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Screening urine protein abnormalities by a novel test paper
ELSEVIER
Ciinica ChimicaActa 240 (1995) 83-88
Short communication
Screening urine protein abnormalities by a novel test paper K i y o k o S h i b a *a, K i y o k o K a n a m o r i b, K a z u m a s a S u g i u r a c aSchool t f Allied Health Sciences, Faculty of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan bDepartment of Clinical Biochemistry, Faculty of Medicine, Tokyo Medical and Dental University Hospital, Tokyo, Japan ¢Toyo Roshi Kaisha, Ltd., Tokyo, Japan
Received 11 August 1993; revision received 2 May 1995; accepted 8 May 1995 Keyword¢: Urine protein test paper; Acid violet 17; Urine albumin; Urine IgG; Bence-Jones
protein
1. Introduction Dipstick method is a well-established routine to detect urinary protein semiqualitatJively. The conventional test paper contains tetrabromphenol blue and citric acid buffer at pH 3 so that its color can change from yellow to blue in reaction with protein. The test, however, produces a strong reaction with albumin, but not with globulin, and so a problem remains in detecting important proteins; missing globulins in qualitative analysis for screening is a serious problem. The use of Coomassie Brilliant Blue G-250 (CBB) for urinary protein was first introduced by Bradford in 1976 [11. We have modified the original method of Bradford and applied this modified method to automatic assay of urinary protein [2]. We reported the use of Acid Violet 17 for staining proteins on cellulose acetate membrane after electrophoresis [3]. Acid violet 17 has a similar structure to CBB. Then, by using Acid Violet 17(AV-17), we developed a new urine protein test paper (UrineTP) [4]. The merit of this, AV-17, is to have equal reactivity to albumin and globulins. Favorable results were obtained on reactivity to albumin and globulins and also for Bence-Jones protein. * Correspondingauthor. 0009-8981/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 00'09-8981 (95)06120-3
84
K. Shiba et al./ Clinica Chimica Acta 240 (1995) 83-88
In the present study, the usefulness of the test was evaluated and compared with conventional protein tests. 2. Materials and methods
2.1. Subjects Portions of the 24-h urine specimens of in-patients with various disorders at Tokyo Medical and Dental University hospital were used. Since Urine-AV-17 was designed for the measurement of trace urine protein, we chose urine specimens of 92 patients who showed total urine protein in the range between 0.5 and 2.0 g/1. 2.2. Method Preparation o f A V-17. A dye solution was prepared by dissolving 0.035 g of AV-17 (SERVA, U.S.A.), 1.4 g of Metolose 60 SH50, a kind of cellulose ether of water soluble type (Shinetsu Chemical Company, Japan), 14 g of oxalic acid and 0.3 g of Pluronic F-87, a kind of polyoxy ethylene-polyoxy-propylene,(Asahi Denka Kogyo, Japan) in 100 ml of distilled water,. A sheet of Glass Filter Paper GC-50 (Toyo Roshi Kaisha, Ltd., Japan) was immersed in the dye solution for several seconds and dried at 60°C. A 5 x 5-mm square was cut out of the dried paper and attached to the tip of a 5 x 85-mm plastic sheet for use as test paper. Test procedure by the use o f A V-17. The test portion of AV-17 was immersed in each urine specimen and was immediately removed. Excess urine was removed and, 30 s later, the color of the test paper was compared with the color tone table to detect a color change (bluish green-dark purple). Six grades of urine protein, 0, 0.2, 0.3, 1, 3 and 10 g/l, were expressed in terms of -, ±, 1+, 2+, 3+, 4+, respectively. Color tone table was prepared by solution, 70% of albumin and 30% of globulin. Conventional test paper. For comparison with AV-17, two commercially available test papers were used, the Combistix (Miles, U.S.A.) and Uropiece (Fujisawa, Japan). According to manufacturer's instructions, examination was performed and the results were classified on six grades: -, ±, 1+, 2+, 3+, and 4+. Quantitative analysis of urine protein. Total protein in urine was determined by the CBB method using the Tonein-TP kit (Otsuka Assay Laboratory, Japan) and SMAC JR. (Teehnicon, U.S.A.) [2]. As a standard solution, Moni-Trol I-X (Dade, U.S.A.) was used by the biuret method using a Hitachi 736 (Hitachi, Japan) and diluted with physiological saline solution to obtain 0.5 g/l. Protein was assayed by separation using cellulose acetate membrane electrophoresis [3]. Reagents for response of the test strip to various kinds of protein. Human serum albumin, oq-acid glycoprotein, IgG were purchased from Sigma Chemical Co., St. Louis, MO (U.S.A.). Bence-Jones K and h were from Gelco Diagnostics, Inc., Shreverport, LS, U.S.A. IgA was from Paesel +Lorei GmbH and Co., Frankfurt, Germany.
3. Results 3.1. Comparison of test results between A V-I 7 and conventional test paper The urine specimens from the 92 samples were tested by both the AV-17 method
K. Shiba et al./ Clinica Chimica Acta 240 (1995) 83-88
~
~
~
-
~
0
0
i ~ + + + + + + ~ ~
i ~ <
+
+
+
+
~
~ ~
85
K. Shiba et al./ Cliniea Chimica Acta 240 (1995) 83-88
86
a n d conventional test p a p e r methods. In all specimens, the results o f the AV-17 method were equal to o r higher than those o f the conventional methods. O f the 92 samples, 14 (15.2%) from the AV-17 m e t h o d were higher than from the conventional methods by 2 grades o r more. One o f the 14 showed a dissociation o f 4 grades or more, two samples showed a dissociation o f 3 grades o r more, and 11 showed a dissociation o f 2 grades o r more. Table 1 shows the total protein levels, grades in each test p a p e r method, and each protein s e p a r a t i o n percentage in the 14 patients. The total protein level by the T o n e i n - T P m e t h o d was < 1.0 g/l in all o f the 14 patients. Patients 1, 10 a n d 11, despite their low a l b u m i n concentration, were positive for protein by the AV-17. F o r patient 1, the -/-globulin separation was 84% a n d it obviously showed the presence o f Bence-Jones protein. F o r patients 10 and 11, the p r o tein separation percentage resulted in higher than 13% in oq-, 16% in c~2- and 35% in fl-globulin separation, respectively
3.2. Sensitivity o f the test strip to various kinds o f protein As urine has various proteins, we examined the sensitivity o f the various test strips to these various kinds o f proteins (Table 2). The protein c o n c e n t r a t i o n o f each soluTable 2 Sensitivity of three test strips to various proteins
Albumin
oq-AG
IgA IgG
Bence-Jones
protein
Concentration (g/l)
AV-17
Uropiece II
Combistix
ZOO 1.00
3+ 2+ l+ 44.
0.50
3+ 2+ 1+
0.25
4-
3+ 1+ ~ 2+ 4. -
2.18
I+
-
1.09
4.
-
4.
0.55
4-
-
-
0.92 0.46
2+ 1+
4. -
1+
2.00 1.00 0.50 0.25
3+ 2 + - 3+ 1+- 2+ 4.
-
44-
2.00 1.00 0.50 0.25 2.00
2+ 1+-2+ 4. 3+
-
4. 4-
1.00
2+ - 3+
-
-
0.50
I+
-
-
0.25
4.
-
-
4.
4-
K. Shiba et al./ Clinica Chimica Acta 240 (1995) 83-88
87
tion was determined by Tonein-TP kit. For albumin, the values obtained by AV-17 and Combistix were the same as the values obtained by quantitative analysis, and the values from the Uropiece were lower in the case of concentrations of 0.50 and 0.25 gill. For IgG, the value of AV-17 was the same as the value obtained by quantitative analysis, but the values from the Combistix and Uropiece were much lower; Uropiece did not detect any concentrations whatsoever. For IgA, the value of AV-17 was the: same as the value obtained by quantitative analysis, but the values of Combistix and Uropiece were lower. For oq-acid glycoprotein, values from all three types of test papers were lower than the value obtained by quantitative analysis. However, when comparing these three types of test papers, the sensitivity of AV-17 was the. highest at concentrations of 2.18, 1.09 and 0.55 g/l. For Bence-Jones protein, the value of AV-17 was the same as the value obtained by quantitative analysis at concentrations of 2.00 and 1.00 g/l, but at concentrations of 0.50 and 0.25 g/l, they were lower. On the other hand, Combistic produced very few reactions, and none were detected on Uropiece at all. 4. Discussion Through study of reaction on principal protein in urine, it was found that, not only albumin, but also IgA and IgG, showed almost the same reaction. In addition, it might be fairly epoch-making if AV- 17 could detect Bence-Jones protein which has not been detected by conventional test paper. It was proved that AV-17 could detect protein in the actual urine if patient urine undergoes globulin separation. The conventional urine dipsticks measure mainly albumin. A new protein dipstick works on the basis of color changing of complex compounds, consisting of positively charged protein and negatively charged agent under acid conditions. The difference in the principle between the conventional urine dipsticks and a new protein dipstick gave a remarkable improvement on the sensitivity of detecting globulin. The protein separa~tion of normal serum is 70% albumin and 30% globulin, and we decided to use thJLs ratio as standard, because urine protein originates from serum protein. However, there was no problem in color/tone even with 100% of albumin. Although there were only two cases out of 92 samples, that is, patients 2 and 3, AV-17 detected them; this must be considered a positive reaction because they produced a negative reaction with conventional test papers. This result was not considered to have been caused by influence of pH, because pH had been recorded at normal levels. However, as there is a possibility that AV-17 detected other contents apart from protein, we are proceeding with further investigations. A weakness with AV-17 is that detecting color-change in reaction can be a little difficult. This weakness is not solved even by varying the reagent formula. Data at present seem to have tendency to result in slightly higher concentrations; also, it was a little difficult to make a clear judgment between 3+ and 4+, because data were checked with the naked eye. Consequently, it is fairly clear that even if reading a reaction with the color-detector is handy and simple, a good repeatability must be expected. However, it must be appreciated that protein was detected regardless of the fact that the concentration was less than 2.0 g/l. Furthermore, such results contribute to clinical value
88
K. Shiba et al. / Clinica Chimica Acta 240 (1995) 83-88
of urine protein separation. By combining two methods, of which one is conventional test paper capable of detecting albumin and the other is the AV-17 method capable of detecting both albumin and globulin, screening for urinary tubule injury and Bence-Jones protein could become more simple and, therefore, we firmly believe that further study could have very valuable results. References [i]
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein binding. Anal Biochem 1976;72:248-254. 12] Sano Shiba K, Kanamori K, Shiba A, Nakao M. Automatic assay of urinary protein using Coomassie Brilliant Blue G-250. Anal Biochem 1981;113:!97-201. 13] Sano Shiba K, Cho H, Nakao M, Soon PL, Shiba A. Highly sensitive method for staining protein fractions on cellulose acetate membrane with Acid Violet 17. Clin Chem 1986;32:2209. [4] Kanamori K, Shiba K, Sugiura K. Development and clinical evaluation of a new urinary protein test paper (Urine-TP). Jpn J Clin Pathol 1991;39:398-404.
Ciinica ChimicaActa 240 (1995) 83-88
Short communication
Screening urine protein abnormalities by a novel test paper K i y o k o S h i b a *a, K i y o k o K a n a m o r i b, K a z u m a s a S u g i u r a c aSchool t f Allied Health Sciences, Faculty of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan bDepartment of Clinical Biochemistry, Faculty of Medicine, Tokyo Medical and Dental University Hospital, Tokyo, Japan ¢Toyo Roshi Kaisha, Ltd., Tokyo, Japan
Received 11 August 1993; revision received 2 May 1995; accepted 8 May 1995 Keyword¢: Urine protein test paper; Acid violet 17; Urine albumin; Urine IgG; Bence-Jones
protein
1. Introduction Dipstick method is a well-established routine to detect urinary protein semiqualitatJively. The conventional test paper contains tetrabromphenol blue and citric acid buffer at pH 3 so that its color can change from yellow to blue in reaction with protein. The test, however, produces a strong reaction with albumin, but not with globulin, and so a problem remains in detecting important proteins; missing globulins in qualitative analysis for screening is a serious problem. The use of Coomassie Brilliant Blue G-250 (CBB) for urinary protein was first introduced by Bradford in 1976 [11. We have modified the original method of Bradford and applied this modified method to automatic assay of urinary protein [2]. We reported the use of Acid Violet 17 for staining proteins on cellulose acetate membrane after electrophoresis [3]. Acid violet 17 has a similar structure to CBB. Then, by using Acid Violet 17(AV-17), we developed a new urine protein test paper (UrineTP) [4]. The merit of this, AV-17, is to have equal reactivity to albumin and globulins. Favorable results were obtained on reactivity to albumin and globulins and also for Bence-Jones protein. * Correspondingauthor. 0009-8981/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 00'09-8981 (95)06120-3
84
K. Shiba et al./ Clinica Chimica Acta 240 (1995) 83-88
In the present study, the usefulness of the test was evaluated and compared with conventional protein tests. 2. Materials and methods
2.1. Subjects Portions of the 24-h urine specimens of in-patients with various disorders at Tokyo Medical and Dental University hospital were used. Since Urine-AV-17 was designed for the measurement of trace urine protein, we chose urine specimens of 92 patients who showed total urine protein in the range between 0.5 and 2.0 g/1. 2.2. Method Preparation o f A V-17. A dye solution was prepared by dissolving 0.035 g of AV-17 (SERVA, U.S.A.), 1.4 g of Metolose 60 SH50, a kind of cellulose ether of water soluble type (Shinetsu Chemical Company, Japan), 14 g of oxalic acid and 0.3 g of Pluronic F-87, a kind of polyoxy ethylene-polyoxy-propylene,(Asahi Denka Kogyo, Japan) in 100 ml of distilled water,. A sheet of Glass Filter Paper GC-50 (Toyo Roshi Kaisha, Ltd., Japan) was immersed in the dye solution for several seconds and dried at 60°C. A 5 x 5-mm square was cut out of the dried paper and attached to the tip of a 5 x 85-mm plastic sheet for use as test paper. Test procedure by the use o f A V-17. The test portion of AV-17 was immersed in each urine specimen and was immediately removed. Excess urine was removed and, 30 s later, the color of the test paper was compared with the color tone table to detect a color change (bluish green-dark purple). Six grades of urine protein, 0, 0.2, 0.3, 1, 3 and 10 g/l, were expressed in terms of -, ±, 1+, 2+, 3+, 4+, respectively. Color tone table was prepared by solution, 70% of albumin and 30% of globulin. Conventional test paper. For comparison with AV-17, two commercially available test papers were used, the Combistix (Miles, U.S.A.) and Uropiece (Fujisawa, Japan). According to manufacturer's instructions, examination was performed and the results were classified on six grades: -, ±, 1+, 2+, 3+, and 4+. Quantitative analysis of urine protein. Total protein in urine was determined by the CBB method using the Tonein-TP kit (Otsuka Assay Laboratory, Japan) and SMAC JR. (Teehnicon, U.S.A.) [2]. As a standard solution, Moni-Trol I-X (Dade, U.S.A.) was used by the biuret method using a Hitachi 736 (Hitachi, Japan) and diluted with physiological saline solution to obtain 0.5 g/l. Protein was assayed by separation using cellulose acetate membrane electrophoresis [3]. Reagents for response of the test strip to various kinds of protein. Human serum albumin, oq-acid glycoprotein, IgG were purchased from Sigma Chemical Co., St. Louis, MO (U.S.A.). Bence-Jones K and h were from Gelco Diagnostics, Inc., Shreverport, LS, U.S.A. IgA was from Paesel +Lorei GmbH and Co., Frankfurt, Germany.
3. Results 3.1. Comparison of test results between A V-I 7 and conventional test paper The urine specimens from the 92 samples were tested by both the AV-17 method
K. Shiba et al./ Clinica Chimica Acta 240 (1995) 83-88
~
~
~
-
~
0
0
i ~ + + + + + + ~ ~
i ~ <
+
+
+
+
~
~ ~
85
K. Shiba et al./ Cliniea Chimica Acta 240 (1995) 83-88
86
a n d conventional test p a p e r methods. In all specimens, the results o f the AV-17 method were equal to o r higher than those o f the conventional methods. O f the 92 samples, 14 (15.2%) from the AV-17 m e t h o d were higher than from the conventional methods by 2 grades o r more. One o f the 14 showed a dissociation o f 4 grades or more, two samples showed a dissociation o f 3 grades o r more, and 11 showed a dissociation o f 2 grades o r more. Table 1 shows the total protein levels, grades in each test p a p e r method, and each protein s e p a r a t i o n percentage in the 14 patients. The total protein level by the T o n e i n - T P m e t h o d was < 1.0 g/l in all o f the 14 patients. Patients 1, 10 a n d 11, despite their low a l b u m i n concentration, were positive for protein by the AV-17. F o r patient 1, the -/-globulin separation was 84% a n d it obviously showed the presence o f Bence-Jones protein. F o r patients 10 and 11, the p r o tein separation percentage resulted in higher than 13% in oq-, 16% in c~2- and 35% in fl-globulin separation, respectively
3.2. Sensitivity o f the test strip to various kinds o f protein As urine has various proteins, we examined the sensitivity o f the various test strips to these various kinds o f proteins (Table 2). The protein c o n c e n t r a t i o n o f each soluTable 2 Sensitivity of three test strips to various proteins
Albumin
oq-AG
IgA IgG
Bence-Jones
protein
Concentration (g/l)
AV-17
Uropiece II
Combistix
ZOO 1.00
3+ 2+ l+ 44.
0.50
3+ 2+ 1+
0.25
4-
3+ 1+ ~ 2+ 4. -
2.18
I+
-
1.09
4.
-
4.
0.55
4-
-
-
0.92 0.46
2+ 1+
4. -
1+
2.00 1.00 0.50 0.25
3+ 2 + - 3+ 1+- 2+ 4.
-
44-
2.00 1.00 0.50 0.25 2.00
2+ 1+-2+ 4. 3+
-
4. 4-
1.00
2+ - 3+
-
-
0.50
I+
-
-
0.25
4.
-
-
4.
4-
K. Shiba et al./ Clinica Chimica Acta 240 (1995) 83-88
87
tion was determined by Tonein-TP kit. For albumin, the values obtained by AV-17 and Combistix were the same as the values obtained by quantitative analysis, and the values from the Uropiece were lower in the case of concentrations of 0.50 and 0.25 gill. For IgG, the value of AV-17 was the same as the value obtained by quantitative analysis, but the values from the Combistix and Uropiece were much lower; Uropiece did not detect any concentrations whatsoever. For IgA, the value of AV-17 was the: same as the value obtained by quantitative analysis, but the values of Combistix and Uropiece were lower. For oq-acid glycoprotein, values from all three types of test papers were lower than the value obtained by quantitative analysis. However, when comparing these three types of test papers, the sensitivity of AV-17 was the. highest at concentrations of 2.18, 1.09 and 0.55 g/l. For Bence-Jones protein, the value of AV-17 was the same as the value obtained by quantitative analysis at concentrations of 2.00 and 1.00 g/l, but at concentrations of 0.50 and 0.25 g/l, they were lower. On the other hand, Combistic produced very few reactions, and none were detected on Uropiece at all. 4. Discussion Through study of reaction on principal protein in urine, it was found that, not only albumin, but also IgA and IgG, showed almost the same reaction. In addition, it might be fairly epoch-making if AV- 17 could detect Bence-Jones protein which has not been detected by conventional test paper. It was proved that AV-17 could detect protein in the actual urine if patient urine undergoes globulin separation. The conventional urine dipsticks measure mainly albumin. A new protein dipstick works on the basis of color changing of complex compounds, consisting of positively charged protein and negatively charged agent under acid conditions. The difference in the principle between the conventional urine dipsticks and a new protein dipstick gave a remarkable improvement on the sensitivity of detecting globulin. The protein separa~tion of normal serum is 70% albumin and 30% globulin, and we decided to use thJLs ratio as standard, because urine protein originates from serum protein. However, there was no problem in color/tone even with 100% of albumin. Although there were only two cases out of 92 samples, that is, patients 2 and 3, AV-17 detected them; this must be considered a positive reaction because they produced a negative reaction with conventional test papers. This result was not considered to have been caused by influence of pH, because pH had been recorded at normal levels. However, as there is a possibility that AV-17 detected other contents apart from protein, we are proceeding with further investigations. A weakness with AV-17 is that detecting color-change in reaction can be a little difficult. This weakness is not solved even by varying the reagent formula. Data at present seem to have tendency to result in slightly higher concentrations; also, it was a little difficult to make a clear judgment between 3+ and 4+, because data were checked with the naked eye. Consequently, it is fairly clear that even if reading a reaction with the color-detector is handy and simple, a good repeatability must be expected. However, it must be appreciated that protein was detected regardless of the fact that the concentration was less than 2.0 g/l. Furthermore, such results contribute to clinical value
88
K. Shiba et al. / Clinica Chimica Acta 240 (1995) 83-88
of urine protein separation. By combining two methods, of which one is conventional test paper capable of detecting albumin and the other is the AV-17 method capable of detecting both albumin and globulin, screening for urinary tubule injury and Bence-Jones protein could become more simple and, therefore, we firmly believe that further study could have very valuable results. References [i]
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein binding. Anal Biochem 1976;72:248-254. 12] Sano Shiba K, Kanamori K, Shiba A, Nakao M. Automatic assay of urinary protein using Coomassie Brilliant Blue G-250. Anal Biochem 1981;113:!97-201. 13] Sano Shiba K, Cho H, Nakao M, Soon PL, Shiba A. Highly sensitive method for staining protein fractions on cellulose acetate membrane with Acid Violet 17. Clin Chem 1986;32:2209. [4] Kanamori K, Shiba K, Sugiura K. Development and clinical evaluation of a new urinary protein test paper (Urine-TP). Jpn J Clin Pathol 1991;39:398-404.