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‘Key paper’ for covalent binding of proteins and its uses
Journal oflmmunologicalMethods, 94 (1986) 263-269
263
Elsevier JIM04129
'Key paper' for covalent binding of proteins and its uses Y. Chemla a, y . Cherny 1, M. Herzberg 1, M. Bracha 2 and J. Sperling 2 10rgenies, Ltd., POB 360, Yavne, and e Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot, Israel
(Received4 April 1986,accepted10 July 1986)
A simPle method for covalent coupling of proteins to filter paper modified with quinone groups is described. This paper, termed 'Key paper' is flexible, stable on storage and does not require any activation before use. Proteins bound to Key paper can be detected by enzyme immunoassay, radioimmunoassay or Coomassie blue staining. Bound enzymes retain their enzymatic activity. Nucleic acids do not bind and do not interfere with the activity of the bound proteins. Because of its mechanical and chemical properties Key paper is a good matrix for electroblotting and for direct in situ analysis of proteins. Key words: Key paper; Proteinbinding; Electrotransfer;Detectionof antigens; Enzyme(Antibodies)
Introduction
One of the most widely used analytical tools in molecular biology and biochemistry is the slab gel for electrophoresis in its numerous matrices and modifications. The identification of gel-separated polypeptides, such as enzymes, antigens, hormone receptors, etc., has presented some technical difficulties. The Southern (1975) blotting technique originally applied to DNA has been shown to be applicable also to proteins. So-called Western blotting is a process comprising the elution of polypeptides from the gel and the immobilization of the eluted material on an insoluble matrix. The most widely used matrices have been nitrocellulose membranes (NC) (Glass et al., 1979; Towbin et al., 1979; Bittner et al., 1980; Bowen et al., 1980; Burnette, 1981) which bind proteins by physical adsorption, probably involving hydrophobic interactions; when covalent binding of protein to the matrix is sought the diazobenzyloxymethyl (DBM) paper (Alwine et al., 1979) or the diazo-phenylthioester (DPT) paper (Reiser and Wardale, 1981; Seed, 1982) are employed; they bind the proteins via azo groups (Renart et al.,
1979). Activation of filter paper by CNBr has also been described (Clarke et al., 1979). Nylon membranes can be modified with tertiary amino groups (Zetabind) (Gershoni and Palade, 1982). These membranes have better binding capacity, but they also strongly bind common cationic dyes (e.g., Coomassie blue). We have developed a new filter paper chemically modified with benzoquinone (Brandt et al., 1975) which covalently binds protein without any prior activation. This Key paper retains its binding properties when stored at room temperature. Since it is important for analytical as well as preparative purposes that chemically or biologically active proteins retain their activities while bound to the paper, we have tested the retention of enzymatic and of antigenic properties of the bound proteins. In other series of tests we transferred gel-separated polypeptides onto Key paper and tested their properties following transfer and binding to the paper. We have also demonstrated that the probes identifying the protein bound to Key paper can be 'erased', leaving behind functional proteins.
0022-1759/86/$03.50 © 1986 ElsevierSciencePublishers B.V.(BiomedicalDivision)
264
Materials and methods
A ctioated paper Whatman 3MM Filter paper, modified by quinone groups according to the technique of Brandt et al. (1975), was obtained from Orgenics (Yavne, Israel) as Key paper.
Binding of proteins to Key paper This was done either by spotting or by transfer from a gel.
Spotting 2 t~l of serial two-fold dilutions, in the range of 20-5000 n g / m l of the test proteins, were spotted on dry Key paper, and kept at room temperature until dry (about 15 rain). The paper was then briefly washed in PBS (5 min) to eliminate excess of proteins.
Electrotransfer Transfer of proteins separated on SDS polyacrylamide gel was carried out with the Transblot Bio-Rad system using either of the following conditions: (1) 0.025 M Tris, 0.192 M glycine, 10% methanol, p H 8.3 at a constant current of 200 mA; (2) 0.025 M sodium phosphate, pH 6.5 at 500 mA. The transfer was carried out with chicken, mouse and human immunoglobulins.
Detection of proteins on Key paper The proteins bound to Key paper can be detected by direct staining, by enzyme immunoassay, by radioimmunoassay or by an assay of the enzymatic activity of proteins that are enzymes. For direct staining the paper was immersed in the staining solution (e.g., 0.1% Coomassie blue R 250, 50% methanol and 10% acetic acid) for 2-5 min, and then destained in a solution of 20% methanol and 10% acetic acid. For enzyme immunoassay (EIA), the proteins were first bound to Key paper by spotting or blotting. The paper with the bound protein was then immersed in the blocking solution (3% BSA in PBS) to saturate the free binding sites. The paper was gently rocked in this solution for 1 h at room temperature, preferably in a sealed plastic bag (to conserve antibody). 20% newborn calf serum or 2% Bacto-tryptone in 20% horse serum
could be substituted for the BSA. Next the paper was washed several times in PBS (each time for 10 min), drained by blotting on filter paper and placed on parafilm. The paper was then covered with the chromogenic substrate and incubated at room temperature until a clear color pattern developed. In our experiments, chicken, human and mouse IgG were detected by peroxidase or alkaline phosphatase-conjugated goat antibodies. Radioimmunoassay was performed with aspartate transcarbamylase (ACTase). This protein was spotted on Key paper, which was then washed and its unoccupied active sites blocked. Next the paper was incubated with anti-ACTase rabbit antibody for 1 h. Excess of antibody was then washed off using PBS, and the paper incubated with 12SI-protein A for 1 h whereupon it was washed extensively in PBS, dried and exposed to X-ray film with an intensifier screen for 13 h at - 7 0 ° C . Detection of enzymatic activity was carried out with fl-galactosidase, horseradish peroxidase and alkaline phosphatase. These enzymes were bound to Key paper by spotting and their activity was tested with their insoluble substrates: for fl-galactosidase X-gal (bromo-chloro-indolyl-fl-D-galactoside), for peroxidase 4-chloro-naphthol, and for alkaline phosphatase naphthol As Mx phosphate + Fast Red were used. The intensity of the insolu: ble colored product was related to the concentration of the enzyme, fl-Galactosidase was fractionated on gel by electrophoresis, electrotransferred to Key paper, and the bands of the enzyme identified by their enzymatic activity on X-gal. These enzymes were also tested for their storage properties in their paper-bound state at various temperatures and times.
Dissociation of antibody from Key paper bound antigen Antibody was dissociated from Key paperbound antigen by the following treatments: (a) 0.1 M glycine HC1, 20 mM Magnesium acetate; 50 mM KC1 (pH 2.2). Incubation for 1.5 h at room temperature. (b) 3 M potassium-thiocyanate, 10 mM TrisHC1, pH 7.5. Incubation for I h, then 30 min in 0.01 M HC1 at 37°C. (c) 8 M urea, 0.1 M 2-mercaptoethanol. 1 h at 60°C.
265
(d) 2% SDS, 0.1 M mercaptoethanol, 130 mM NaC1, 50 mM sodium phosphate, pH 7.4 (1 h) at 60 ° C. Any of these treatments will remove the antibody from the bound antigen and restore the antigen-bound key paper to its original antibody binding capacity.
Key paper, as determined by this assay was 0.1 m g / c m 2. The experiment was repeated with various amounts of a mixture of BSA labeled with cold iodine (conjugation of iodine to BSA was performed under conditions identical to the ones used with radioactive iodine) and a fixed amount of 125I-BSA. The results were identical to the ones obtained in previous experiments.
Results
Capacity of Key paper Increasing amounts (5-500 /~g) of BSA were mixed with fixed amounts of 125I-labeled BSA in a total volume of 50/~1 of 0.1 M N a H C O 3 at pH 8, and spotted onto Key Paper circles (1 cm 2) each. The circles were washed extensively with 0.1 M NaHCO3/1 M NaC1 and counted in a gamma counter. Saturation of the filters occurred at an input level of 100-125 ~tg. The efficiency of binding of BSA quantities lower than 100/~g/cm 2 was in the range of 90-100%. The binding capacity of
Fig. 1. Coomassie blue staining of chicken IgG bound on Key paper. After spotting serial dilutions of the protein (5/Lg to 20 ng), the paper was immersed for 2 min in the staining solution and then 5 min in the destaining solution.
Detection of proteins by direct staining or by enzyme immunoassay Chicken, human or mouse IgG diluted in PBS within the range of 0.02-5 # g / s p o t were spotted on Key paper and either stained with Coomassie blue (Fig. 1) or tested for their antigenicity by
Fig. 2. Detection of b o u n d antigen by EIA. Serial dilutions of chicken IgG (5 ~g to 20 ng) were spotted. The active sites were blocked in blocking solution and then the paper was incubated with goat anti-chicken IgG diluted 1 / 5 0 in the blocking solution. After several washes in PBS, the chromogen chloronaphthol was added and specific coloration was obtained without background.
266
EIA (Fig. 2). The minimal detectable quantity was 0.08/~g by Coomassie blue and 0.02/~g by EIA. The same proteins were run on a 10% SDSpolyacrylamide gel (3 h, 100 V). The proteins were then electrotransferred (2.5 h, 0.5 A in sodium phosphate buffer) to Key paper (Fig. 3). Application of the EIA technique to such Key paper permitted the specific detection of these immunoglobulins.
Detection of proteins (antigens by radioimmunoassay Aspartate transcarbamylase (ATCase), was spotted on Key paper and detected there with the
ai~t of a specific antibody followed by a25I-protein A and autoradiography (Fig. 4). The assay on Key paper was specific inasmuch as no autoradiographic stain appeared when the immunoglobulin used was not directed against ACTase. Treatment of the Key paper with ACTase and its bound antibody either by glycine HC1 buffer, pH 2.2, or by SDS, or by 3 M KCNS or by 8 M urea, released the antibody completely from the bound protein (Fig. 4b-e, two upper squares). These dissociation treatments did not affect the binding and the antigenicity of the bound protein to Key paper because it could be redetected with the same specificity (Fig. 4b-e, lower right squares).
Actioity and stability of bound enzymes Serial dilutions of/~-galactosidase, horseradish peroxidase or alkaline phosphatase were spotted on Key paper and stored for 20 days at room temperature, 4°C or - 2 0 ° C . The activity of the bound enzymes was tested using their corresponding substrates following various storage conditions. /~-galactosidase lost its activity after 1 day incubation at any temperature. Peroxidase retained its activity for 20 days following application of a solution of 0.5 m g / m l when stored at 20 o C. Alkaline phosphatase was the most stable enzyme and could be detected on Key paper even -
Fig. 3. Detection of transferred protein on Key paper by EIA: 5-0.15 /zg of chicken IgG were electrophoresed in 10% SDS polyacrylamide gel and electrotransferred in Tris-glycine methanol for 4 h at 0.2 A (constant current). Following transfer, Key paper was incubated in blocking solution and then the antigen was revealed by EIA.
Fig. 4. Detection of aspartate transcarbamylase (ACTase) b o u n d to Key paper. Key paper filters (2 × 2 cm) were divided into 1 × 1 cm squares and each square was spotted with 100 # g of ACTase. The filters were treated with anti-ACTase antibodies and with 125I-protein as described in the materials and methods section. Filter (a) was autoradiographed without further treatment. The 125I label was removed from the remaining filters by the following treatments: (b) low pH; (c) potassium thiocyanate; ( d ) SDS and mercaptoethanol; (e) urea and mercaptoethanol, as described in the materials and methods section. The upper halves of filters b - e were autoradiographed without further treatment, The lower left squares of each of the filters were treated with 125I-protein A; the lower right squares were treated with anti-ACTase antibodies followed by 125I-protein A, and autoradiographed.
267 after 20 days of storage at r o o m temperature and at concentration as low as 0.01 m g / m l . 1.5, 3 and 6 U of active fl-galactosidase were run on 5% SDS polyacrylamide gel and then electrotransferred to Key paper. Enzymatic activity was apparent after 4 h of electrotransfer. All subunits of the enzyme were detected on Key paper after 24 h of transfer. The enzyme was detected specifically and immediately on the paper u p o n the addition of substrate solution (Fig. 5).
1 /xl of serial dilutions of purified goat antichicken I g G antibodies at concentrations ranging
f r o m 0.03 to 4 /~g were spotted onto K e y paper. K e y paper was first incubated in the blocking solution for 1 h and then for 2 h in the antigen solution containing 0.5 mg of chicken I g G per ml of blocking solution (Fig. 6). After washing in PBS, the b o u n d antigen was detected by incubation with peroxidase-conjugated chicken anti-goat I g G followed b y washing with PBS and addition of the chromogen (Fig. 7). The results show that an a n t i b o d y attached to Key paper specifically binds its antigen; no chromogenic reaction occurred when no antibody or antigen were present or when the conjugated antibody was not relevant.
Fig. 5. Electroblotting of fl-galactosidase to Key paper and detection by enzymatic activity. 1.5, 3 and 6 U (slots 1, 2 and 3 respectively) of fl-galactosidase were electrophoresed in a 7.5% acrylamide gel. After transfer in 25 mM sodium phosphate buffer pH 6.5 at 0.5 A (constant current), the enzyme and its subunits were detected by direct addition of the insoluble substrate (X-gal).
Fig. 6. Direct binding of antibody to Key paperl purified goat anti-chicken IgG (4-0.03 #g) was bound to Key paper. After blocking, Key paper was incubated with the specific antigen (Chicken IgG 0.5 mg/ml), and the specific binding revealed by incubation with peroxidase-conjugated goat anti-chicken IgG and the substrate.
Activity of antibody bound to Key paper
268
Fig. 7. Specificity of antibody binding: 1 /*g and 0.5 #g of goat anti-chicken IgG were spotted on Key paper. The binding of the antibody was revealed by specific immunoassay (a). Negative controls comprised the following test strips: (b) without test antibody (goat antichicken IgG); (c) without specific antigen (chicken IgG); (d) with non-specific antibody conjugate: peroxidase-conjugated rabbit anti-mouse IgG instead of peroxidase-conjugated goat anti-chicken IgG.
Discussion
As opposed to other matrices, Key paper is ready for use and does not need any activation. It is flexible, not breakable, and thus fulfills chemical and mechanical conditions required for a suitable matrix for immobilization of proteins. Key paper binds proteins at room temperature. The binding is very fast, does not need any specific buffers or conditions, and is independent of molecular size. Immobilization can be done by direct spotting on the paper or by transfer from acrylamide gel. Proteins bind covalently to Key paper. Washing of the paper in PBS, Brij 0.1%, SDS 0.1% or even heating to 65°C does not affect the binding of the protein or its antigenicity. Heating the paper to 100°C, however, affects the antigenicity of the proteins which remain bound to Key paper. Proteins separated on SDS polyacrylamide gel electrophoresis can be transferred to Key paper. Proteins of different molecular weights in the range of 100 000-14000 bound to the paper to the same extent (data not shown). Proteins bound to Key paper can be detected as antigens, antibodies or enzymes by suitable reagents and assays. In the radioimmunoassay the antibody reacting with the bound antigen can be washed off using
suitable treatments, such as elution with glycine at low p H or by denaturing agents. These reagents do not affect the covalent binding of the protein itself to the Key paper and do not affect its antigenicity. Thus, the same paper to which the antigen is still bound can be re-used for further examination. Though initial screening for the presence of protein can be done by staining with Coomassie blue (which though less sensitive is very rapid) specific detection requires either RIA or EIA assays. Some enzymes bound to Key paper can preserve their enzymatic activity. The enzymatic activity of some enzymes can be demonstrated on the paper even after a long period of storage (several days to several weeks). The fact that Key paper does not bind nucleic acids allows the detection of trace amounts of proteins in the presence of large amounts of nucleic acids. This property is of crucial importance when analyzing proteins derived from whole cell lysates since the nucleic acids do not compete with proteins for binding sites on the solid matrix. Because of these properties, Key paper has potential applications in many fields, e.g., identification of monoclonal antibodies secreted by hybridomas, tests for the presence of a specific protein, immobilization of proteins on a solid matrix,
269
development of chromatography-like paper for binding and purification of antibodies or antigens and functional tests of specific proteins (binding to specific molecules or enzymatic activity).
Acknowledgement We gratefully acknowledge the excellent technical help of Mrs. Hanna Yakim.
References Alwine, J.C., D.J. Kemp, B.A. Parker, J. Reiser, G.R. Stark and G.M. Wahl, 1979, in: Methods in Enzymology, Vol. 68, ed. R. Wu (Academic Press, New York) pp. 220-242. Bittner, M., P. Kupferer and C.F. Morris, 1980, Anal. Biochem. 102, 459.
Bowen, B., J. Steinberg, U.K. Laemmli and H. Weintraub, 1980, Nucleic Acids Res. 8, 1. Brandt, J., L. Andersson and J. Porath, 1975, Biochim. Biophys. Acta 386, 196. Burnette, W.N., 1981, Anal. Biochem. 112, 195. Clarke, L., R. Hitzeman and J. Carbon, 1979, in: Methods in Enzymology, vol. 68, ed. R. Wu (Academic Press, New York) pp. 436-442. Gershoni, J.M. and G.E. Palade, 1982, Anal. Biochem. 124, 396. Glass, W.F., R.C. Briggs and L.S. Hnilica, 1981, Anal. Biochem. 115, 219. Reiser, J. and J. Wardale, 1981, Eur. J. Biochem. 114, 569. Renart, J., J. Reiser and G.R. Stark, 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 3116. Seed, B., 1982, Nucleic Acids Res. 10, 1799. Southern, E., 1975, J. Mol. Biol. 98, 503. Towbin, H., T. Staehelin and J. Gordon, 1979, Proc. Natl. Aead. Sci. U.S.A. 76, 4350.
263
Elsevier JIM04129
'Key paper' for covalent binding of proteins and its uses Y. Chemla a, y . Cherny 1, M. Herzberg 1, M. Bracha 2 and J. Sperling 2 10rgenies, Ltd., POB 360, Yavne, and e Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot, Israel
(Received4 April 1986,accepted10 July 1986)
A simPle method for covalent coupling of proteins to filter paper modified with quinone groups is described. This paper, termed 'Key paper' is flexible, stable on storage and does not require any activation before use. Proteins bound to Key paper can be detected by enzyme immunoassay, radioimmunoassay or Coomassie blue staining. Bound enzymes retain their enzymatic activity. Nucleic acids do not bind and do not interfere with the activity of the bound proteins. Because of its mechanical and chemical properties Key paper is a good matrix for electroblotting and for direct in situ analysis of proteins. Key words: Key paper; Proteinbinding; Electrotransfer;Detectionof antigens; Enzyme(Antibodies)
Introduction
One of the most widely used analytical tools in molecular biology and biochemistry is the slab gel for electrophoresis in its numerous matrices and modifications. The identification of gel-separated polypeptides, such as enzymes, antigens, hormone receptors, etc., has presented some technical difficulties. The Southern (1975) blotting technique originally applied to DNA has been shown to be applicable also to proteins. So-called Western blotting is a process comprising the elution of polypeptides from the gel and the immobilization of the eluted material on an insoluble matrix. The most widely used matrices have been nitrocellulose membranes (NC) (Glass et al., 1979; Towbin et al., 1979; Bittner et al., 1980; Bowen et al., 1980; Burnette, 1981) which bind proteins by physical adsorption, probably involving hydrophobic interactions; when covalent binding of protein to the matrix is sought the diazobenzyloxymethyl (DBM) paper (Alwine et al., 1979) or the diazo-phenylthioester (DPT) paper (Reiser and Wardale, 1981; Seed, 1982) are employed; they bind the proteins via azo groups (Renart et al.,
1979). Activation of filter paper by CNBr has also been described (Clarke et al., 1979). Nylon membranes can be modified with tertiary amino groups (Zetabind) (Gershoni and Palade, 1982). These membranes have better binding capacity, but they also strongly bind common cationic dyes (e.g., Coomassie blue). We have developed a new filter paper chemically modified with benzoquinone (Brandt et al., 1975) which covalently binds protein without any prior activation. This Key paper retains its binding properties when stored at room temperature. Since it is important for analytical as well as preparative purposes that chemically or biologically active proteins retain their activities while bound to the paper, we have tested the retention of enzymatic and of antigenic properties of the bound proteins. In other series of tests we transferred gel-separated polypeptides onto Key paper and tested their properties following transfer and binding to the paper. We have also demonstrated that the probes identifying the protein bound to Key paper can be 'erased', leaving behind functional proteins.
0022-1759/86/$03.50 © 1986 ElsevierSciencePublishers B.V.(BiomedicalDivision)
264
Materials and methods
A ctioated paper Whatman 3MM Filter paper, modified by quinone groups according to the technique of Brandt et al. (1975), was obtained from Orgenics (Yavne, Israel) as Key paper.
Binding of proteins to Key paper This was done either by spotting or by transfer from a gel.
Spotting 2 t~l of serial two-fold dilutions, in the range of 20-5000 n g / m l of the test proteins, were spotted on dry Key paper, and kept at room temperature until dry (about 15 rain). The paper was then briefly washed in PBS (5 min) to eliminate excess of proteins.
Electrotransfer Transfer of proteins separated on SDS polyacrylamide gel was carried out with the Transblot Bio-Rad system using either of the following conditions: (1) 0.025 M Tris, 0.192 M glycine, 10% methanol, p H 8.3 at a constant current of 200 mA; (2) 0.025 M sodium phosphate, pH 6.5 at 500 mA. The transfer was carried out with chicken, mouse and human immunoglobulins.
Detection of proteins on Key paper The proteins bound to Key paper can be detected by direct staining, by enzyme immunoassay, by radioimmunoassay or by an assay of the enzymatic activity of proteins that are enzymes. For direct staining the paper was immersed in the staining solution (e.g., 0.1% Coomassie blue R 250, 50% methanol and 10% acetic acid) for 2-5 min, and then destained in a solution of 20% methanol and 10% acetic acid. For enzyme immunoassay (EIA), the proteins were first bound to Key paper by spotting or blotting. The paper with the bound protein was then immersed in the blocking solution (3% BSA in PBS) to saturate the free binding sites. The paper was gently rocked in this solution for 1 h at room temperature, preferably in a sealed plastic bag (to conserve antibody). 20% newborn calf serum or 2% Bacto-tryptone in 20% horse serum
could be substituted for the BSA. Next the paper was washed several times in PBS (each time for 10 min), drained by blotting on filter paper and placed on parafilm. The paper was then covered with the chromogenic substrate and incubated at room temperature until a clear color pattern developed. In our experiments, chicken, human and mouse IgG were detected by peroxidase or alkaline phosphatase-conjugated goat antibodies. Radioimmunoassay was performed with aspartate transcarbamylase (ACTase). This protein was spotted on Key paper, which was then washed and its unoccupied active sites blocked. Next the paper was incubated with anti-ACTase rabbit antibody for 1 h. Excess of antibody was then washed off using PBS, and the paper incubated with 12SI-protein A for 1 h whereupon it was washed extensively in PBS, dried and exposed to X-ray film with an intensifier screen for 13 h at - 7 0 ° C . Detection of enzymatic activity was carried out with fl-galactosidase, horseradish peroxidase and alkaline phosphatase. These enzymes were bound to Key paper by spotting and their activity was tested with their insoluble substrates: for fl-galactosidase X-gal (bromo-chloro-indolyl-fl-D-galactoside), for peroxidase 4-chloro-naphthol, and for alkaline phosphatase naphthol As Mx phosphate + Fast Red were used. The intensity of the insolu: ble colored product was related to the concentration of the enzyme, fl-Galactosidase was fractionated on gel by electrophoresis, electrotransferred to Key paper, and the bands of the enzyme identified by their enzymatic activity on X-gal. These enzymes were also tested for their storage properties in their paper-bound state at various temperatures and times.
Dissociation of antibody from Key paper bound antigen Antibody was dissociated from Key paperbound antigen by the following treatments: (a) 0.1 M glycine HC1, 20 mM Magnesium acetate; 50 mM KC1 (pH 2.2). Incubation for 1.5 h at room temperature. (b) 3 M potassium-thiocyanate, 10 mM TrisHC1, pH 7.5. Incubation for I h, then 30 min in 0.01 M HC1 at 37°C. (c) 8 M urea, 0.1 M 2-mercaptoethanol. 1 h at 60°C.
265
(d) 2% SDS, 0.1 M mercaptoethanol, 130 mM NaC1, 50 mM sodium phosphate, pH 7.4 (1 h) at 60 ° C. Any of these treatments will remove the antibody from the bound antigen and restore the antigen-bound key paper to its original antibody binding capacity.
Key paper, as determined by this assay was 0.1 m g / c m 2. The experiment was repeated with various amounts of a mixture of BSA labeled with cold iodine (conjugation of iodine to BSA was performed under conditions identical to the ones used with radioactive iodine) and a fixed amount of 125I-BSA. The results were identical to the ones obtained in previous experiments.
Results
Capacity of Key paper Increasing amounts (5-500 /~g) of BSA were mixed with fixed amounts of 125I-labeled BSA in a total volume of 50/~1 of 0.1 M N a H C O 3 at pH 8, and spotted onto Key Paper circles (1 cm 2) each. The circles were washed extensively with 0.1 M NaHCO3/1 M NaC1 and counted in a gamma counter. Saturation of the filters occurred at an input level of 100-125 ~tg. The efficiency of binding of BSA quantities lower than 100/~g/cm 2 was in the range of 90-100%. The binding capacity of
Fig. 1. Coomassie blue staining of chicken IgG bound on Key paper. After spotting serial dilutions of the protein (5/Lg to 20 ng), the paper was immersed for 2 min in the staining solution and then 5 min in the destaining solution.
Detection of proteins by direct staining or by enzyme immunoassay Chicken, human or mouse IgG diluted in PBS within the range of 0.02-5 # g / s p o t were spotted on Key paper and either stained with Coomassie blue (Fig. 1) or tested for their antigenicity by
Fig. 2. Detection of b o u n d antigen by EIA. Serial dilutions of chicken IgG (5 ~g to 20 ng) were spotted. The active sites were blocked in blocking solution and then the paper was incubated with goat anti-chicken IgG diluted 1 / 5 0 in the blocking solution. After several washes in PBS, the chromogen chloronaphthol was added and specific coloration was obtained without background.
266
EIA (Fig. 2). The minimal detectable quantity was 0.08/~g by Coomassie blue and 0.02/~g by EIA. The same proteins were run on a 10% SDSpolyacrylamide gel (3 h, 100 V). The proteins were then electrotransferred (2.5 h, 0.5 A in sodium phosphate buffer) to Key paper (Fig. 3). Application of the EIA technique to such Key paper permitted the specific detection of these immunoglobulins.
Detection of proteins (antigens by radioimmunoassay Aspartate transcarbamylase (ATCase), was spotted on Key paper and detected there with the
ai~t of a specific antibody followed by a25I-protein A and autoradiography (Fig. 4). The assay on Key paper was specific inasmuch as no autoradiographic stain appeared when the immunoglobulin used was not directed against ACTase. Treatment of the Key paper with ACTase and its bound antibody either by glycine HC1 buffer, pH 2.2, or by SDS, or by 3 M KCNS or by 8 M urea, released the antibody completely from the bound protein (Fig. 4b-e, two upper squares). These dissociation treatments did not affect the binding and the antigenicity of the bound protein to Key paper because it could be redetected with the same specificity (Fig. 4b-e, lower right squares).
Actioity and stability of bound enzymes Serial dilutions of/~-galactosidase, horseradish peroxidase or alkaline phosphatase were spotted on Key paper and stored for 20 days at room temperature, 4°C or - 2 0 ° C . The activity of the bound enzymes was tested using their corresponding substrates following various storage conditions. /~-galactosidase lost its activity after 1 day incubation at any temperature. Peroxidase retained its activity for 20 days following application of a solution of 0.5 m g / m l when stored at 20 o C. Alkaline phosphatase was the most stable enzyme and could be detected on Key paper even -
Fig. 3. Detection of transferred protein on Key paper by EIA: 5-0.15 /zg of chicken IgG were electrophoresed in 10% SDS polyacrylamide gel and electrotransferred in Tris-glycine methanol for 4 h at 0.2 A (constant current). Following transfer, Key paper was incubated in blocking solution and then the antigen was revealed by EIA.
Fig. 4. Detection of aspartate transcarbamylase (ACTase) b o u n d to Key paper. Key paper filters (2 × 2 cm) were divided into 1 × 1 cm squares and each square was spotted with 100 # g of ACTase. The filters were treated with anti-ACTase antibodies and with 125I-protein as described in the materials and methods section. Filter (a) was autoradiographed without further treatment. The 125I label was removed from the remaining filters by the following treatments: (b) low pH; (c) potassium thiocyanate; ( d ) SDS and mercaptoethanol; (e) urea and mercaptoethanol, as described in the materials and methods section. The upper halves of filters b - e were autoradiographed without further treatment, The lower left squares of each of the filters were treated with 125I-protein A; the lower right squares were treated with anti-ACTase antibodies followed by 125I-protein A, and autoradiographed.
267 after 20 days of storage at r o o m temperature and at concentration as low as 0.01 m g / m l . 1.5, 3 and 6 U of active fl-galactosidase were run on 5% SDS polyacrylamide gel and then electrotransferred to Key paper. Enzymatic activity was apparent after 4 h of electrotransfer. All subunits of the enzyme were detected on Key paper after 24 h of transfer. The enzyme was detected specifically and immediately on the paper u p o n the addition of substrate solution (Fig. 5).
1 /xl of serial dilutions of purified goat antichicken I g G antibodies at concentrations ranging
f r o m 0.03 to 4 /~g were spotted onto K e y paper. K e y paper was first incubated in the blocking solution for 1 h and then for 2 h in the antigen solution containing 0.5 mg of chicken I g G per ml of blocking solution (Fig. 6). After washing in PBS, the b o u n d antigen was detected by incubation with peroxidase-conjugated chicken anti-goat I g G followed b y washing with PBS and addition of the chromogen (Fig. 7). The results show that an a n t i b o d y attached to Key paper specifically binds its antigen; no chromogenic reaction occurred when no antibody or antigen were present or when the conjugated antibody was not relevant.
Fig. 5. Electroblotting of fl-galactosidase to Key paper and detection by enzymatic activity. 1.5, 3 and 6 U (slots 1, 2 and 3 respectively) of fl-galactosidase were electrophoresed in a 7.5% acrylamide gel. After transfer in 25 mM sodium phosphate buffer pH 6.5 at 0.5 A (constant current), the enzyme and its subunits were detected by direct addition of the insoluble substrate (X-gal).
Fig. 6. Direct binding of antibody to Key paperl purified goat anti-chicken IgG (4-0.03 #g) was bound to Key paper. After blocking, Key paper was incubated with the specific antigen (Chicken IgG 0.5 mg/ml), and the specific binding revealed by incubation with peroxidase-conjugated goat anti-chicken IgG and the substrate.
Activity of antibody bound to Key paper
268
Fig. 7. Specificity of antibody binding: 1 /*g and 0.5 #g of goat anti-chicken IgG were spotted on Key paper. The binding of the antibody was revealed by specific immunoassay (a). Negative controls comprised the following test strips: (b) without test antibody (goat antichicken IgG); (c) without specific antigen (chicken IgG); (d) with non-specific antibody conjugate: peroxidase-conjugated rabbit anti-mouse IgG instead of peroxidase-conjugated goat anti-chicken IgG.
Discussion
As opposed to other matrices, Key paper is ready for use and does not need any activation. It is flexible, not breakable, and thus fulfills chemical and mechanical conditions required for a suitable matrix for immobilization of proteins. Key paper binds proteins at room temperature. The binding is very fast, does not need any specific buffers or conditions, and is independent of molecular size. Immobilization can be done by direct spotting on the paper or by transfer from acrylamide gel. Proteins bind covalently to Key paper. Washing of the paper in PBS, Brij 0.1%, SDS 0.1% or even heating to 65°C does not affect the binding of the protein or its antigenicity. Heating the paper to 100°C, however, affects the antigenicity of the proteins which remain bound to Key paper. Proteins separated on SDS polyacrylamide gel electrophoresis can be transferred to Key paper. Proteins of different molecular weights in the range of 100 000-14000 bound to the paper to the same extent (data not shown). Proteins bound to Key paper can be detected as antigens, antibodies or enzymes by suitable reagents and assays. In the radioimmunoassay the antibody reacting with the bound antigen can be washed off using
suitable treatments, such as elution with glycine at low p H or by denaturing agents. These reagents do not affect the covalent binding of the protein itself to the Key paper and do not affect its antigenicity. Thus, the same paper to which the antigen is still bound can be re-used for further examination. Though initial screening for the presence of protein can be done by staining with Coomassie blue (which though less sensitive is very rapid) specific detection requires either RIA or EIA assays. Some enzymes bound to Key paper can preserve their enzymatic activity. The enzymatic activity of some enzymes can be demonstrated on the paper even after a long period of storage (several days to several weeks). The fact that Key paper does not bind nucleic acids allows the detection of trace amounts of proteins in the presence of large amounts of nucleic acids. This property is of crucial importance when analyzing proteins derived from whole cell lysates since the nucleic acids do not compete with proteins for binding sites on the solid matrix. Because of these properties, Key paper has potential applications in many fields, e.g., identification of monoclonal antibodies secreted by hybridomas, tests for the presence of a specific protein, immobilization of proteins on a solid matrix,
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development of chromatography-like paper for binding and purification of antibodies or antigens and functional tests of specific proteins (binding to specific molecules or enzymatic activity).
Acknowledgement We gratefully acknowledge the excellent technical help of Mrs. Hanna Yakim.
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