Journal of Immunological Methods, 23 (1978) 109--116
109
© Elsevier/North-Holland Biomedical Press
MEASUREMENT OF IMMUNOPEROXIDASE ACTIVITY. A RAPID AND REPRODUCIBLE IMMUNOASSAY FOR QUANTITATION OF C E L L U L A R ANTIGENS
F. VAN LEUVEN, J.J. CASSIMAN and H. VAN DEN BERGHE Division of Human Genetics, Department of Human Biology, Minderbroederstraat 12, B-3000 Louvain, Belgium
(Received 6 January 1978, accepted 20 March 1978) A procedure is described making it possible to obtain quantitative data with the immunohistochemical peroxidase anti--peroxidase (PAP) technique. After applying PAP on fixed fibroblast preparations, peroxidase enzymic activity is measured on solubilized immune-complexes. The results show this technique to be sensitive and reproducible. Differences in the amounts of the same antigen (t~2-macroglobulin) are detected in differently treated fibroblast preparations. The value of the technique for the demonstration of cellular antigens is discussed.
INTRODUCTION The peroxidase anti--peroxidase (PAP) technique is becoming a widely used immunohistochemical technique for localization of specific cellular antigens with the light or electron microscope (Sternberger et al., 1970; Weir et al., 1974; De Mey et al., 1976). The technique involves double immunochemical complexing as follows: antigen, specific antibody, antiIgG, peroxidase anti--peroxidase (PAP) complex. The antigen is detected and localized in frozen or fixed specimens by in situ generation of a colored precipitate by the action of the peroxidase on the substrate in the presence of H202 (Graham and Karnovsky, 1966). This procedure gives excellent results. Only qualitative data are obtained, however, for serial dilution of the specific antibody cannot give reliable quantitative data, since the density of the colored precipitate generated is amplified by osmium tetroxide postfixation. The present study describes a procedure to make the PAP technique quantitative by solubilizing the peroxidase before the staining step. Peroxidase enzyme activity is measured in solution, so that the a m o u n t of the antigen present in a particular preparation is indirectly determined. Variable concentrations of two antigens, ~2-macroglobulin (a2M) and tubulin, known to be present in fibroblast cultures (Van Leuven et al., 1977; De Mey et al., 1976) are shown to be detectable with this assay.
110 MATERIALS AND METHODS
Materials Soluble complexes of peroxidase anti--peroxidase (PAP) and rabbit-anti-human a2M-antibodies were purchased from Dakopatts A/S, Denmark. Rabbit anti-tubulin was a gift from M. Joniau. The non-ionic detergent Berol-043 was a gift from Berol Kemi AB, Sweden. Ortho-dianisidine dihydrochloride and 3,3'-diaminobenzidine tetrahydrochloride were obtained from Sigma Chem. Co., St. Louis (U.S.A.}. All other chemicals were of the highest purity available. Cell culture and dissociation Human diploid skin fibroblasts (NHF) were cultured as previously described (Van der Schueren et al., 1976). The medium consisted of Dulbecco's modified Eagle's medium (DME), containing 10% (v/v) heat-inactivated newborn calf serum (NCS), 1 g/1 NaHCO3, 15 mM N-Tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES) and 15 mM N-2-hydroxyethyl piperazine-N-2-ethane sulfonic acid (HEPES) buffered to pH 7.5 with 1 N NaOH. In some experiments NCS was treated with trypsin (crystalline; Sigma Chem. Co.) at 2.40 mg/ml. This is sufficient to saturate all a2M present in the serum. Excess trypsin was inactivated with Trasylol (Behringwerke AG) 0.67 mg/ml. For PAP tests, fibroblast cultures were dissociated by a solution containing 0.05% crude trypsin (Difco 1 : 200) and 0.02% EDTA in Tris buffered saline (TBS, 0.02 M Tris--HC1 containing 8 g NaC1 and 0.4 g KC1/1), pH 7.4. The cells were washed in growth medium, counted in an electronic particle counter (Coulter Electronics, Model ZBI), and diluted to 100,000 cells/ml in medium. One millilitre of this suspension was plated in disposable trays (Linbro Co.) containing 13 mm round glass coverslips (Assistent Co.). Cells were allowed to grow for at least 48 h before being used in any assay. Incorporation o f [3H]leucine [3H]Leucine (Amersham, S.A. 1 Ci/mM) was added to the medium (10 pCi/ml) and after 48 h culture, the cells were washed three times and incubated in fresh medium for 24 h. Sonicates of the labeled cells were processed on a Skatron apparatus (Skatron AS, Lierbyen, Norway). The filters were immersed in scintillation fluid (Instagel, Packard Co.) and counted in a Tricarb scintillation counter (Packard). By external standardization no differences in counting efficiency were noted, so the data were not corrected. PAP and peroxidase assay PAP was performed essentially as described by De Mey et al. (1976). N H F cultures grown on round glass coverslips were fixed for 15 min with 0.3%
111 glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4), at room temperature. After washing in the same buffer for 30 min, the cells were postfixed at 4°C for 5 min each with 50%, 100% and 50% acetone consecutively to increase plasma membrane permeability. The specific antibody was added (diluted as indicated in TBS) and left in contact for 30 min at room temperature. Swine--anti-rabbit IgG (1 : 20 in TBS) was added for 30 min at the PAP complex (1 : 300 in TBS) for 30 min. In between antibodies, the coverslips were soaked for 30 min in three changes of TBS. At this stage the preparations were incubated with 3,3'-diaminobenzidine tetrahydrochloride 5 mg/10 ml in 0.05 M Tris--HC1 buffer (pH 7), containing 0.01% hydrogen peroxide, for 30 min, post-stained for 10 min with 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4), at 4°C, m o u n t e d and examined by light microscopy. F o r quantitative measurement of the peroxidase activity, o-dianisidine was chosen as a more soluble substrate yielding a soluble reaction product. The diaminobenzidine and following steps were therefore omitted. The slides were washed with phosphate buffer (20 mM, pH 6.0) containing 1% Berol. Two millilitres of the same buffer were added to each slide, and the contents sonicated for 10 sec at 30% of maximal energy in a Virsonic cell disrupter (Virtis Co.) on ice. Between 0.8 ml and 1.6 ml of the total volume was used for determination of peroxidase activity. The assay mixture contained the following components (final concentration): sodium phosphate buffer (pH 6.0), 20 mM; non-ionic detergent Berol-0.43, 0.73 (w/v); o-dianisidine (added as a methanol solution) 1.43 mM, 0.0027% H202 and enzyme, in a final volume of 2.2 ml.
Rocket immunoelectrophoresis (RIEF) R I E F was performed according to the methods described by Weeke {1973). Agarose gels, 1% (w/v) in sodium veronal buffer (75 mM, pH 8.6) containing 2 mM calcium lactate and 15 mM sodium azide were used. These gels contained 1% (w/v) of the non-ionic detergent Berol-0.43. The antibody concentration in the gel was as indicated in the results section. Electrophoresis was performed at 4 V/cm for 18 h at 15°C. Cell samples were solubilized by sonication on ice in gel buffer containing 1% Berol {w/v). Precipitates were stained with Coomassie brilliant blue after appropriate removal of non-precipitated proteins. RESULTS AND DISCUSSION
PAP-complex enzymatic activity measurement The characterization of the assay and some results obtained with it will be described and discussed in this section. To aid solubilization of the specimens after double labeling with antibodies, the addition of a non-ionic detergent, Berol-0.43 proved helpful. Berol was therefore added to all enzyme assay mixtures in a final concentration of 0.73%. This however
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109
© Elsevier/North-Holland Biomedical Press
MEASUREMENT OF IMMUNOPEROXIDASE ACTIVITY. A RAPID AND REPRODUCIBLE IMMUNOASSAY FOR QUANTITATION OF C E L L U L A R ANTIGENS
F. VAN LEUVEN, J.J. CASSIMAN and H. VAN DEN BERGHE Division of Human Genetics, Department of Human Biology, Minderbroederstraat 12, B-3000 Louvain, Belgium
(Received 6 January 1978, accepted 20 March 1978) A procedure is described making it possible to obtain quantitative data with the immunohistochemical peroxidase anti--peroxidase (PAP) technique. After applying PAP on fixed fibroblast preparations, peroxidase enzymic activity is measured on solubilized immune-complexes. The results show this technique to be sensitive and reproducible. Differences in the amounts of the same antigen (t~2-macroglobulin) are detected in differently treated fibroblast preparations. The value of the technique for the demonstration of cellular antigens is discussed.
INTRODUCTION The peroxidase anti--peroxidase (PAP) technique is becoming a widely used immunohistochemical technique for localization of specific cellular antigens with the light or electron microscope (Sternberger et al., 1970; Weir et al., 1974; De Mey et al., 1976). The technique involves double immunochemical complexing as follows: antigen, specific antibody, antiIgG, peroxidase anti--peroxidase (PAP) complex. The antigen is detected and localized in frozen or fixed specimens by in situ generation of a colored precipitate by the action of the peroxidase on the substrate in the presence of H202 (Graham and Karnovsky, 1966). This procedure gives excellent results. Only qualitative data are obtained, however, for serial dilution of the specific antibody cannot give reliable quantitative data, since the density of the colored precipitate generated is amplified by osmium tetroxide postfixation. The present study describes a procedure to make the PAP technique quantitative by solubilizing the peroxidase before the staining step. Peroxidase enzyme activity is measured in solution, so that the a m o u n t of the antigen present in a particular preparation is indirectly determined. Variable concentrations of two antigens, ~2-macroglobulin (a2M) and tubulin, known to be present in fibroblast cultures (Van Leuven et al., 1977; De Mey et al., 1976) are shown to be detectable with this assay.
110 MATERIALS AND METHODS
Materials Soluble complexes of peroxidase anti--peroxidase (PAP) and rabbit-anti-human a2M-antibodies were purchased from Dakopatts A/S, Denmark. Rabbit anti-tubulin was a gift from M. Joniau. The non-ionic detergent Berol-043 was a gift from Berol Kemi AB, Sweden. Ortho-dianisidine dihydrochloride and 3,3'-diaminobenzidine tetrahydrochloride were obtained from Sigma Chem. Co., St. Louis (U.S.A.}. All other chemicals were of the highest purity available. Cell culture and dissociation Human diploid skin fibroblasts (NHF) were cultured as previously described (Van der Schueren et al., 1976). The medium consisted of Dulbecco's modified Eagle's medium (DME), containing 10% (v/v) heat-inactivated newborn calf serum (NCS), 1 g/1 NaHCO3, 15 mM N-Tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES) and 15 mM N-2-hydroxyethyl piperazine-N-2-ethane sulfonic acid (HEPES) buffered to pH 7.5 with 1 N NaOH. In some experiments NCS was treated with trypsin (crystalline; Sigma Chem. Co.) at 2.40 mg/ml. This is sufficient to saturate all a2M present in the serum. Excess trypsin was inactivated with Trasylol (Behringwerke AG) 0.67 mg/ml. For PAP tests, fibroblast cultures were dissociated by a solution containing 0.05% crude trypsin (Difco 1 : 200) and 0.02% EDTA in Tris buffered saline (TBS, 0.02 M Tris--HC1 containing 8 g NaC1 and 0.4 g KC1/1), pH 7.4. The cells were washed in growth medium, counted in an electronic particle counter (Coulter Electronics, Model ZBI), and diluted to 100,000 cells/ml in medium. One millilitre of this suspension was plated in disposable trays (Linbro Co.) containing 13 mm round glass coverslips (Assistent Co.). Cells were allowed to grow for at least 48 h before being used in any assay. Incorporation o f [3H]leucine [3H]Leucine (Amersham, S.A. 1 Ci/mM) was added to the medium (10 pCi/ml) and after 48 h culture, the cells were washed three times and incubated in fresh medium for 24 h. Sonicates of the labeled cells were processed on a Skatron apparatus (Skatron AS, Lierbyen, Norway). The filters were immersed in scintillation fluid (Instagel, Packard Co.) and counted in a Tricarb scintillation counter (Packard). By external standardization no differences in counting efficiency were noted, so the data were not corrected. PAP and peroxidase assay PAP was performed essentially as described by De Mey et al. (1976). N H F cultures grown on round glass coverslips were fixed for 15 min with 0.3%
111 glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4), at room temperature. After washing in the same buffer for 30 min, the cells were postfixed at 4°C for 5 min each with 50%, 100% and 50% acetone consecutively to increase plasma membrane permeability. The specific antibody was added (diluted as indicated in TBS) and left in contact for 30 min at room temperature. Swine--anti-rabbit IgG (1 : 20 in TBS) was added for 30 min at the PAP complex (1 : 300 in TBS) for 30 min. In between antibodies, the coverslips were soaked for 30 min in three changes of TBS. At this stage the preparations were incubated with 3,3'-diaminobenzidine tetrahydrochloride 5 mg/10 ml in 0.05 M Tris--HC1 buffer (pH 7), containing 0.01% hydrogen peroxide, for 30 min, post-stained for 10 min with 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4), at 4°C, m o u n t e d and examined by light microscopy. F o r quantitative measurement of the peroxidase activity, o-dianisidine was chosen as a more soluble substrate yielding a soluble reaction product. The diaminobenzidine and following steps were therefore omitted. The slides were washed with phosphate buffer (20 mM, pH 6.0) containing 1% Berol. Two millilitres of the same buffer were added to each slide, and the contents sonicated for 10 sec at 30% of maximal energy in a Virsonic cell disrupter (Virtis Co.) on ice. Between 0.8 ml and 1.6 ml of the total volume was used for determination of peroxidase activity. The assay mixture contained the following components (final concentration): sodium phosphate buffer (pH 6.0), 20 mM; non-ionic detergent Berol-0.43, 0.73 (w/v); o-dianisidine (added as a methanol solution) 1.43 mM, 0.0027% H202 and enzyme, in a final volume of 2.2 ml.
Rocket immunoelectrophoresis (RIEF) R I E F was performed according to the methods described by Weeke {1973). Agarose gels, 1% (w/v) in sodium veronal buffer (75 mM, pH 8.6) containing 2 mM calcium lactate and 15 mM sodium azide were used. These gels contained 1% (w/v) of the non-ionic detergent Berol-0.43. The antibody concentration in the gel was as indicated in the results section. Electrophoresis was performed at 4 V/cm for 18 h at 15°C. Cell samples were solubilized by sonication on ice in gel buffer containing 1% Berol {w/v). Precipitates were stained with Coomassie brilliant blue after appropriate removal of non-precipitated proteins. RESULTS AND DISCUSSION
PAP-complex enzymatic activity measurement The characterization of the assay and some results obtained with it will be described and discussed in this section. To aid solubilization of the specimens after double labeling with antibodies, the addition of a non-ionic detergent, Berol-0.43 proved helpful. Berol was therefore added to all enzyme assay mixtures in a final concentration of 0.73%. This however
112 1.0 bJ U Z
