MATERIAL AND METHODS added as a preservative. Wells were 6 mm. Preparation of Extracts. Human pros- in diameter, 3 mm. deep, and separated tate glands from a variety of clinical cases edge to edge by 3 mm. After permitting other than diseases of the prostate were precipitation to proceed for 48 hr., plates removed at autopsy some hours after were washed in a saline (0.15 M NaCl) death and stored at -20° C. until use. bath at 4 o C. for 5 days, stained for the Individual prostate glands were partially presence of acid phosphatase by using a thawed, decapsulated, and trimmed of fat modification of the Gomori histochemical * Supported by a grant from the John A. procedure,5 and photographed. Hartford Foundation, Inc. Staining. Specific staining for the prest Address requests for reprints to: R. J. Ahlin, ence of prostatic acid phosphatase was Division of Immunology, Millard Fillmore Hospicarried out through the modification 7 of tal, Buffalo, N.Y. 14209. 344
April1970
PROSTATIC ACID PHOSPHATASE
the Gomori histochemical procedure and through inhibition of staining by L-tartaric acid/ utilizing the following reagents: (1) buffer: 0.05 M acetic acid-sodium acetate, pH 5.0, with lead nitrate incorporated at a concentration of 1.33 gm./L.; (2) substrate: 3% (w/v) sodium glycerophosphate, freshly prepared prior to usage; (3) rinse: 1% (w/v) acetic acid; (4) stain: 2% (w/v) ammonium sulfide; and (5) 0.01 M L-tartaric acid. Previously washed gel diffusion plates were: (1) incubated in the buffer-substrate mixture (10: 1 v /v) at 37° C. for approximately 30 min.; (2) rinsed in distilled
anti -HPE
HPE
HPE
water, 1% acetic acid, and distilled water; and (3) stained for approximately 1-2 min. with 2% ammonium sulfide. Identification of the stained precipitates as prostatic acid phosphatase was accomplished by incorporating L-tartaric acid at a final concentration of 0.01 M in the buffer-substrate mixture. Staining was then carried out as described above. RESULTS
Rabbit antisera to HPE revealed three to six bands of preq!,pJ.t_ate, depending upon the particular extract preparation and trial bleeding used. A representative gel pre-
NHS
~-0
anti-HPE
345
1
NHS
2
. FIG. 1. Gel diffusion precipitation pattern obtained between saline extract of normal human prostatic tissue (HPE No.3), rabbit antiserum to HPE (R531j), and normal human serum (NHS). FIG. 2. Gel diffusion precipitation reaction between HPE No.3, rabbit anti-HPE serum R531j, and NHS, as seen following staining of precipitin bands for acid phosphatase.
Vol. 21
PFEIFFER ET AL.
346
anti-HPE
NHS
HPE Fw. 3. Same reactants as in Figs. 1 and 2, illustrating inhibition of staining of precipitin bands by L-tartaric acid, which demonstrates the presence of antibodies to prostatic acid phosphatase.
cipitation pattern obtained between rabbit anti-HPE serum R531j and HPE No. 3 is shown in Fig. 1. It may be seen in Fig. 2 that two of the four bands of precipitate stained for acid phosphatase. One of these bands was a dense broad band located on or adjacent to the periphery of the antibody-contain ing well. The second band, of equal density but considerably thinner in appearance, was on the periphery of the antigen-conta ining well. Both of these bands of precipitate were brown in the original agar pattern. As shown in Fig. 3, the phosphatase staining of both of these bands was inhibited by the addition of 0.01 M L-tartaric acid to the buffer-substrate mixture. Block titrations of rabbit anti-HPE and HPE revealed end-points in the range of 1:8-1:16 and 1:8-1:16, respectively. In one series of experiments, HPE still gave a positive reaction at a dilution of 1: 1024 with undiluted antiserum. Normal human serum exhibited two to three bands of precipitate when tested with rabbit anti-HPE sera (Fig. 1). However, none of these bands of precipitate stained for acid phosphatase, even if the time of incubation with substrate was extended to 1 hr. (Fig. 2). Absorption of antiHPE with 30 mg. of lyophilized normal
human serum was sufficient to remove the reaction with normal human serum but did not remove the two bands of precipitate staining for acid phosphatase. Preparations of other human tissues (heart, liver, lung, kidney, and seminal vesicle) and human fluids (prostatic fluid and seminal plasma) yielded precipitation reactions with rabbit anti-HPE sera but, with the exception of prostatic fluid, they did not develop bands of precipitate staining for acid phosphatase. Prostatic fluid yielded one and occasionally two bands of precipitation. Preliminary gel diffusion precipitation studies employing several sera of patients with benign and malignant tumors of the prostate have yielded precipitation reactions accompanied by staining for prostatic acid phosphatase. However, the relevance of these reactions remains to be determined. DISCUSSION
The possible utilization of a modification7 of the Gomori histochemical procedure for the demonstration of acid phosphatase activity in precipitates obtained by the reaction of saline extracts of human prostatic tissue and rabbit antiserum to human prostatic tissue has been presented.
April1970
PROSTATIC ACID PHOSPHATASE
As the staining was inhibited by L-tartaric acid, which is specific for prostatic acid phosphatase, but not for plasma or erythrocytic phosphatase, the data indicate that specific staining of this prostatic enzyme has been demonstrated. Similar testing of other human tissue and fluid preparations (heart, liver, lung, kidney, seminal vesicle, prostatic fluid, and seminal plasma) for phosphatase activity were negative, with the exception of prostatic fluid. Such findings appear to be in agreement with previous studies.l 0 Presently, gel diffusion precipitation reactions obtained with sera of patients with benign and malignant tumors of the prostate, in addition to the isolation of prostatic acid phosphatase from such cases for the purpose of preparing specific antisera, are m progress. SUMMARY
The adjunct specificity of antigen-antibody interaction, as observed in gel diffusion precipitation and enzymatic analysis, has been employed in the study of rabbit antisera to saline extracts of normal human prostatic tissue for the demonstration of antibodies to human prostatic acid phosphatase. Preliminary gel diffusion precipitation studies employing sera of patients with benign and malignant tumors of the prostate also yield precipitation reactions accompanied by staining for prostatic acid phosphatase. Acknowledgments. The authors gratefully acknowledge the helpful assistance of Miss Eva Jane Brandt and Mr. Paul Bronson. REFERENCES 1. ABLIN, R. J., WITEBSKY, E., GONDER, M. J., AND SoANES, W. A. The serologic specificity
347
of extracts of normal human prostatic tissue. I. In preparation. 2. BARNES, G. W., SHULMAN, s., GONDER, M. J., AND SoANES, W. A. Further studies of the immunological properties of human prostatic fluid. J Lab Clin Med 66:741, 1965. 3. BARNEs, G. W., SoANEs, W. A., GoNDER, M. J., MAMROD, L., AND SHULMAN, S. Immunologic properties of human prostatic fluid. J Lab Clin Med 61:578, 1963. 4. FISHMAN, W. H., AND LERNER, F. Method for estimating serum acid phosphatase of prostatic origin. J Biol Chem 200:89, 1953. 5. GoMORI, G. Microtechnical demonstration of phosphatase in tissue sections. Proc Soc Exp Biol Med 42:23, 1939. 6. GoRNALL, A., BARDAWILL, C., AND DAviD, M. Determination of serum proteins by means of the biuret reaction. J Biol Chem 177:751, 1949. 7. MAcMANUS, J. F., AND MowDY, R. Staining Methods: Hi8tological and Hi8tochemical. Roeber, New York, 1960, p. 163. 8. OucHTERLONY, 0. In vitro method for testing the toxin-producing capacity of diphtheria bacteria. Acta Path Microbial Scand 25:186, 1948. 9. SHuLMAN, S., BRoNsoN, P., GoNDER, M. J., AND SoANEs, W. A. Ultracentrifugal constituents of human and canine prostatic fluid. Nature (London) 197:58, 1963. 10. SHULMAN, S., MAMROD, L., GoNDER, M. J., AND SoANEs, W. A. Measurement of prostatic acid phosphatase by antibody reactions in gel diffusion. J Immun 93:474, 1964. 11. SHULMAN, s., MAMROD, L., LANG, R. W., GoNDER, M. J., AND SoANES, W. A. Measurement of prostatic acid phosphatase by gel diffusion methods. J Reprod Fertil 10:55, 1965. 12. SOANES, w. A., GABRIELLI, E., AND FELCH, K. Biochemical classification of prostatitis: Based on electrophoretic study of prostatic fluid. J Ural 85:621, 1961. 13. SoANEs, W. A., SHULMAN, S., MAMROD, L., BARNES, G. W., AND GoNDER, M. J. Electrophoretic analysis of prostatic fluid. Invest Urol1 :269, 1963.