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Quantification of proteins down to nanograms per millilitre using zone immunoelectrophoresis assay (ZIA) — use of two antibodies for assay of α-fetoprotein (AFP)
Climca Chlmrca Acta. 132 (1983) 209-212 Elsewer
209
CCA 2582
Brief technical note
Quantification of proteins down to nanograms per millilitre using zone immunoelectrophoresis assay (ZIA) - use of two antibodies for assay of a-fetoprotein (AFP) Olof Vesterberg Chemrsrry Dwisron, National Board (Received
of Occupatronal Safety and Health, S - I71 84 Solna (Sweden)
July 9th. 1982; revision March 29th, 1983)
Introduction Quantitative determination of the concentrations of proteins in biological fluids is of growing importance. Using radial immunodiffusion and rocket immunoelectrophoresis, proteins can be quantified down to about 5 mg/l. With zone immunoelectrophoresis assay (ZIA) and staining of the immunoprecipitates, it was possible to measure specific proteins down to about 0.1 mg/l [ 11. We tried many procedures to lower the detection limit. When the antiserum concentration in the gel was reduced the extension of the immunoprecipitates often increased in inverse proportion [2]. However, below a certain antiserum concentration the precipitates were not visible by protein staining. Special staining procedures and staining at 40°C gave some improvement [l], as did addition of a second antibody after electrophoresis to increase the amount of protein in the immunoprecipitates. To lower further the detection limit, the immunoprecipitates formed in ZIA were reacted with enzyme-conjugated antibodies. Use of substrate and chromogen then produced coloured zones in the gels. We have modified a peroxidase staining procedure [ 31. Material and methods Triton X-100, polyethylene glycol (PEG) (M, 20000) and 3-amino-9-ethyl carbazole were from Sigma (St. Louis, MO, USA). Antibodies, normal swine serum and standard a-fetoprotein (AFP) were purchased from DAK0 (Copenhagen, Denmark). Equipment for ZIA (Quantiphor) was from Desaga (Heidelberg, FRG). Serum samples were collected from pregnant women at the Karolinska Hospital, Stockholm. The AFP concentrations were determined with a RIA kit from Behring-Werke (Marburg, FRG) and also with ZIA. ZIA was performed in principle as previously described [ 11. The gel-forming solution contained per ml 13 mg of agarose, 20 mg of 0009~8981/83/$03.00
0 1983 Elsevier Science Publishers
B.V.
PEG and 1 I_LIof rabbit antl-human a-fetoprotein Ig(i fraction. Samples were usually applied undiluted. i?O ~1 of each. If the AFP concentration was > 150 pg/l they were diluted. Standard containing 109 mg/l was diluted 1 : 300, 1 : 600, 1 : 1200, 1 :4X00 and I : 9600. All dilutions were made in a miuturr of 9 parts electrophoresis buffer with sucrose 89, (w/v) and 1 part normal herum. Serum was used to protect against protein losses. ZIA was usually performed overnight at 30 V. Then the following steps were taken. (1) The gels were placed on the Gel Bond foil resting on a table and covered with a 1.5-mm thick chromatography paper moistened with water. On top of this was placed a glass plate and a 650-g weight. Pressure was continued for 7 min and repeated after steps 3 and 4. (2) To fix the gels to the foil their lower ends were heated against the rim of a heating plate at 100°C. (3) To remove unwanted protein the gels were washed for 150 min at 40°C m electrophoresis buffer with 1 g/l Tween 80. (4) To attach enzyme-conjugated antibody the gels were incubated at 40°C for 8 min in a buffer as above with 10 pi/ml of peroxidase-conjugated swine anti-rabbit IgG and 40 Ill/ml of normal swine serum. If stored at 4°C this solution could be reused at least four times during a week. (5) After pressing. the gels could either be dried at a maximum of 60°C or directly incubated at 40°C for 4 min m chromogen soiution. This was made by dissolving 10 mg of 3-amino-9-ethyl carbazole in 2 ml of acetone and then mixing the solution with 38 ml of 0.04 mol/l of sodium acetate buffer. This mixture was filtered and 4 ~1 of hydrogen peroxide (30%) was added just before the gels were incubated at 40°C. The immunoprecipitate fronts. appearing as red bands, usually became visible after about 2 min. Too long an incubation time gave disturbing background staining. To stop the process the gels. still fixed to the foil, were transferred to a water bath. They could be kept there for weeks or be dried in the air. The distance from the upper gel surface to the front of the immunoprecipitate in each gel was measured preferably on the wet gels. Standard curves were constructed (cf. Fig. 1) and the concentration of AFP in the samples calculated. If the washings and incubations were performed at 20°C each procedure must be prolonged for at least twice the recommended times. This was also the case if higher antiserum concentrations were used for ZIA. If this concentration was lowered to 0.5 PI/ml gel, the time of washing before steps 4 and 5 could be halved. This low concentration did. however, give faint precipitates. To save time another pressing could be added in step 3, the shortening of the washing time being guided by the background stain intensity obtained. Results and discussion Typical results can be seen in Fig. 1. The relative standard deviation was typically about 5%. The detection limit (i.e. 1 mm precipitate extension from the upper gel surface) was 8 pg/l of AFP when applying 20 ~1 sample. With larger
211
APP
83-02
r= 0.5998
Fig. 1. A typical calibration curve for ZIA using standard and rabbit anti-human a-fetoprotein DAKO. The immunoprecipitates were visualised with peroxtdase-conjugated second antibodies chromogen.
from and
sample volumes up to 100 ~1, the detection limit was lowered correspondingly to about 2 pg/l. A good agreement between a standard from DAK0 and Behringwerke was obtained. The correlation with RIA was also good, as shown in Fig. 2. We
Fig. 2. Comparison of quantification of a-fetoprotein (pg/l) in 25 serum samples with ZIA. The sera were taken at various times in early pregnancy. The correlation coefficient was 0.972.
started to work with enzyme-labelled antibodies using much longer times for washings and incubations than described above. Many experimenters working with similar procedures, i.e. immunofixation, use 24 or 48 h to wash unwanted protein out of gels [4]. However, when the temperature is doubled from 20°C to 40°C. the diffusion rate is increased several fold. This allowed considerable decrease of the times needed for the different steps. This was also aided by incorporati~~n into the washing and incubation solutions of homologous normal swine serum and Triton X-100 to decreased protein-unspecific absorption. The reduced time requirement for washing may also be explained by the more favourable geometry of compressed thrn gel rods compared to gel slabs. Earlier attempts were made to lower the detection limit of rocket electrophoresis by using radiolabelled AFP [5] or a second antibody conjugated with peroxidase [4]. However, these procedure are lengthy. requiring up to 88 h. Furthermore, with these procedures it is difficult to quantify AFP below 0.1 “g/l. AFP concentrations at this level and above can be determined with ZIA simply by visualizing the immunoprecipitates with protein staining [II. More details will be published soon. Acknowledgements Thanks
are due to Miss F-I. Saranius
for skilful technical
assistance.
References Vesterberg 0. Quantification of proteins with a new sensttive method - zone immunoelectrophoresls assay @IA). Hoppe Seyler’s 2 Physiol Chem 1980; 36 1: 617-624. Vesterberg 0. Quantification of proteins with zone immunoelectrophoresis assay (ZIA) in: Allen B. Arnaud P, eds. Electrophoresis ‘81. Berlm. New York: Walter de Gruyter & Co.. 1981: X9- 101. Poolman JT, Hopman CTH, Zanen HC. lmmunochemtcal charactertzation of outer membrane complexes from Neissena Menrngrtrdrs by the SDS-polyacrylamide-gel-electrophores~s-immunoperox~dase technique (SGIP). FEMS Microbial Lett 1978; 4: 245-248. Alpert F, Coston R, Perretto J. Immuno enzymatic assay for AFP Lancet 1974; 1. 626. Norgaard-Pedersen B. A hrghly sensttive radioimmuno electrophorettc quantification of human AFP Clin Chim Acta 1973; 48: 345.
209
CCA 2582
Brief technical note
Quantification of proteins down to nanograms per millilitre using zone immunoelectrophoresis assay (ZIA) - use of two antibodies for assay of a-fetoprotein (AFP) Olof Vesterberg Chemrsrry Dwisron, National Board (Received
of Occupatronal Safety and Health, S - I71 84 Solna (Sweden)
July 9th. 1982; revision March 29th, 1983)
Introduction Quantitative determination of the concentrations of proteins in biological fluids is of growing importance. Using radial immunodiffusion and rocket immunoelectrophoresis, proteins can be quantified down to about 5 mg/l. With zone immunoelectrophoresis assay (ZIA) and staining of the immunoprecipitates, it was possible to measure specific proteins down to about 0.1 mg/l [ 11. We tried many procedures to lower the detection limit. When the antiserum concentration in the gel was reduced the extension of the immunoprecipitates often increased in inverse proportion [2]. However, below a certain antiserum concentration the precipitates were not visible by protein staining. Special staining procedures and staining at 40°C gave some improvement [l], as did addition of a second antibody after electrophoresis to increase the amount of protein in the immunoprecipitates. To lower further the detection limit, the immunoprecipitates formed in ZIA were reacted with enzyme-conjugated antibodies. Use of substrate and chromogen then produced coloured zones in the gels. We have modified a peroxidase staining procedure [ 31. Material and methods Triton X-100, polyethylene glycol (PEG) (M, 20000) and 3-amino-9-ethyl carbazole were from Sigma (St. Louis, MO, USA). Antibodies, normal swine serum and standard a-fetoprotein (AFP) were purchased from DAK0 (Copenhagen, Denmark). Equipment for ZIA (Quantiphor) was from Desaga (Heidelberg, FRG). Serum samples were collected from pregnant women at the Karolinska Hospital, Stockholm. The AFP concentrations were determined with a RIA kit from Behring-Werke (Marburg, FRG) and also with ZIA. ZIA was performed in principle as previously described [ 11. The gel-forming solution contained per ml 13 mg of agarose, 20 mg of 0009~8981/83/$03.00
0 1983 Elsevier Science Publishers
B.V.
PEG and 1 I_LIof rabbit antl-human a-fetoprotein Ig(i fraction. Samples were usually applied undiluted. i?O ~1 of each. If the AFP concentration was > 150 pg/l they were diluted. Standard containing 109 mg/l was diluted 1 : 300, 1 : 600, 1 : 1200, 1 :4X00 and I : 9600. All dilutions were made in a miuturr of 9 parts electrophoresis buffer with sucrose 89, (w/v) and 1 part normal herum. Serum was used to protect against protein losses. ZIA was usually performed overnight at 30 V. Then the following steps were taken. (1) The gels were placed on the Gel Bond foil resting on a table and covered with a 1.5-mm thick chromatography paper moistened with water. On top of this was placed a glass plate and a 650-g weight. Pressure was continued for 7 min and repeated after steps 3 and 4. (2) To fix the gels to the foil their lower ends were heated against the rim of a heating plate at 100°C. (3) To remove unwanted protein the gels were washed for 150 min at 40°C m electrophoresis buffer with 1 g/l Tween 80. (4) To attach enzyme-conjugated antibody the gels were incubated at 40°C for 8 min in a buffer as above with 10 pi/ml of peroxidase-conjugated swine anti-rabbit IgG and 40 Ill/ml of normal swine serum. If stored at 4°C this solution could be reused at least four times during a week. (5) After pressing. the gels could either be dried at a maximum of 60°C or directly incubated at 40°C for 4 min m chromogen soiution. This was made by dissolving 10 mg of 3-amino-9-ethyl carbazole in 2 ml of acetone and then mixing the solution with 38 ml of 0.04 mol/l of sodium acetate buffer. This mixture was filtered and 4 ~1 of hydrogen peroxide (30%) was added just before the gels were incubated at 40°C. The immunoprecipitate fronts. appearing as red bands, usually became visible after about 2 min. Too long an incubation time gave disturbing background staining. To stop the process the gels. still fixed to the foil, were transferred to a water bath. They could be kept there for weeks or be dried in the air. The distance from the upper gel surface to the front of the immunoprecipitate in each gel was measured preferably on the wet gels. Standard curves were constructed (cf. Fig. 1) and the concentration of AFP in the samples calculated. If the washings and incubations were performed at 20°C each procedure must be prolonged for at least twice the recommended times. This was also the case if higher antiserum concentrations were used for ZIA. If this concentration was lowered to 0.5 PI/ml gel, the time of washing before steps 4 and 5 could be halved. This low concentration did. however, give faint precipitates. To save time another pressing could be added in step 3, the shortening of the washing time being guided by the background stain intensity obtained. Results and discussion Typical results can be seen in Fig. 1. The relative standard deviation was typically about 5%. The detection limit (i.e. 1 mm precipitate extension from the upper gel surface) was 8 pg/l of AFP when applying 20 ~1 sample. With larger
211
APP
83-02
r= 0.5998
Fig. 1. A typical calibration curve for ZIA using standard and rabbit anti-human a-fetoprotein DAKO. The immunoprecipitates were visualised with peroxtdase-conjugated second antibodies chromogen.
from and
sample volumes up to 100 ~1, the detection limit was lowered correspondingly to about 2 pg/l. A good agreement between a standard from DAK0 and Behringwerke was obtained. The correlation with RIA was also good, as shown in Fig. 2. We
Fig. 2. Comparison of quantification of a-fetoprotein (pg/l) in 25 serum samples with ZIA. The sera were taken at various times in early pregnancy. The correlation coefficient was 0.972.
started to work with enzyme-labelled antibodies using much longer times for washings and incubations than described above. Many experimenters working with similar procedures, i.e. immunofixation, use 24 or 48 h to wash unwanted protein out of gels [4]. However, when the temperature is doubled from 20°C to 40°C. the diffusion rate is increased several fold. This allowed considerable decrease of the times needed for the different steps. This was also aided by incorporati~~n into the washing and incubation solutions of homologous normal swine serum and Triton X-100 to decreased protein-unspecific absorption. The reduced time requirement for washing may also be explained by the more favourable geometry of compressed thrn gel rods compared to gel slabs. Earlier attempts were made to lower the detection limit of rocket electrophoresis by using radiolabelled AFP [5] or a second antibody conjugated with peroxidase [4]. However, these procedure are lengthy. requiring up to 88 h. Furthermore, with these procedures it is difficult to quantify AFP below 0.1 “g/l. AFP concentrations at this level and above can be determined with ZIA simply by visualizing the immunoprecipitates with protein staining [II. More details will be published soon. Acknowledgements Thanks
are due to Miss F-I. Saranius
for skilful technical
assistance.
References Vesterberg 0. Quantification of proteins with a new sensttive method - zone immunoelectrophoresls assay @IA). Hoppe Seyler’s 2 Physiol Chem 1980; 36 1: 617-624. Vesterberg 0. Quantification of proteins with zone immunoelectrophoresis assay (ZIA) in: Allen B. Arnaud P, eds. Electrophoresis ‘81. Berlm. New York: Walter de Gruyter & Co.. 1981: X9- 101. Poolman JT, Hopman CTH, Zanen HC. lmmunochemtcal charactertzation of outer membrane complexes from Neissena Menrngrtrdrs by the SDS-polyacrylamide-gel-electrophores~s-immunoperox~dase technique (SGIP). FEMS Microbial Lett 1978; 4: 245-248. Alpert F, Coston R, Perretto J. Immuno enzymatic assay for AFP Lancet 1974; 1. 626. Norgaard-Pedersen B. A hrghly sensttive radioimmuno electrophorettc quantification of human AFP Clin Chim Acta 1973; 48: 345.