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Automatic assay of urinary protein using Coomassie Brilliant Blue G-250
ANALYTICAL
BIOCHEMISTRY
Automatic
KIYOKO
SANO,*
*Department University
113, 197-201 (1981)
Assay of Urinary Protein Using Coomassie Blue G-250 KIYOKO
KANAMORI,*
AKIHIKO
SHIB&t
AND MAKOTO
Brilliant
NAKAOS
of Clinical Biochemistry, Faculty of Medicine, Tokyo Medical and Dental Hospital, I-S-45. Yushima, Bunkyo-Ku. Tokyo. Japan; TDepartment of Prosthetics II, Faculty of Dentistry, Showa University, and #Department of Biochemistry I, Faculty of Medicine, Tokyo Medical and Dental University, Tokyo, Japan
Received November 5, 1980 An automatic method for the protein assay using Coomassie Brilliant Blue G-250 was developed and applied to the assay of urinary proteins. In developing the automatic system, the adhesion of protein-bound dye to the walls of the flow cell and tubes was found to be the most troublesome problem, by which the baseline was shifted upwardly to give positive errors. For the purpose of preventing such adhesion, the concentration of CBB was reduced to half of that used in the manual method, glass tubes and glass coils were changed to those made of Kel-F material, and the flow cell was coated with fluorine resin. As a result, the staining with protein-bound dye was nearly completely eliminated. The final system showed satisfactory ability in performance, namely, the value of a coefficient variation for the reproducibility within run was 1.3%, that for the carry over was O-1.1%, and the recovery was 98.8%. The calibration curve was linear in a range of O-1000 @ml, and 80 samples could be processed in 1 h. Thus, the present method may serve as an efficient automatic protein analyzer for routine clinical tests of urine samples.
The use of Coomassie Brilliant Blue G250 (CBB) for microprotein assay was first introduced by Bradford in 1976 (1). Since this method is simple in performance and has high sensitivity, it has been applied to the assay of proteins in urine and cerebrospinal fluid (2,3). We have modified the original method of Bradford so as to showya linear calibration curve from 0 to 1000 CLg/ml altering the mixing ratio of a sample and reagent solution, and applied this modified method to manual routine assays of urinary protein (4). As the number of samples, which could be assayed by such a manual method was limited, we have developed the flow diagram for the autoanalyzer-ll (AAll).’ Particular, precautions were taken to 1 Abbreviations used: AA-l 1, autoanalyzer 11; CBB, Coomassie Brilliant Blue G-250; SDS, sodium dodecyl sulfate. 197
prevent the adhesion of protein-bound dye to the cell and tubes in the AA-11 system, and finally the efficient automatic system capable of assaying 80 urine samples per hour was successfully developed. The detail of the method is presented in this paper. METHODS AND MATERIALS
Materials CBB used was obtained from Sigma lot No. 57C-0126. Protein standard solution used was Monitrol IX from DADE. Human albumin and human globulin were purchased from the Institute for Chemo- and Sero-Therapies, and their protein concentrations were determined by Biuret method using the SMAC system (Technicon). For the coating of the flow cell, the fluorine resin spray, Scotchgard, (Sumitomo 3M, 0003-269718 l/070197-05302.00/O Copyright Q 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
198
SAN0 ET AL.
cell using an injector connected to the flow cell via a tube, then the resin solution was removed after 30 s, and the cell was dried at room temperature over night. Operuting Methods 1. Automatic method. Figure 1 shows FIG. I. Flow diagram for the assay of proteins in the flow diagram, in which a sample was body fluids using Coomassie Brilliant Blue G-250. first mixed with reagent solution separated by air bubbles, and then further mixed with Japan) was used. Surface active substances the reagent solution. A mixing coil, made of were Brij 35 (30%), Brij 99 (20%), Triton XKel-F tube, 2 cm in coil diameter, 55 turns, 405, Triton X-100, ARW-7, FC-134 (0.2%), was used for the mixing. The Kel-F tube Wetting Agent A, NWA, Tegitol NPX, was also used for the tubing line up to the Tween 80 and Tween 20, all from Techniphotometer cell. The flow cell used had a con, Teepol AS36 (Dai-ichi Kagaku, Japan) IS-mm light path, which was coated with and SDS (15%) from Wako Pure Chemical. the fluorine resin. The filter for 600 nm was Ethanol and phosphoric acid were of reused. The speed of assay was chosen to be agent grade from Dai-ici Kagaku. 80 samples/h, and the wash time was set to Instruments used were an autoanalyzer, be 10 s. The time required from the aspiraModel AA-II, Technicon, equipped with a tion of a sample to the photometric meadata converter, and a spectrophotometer, surement was about 6.5 min. The range of Model 100-20, Hitachi. the recorder was set to be 200 by applying the DAMP 1, and the protein standard soluMethods tion, 500 pg/ml, was calibrated to place on the 25th line of the chart paper, then the an1. Preparation of dye solutions. The original method of Bradford (1) was used alyzer was operated as usual. After comexcept that the amount of CBB was re- pleting the assay of 80 samples, distilled duced to half. Briefly, CBB 50 mg was water was introduced via the reagent line, added to 50 ml of 95% ethanol, stirred for 3 and the system was washed for 20 min. h, then 100 ml of phosphoric acid was When the baseline was elevated or no welldefined peak became available on the readded and diluted to 1 liter with distilled cording, 10% New Contrad 70 (Harleco) water. Undissolved dye was removed by was introduced through the reagent line for filtration. For the manual method, CBB was simply increased to 100 mg. The re- 5 min, followed by distilled water for 20 agents thus prepared were stable for at least min. 2. Manual method. A urine sample, 0.05 2 weeks. ml, was mixed well with 5.0 ml of the dye 2. Preparation of protein stundard solusolution. Within a period from 2 to 30 min tions. Monitrol IX, containing 66 mg/ml of after the mixing, the absorption at 590 nm total serum protein and 46 mg/ml of albuwas measured with use of the spectrophomin, was diluted with physiological saline tometer using the reagent blank (0.05 ml of to give standard solutions with concentraphysiological saline + 5.0 ml of the dye sotions of 100-1000 fig/ml. Calibration curves lution) as a reference. were drawn using these solutions for both automatic and manual methods. RESULTS AND DISCUSSION 3. Coating of the flOw cell with @ovine 1. Removal of adhering protein-bound resin. The solution of the fluorine resin, Scotchgard, was introduced into the flow dye. As the CBB combined with protein < to waste
=Yel -Blue ,rom k ’ ‘0 “k,.
URINARY
PROTEIN
easily adhered to tubes, mixing coils, and flow cell, etc., the baseline level was shifted upward, resulting in causing positive errors. In order to avoid these errors, surface active agents were tested. However, a final concentration of O.l%, Brij 35, Brij 99, Triton X-405, Triton X-100, Teepol AS36, Tergitol NPX, SDS, Tween 80, or Tween 20, was found to make the dye solution to be strongly colored in blue. Although the addition of ARW-7, FC-134, Wetting Agent A, or NWA did not produce intense blue color, the sensitivity was lowered not to give a linearity up to 1000 pg/ml. New Contrad 70 is known as an effective remover of protein-bound dyes from vessel walls similar to that of ARW-7. The reduction of the reagent concentration of l/10, l/5, and l/3 of 0.01% was attempted. However, the STD Cal. had to be set at high gain, and noise levels were also enlarged, because of insufficient reactivity. The use of the concentration of one-half, 500 pg/ml, on the other hand, was found to give the STD. Cal. value of 450, and to be in the best operating condition of the instrument. Moreover, very good steady state could be obtained. According to these results, it was dicided not to use any detergents, but to change the CBB concentration to 500 kg/ml. In spite of such reduction in the dye concentration, protein-bound dye was found to still adhere to the walls of the glass-made cell, coil, and tubes. In order to minimize this adhesion, the following three changes were made: the use of Kel-F tube instead of transfer tube, which is known to be less adhered by dyes; the use of Kel-F coil instead of glass coil, and the coating of the flow cell with fluorine resin. Although the extent of the adhesion with protein bound dye to vessel walls was considerably reduced by these alterations, the baseline was still shifted upwardly by about 20 pg/ml equivalent after the continuous assay of 10 urine samples. Therefore, 2 water samples were inserted following every 10 samples, and the baseline was readjusted
199
ASSAY
Concentration FIG.
2. Calibration
of
Protein curves.
when the second water sample was being processed. The silicon resin which is known to be a stronger coating material than the fluorine resin had not been used in this method, because it requires heating up to quite high temperature, probably resulting in the burning out of cell accessaries. The coating with the fluorine resin, on the other hand, does not require any heating. Recoating once a week with the fluorine resin was found to be sufficient for routine assays. 2. Linearity. In the manual method the ratio of the sample volume to the reagent volume was 1: 100, namely, 0.05 ml of a sample and 5.0 ml of a reagent solution. Under this condition the calibration curve showed good linearity (4). Therefore, the flow diagram for the present automatic system was designed based on this manual condition. As the volume of a sample was much smaller than that of a reagent, the how diagram was first designed in the following manner. A sample was first diluted by 10 times using a prediluting line, then mixed with a reagent solution at a ratio of 1 : 10, thus finally giving the same samplereagent ratio, i.e., 1: 100. However, it was found difficult to achieve a steady state and to restore the original baseline level in this prediluting system. Therefore, the predilution process was omitted, and the finest tube, namely 0.03 mUmin (orange-red), was
200
SAN0 ET AL.
,..................................
,.......................,............
...................................-..*
FIG. 3. Traced records in the assay of urinary protein using Coomassie Brilliant Blue G-250. 1. Calibration curves. 2. Reproducibility within run. 3. Urine samples.
employed as a sampling line and two tubes with a size of 2.00 ml/min (green-green) were used for a reagent line, the final dilution factor being 1: 133. As shown in Figs. 2 and 3-1, the calibration curve showed a good linearity in a range of O-1000 pg/ml in this tubing system. Application Protein
to the Assay of Urinary
1. Reproducibility within run. The value of the coefficient variation for the reproducibility within run was estimated to be 1.3%, indicating good reproducibility (Fig. 3-2). 2. Recovery. Recoveries were measured on samples containing 0.9 ml of urine, 0.1 ml of physiological saline, and 0.1 ml of human albumin solution at concentrations between 1 and 10 mg/ml. An average of 98.8% was obtained as the recovery of the albumin. 3. Carry over. Three standard samples for each of 100, 1000, and 100 pg/ml con-
I low 6
9cm kc
ml zbo
700 $00
600 200
500 Coo
100 kc
300 ice
203 ebo
100 9.m
centrations were assayed to estimate the degree of the carry over. The coefficient variation of the carry over from a low protein sample to a high protein sample was O%, while that from a high protein sample to a low protein sample was l.l%, indicating almost no carry over. 4. Reactivities to albumin and y-globufin. Sample solutions containing human albumin and human y-globulin in various mixing ratios in a range from 0 to 1000 pg/ml for each were prepared, and reactivities to these proteins examined. The reactivities expressed as the percentage of the reactivity to 1000 pg/ml albumin are shown in Fig. 4. The reactivity was found to decrease with increasing y-globulin content in the mixture, and the presence of y-globulin alone showed a low value of 50%. In Meulemans’ method (5), a turbidometry, however, the reactivity to y-globulin had been reported to be only 35% of that to albumin. Because it was considered smaller the reac-
0 Alb 16m,;;y
FIG. 4. Reactivities to albumin and y-globulin. CBB method. 2. Meulemans’ method.
500
1.
FIG. 5. Correlation and manual method.
1000 rag/ml
between the automatic method
URINARY
tivity difference, the more reliable the method, the method using CBB appeared to be superior to the turbidometric method. Since urinary proteins originate from serum proteins, Monitrol IX which has a similar protein ratio to that of serum, namely, 60% albumin and 40% globulin, was employed in the present method. 5. Effects of the pH of urine. No effect of the change of the pH of urine in a range from 2 to 11 was observed on the protein values assayed. This might be due to the low pH value, i. e., < 1.0, of the dye solution and also to the minute volume of a sample solution aspirated. Correlation
to the Manual
201
PROTEIN ASSAY
gression equation obtained was Y = 1.03 * X -45.5, where Y is the value by the automatic method, while X is that by the manual method, indicating a slightly lower value in the automatic method. This slight difference may be considered to be caused by greater adhesion of protein-bound dye to the cell wall in the manual method, in which higher CBB concentration was used. ACKNOWLEDGMENT We wish to express our sincere thanks to Mr. Kinichi Nakagawa of Technicon Japan for his skillful technical assistance.
REFERENCES
Method
The values obtained for 130 urine samples by the automatic method were comuared with those by the manual method. Figure 3-3 shows the chart for individual urine samples. As shown in Fig. 5, there was a good correlation between these two methods, as indicated by r = 0.974. The re-
1. Bradford, M. M. (1976) Anal.
Biochem.
72, 248-
254.
2. McIntosh, J. C. (1977)C’lin. Chem. 23, 1939-1940. 3. Johnson, J. A., Lott, J. A. (1978) Clin. Chem. 24, 1931-1933. 4. Kanamori, K., Sano, K. (1980) Japan J. C/in. Pathol.
5. Meulemans, 761.
28, 235-238.
0. (1960) C/in. Chem.
Acta
5, 757-
BIOCHEMISTRY
Automatic
KIYOKO
SANO,*
*Department University
113, 197-201 (1981)
Assay of Urinary Protein Using Coomassie Blue G-250 KIYOKO
KANAMORI,*
AKIHIKO
SHIB&t
AND MAKOTO
Brilliant
NAKAOS
of Clinical Biochemistry, Faculty of Medicine, Tokyo Medical and Dental Hospital, I-S-45. Yushima, Bunkyo-Ku. Tokyo. Japan; TDepartment of Prosthetics II, Faculty of Dentistry, Showa University, and #Department of Biochemistry I, Faculty of Medicine, Tokyo Medical and Dental University, Tokyo, Japan
Received November 5, 1980 An automatic method for the protein assay using Coomassie Brilliant Blue G-250 was developed and applied to the assay of urinary proteins. In developing the automatic system, the adhesion of protein-bound dye to the walls of the flow cell and tubes was found to be the most troublesome problem, by which the baseline was shifted upwardly to give positive errors. For the purpose of preventing such adhesion, the concentration of CBB was reduced to half of that used in the manual method, glass tubes and glass coils were changed to those made of Kel-F material, and the flow cell was coated with fluorine resin. As a result, the staining with protein-bound dye was nearly completely eliminated. The final system showed satisfactory ability in performance, namely, the value of a coefficient variation for the reproducibility within run was 1.3%, that for the carry over was O-1.1%, and the recovery was 98.8%. The calibration curve was linear in a range of O-1000 @ml, and 80 samples could be processed in 1 h. Thus, the present method may serve as an efficient automatic protein analyzer for routine clinical tests of urine samples.
The use of Coomassie Brilliant Blue G250 (CBB) for microprotein assay was first introduced by Bradford in 1976 (1). Since this method is simple in performance and has high sensitivity, it has been applied to the assay of proteins in urine and cerebrospinal fluid (2,3). We have modified the original method of Bradford so as to showya linear calibration curve from 0 to 1000 CLg/ml altering the mixing ratio of a sample and reagent solution, and applied this modified method to manual routine assays of urinary protein (4). As the number of samples, which could be assayed by such a manual method was limited, we have developed the flow diagram for the autoanalyzer-ll (AAll).’ Particular, precautions were taken to 1 Abbreviations used: AA-l 1, autoanalyzer 11; CBB, Coomassie Brilliant Blue G-250; SDS, sodium dodecyl sulfate. 197
prevent the adhesion of protein-bound dye to the cell and tubes in the AA-11 system, and finally the efficient automatic system capable of assaying 80 urine samples per hour was successfully developed. The detail of the method is presented in this paper. METHODS AND MATERIALS
Materials CBB used was obtained from Sigma lot No. 57C-0126. Protein standard solution used was Monitrol IX from DADE. Human albumin and human globulin were purchased from the Institute for Chemo- and Sero-Therapies, and their protein concentrations were determined by Biuret method using the SMAC system (Technicon). For the coating of the flow cell, the fluorine resin spray, Scotchgard, (Sumitomo 3M, 0003-269718 l/070197-05302.00/O Copyright Q 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
198
SAN0 ET AL.
cell using an injector connected to the flow cell via a tube, then the resin solution was removed after 30 s, and the cell was dried at room temperature over night. Operuting Methods 1. Automatic method. Figure 1 shows FIG. I. Flow diagram for the assay of proteins in the flow diagram, in which a sample was body fluids using Coomassie Brilliant Blue G-250. first mixed with reagent solution separated by air bubbles, and then further mixed with Japan) was used. Surface active substances the reagent solution. A mixing coil, made of were Brij 35 (30%), Brij 99 (20%), Triton XKel-F tube, 2 cm in coil diameter, 55 turns, 405, Triton X-100, ARW-7, FC-134 (0.2%), was used for the mixing. The Kel-F tube Wetting Agent A, NWA, Tegitol NPX, was also used for the tubing line up to the Tween 80 and Tween 20, all from Techniphotometer cell. The flow cell used had a con, Teepol AS36 (Dai-ichi Kagaku, Japan) IS-mm light path, which was coated with and SDS (15%) from Wako Pure Chemical. the fluorine resin. The filter for 600 nm was Ethanol and phosphoric acid were of reused. The speed of assay was chosen to be agent grade from Dai-ici Kagaku. 80 samples/h, and the wash time was set to Instruments used were an autoanalyzer, be 10 s. The time required from the aspiraModel AA-II, Technicon, equipped with a tion of a sample to the photometric meadata converter, and a spectrophotometer, surement was about 6.5 min. The range of Model 100-20, Hitachi. the recorder was set to be 200 by applying the DAMP 1, and the protein standard soluMethods tion, 500 pg/ml, was calibrated to place on the 25th line of the chart paper, then the an1. Preparation of dye solutions. The original method of Bradford (1) was used alyzer was operated as usual. After comexcept that the amount of CBB was re- pleting the assay of 80 samples, distilled duced to half. Briefly, CBB 50 mg was water was introduced via the reagent line, added to 50 ml of 95% ethanol, stirred for 3 and the system was washed for 20 min. h, then 100 ml of phosphoric acid was When the baseline was elevated or no welldefined peak became available on the readded and diluted to 1 liter with distilled cording, 10% New Contrad 70 (Harleco) water. Undissolved dye was removed by was introduced through the reagent line for filtration. For the manual method, CBB was simply increased to 100 mg. The re- 5 min, followed by distilled water for 20 agents thus prepared were stable for at least min. 2. Manual method. A urine sample, 0.05 2 weeks. ml, was mixed well with 5.0 ml of the dye 2. Preparation of protein stundard solusolution. Within a period from 2 to 30 min tions. Monitrol IX, containing 66 mg/ml of after the mixing, the absorption at 590 nm total serum protein and 46 mg/ml of albuwas measured with use of the spectrophomin, was diluted with physiological saline tometer using the reagent blank (0.05 ml of to give standard solutions with concentraphysiological saline + 5.0 ml of the dye sotions of 100-1000 fig/ml. Calibration curves lution) as a reference. were drawn using these solutions for both automatic and manual methods. RESULTS AND DISCUSSION 3. Coating of the flOw cell with @ovine 1. Removal of adhering protein-bound resin. The solution of the fluorine resin, Scotchgard, was introduced into the flow dye. As the CBB combined with protein < to waste
=Yel -Blue ,rom k ’ ‘0 “k,.
URINARY
PROTEIN
easily adhered to tubes, mixing coils, and flow cell, etc., the baseline level was shifted upward, resulting in causing positive errors. In order to avoid these errors, surface active agents were tested. However, a final concentration of O.l%, Brij 35, Brij 99, Triton X-405, Triton X-100, Teepol AS36, Tergitol NPX, SDS, Tween 80, or Tween 20, was found to make the dye solution to be strongly colored in blue. Although the addition of ARW-7, FC-134, Wetting Agent A, or NWA did not produce intense blue color, the sensitivity was lowered not to give a linearity up to 1000 pg/ml. New Contrad 70 is known as an effective remover of protein-bound dyes from vessel walls similar to that of ARW-7. The reduction of the reagent concentration of l/10, l/5, and l/3 of 0.01% was attempted. However, the STD Cal. had to be set at high gain, and noise levels were also enlarged, because of insufficient reactivity. The use of the concentration of one-half, 500 pg/ml, on the other hand, was found to give the STD. Cal. value of 450, and to be in the best operating condition of the instrument. Moreover, very good steady state could be obtained. According to these results, it was dicided not to use any detergents, but to change the CBB concentration to 500 kg/ml. In spite of such reduction in the dye concentration, protein-bound dye was found to still adhere to the walls of the glass-made cell, coil, and tubes. In order to minimize this adhesion, the following three changes were made: the use of Kel-F tube instead of transfer tube, which is known to be less adhered by dyes; the use of Kel-F coil instead of glass coil, and the coating of the flow cell with fluorine resin. Although the extent of the adhesion with protein bound dye to vessel walls was considerably reduced by these alterations, the baseline was still shifted upwardly by about 20 pg/ml equivalent after the continuous assay of 10 urine samples. Therefore, 2 water samples were inserted following every 10 samples, and the baseline was readjusted
199
ASSAY
Concentration FIG.
2. Calibration
of
Protein curves.
when the second water sample was being processed. The silicon resin which is known to be a stronger coating material than the fluorine resin had not been used in this method, because it requires heating up to quite high temperature, probably resulting in the burning out of cell accessaries. The coating with the fluorine resin, on the other hand, does not require any heating. Recoating once a week with the fluorine resin was found to be sufficient for routine assays. 2. Linearity. In the manual method the ratio of the sample volume to the reagent volume was 1: 100, namely, 0.05 ml of a sample and 5.0 ml of a reagent solution. Under this condition the calibration curve showed good linearity (4). Therefore, the flow diagram for the present automatic system was designed based on this manual condition. As the volume of a sample was much smaller than that of a reagent, the how diagram was first designed in the following manner. A sample was first diluted by 10 times using a prediluting line, then mixed with a reagent solution at a ratio of 1 : 10, thus finally giving the same samplereagent ratio, i.e., 1: 100. However, it was found difficult to achieve a steady state and to restore the original baseline level in this prediluting system. Therefore, the predilution process was omitted, and the finest tube, namely 0.03 mUmin (orange-red), was
200
SAN0 ET AL.
,..................................
,.......................,............
...................................-..*
FIG. 3. Traced records in the assay of urinary protein using Coomassie Brilliant Blue G-250. 1. Calibration curves. 2. Reproducibility within run. 3. Urine samples.
employed as a sampling line and two tubes with a size of 2.00 ml/min (green-green) were used for a reagent line, the final dilution factor being 1: 133. As shown in Figs. 2 and 3-1, the calibration curve showed a good linearity in a range of O-1000 pg/ml in this tubing system. Application Protein
to the Assay of Urinary
1. Reproducibility within run. The value of the coefficient variation for the reproducibility within run was estimated to be 1.3%, indicating good reproducibility (Fig. 3-2). 2. Recovery. Recoveries were measured on samples containing 0.9 ml of urine, 0.1 ml of physiological saline, and 0.1 ml of human albumin solution at concentrations between 1 and 10 mg/ml. An average of 98.8% was obtained as the recovery of the albumin. 3. Carry over. Three standard samples for each of 100, 1000, and 100 pg/ml con-
I low 6
9cm kc
ml zbo
700 $00
600 200
500 Coo
100 kc
300 ice
203 ebo
100 9.m
centrations were assayed to estimate the degree of the carry over. The coefficient variation of the carry over from a low protein sample to a high protein sample was O%, while that from a high protein sample to a low protein sample was l.l%, indicating almost no carry over. 4. Reactivities to albumin and y-globufin. Sample solutions containing human albumin and human y-globulin in various mixing ratios in a range from 0 to 1000 pg/ml for each were prepared, and reactivities to these proteins examined. The reactivities expressed as the percentage of the reactivity to 1000 pg/ml albumin are shown in Fig. 4. The reactivity was found to decrease with increasing y-globulin content in the mixture, and the presence of y-globulin alone showed a low value of 50%. In Meulemans’ method (5), a turbidometry, however, the reactivity to y-globulin had been reported to be only 35% of that to albumin. Because it was considered smaller the reac-
0 Alb 16m,;;y
FIG. 4. Reactivities to albumin and y-globulin. CBB method. 2. Meulemans’ method.
500
1.
FIG. 5. Correlation and manual method.
1000 rag/ml
between the automatic method
URINARY
tivity difference, the more reliable the method, the method using CBB appeared to be superior to the turbidometric method. Since urinary proteins originate from serum proteins, Monitrol IX which has a similar protein ratio to that of serum, namely, 60% albumin and 40% globulin, was employed in the present method. 5. Effects of the pH of urine. No effect of the change of the pH of urine in a range from 2 to 11 was observed on the protein values assayed. This might be due to the low pH value, i. e., < 1.0, of the dye solution and also to the minute volume of a sample solution aspirated. Correlation
to the Manual
201
PROTEIN ASSAY
gression equation obtained was Y = 1.03 * X -45.5, where Y is the value by the automatic method, while X is that by the manual method, indicating a slightly lower value in the automatic method. This slight difference may be considered to be caused by greater adhesion of protein-bound dye to the cell wall in the manual method, in which higher CBB concentration was used. ACKNOWLEDGMENT We wish to express our sincere thanks to Mr. Kinichi Nakagawa of Technicon Japan for his skillful technical assistance.
REFERENCES
Method
The values obtained for 130 urine samples by the automatic method were comuared with those by the manual method. Figure 3-3 shows the chart for individual urine samples. As shown in Fig. 5, there was a good correlation between these two methods, as indicated by r = 0.974. The re-
1. Bradford, M. M. (1976) Anal.
Biochem.
72, 248-
254.
2. McIntosh, J. C. (1977)C’lin. Chem. 23, 1939-1940. 3. Johnson, J. A., Lott, J. A. (1978) Clin. Chem. 24, 1931-1933. 4. Kanamori, K., Sano, K. (1980) Japan J. C/in. Pathol.
5. Meulemans, 761.
28, 235-238.
0. (1960) C/in. Chem.
Acta
5, 757-