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Lectin and serum-PSA interaction as a screening test for prostate cancer
Clinical Biochemistry 36 (2003) 373–376
Lectin and serum-PSA interaction as a screening test for prostate cancer Pranab S. Basua,*, Ramdhan Majhia, Sandip K. Batabyalb a
Indian Institute Of Chemical Biology 4, Raja S.C. Mullick Road, Jadavpur, Calcutta 700 032, India b B.R. Singh Hospital and Centre for Medical Education and Research, Calcutta 700 14, India
Abstract Objectives: The present investigation was designed to distinguish prostate cancer and benign prostate hyperplasia by lectin-prostate specific antigen (PSA) binding. Design & Methods: The quantitative precipitin method of concanavalin A (Con A)-carbohydrate interaction was explored with the serum PSA of patients suffering from prostatic complications. Results: The carbohydrate content in the precipitate after binding of Con A with serum PSA of prostate cancer was significantly lower than that of benign prostate hyperplasia. This may be due to altered sugar chain structure or less glycosylation of PSA in prostate cancer. Conclusions: We conclude that a serum value ⬍3.0 g/mL of the carbohydrate content of Con A-PSA precipitate indicates strong suspicion for prostate cancer and this cut off level is effective in reducing the rate of unnecessary biopsies in men with total PSA value between 4.0 to 10.0 ng/mL. © 2003 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Prostate cancer; BPH; PSA; Lectin
1. Introduction Prostate Specific Antigen (PSA) in human serum has been recently demonstrated to be more sensitive and specific than Prostatic Acid Phosphatase (PAP) for diagnosing prostate cancer [1]. A low serum PSA cut-off level of 4.0 ng/ml is used during screening to detect prostate cancer at an early stage [2]. However, an appreciable risk of false positive results with this low cut-off value was observed resulting in unnecessary biopsies for those with benign prostatic hyperplasia (BPH) [2]. Thus the techniques currently used in the immunodetection of serum PSA concentrations are of limited clinical value in the early detection of prostate cancer (PC) and its distinction from BPH. PSA is a glycoprotein and a few studies have discussed the changes in PSA sugar-chain structures or glycosylation of PSA during malignant transformation [1,3]. There were some controversy about glycosylation pattern of PSA during PC [4,5,6]. However, Concanavalin A, a nonglycoprotein lectin, binds the terminal sugar residues of glycoproteins. In this study, the quantitative precipitin method of
* Corresponding author. Tel.: ⫹2473-3491 (extn.115). E-mail address: [email protected].
Con A-carbohydrate interaction [7] was explored for differentiation of PC and BPH. 2. Methods & materials Con A was purchased from Sigma Chemical Co. All other chemicals were of analytical grade. Standard PSA were obtained from M/s Diagnostic Automation Inc, California, and USA. Serum samples of 12 male histologically proven prostate cancer patients (age between 45–75 yr) and 10 benign prostatic hyperplasia (age between 40 –70 yr) were collected from Urology Clinic of B.R.Singh Railway Hospital, Govt of India, Calcutta. 8 elderly healthy male subjects (age 45–75 yr) without any prostatic complication were also included in this work. Patients underwent digital rectal examination, transrectal ultrasound, and transrectal ultrasound- guided biopsy of the prostate.8 normal female subjects (age between 42– 60 yr) were also collected from the hospital as control. Venous blood samples for PSA measurements were drawn before rectal examination or instrumentation and stored at ⫺20°C until assays were performed. Concentrations of serum-PSA in BPH and PC were assayed by Enzyme Immunoassay (EIA) method in the
0009-9120/03/$ – see front matter © 2003 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/S0009-9120(03)00050-X
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hospital.The PSA test by EIA is a solid phase two-site immunoassay. Rabbit anti-PSA is coated on the surface of the microtitre wells and another anti-PSA monoclonal antibody labeled with horseradish peroxidase is used as the tracer. The PSA molecules present in the standard solution or serum are sandwiched between the two antibodies. Following the formation of the coated antibody-antigen-antibody-enzyme complex, the unbound antibody-enzyme tracers are removed by washing. The horseradish peroxidase activity bound in the wells is then assayed by a colorimetric reaction at 450 nm. The intensity of the color formed is proportional to the concentration of PSA present in the sample. Precipitation reactions of 0.25 ml ConA (2 mg/mL) with standard PSA of different concentrations (0.5, 1.0, 5.0, 10, 20 ng/mL) and with 0.40 ml serum PSA were performed as described by So and Goldstein [8] in presence of 0.15 M phosphate buffered saline pH 7.2 (PBS). To correct the value for the precipitation between Con A and other glycoproteins present in serum, normal female serum devoid of PSA, was used. Con A only was kept as blank. The reaction mixtures were incubated at 0°C for 60 min. After incubation period, the reaction mixtures were centrifuged at 4000 rpm to sediment the precipitate and it was washed by PBS. Supernatant was decanted; tubes were kept upside down on the filter paper to remove the last drop. The precipitate was estimated spectrophotomerically at 480 nm in terms of carbohydrate by the method of Dubois et al. [9] using mannose as standard.
3. Results The carbohydrate content obtained in the precipitate of the interaction between Con A and standard PSA confirmed the Con A-PSA binding. Con A can also form precipitate after binding with other serum glycoproteins. In the present study, the estimated mean values of carbohydrate precipitate after binding with Con A for normal female, BPH and PC sera were 9.2 ⫾ 1.0 g/mL, 21.2 ⫾ 1.6 g/mL and 12.2 ⫾ 1.6 g/mL (Figure. 1). On the other hand, total serum PSA concentration determined by Enzyme Immunoassay was negligible (0.08 ⫾ 0.02 ng/ml) in normal female (Figure. 1). Serum PSA level in normal male never exceeds 4.0 ng/mL in our laboratory and this value was taken to be the cut off level for normal male. The mean value of serum PSA in ten BPH and twelve PC samples were found (Figure. 1) to be 5.2 ⫾ 1.8 ng/mL and 6.8 ⫾ 3.2 ng/mL respectively (p ⬍ 0.10). Normal female serum which is almost devoid of PSA was taken to get the actual binding of serum PSA with Con A in BPH and PC. The mean value (9.2 ⫾ 1.0 g/mL) of carbohydrate precipitate of female serum was subtracted from the total precipitate formed between Con A and normal elderly male serum, Con A and BPH serum, and Con A and PC serum glycoproteins (Table 1).
Fig. 1. Mean carbohydrate content in precipitate of Con A-Serum glycoprotein interaction and mean Serum PSA level. (1) Normal female, (2) Benign Prostatic Hyperplasia (BPH), (3) Prostate Cancer (PC).
It was observed that the carbohydrate content (3.0 ⫾ 0.8 g/mL) in precipitate after binding of Con A with serum PSA of PC patient was significantly (p ⬍ 0.001) lower than that of BPH (12.0 ⫾ 1.4 g/mL) and that of normal elderly male (13.7 ⫾ 1.8 g/mL) but this mean carbohydrate precipitate value (13.7 ⫾ 1.8 g/mL) of normal elderly male did not show any significance with the mean carbohydrate precipitate (12.0 ⫾ 1.4 g/mL) of BPH patient (Table 1). The diagram presented graphically (Figure. 2A) with the carbohydrate precipitate values for ten individual BPH and twelve individual PC samples showed that the precipitate values of the malignant patients were significantly lower than that of benign patients. There was no overlapping of the data between PC and BPH groups. The cut off level of the precipitate of PC was ⬍3.0 g/mL.
4. Discussion The difference in Con A binding pattern with PSA in BPH and in PC patients may be due to altered posttranslational glycosylation or less glycosylation of PSA during malignant transformation of the human prostate [3,4]. The value of Con A-PSA binding in malignant PC was significantly less (p ⬍ 0.001) than that of BPH in our experiment. The carbohydrate precipitate values were segregated into two distinctly separate groups of the benign and the prostate cancer (Figure. 2A). It was observed that there was an overlap of the data of serum PSA values between PC and BPH (Figure. 2B) and hence the specificity was too low in these two groups. This implied that false positive rate was significantly high (p ⬍ 0.001) in case of BPH. So the total serum PSA assay may not be effective for diagnostic purpose. Shuhei et al. [3] used Con A, PHA-E4, PHA-L4, PS, and WGA serial lectin
P. S. Basu et al. / Clinical Biochemistry 36 (2003) 373–376
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Table 1 Mean carbohydrate content in precipitate for the interaction of Con A with total serum glycoproteins and with serum PSA Mean carbohydrate content in precipitate
Normal Female (8)† Normal Male (8)† BPH (10)† PC (12)†
Con A-serum glycoproteins (g/ml)
Con A-serum PSA* (g/ml)
9.2 ⫾ 1.0 (A) 22.9 ⫾ 1.8 (A⬘) 21.2 ⫾ 1.6 (B) 12.2 ⫾ 1.6 (C)
– 13.7 ⫾ 1.8 12.0 ⫾ 1.4 3.0 ⫾ 0.8
* Con A-serum PSA values of Normal elderly male, BPH and PC sera were obtained by subtracting (A⬘-A), (B-A) and (C-A), respectively. † Indicates the number of sample taken.
affinity column for binding of PSA from BPH and PC tissue extracts separately. In each case (BPH & PC) seven fractions were obtained according to lower and higher binding affinity of PSA to various lectins. Shuhei et al. observed that PSA in fractions 4 & 5 after binding to Con A affinity column were significantly lower in PC than BPH. Results of the present study are similar to the observations of Barak et al. [9] although the method described in this paper was different from the methodology described by them. Barak et al. [9] incubated serum
sample for both PC and BPH with Con A-Sepharose 4B and only with Sepharose 4B.The unbound nonglycosylated serum PSA in both cases were measured by TANDEM-EPSA (Hybritech) immunoenzymatic assay method [10]. They observed that the mean percentage of nonglycosylated PSA was significantly higher in PC than that in BPH but no cut off level was mentioned irrespective of total serum PSA cut off limit ⬎4.0 ng/mL for PC. Some PSA was nonspecfically bound by Sepharose 4B. Con A-Sepharose 4B preparation is also laborious and varies from preparation to preparation. So unbound nonglycosylated PSA may vary but such difficulties will not arise in carbohydrate precipitate method as described by us. We, therefore, suggest that there is a striking difference in glycosylation pattern of PSA in benign prostatic complications and prostate cancer, and conclude that the cut off value ⬍3.0 g/mL carbohydrate content in the precipitate for the interaction between Con A and serum PSA is a strong indication of malignant process. This value may be taken as a guideline in differentiating PC and BPH irrespective of total serum PSA cut off level ⬎4.0 ng/mL and thus reducing the rate of unnecessary biopsies for men with prostatic disorders.
Acknowledgments Research work was supported by funds from the Indian Institute of Chemical Biology.
References
Fig. 2. (A) The graphical representation of individual carbohydrate content of the precipitate of ten BPH samples (⽧) and twelve PC samples ( ). (B) The graphical representation of individual serum PSA of ten BPH sample (⽧) and twelve PC samples ( ).
[1] Hara M, Kimura H. Two prostate-specific antigens, ␥-seminoprotein and –macroseminoprotein. J Lab Clin Med 1989;113:541– 48. [2] Catalona WJ, Smith DS, Ratliff TL, et al. Measurement of prostatespecific antigen in serum as a screening test for prostate cancer. N Eng J Med 1991;324:1156 – 61. [3] Shuhei S, Kyoko A, Satoshi K, Ken-ichiro Y. Serial lectin affinity chromatography demonstrates altered asparagine-linked sugar-chain structures of prostate-specific antigen in human prostate carcinoma. J chromatogr B 1999;727:9 –14; 339:619-20. [4] Chan DW, Gao YM. Variants of prostate specific antigen separated by Concanavalin A. Clin Chem 1991;37:1133–34.
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[5] Marrink J, Klip H, De Jong R. Prostate-specific antigen-Con A binding ratio in benign prostate hyperplasia and prostate cancer. The Lancet 1992;339:619 –20. [6] van Pelt J, van Dieijen-Visser MP. PSA-con A binding ratio in benign prostate hyperplasia, and prostate cancer. The Lancet 1992;340:174 – 75. [7] So LL, Goldstein J. Application of the quantitative precipitin method to polysaccharide-concanavalin A interaction. J Biol Chem 1967;242: 1617–22.
[8] Dubois M, Gilles␣´ KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem 1956;28:350 –56. [9] Barak M, Mecz Y, Lurie A, Gruener N. Binding of serum prostate antigen to concanavalin A in patients with cancer or hyperplasia of the prostate. Oncology 1989;46:375–77. [10] Myrtle JF, Klimley PG, Ivor LP, Bruni JF. Clinical utility of PSA in the management of prostate cancer. Adv Cancer Diagnostics Hybritech 1986;6:1– 6.
Lectin and serum-PSA interaction as a screening test for prostate cancer Pranab S. Basua,*, Ramdhan Majhia, Sandip K. Batabyalb a
Indian Institute Of Chemical Biology 4, Raja S.C. Mullick Road, Jadavpur, Calcutta 700 032, India b B.R. Singh Hospital and Centre for Medical Education and Research, Calcutta 700 14, India
Abstract Objectives: The present investigation was designed to distinguish prostate cancer and benign prostate hyperplasia by lectin-prostate specific antigen (PSA) binding. Design & Methods: The quantitative precipitin method of concanavalin A (Con A)-carbohydrate interaction was explored with the serum PSA of patients suffering from prostatic complications. Results: The carbohydrate content in the precipitate after binding of Con A with serum PSA of prostate cancer was significantly lower than that of benign prostate hyperplasia. This may be due to altered sugar chain structure or less glycosylation of PSA in prostate cancer. Conclusions: We conclude that a serum value ⬍3.0 g/mL of the carbohydrate content of Con A-PSA precipitate indicates strong suspicion for prostate cancer and this cut off level is effective in reducing the rate of unnecessary biopsies in men with total PSA value between 4.0 to 10.0 ng/mL. © 2003 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Prostate cancer; BPH; PSA; Lectin
1. Introduction Prostate Specific Antigen (PSA) in human serum has been recently demonstrated to be more sensitive and specific than Prostatic Acid Phosphatase (PAP) for diagnosing prostate cancer [1]. A low serum PSA cut-off level of 4.0 ng/ml is used during screening to detect prostate cancer at an early stage [2]. However, an appreciable risk of false positive results with this low cut-off value was observed resulting in unnecessary biopsies for those with benign prostatic hyperplasia (BPH) [2]. Thus the techniques currently used in the immunodetection of serum PSA concentrations are of limited clinical value in the early detection of prostate cancer (PC) and its distinction from BPH. PSA is a glycoprotein and a few studies have discussed the changes in PSA sugar-chain structures or glycosylation of PSA during malignant transformation [1,3]. There were some controversy about glycosylation pattern of PSA during PC [4,5,6]. However, Concanavalin A, a nonglycoprotein lectin, binds the terminal sugar residues of glycoproteins. In this study, the quantitative precipitin method of
* Corresponding author. Tel.: ⫹2473-3491 (extn.115). E-mail address: [email protected].
Con A-carbohydrate interaction [7] was explored for differentiation of PC and BPH. 2. Methods & materials Con A was purchased from Sigma Chemical Co. All other chemicals were of analytical grade. Standard PSA were obtained from M/s Diagnostic Automation Inc, California, and USA. Serum samples of 12 male histologically proven prostate cancer patients (age between 45–75 yr) and 10 benign prostatic hyperplasia (age between 40 –70 yr) were collected from Urology Clinic of B.R.Singh Railway Hospital, Govt of India, Calcutta. 8 elderly healthy male subjects (age 45–75 yr) without any prostatic complication were also included in this work. Patients underwent digital rectal examination, transrectal ultrasound, and transrectal ultrasound- guided biopsy of the prostate.8 normal female subjects (age between 42– 60 yr) were also collected from the hospital as control. Venous blood samples for PSA measurements were drawn before rectal examination or instrumentation and stored at ⫺20°C until assays were performed. Concentrations of serum-PSA in BPH and PC were assayed by Enzyme Immunoassay (EIA) method in the
0009-9120/03/$ – see front matter © 2003 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/S0009-9120(03)00050-X
374
P. S. Basu et al. / Clinical Biochemistry 36 (2003) 373–376
hospital.The PSA test by EIA is a solid phase two-site immunoassay. Rabbit anti-PSA is coated on the surface of the microtitre wells and another anti-PSA monoclonal antibody labeled with horseradish peroxidase is used as the tracer. The PSA molecules present in the standard solution or serum are sandwiched between the two antibodies. Following the formation of the coated antibody-antigen-antibody-enzyme complex, the unbound antibody-enzyme tracers are removed by washing. The horseradish peroxidase activity bound in the wells is then assayed by a colorimetric reaction at 450 nm. The intensity of the color formed is proportional to the concentration of PSA present in the sample. Precipitation reactions of 0.25 ml ConA (2 mg/mL) with standard PSA of different concentrations (0.5, 1.0, 5.0, 10, 20 ng/mL) and with 0.40 ml serum PSA were performed as described by So and Goldstein [8] in presence of 0.15 M phosphate buffered saline pH 7.2 (PBS). To correct the value for the precipitation between Con A and other glycoproteins present in serum, normal female serum devoid of PSA, was used. Con A only was kept as blank. The reaction mixtures were incubated at 0°C for 60 min. After incubation period, the reaction mixtures were centrifuged at 4000 rpm to sediment the precipitate and it was washed by PBS. Supernatant was decanted; tubes were kept upside down on the filter paper to remove the last drop. The precipitate was estimated spectrophotomerically at 480 nm in terms of carbohydrate by the method of Dubois et al. [9] using mannose as standard.
3. Results The carbohydrate content obtained in the precipitate of the interaction between Con A and standard PSA confirmed the Con A-PSA binding. Con A can also form precipitate after binding with other serum glycoproteins. In the present study, the estimated mean values of carbohydrate precipitate after binding with Con A for normal female, BPH and PC sera were 9.2 ⫾ 1.0 g/mL, 21.2 ⫾ 1.6 g/mL and 12.2 ⫾ 1.6 g/mL (Figure. 1). On the other hand, total serum PSA concentration determined by Enzyme Immunoassay was negligible (0.08 ⫾ 0.02 ng/ml) in normal female (Figure. 1). Serum PSA level in normal male never exceeds 4.0 ng/mL in our laboratory and this value was taken to be the cut off level for normal male. The mean value of serum PSA in ten BPH and twelve PC samples were found (Figure. 1) to be 5.2 ⫾ 1.8 ng/mL and 6.8 ⫾ 3.2 ng/mL respectively (p ⬍ 0.10). Normal female serum which is almost devoid of PSA was taken to get the actual binding of serum PSA with Con A in BPH and PC. The mean value (9.2 ⫾ 1.0 g/mL) of carbohydrate precipitate of female serum was subtracted from the total precipitate formed between Con A and normal elderly male serum, Con A and BPH serum, and Con A and PC serum glycoproteins (Table 1).
Fig. 1. Mean carbohydrate content in precipitate of Con A-Serum glycoprotein interaction and mean Serum PSA level. (1) Normal female, (2) Benign Prostatic Hyperplasia (BPH), (3) Prostate Cancer (PC).
It was observed that the carbohydrate content (3.0 ⫾ 0.8 g/mL) in precipitate after binding of Con A with serum PSA of PC patient was significantly (p ⬍ 0.001) lower than that of BPH (12.0 ⫾ 1.4 g/mL) and that of normal elderly male (13.7 ⫾ 1.8 g/mL) but this mean carbohydrate precipitate value (13.7 ⫾ 1.8 g/mL) of normal elderly male did not show any significance with the mean carbohydrate precipitate (12.0 ⫾ 1.4 g/mL) of BPH patient (Table 1). The diagram presented graphically (Figure. 2A) with the carbohydrate precipitate values for ten individual BPH and twelve individual PC samples showed that the precipitate values of the malignant patients were significantly lower than that of benign patients. There was no overlapping of the data between PC and BPH groups. The cut off level of the precipitate of PC was ⬍3.0 g/mL.
4. Discussion The difference in Con A binding pattern with PSA in BPH and in PC patients may be due to altered posttranslational glycosylation or less glycosylation of PSA during malignant transformation of the human prostate [3,4]. The value of Con A-PSA binding in malignant PC was significantly less (p ⬍ 0.001) than that of BPH in our experiment. The carbohydrate precipitate values were segregated into two distinctly separate groups of the benign and the prostate cancer (Figure. 2A). It was observed that there was an overlap of the data of serum PSA values between PC and BPH (Figure. 2B) and hence the specificity was too low in these two groups. This implied that false positive rate was significantly high (p ⬍ 0.001) in case of BPH. So the total serum PSA assay may not be effective for diagnostic purpose. Shuhei et al. [3] used Con A, PHA-E4, PHA-L4, PS, and WGA serial lectin
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375
Table 1 Mean carbohydrate content in precipitate for the interaction of Con A with total serum glycoproteins and with serum PSA Mean carbohydrate content in precipitate
Normal Female (8)† Normal Male (8)† BPH (10)† PC (12)†
Con A-serum glycoproteins (g/ml)
Con A-serum PSA* (g/ml)
9.2 ⫾ 1.0 (A) 22.9 ⫾ 1.8 (A⬘) 21.2 ⫾ 1.6 (B) 12.2 ⫾ 1.6 (C)
– 13.7 ⫾ 1.8 12.0 ⫾ 1.4 3.0 ⫾ 0.8
* Con A-serum PSA values of Normal elderly male, BPH and PC sera were obtained by subtracting (A⬘-A), (B-A) and (C-A), respectively. † Indicates the number of sample taken.
affinity column for binding of PSA from BPH and PC tissue extracts separately. In each case (BPH & PC) seven fractions were obtained according to lower and higher binding affinity of PSA to various lectins. Shuhei et al. observed that PSA in fractions 4 & 5 after binding to Con A affinity column were significantly lower in PC than BPH. Results of the present study are similar to the observations of Barak et al. [9] although the method described in this paper was different from the methodology described by them. Barak et al. [9] incubated serum
sample for both PC and BPH with Con A-Sepharose 4B and only with Sepharose 4B.The unbound nonglycosylated serum PSA in both cases were measured by TANDEM-EPSA (Hybritech) immunoenzymatic assay method [10]. They observed that the mean percentage of nonglycosylated PSA was significantly higher in PC than that in BPH but no cut off level was mentioned irrespective of total serum PSA cut off limit ⬎4.0 ng/mL for PC. Some PSA was nonspecfically bound by Sepharose 4B. Con A-Sepharose 4B preparation is also laborious and varies from preparation to preparation. So unbound nonglycosylated PSA may vary but such difficulties will not arise in carbohydrate precipitate method as described by us. We, therefore, suggest that there is a striking difference in glycosylation pattern of PSA in benign prostatic complications and prostate cancer, and conclude that the cut off value ⬍3.0 g/mL carbohydrate content in the precipitate for the interaction between Con A and serum PSA is a strong indication of malignant process. This value may be taken as a guideline in differentiating PC and BPH irrespective of total serum PSA cut off level ⬎4.0 ng/mL and thus reducing the rate of unnecessary biopsies for men with prostatic disorders.
Acknowledgments Research work was supported by funds from the Indian Institute of Chemical Biology.
References
Fig. 2. (A) The graphical representation of individual carbohydrate content of the precipitate of ten BPH samples (⽧) and twelve PC samples ( ). (B) The graphical representation of individual serum PSA of ten BPH sample (⽧) and twelve PC samples ( ).
[1] Hara M, Kimura H. Two prostate-specific antigens, ␥-seminoprotein and –macroseminoprotein. J Lab Clin Med 1989;113:541– 48. [2] Catalona WJ, Smith DS, Ratliff TL, et al. Measurement of prostatespecific antigen in serum as a screening test for prostate cancer. N Eng J Med 1991;324:1156 – 61. [3] Shuhei S, Kyoko A, Satoshi K, Ken-ichiro Y. Serial lectin affinity chromatography demonstrates altered asparagine-linked sugar-chain structures of prostate-specific antigen in human prostate carcinoma. J chromatogr B 1999;727:9 –14; 339:619-20. [4] Chan DW, Gao YM. Variants of prostate specific antigen separated by Concanavalin A. Clin Chem 1991;37:1133–34.
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[5] Marrink J, Klip H, De Jong R. Prostate-specific antigen-Con A binding ratio in benign prostate hyperplasia and prostate cancer. The Lancet 1992;339:619 –20. [6] van Pelt J, van Dieijen-Visser MP. PSA-con A binding ratio in benign prostate hyperplasia, and prostate cancer. The Lancet 1992;340:174 – 75. [7] So LL, Goldstein J. Application of the quantitative precipitin method to polysaccharide-concanavalin A interaction. J Biol Chem 1967;242: 1617–22.
[8] Dubois M, Gilles␣´ KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem 1956;28:350 –56. [9] Barak M, Mecz Y, Lurie A, Gruener N. Binding of serum prostate antigen to concanavalin A in patients with cancer or hyperplasia of the prostate. Oncology 1989;46:375–77. [10] Myrtle JF, Klimley PG, Ivor LP, Bruni JF. Clinical utility of PSA in the management of prostate cancer. Adv Cancer Diagnostics Hybritech 1986;6:1– 6.