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Immunodetection with streptavidin-acid phosphatase complex on Western blots
ANALYTICAL
BIOCHEMISTRY
147, 382-386
(1985)
lmmunodetection with Streptavidin-Acid Phosphatase Complex on Western Blots MARK S. BROWER, CHRISTINE L. BRAKEL, AND KIMBERLY
GARRY
Division of Hematology-Oncology, Department of Medicine and the Specialized Center fbr Research in Thrombosis, The New York Hospital-Cornell Medical Center, New York, New York, and Enzo Biochem, Inc.. New York, New York Received July 12, 1984 A technique for the detection of nanogram amounts of protein blotted onto nitrocellulose membranes has been developed using nonradioactive probes. Protein transferred to nitrocellulose membranes is detected by a specific antibody followed by incubation with biotinylated antiantibody. After addition of streptavidin-acid phosphatase complex, incubation with fast violet B salt produces sharp magenta bands. This method allows detection of bands containing less than 20 ng of protein. The procedure does not use radioactive or carcinogenic materials. 0 1985 Academic KEY
WORDS:
Press. Inc.
Western blots; immunoblot; immune assays;biotin; avidin: acid phosphatase.
The detection of antigens by nonradioactive probes has become an important analytic technique. One method, the “Western blot” (1) transfers antigen onto nitrocellulose membranes to facilitate detection. Antibodyantigen complexes on nitrocellulose membranes have been detected using radiolabeled protein A (2) or a radiolabeled anti-antibody (3). More recently, nonradioactive probes for the detection of antibody-antigen complexes have been developed. These techniques use an enzyme coupled to either protein A or to an anti-antibody (4-9). However, immunoenzymatic assays frequently are not as sensitive as similar methods using radioactive probes. Some use substances believed to be carcinogenic, such as diaminobenzidine. Additionally, many of these techniques result in the production of significant background staining. We describe herein a method for the detection of biotinylated antibody-antigen complexes on nitrocellulose membranes using streptavidin-acid phosphatase complex. Neither radioactive nor carcinogenic materials are used in this procedure. The sensitivity of this method appears to equal or exceed that 0003-2697185 Copytight ,411 rights
$3.00
0 1985 by Academic Press. Inc. of reproduction in any form reserved.
of similar techniques using radiolabeled probes. The resulting background staining is minimal. MATERIALS
AND METHODS
Antibodies and antigens. a,-Antitrypsin was purified from plasma as previously described and its concentration was determined by its extinction coefficient of 5.3 ( 10,ll). The final inhibitor preparation was free of other contaminating plasma proteinase inhibitors as analyzed by double diffusion in agarose gels with specific antibodies against az-macroglobulin, Cl -inactivator, antithrombin III, inter-cY-trypsin inhibitor, or chymotrypsin inhibitor obtained from Calbiochem-Behring Corp. Samples (7.1 mg/ ml) were stored at -70°C prior to use. Rabbit anti-human cur-antitrypsin, immunoglobulin G (IgG)’ fraction, was from Sigma Chemical Corporation. ’ Abbreviations used: IgG, immunoglobulin G; PBS, phosphate-buffered saline (100 mM NaCl, 38 mM Na2HP04, 12.5 mM NaH2P04, pH 7.4); PBS-Tween, PBS containing 0.5% (vol/vol) Tween 20; Tris-saline, 0.15 M NaCl, 10 mM Tris, pH 7.4. 382
IMMUNOBLOT
WITH STREPTAVIDIN-ACID
Sodium dodecyl sulJate-polyacrylamide slab gel electrophoresis and electroblotting technique. cYi-Antitrypsin (1.4 pg/ml) in 0.25 M Tris, pH 7.4, was mixed with an equal volume of 0.06 M Tris, 2% sodium dodecyl sulfate, 5% glycerol, 0.00 1% bromphenol blue, and 2% dithiothreitol prior to heating at 100°C for 3 min. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis ( 1- 100 ng protein/lane) was performed by the method of Laemmli (12) using a 3.8% stacking gel and a 9% separation gel. Proteins were transferred onto nitrocellulose by standard technique (13). The aIantitrypsin was electrophoretically transferred (23°C) over 1 h at 50 V/cm onto nitrocellulose paper (BA85, Schleicher and Schuell). After electroblotting was performed, the nitrocellulose was incubated overnight at 4°C in phosphate-buffered saline (PBS, 100 mM NaCl, 38 mM Na2HP04, 12.5 mM NaH2P04, pH 7.4) containing 5% bovine serum albumin (Sigma, essentially fatty acid free) and 3 mM NaN3. After it was incubated an additional 30 min in the above buffer containing 0.5% (vol/vol) Tween 20 (PBS-Tween), the blocked nitrocellulose membrane was incubated with primary antibody (rabbit anti-human al-antitrypsin) diluted 1: 100 in PBS-Tween for 1 h at 37°C with gentle rocking. The membrane was washed three times with PBS-Tween. Immunoenzymatic detection with streptavidin-acid phosphatase complex. The nitrocellulose membrane was incubated with biotinylated goat anti-rabbit IgG (Enzo Biochem) diluted 1: 1000 in PBS-Tween for 1 h at 37°C with gentle rocking. The membrane was washed three times with PBS-Tween, rinsed once in PBS, and incubated 30 min at 23°C in PBS containing 5 mM trisodium EDTA, 2% bovine serum albumin, and 0.05% (vol/ vol) Triton X- 100. Portions of the membrane were incubated with streptavidin-acid phosphatase complex [Ref. ( 14); Enzo Biochem, 19 pg/ml acid phosphatase coupled to 15.7 pg/ml streptavidin in 0.13 M NaCl, 7 mM Na2HP04, 3 mM NaH2P04, pH 7.41 diluted 1:200 in PBS, 5
PHOSPHATASE
383
mM trisodium EDTA for 1 h at 23°C (0.015 ml/cm*). After it was washed five times (5 min per wash) in 10 mM K2HP04, 0.5 M NaCl, 1 mM EDTA, pH 6.5, containing 2% bovine serum albumin and 0.5% Triton X100, the membrane was rinsed twice (2 min per rinse) in a clean dish with 0.2 M acetic acid, 0.2 M sodium acetate, pH 5.8. To detect the complex the membrane was incubated 5 min with 1 mM naphthol AS-MX phosphate (Sigma), 0.2 M sodium acetate, pH 5.8 (10 ml; approximately 0.18 ml/cm2) before 0.1 ml (1.8 &cm*) fast violet B salt (Sigma; 4 mg/ml in 0.2 M sodium acetate, pH 5.8) was added. Color development at 23°C was first observed after 5-20 min. After l-4 h, the membrane was removed from the dye solution, rinsed in tapwater, and air-dried. Alternatively, the membrane was incubated overnight (4°C) prior to rinsing and drying. Immunoenzymatic detection with streptavidin-horseradish peroxidase complex. After the addition of the primary antibody and the washing, portions of the membrane were incubated with biotinylated goat anti-rabbit IgG as above and washed three times in PBS-Tween, followed by one rinse in PBS. The membrane was incubated for 1 h at 37°C with streptavidin-horseradish peroxidase complex [( 14) Enzo Biochem, 0.5 mg/ ml horseradish peroxidase coupled to 1.25 mg/ml streptavidin in 0.13 M NaCl, 7 mM Na2HP04, 3 mM NaH2P04, pH 7.4, containing 1% BSA] diluted 1:250 in the same buffer (0.18 ml/cm*). After it was washed three times for 5 min each in 0.5 M NaCl, 10 mM NaH2P04, pH 6.5, containing 0.1% bovine serum albumin and 0.5% Tween 20, the membrane was washed twice in 0.3 M NaCl, 0.03 M sodium citrate, 0.1% BSA, pH 8.6. To detect the complex, 10 ml of freshly prepared diaminobenzidine (Polysciences; 0.5 mg/ml in 10 mM Tris, pH 7.5) was mixed with 0.2 ml CoClz (10 mg/ml) and incubated in the dark for 10 min at 4°C. After 0.2 ml of hydrogen peroxide (1%) was added, the diaminobenzidine preparation was incubated with the membrane (0.18 ml/cm*) in the
384
BROWER,
BRAKEL,
dark. Color development at 23°C was first observed in 2- 10 min. After l-2 h, the membrane was removed from the dye solution, rinsed in tapwater, and air-dried. Prolonged incubation increased the background staining of the membrane without increasing the intensity of the stained bands. The color of the bands on some nitrocellulose membranes faded upon drying. The addition of water or buffer (neutral or slightly acidic) restored color.
Immunodetection with ‘2’I-labeled immunoglobulin. After the incubation with the primary antibody and the washing, portions of the membrane were incubated (23°C) for 30 min with rotation in 0.15 M NaCl, 10 mM Tris, pH 7.4 (Tris-saline), 5% bovine serum albumin, 0.02% NaN3 containing 5 X 10’ cpm/ml donkey anti-rabbit ‘251-F(ab’)2 (7.5 pCi/pg; Amersham). The solution was aspirated and the membrane washed (10 min per wash) with 200 ml of T&saline, then T&saline containing 0.05% Tween 20 (twice), and was then washed again with Tris-saline. The membrane was blotted briefly with filter paper, and radioautography was performed as detailed (15). RESULTS
a,-Antitrypsin was electroblotted following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrotransfer, the nitrocellulose membrane was blocked with bovine serum albumin and incubated with rabbit anti-human cur-antitrypsin. One-third of the membrane was then incubated with [1251]IgG prior to radioautography. The second third of the membrane was incubated with biotinylated goat anti-rabbit IgG, and the biotinylated probe was detected by strep tavidin-horseradish peroxidase complex followed by the addition of diaminobenzidine. The final third of the membrane also was incubated with biotinylated goat anti-rabbit IgG, followed by streptavidin-acid phosphatase complex and fast violet B salt. The results of immunodetection with
AND
GARRY
[‘251]IgG are shown in Fig. 1, lanes l-5. After the film is incubated with the nitrocellulose membrane for 7 days, a minimum of 10 to 30 ng a,-antitrypsin per lane is necessary for detection by radioautogram. Similar quantities of protein are required for detection by streptavidin-horseradish peroxidase complex (2 pg/ml horseradish peroxidase) upon incubation for 2 h with diaminobenzidine (Fig. 1, lanes 6-10). (Prolonged incubations resulted in increased background without increased sensitivity.) The results of detection by streptavidin-acid phosphatase (95 rig/ml acid phosphatase) upon incubation with fast violet B salt for 18 h at 4°C are shown in Fig. 1, lanes 1 l- 15. Between 3 and 10 ng protein per lane are detected. There is less background staining than with the horseradish peroxidase technique. DlSCUSSlON This study demonstrates that the streptavidin-acid phosphatase method for the detection of proteins following Western blot is a relatively sensitive and specific technique capable of detecting nanogram amounts of protein. As demonstrated in Fig. 1, results can be more sensitive than those obtained with radiolabeled probes such as [1251]IgG or with similar enzymatic probes using horseradish peroxidase. Similar results have been achieved with other antigens on Western blots [platelet membrane glycoproteins IIb and IIIa; antigens and corresponding antibodies were the kind gift of Dr. L. Leung, Cornell University Medical Center (16)]. In addition, the background staining of the nitrocellulose membrane is less intense than that observed with horseradish peroxidase. Both the increase in sensitivity and the decrease in background are achieved without the use of radioactive or carcinogenic materials. In contrast both to radioautography and to the horseradish peroxidase-diaminobenzidine immunoenzymatic assay, the production of color by the acid phosphatase-fast
IMMUNOBLOT
IMMUNODETECTION AND
12346
WITH STREPTAVIDIN-ACID
BY ‘251Q#3, AVIDIN-HORSERADISH
6
PHOSPHATASE
385
AVIDIN-ACID PHOSPHATASE, PEROXIDASE
7 8 9 IO
It 12 13 14 lb
FIG. 1. Immunodetection of cYI-antitrypsin by [‘*‘I]IgG, streptavidin-horseradish peroxidase, and streptatidin-acid phosphatase. a-l-Antitrypsin was reduced prior to sodium dodecyl sulfate-plyxryhmide gel electrophoresis (I-100 n&lane). After electrotransfer, the a,-antitrypsin was detected (lanes 1-5) by [‘Z5I]IgG (5 X lo5 cpm/ml); (lanes 6-10) streptavidin-horseradish peroxidase complex (2 &ml horseradish peroxidase); or (lanes 1l-15) streptavidin-acid phosphatase complex (95 rig/ml acid phosphatase) as detailed under Materials and Methods. Lanes l-5, radioautogram of electroblot from gel containing 1, 3, 10, 30, and 100 ng protein/lane, respectively. Lanes 6-10, electroblot onto nitrocellulose from the same gel containing 1, 3, 10, 30, and 100 na/lane. Lanes I I-15, electroblot onto nitrocellulose from the same gel containing 1, 3, 10, 30, and 100 n&ane.
violet B salt reaction is not light sensitive. This permits constant observation of the membrane, preventing under- or overdevelopment of color as the protein bands are visualized. Color development using the acid phosphatase-fast violet B salt interaction is both time and temperature dependent. Color intensifies over approximately 12 to 24 h without overstaining of the membrane background. Color development can be increased by incubation at 37°C or slowed by incubation at 4°C. To prevent overdevelopment, lanes with a heavy protein inoculum can be cut from the membrane and removed from the substrate as color intensification in un-
derdeveloped lanes continues. Thus, the detection of a broad range of protein cvncentrations on the same membrane is possible. ACKNOWLEDGMENTS This study was supported by U. S. Public Health Service Grant H I- 18828 (Specialized Center of Research in Thrombosis) and ROI HL-32166, and a grant from the New York Community Trust. Dr. Brower is the recipient of a William S. Paley Fellowship in Academic Medicine. REFERENCES 1. Towbin, H., Staehelin, T., and Gordon, 3. (1979) Proc. Natl. Acad.
Sci. USA 76, 4350-4355.
2. Dimond, R. L., and Loomis, W. F. (1976) J. Biol Chem. 251.2680.
386
BROWER,
BRAKEL,
3. Gregory Lee, C.-Y., Huang, Y.-S., Hu, P.-C., Gomel, V., and Menge, A. C. (1982) Anal. Biochem. 123, 14-22. 4.
5.
6. 7. 8. 9.
O’Connor, C. G., and Ashman, L. K. (1982) J. Immunol. Methods 54, 267-27 1. Blake, M. S., Johnston, K. H., Russell-Jones, G. J., and Gotschlich, E. C. (1984) Anal. Biochem. 136, 175-179. Knecht, D. A., and Dimond, R. L. (1984) Anal. Biochem. 136, 180-184. De Blas, A. L., and Cherwinski, H. M. (1983) Anal. Biochem. 133, 2 14-2 19. Wojtkowiak, Z., Briggs, R. C., and Hnilica, L. S. (1983) Anal. Biochem. 129,486-489. Glass, W. F., Brings, R. C., and Hnilica, L. S. (1981) Science (Washington, D. C.) 211, 70-72.
AND CARRY 10. Pannell, R., Johnson, D., and Travis, J. (1974) Biochemistry 13, 5439-5445. Il. Brewer, M. S., and Harpel, P. C. (1973) Blood 61, 842-849. 12.
Laemmli, U. K. (1970) Nature (London) 227, 680-
13.
Burnette. W. N. (1981) Anal. Biochem. 112, 195-
684. 203.
Brakel, C. L., and Englehardt, D. L. (1985) in Symposium on Rapid Detection and Identification of Infectious Agents (Kingsbury, D. T., and Falkow, S., eds.), Academic Press, New York, in press. 1.5. Kinoshita, T., Nachman, R.. and Minick, R. (1979) J. Cell Biol. 82, 688-696. 16. Nachman, R. L., and Leung, L. L. K. (1982) J. Clin. Invest. 69, 263-269. 14.
BIOCHEMISTRY
147, 382-386
(1985)
lmmunodetection with Streptavidin-Acid Phosphatase Complex on Western Blots MARK S. BROWER, CHRISTINE L. BRAKEL, AND KIMBERLY
GARRY
Division of Hematology-Oncology, Department of Medicine and the Specialized Center fbr Research in Thrombosis, The New York Hospital-Cornell Medical Center, New York, New York, and Enzo Biochem, Inc.. New York, New York Received July 12, 1984 A technique for the detection of nanogram amounts of protein blotted onto nitrocellulose membranes has been developed using nonradioactive probes. Protein transferred to nitrocellulose membranes is detected by a specific antibody followed by incubation with biotinylated antiantibody. After addition of streptavidin-acid phosphatase complex, incubation with fast violet B salt produces sharp magenta bands. This method allows detection of bands containing less than 20 ng of protein. The procedure does not use radioactive or carcinogenic materials. 0 1985 Academic KEY
WORDS:
Press. Inc.
Western blots; immunoblot; immune assays;biotin; avidin: acid phosphatase.
The detection of antigens by nonradioactive probes has become an important analytic technique. One method, the “Western blot” (1) transfers antigen onto nitrocellulose membranes to facilitate detection. Antibodyantigen complexes on nitrocellulose membranes have been detected using radiolabeled protein A (2) or a radiolabeled anti-antibody (3). More recently, nonradioactive probes for the detection of antibody-antigen complexes have been developed. These techniques use an enzyme coupled to either protein A or to an anti-antibody (4-9). However, immunoenzymatic assays frequently are not as sensitive as similar methods using radioactive probes. Some use substances believed to be carcinogenic, such as diaminobenzidine. Additionally, many of these techniques result in the production of significant background staining. We describe herein a method for the detection of biotinylated antibody-antigen complexes on nitrocellulose membranes using streptavidin-acid phosphatase complex. Neither radioactive nor carcinogenic materials are used in this procedure. The sensitivity of this method appears to equal or exceed that 0003-2697185 Copytight ,411 rights
$3.00
0 1985 by Academic Press. Inc. of reproduction in any form reserved.
of similar techniques using radiolabeled probes. The resulting background staining is minimal. MATERIALS
AND METHODS
Antibodies and antigens. a,-Antitrypsin was purified from plasma as previously described and its concentration was determined by its extinction coefficient of 5.3 ( 10,ll). The final inhibitor preparation was free of other contaminating plasma proteinase inhibitors as analyzed by double diffusion in agarose gels with specific antibodies against az-macroglobulin, Cl -inactivator, antithrombin III, inter-cY-trypsin inhibitor, or chymotrypsin inhibitor obtained from Calbiochem-Behring Corp. Samples (7.1 mg/ ml) were stored at -70°C prior to use. Rabbit anti-human cur-antitrypsin, immunoglobulin G (IgG)’ fraction, was from Sigma Chemical Corporation. ’ Abbreviations used: IgG, immunoglobulin G; PBS, phosphate-buffered saline (100 mM NaCl, 38 mM Na2HP04, 12.5 mM NaH2P04, pH 7.4); PBS-Tween, PBS containing 0.5% (vol/vol) Tween 20; Tris-saline, 0.15 M NaCl, 10 mM Tris, pH 7.4. 382
IMMUNOBLOT
WITH STREPTAVIDIN-ACID
Sodium dodecyl sulJate-polyacrylamide slab gel electrophoresis and electroblotting technique. cYi-Antitrypsin (1.4 pg/ml) in 0.25 M Tris, pH 7.4, was mixed with an equal volume of 0.06 M Tris, 2% sodium dodecyl sulfate, 5% glycerol, 0.00 1% bromphenol blue, and 2% dithiothreitol prior to heating at 100°C for 3 min. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis ( 1- 100 ng protein/lane) was performed by the method of Laemmli (12) using a 3.8% stacking gel and a 9% separation gel. Proteins were transferred onto nitrocellulose by standard technique (13). The aIantitrypsin was electrophoretically transferred (23°C) over 1 h at 50 V/cm onto nitrocellulose paper (BA85, Schleicher and Schuell). After electroblotting was performed, the nitrocellulose was incubated overnight at 4°C in phosphate-buffered saline (PBS, 100 mM NaCl, 38 mM Na2HP04, 12.5 mM NaH2P04, pH 7.4) containing 5% bovine serum albumin (Sigma, essentially fatty acid free) and 3 mM NaN3. After it was incubated an additional 30 min in the above buffer containing 0.5% (vol/vol) Tween 20 (PBS-Tween), the blocked nitrocellulose membrane was incubated with primary antibody (rabbit anti-human al-antitrypsin) diluted 1: 100 in PBS-Tween for 1 h at 37°C with gentle rocking. The membrane was washed three times with PBS-Tween. Immunoenzymatic detection with streptavidin-acid phosphatase complex. The nitrocellulose membrane was incubated with biotinylated goat anti-rabbit IgG (Enzo Biochem) diluted 1: 1000 in PBS-Tween for 1 h at 37°C with gentle rocking. The membrane was washed three times with PBS-Tween, rinsed once in PBS, and incubated 30 min at 23°C in PBS containing 5 mM trisodium EDTA, 2% bovine serum albumin, and 0.05% (vol/ vol) Triton X- 100. Portions of the membrane were incubated with streptavidin-acid phosphatase complex [Ref. ( 14); Enzo Biochem, 19 pg/ml acid phosphatase coupled to 15.7 pg/ml streptavidin in 0.13 M NaCl, 7 mM Na2HP04, 3 mM NaH2P04, pH 7.41 diluted 1:200 in PBS, 5
PHOSPHATASE
383
mM trisodium EDTA for 1 h at 23°C (0.015 ml/cm*). After it was washed five times (5 min per wash) in 10 mM K2HP04, 0.5 M NaCl, 1 mM EDTA, pH 6.5, containing 2% bovine serum albumin and 0.5% Triton X100, the membrane was rinsed twice (2 min per rinse) in a clean dish with 0.2 M acetic acid, 0.2 M sodium acetate, pH 5.8. To detect the complex the membrane was incubated 5 min with 1 mM naphthol AS-MX phosphate (Sigma), 0.2 M sodium acetate, pH 5.8 (10 ml; approximately 0.18 ml/cm2) before 0.1 ml (1.8 &cm*) fast violet B salt (Sigma; 4 mg/ml in 0.2 M sodium acetate, pH 5.8) was added. Color development at 23°C was first observed after 5-20 min. After l-4 h, the membrane was removed from the dye solution, rinsed in tapwater, and air-dried. Alternatively, the membrane was incubated overnight (4°C) prior to rinsing and drying. Immunoenzymatic detection with streptavidin-horseradish peroxidase complex. After the addition of the primary antibody and the washing, portions of the membrane were incubated with biotinylated goat anti-rabbit IgG as above and washed three times in PBS-Tween, followed by one rinse in PBS. The membrane was incubated for 1 h at 37°C with streptavidin-horseradish peroxidase complex [( 14) Enzo Biochem, 0.5 mg/ ml horseradish peroxidase coupled to 1.25 mg/ml streptavidin in 0.13 M NaCl, 7 mM Na2HP04, 3 mM NaH2P04, pH 7.4, containing 1% BSA] diluted 1:250 in the same buffer (0.18 ml/cm*). After it was washed three times for 5 min each in 0.5 M NaCl, 10 mM NaH2P04, pH 6.5, containing 0.1% bovine serum albumin and 0.5% Tween 20, the membrane was washed twice in 0.3 M NaCl, 0.03 M sodium citrate, 0.1% BSA, pH 8.6. To detect the complex, 10 ml of freshly prepared diaminobenzidine (Polysciences; 0.5 mg/ml in 10 mM Tris, pH 7.5) was mixed with 0.2 ml CoClz (10 mg/ml) and incubated in the dark for 10 min at 4°C. After 0.2 ml of hydrogen peroxide (1%) was added, the diaminobenzidine preparation was incubated with the membrane (0.18 ml/cm*) in the
384
BROWER,
BRAKEL,
dark. Color development at 23°C was first observed in 2- 10 min. After l-2 h, the membrane was removed from the dye solution, rinsed in tapwater, and air-dried. Prolonged incubation increased the background staining of the membrane without increasing the intensity of the stained bands. The color of the bands on some nitrocellulose membranes faded upon drying. The addition of water or buffer (neutral or slightly acidic) restored color.
Immunodetection with ‘2’I-labeled immunoglobulin. After the incubation with the primary antibody and the washing, portions of the membrane were incubated (23°C) for 30 min with rotation in 0.15 M NaCl, 10 mM Tris, pH 7.4 (Tris-saline), 5% bovine serum albumin, 0.02% NaN3 containing 5 X 10’ cpm/ml donkey anti-rabbit ‘251-F(ab’)2 (7.5 pCi/pg; Amersham). The solution was aspirated and the membrane washed (10 min per wash) with 200 ml of T&saline, then T&saline containing 0.05% Tween 20 (twice), and was then washed again with Tris-saline. The membrane was blotted briefly with filter paper, and radioautography was performed as detailed (15). RESULTS
a,-Antitrypsin was electroblotted following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrotransfer, the nitrocellulose membrane was blocked with bovine serum albumin and incubated with rabbit anti-human cur-antitrypsin. One-third of the membrane was then incubated with [1251]IgG prior to radioautography. The second third of the membrane was incubated with biotinylated goat anti-rabbit IgG, and the biotinylated probe was detected by strep tavidin-horseradish peroxidase complex followed by the addition of diaminobenzidine. The final third of the membrane also was incubated with biotinylated goat anti-rabbit IgG, followed by streptavidin-acid phosphatase complex and fast violet B salt. The results of immunodetection with
AND
GARRY
[‘251]IgG are shown in Fig. 1, lanes l-5. After the film is incubated with the nitrocellulose membrane for 7 days, a minimum of 10 to 30 ng a,-antitrypsin per lane is necessary for detection by radioautogram. Similar quantities of protein are required for detection by streptavidin-horseradish peroxidase complex (2 pg/ml horseradish peroxidase) upon incubation for 2 h with diaminobenzidine (Fig. 1, lanes 6-10). (Prolonged incubations resulted in increased background without increased sensitivity.) The results of detection by streptavidin-acid phosphatase (95 rig/ml acid phosphatase) upon incubation with fast violet B salt for 18 h at 4°C are shown in Fig. 1, lanes 1 l- 15. Between 3 and 10 ng protein per lane are detected. There is less background staining than with the horseradish peroxidase technique. DlSCUSSlON This study demonstrates that the streptavidin-acid phosphatase method for the detection of proteins following Western blot is a relatively sensitive and specific technique capable of detecting nanogram amounts of protein. As demonstrated in Fig. 1, results can be more sensitive than those obtained with radiolabeled probes such as [1251]IgG or with similar enzymatic probes using horseradish peroxidase. Similar results have been achieved with other antigens on Western blots [platelet membrane glycoproteins IIb and IIIa; antigens and corresponding antibodies were the kind gift of Dr. L. Leung, Cornell University Medical Center (16)]. In addition, the background staining of the nitrocellulose membrane is less intense than that observed with horseradish peroxidase. Both the increase in sensitivity and the decrease in background are achieved without the use of radioactive or carcinogenic materials. In contrast both to radioautography and to the horseradish peroxidase-diaminobenzidine immunoenzymatic assay, the production of color by the acid phosphatase-fast
IMMUNOBLOT
IMMUNODETECTION AND
12346
WITH STREPTAVIDIN-ACID
BY ‘251Q#3, AVIDIN-HORSERADISH
6
PHOSPHATASE
385
AVIDIN-ACID PHOSPHATASE, PEROXIDASE
7 8 9 IO
It 12 13 14 lb
FIG. 1. Immunodetection of cYI-antitrypsin by [‘*‘I]IgG, streptavidin-horseradish peroxidase, and streptatidin-acid phosphatase. a-l-Antitrypsin was reduced prior to sodium dodecyl sulfate-plyxryhmide gel electrophoresis (I-100 n&lane). After electrotransfer, the a,-antitrypsin was detected (lanes 1-5) by [‘Z5I]IgG (5 X lo5 cpm/ml); (lanes 6-10) streptavidin-horseradish peroxidase complex (2 &ml horseradish peroxidase); or (lanes 1l-15) streptavidin-acid phosphatase complex (95 rig/ml acid phosphatase) as detailed under Materials and Methods. Lanes l-5, radioautogram of electroblot from gel containing 1, 3, 10, 30, and 100 ng protein/lane, respectively. Lanes 6-10, electroblot onto nitrocellulose from the same gel containing 1, 3, 10, 30, and 100 na/lane. Lanes I I-15, electroblot onto nitrocellulose from the same gel containing 1, 3, 10, 30, and 100 n&ane.
violet B salt reaction is not light sensitive. This permits constant observation of the membrane, preventing under- or overdevelopment of color as the protein bands are visualized. Color development using the acid phosphatase-fast violet B salt interaction is both time and temperature dependent. Color intensifies over approximately 12 to 24 h without overstaining of the membrane background. Color development can be increased by incubation at 37°C or slowed by incubation at 4°C. To prevent overdevelopment, lanes with a heavy protein inoculum can be cut from the membrane and removed from the substrate as color intensification in un-
derdeveloped lanes continues. Thus, the detection of a broad range of protein cvncentrations on the same membrane is possible. ACKNOWLEDGMENTS This study was supported by U. S. Public Health Service Grant H I- 18828 (Specialized Center of Research in Thrombosis) and ROI HL-32166, and a grant from the New York Community Trust. Dr. Brower is the recipient of a William S. Paley Fellowship in Academic Medicine. REFERENCES 1. Towbin, H., Staehelin, T., and Gordon, 3. (1979) Proc. Natl. Acad.
Sci. USA 76, 4350-4355.
2. Dimond, R. L., and Loomis, W. F. (1976) J. Biol Chem. 251.2680.
386
BROWER,
BRAKEL,
3. Gregory Lee, C.-Y., Huang, Y.-S., Hu, P.-C., Gomel, V., and Menge, A. C. (1982) Anal. Biochem. 123, 14-22. 4.
5.
6. 7. 8. 9.
O’Connor, C. G., and Ashman, L. K. (1982) J. Immunol. Methods 54, 267-27 1. Blake, M. S., Johnston, K. H., Russell-Jones, G. J., and Gotschlich, E. C. (1984) Anal. Biochem. 136, 175-179. Knecht, D. A., and Dimond, R. L. (1984) Anal. Biochem. 136, 180-184. De Blas, A. L., and Cherwinski, H. M. (1983) Anal. Biochem. 133, 2 14-2 19. Wojtkowiak, Z., Briggs, R. C., and Hnilica, L. S. (1983) Anal. Biochem. 129,486-489. Glass, W. F., Brings, R. C., and Hnilica, L. S. (1981) Science (Washington, D. C.) 211, 70-72.
AND CARRY 10. Pannell, R., Johnson, D., and Travis, J. (1974) Biochemistry 13, 5439-5445. Il. Brewer, M. S., and Harpel, P. C. (1973) Blood 61, 842-849. 12.
Laemmli, U. K. (1970) Nature (London) 227, 680-
13.
Burnette. W. N. (1981) Anal. Biochem. 112, 195-
684. 203.
Brakel, C. L., and Englehardt, D. L. (1985) in Symposium on Rapid Detection and Identification of Infectious Agents (Kingsbury, D. T., and Falkow, S., eds.), Academic Press, New York, in press. 1.5. Kinoshita, T., Nachman, R.. and Minick, R. (1979) J. Cell Biol. 82, 688-696. 16. Nachman, R. L., and Leung, L. L. K. (1982) J. Clin. Invest. 69, 263-269. 14.