J. Periodontal Res. 10: 224-229
Immunoradiometnc assay for quantification of serum antibodies to dental plaque antigen in immunized dogs STAFFAN AHLSTEDT AND HARALD RYLANDER
Department of Immunology, Institute of Medical Microbiology and Department of Periodontology, Faculty of Odontology, University of Goteborg, Gbteborg, Sweden An immunoradiometric assay (IRMA) was used for quantifying dog serum antibodies to antigens from dental plaque collected from full-grown dogs. The antigens were adsorbed onto the inner surface of plastic tubes and then incubated with dog-anti-plaque serum. I'^I-labelled anti-dog plasma-immunoglobulin was used for quantification of the specific antibodies. Four 10 months old Beagle dogs in excellent gingival health were immunized for 10 weeks with ultrasonicated dog dental plaque. The anibody levels in antisera sampled 6, 8, 10 and 11 weeks after the first antigen injection were 2 to 5 times as high as those recorded before the immunizing period. Tbe variability of the assay as judged from the difference between duplicate samples was found to be 18 % ± 4 (p < 0.01) of the mean value and the variability between the same serum ran on different test occasions 13 % + 7 (p < 0,01). The specificity of the antigen-antibody reaction in the immuno assay was tested by inhibition experiments, Preincubation of the antisera with dental plaque antigen significantly inhibited the antigenantibody reaction in .tbe IRMA, while bovine serum albumin did not, (Received for publication December 10, 1974; accepted Apr. 4,1975}
° Specific antibodies formed locally in the dento-gingival region or present io serum against dentai plaque antigens have been suggested to be of importance for the development of tbe gingival inflammation (Berglund 1971), The tissue alterations have been regarded as an immunopathological reaction to dental plaque antigens (Mergenhagen et al, 1970, Berglund 1971), Thus Rizzo & Mitchell (1966), Ranney & Zander (1970), McDougall (1974) found that application of antigen onto the gingiva of
hyperimmunized animals induced inflammatory tissue alterations similar to the lesion seen in naturaiiy occurring gingivitis induced by accumulation of dental plaque (Theilade et al, 1966, Attstrom & Egelberg 1967, Lindhe, Hamp & Loe 1973), A chronic gingivai lesion is characterized by a subepithelial zone of infiltrating leukocytes, the majority of which can be morphologically classified as cells belonging to the lymphocyte line (Zachrisson 1968, Schroeder, Munzel-Pedrazzoli 1973, Page & Schroeder 1974), Since these cells have the capacity to produce immunoglobulins
SERUM
A N T I B O D I E S
(Brandzaeg 1973) the histoiogicai picture thus supports the hypothesis that immunopathological reactions might contribute to the destruction of the periodontal tissue, i, e, through antigen-antibody complexes and complement activation. This hypothesis is supported also by recent demonstration of antibodies to plaque microorganisms from diseased human gingival cells as well as from sertim (Berglund 1971, Mergenhagen et al, 1970), This paper concerns a) a method (immuno-radio-metric-assay, IRMA) for quantifying serum antibodies to dental plaque antigens and b) estimation of the levels of serum antibodies to dental plaque antigens during experimental immunization of Beagle dogs with such antigen. Materials and Methods
Antigens Dental plaque was collected from full-grown Beagle dogs. The animals had for several years been fed a standardized diet (Hamp, Lindhe & Loe 1973) and had accumulated abundant plaque on their tooth surfaces. The gingiva showed clinical sigos of severe chronic inflammation. Following plaque sampling the different plaques were pooled and suspended in phosphate buffer saline, PBS (0,14 M NaCl, 0,01 M phosphate: pH 7,2), The pooled plaque antigen was dispensed using a Whirlimixer® (Fisons, England) at maximal speed for about 2 minutes. The suspension was then ultrasonicated (20 khz) in a cell disrupter, ModeU B-12 Soaifier® (Branson Sonic Power Company, USA) for 15 minutes in 2 minute intervals
Abbreviations used in this paper: BSA = bovine serum albumin, CPM = counts per minute, FCA = Freund's complete adjuvant, IRMA = Immunoradiometric assay, PBS = phosphate buffered saline.
TO
PLAOUE
ANTIGEN
225
with cooling in ice-water (Lehner, Wilton & Ward 1970), After centrifugation at 12,000 g for one hour at +4°C the supernatant was sterilized by filtering through a 0,45 pm Millipore filter. The protein content of the supernatant was determined according to Lowry et al, (1951) with thyrosine as a standard and adjusted to 0,5 mg protein/ml by PBS, This solution was stored at —20°C until used. The pellet was suspended in 0,5 % formalin over night and then washed three times in PBS at pH 7,2, Also this suspension was stored at —20°C until used, Antisera Four Beagle dogs, about 10 months old, and in excellent gingival health as evaluated by clinical and histoiogicai inspection (Lindhe, Hamp & Loe 1973), were immunized with dental plaque antigen. The antigen used for immunization consisted of 2,0 mg protein (Lowrj' et al, 1951) from ultrasonicated plaque substances and 50 mg pellet in 4 ml PBS, Together with an equal volume of Freund's complete adjuvant (FCA) the antigen extract was injected stibcixtaneotisly close to lymph nodes in four different sites in the back of the animal. Immunization was repeated 3, 6, 8 and 10 weeks later without FCA, In order to study the antibody response to the plaque antigen, antisera from the dogs were sampled before and 6, 8, 10 and 11 weeks after the initial injection. Labelling of anti-dog serum with '^'Iodine The j-globulin fraction of 2 ml anti-dog plasma protein serum (Behringewerke, Marburg, Germany) was prepared by precipitation of the serum with an equal volume of saturated ammonium-sulphate. The precipitate was suspended in 2 ml PBS (pH = 7,6) and dialysed for 24 bours against the same PBS, The antiserum was labelled essentially according to Hunter & Greenwood (1962). Thtis, 2 mCi of Nai^si was added to 2 ml of the dialysed antiserum (16,4 mg/ml) and
226
AHLSTEDT
AND
25 /ji chloramine T solution (8 mg/ml in PBS pH = 7,6) was added for iodination. The reaction was allowed to continue for 4 minutes, after which it was terminated by addition of 100 ^1 sodium metabisulphite (4,8 mg/ml in PBS pH = 7.6), The labelled protein was dialysed free from unreacted 1251 after addition of 100 ^1 KI (20 mg/ml) in PBS pH = 7,6 as carrier and then stored at -20°C utitil used in the IRMA, The activity of all radio-iodinated anti-dog sera were checked by coating polystyrene tubes with ammonium-sulphate precipitated dog y-globulin, followed by incubation with the labelled anti-dog serum as described helow. Immunoradiometric Assay (IRMA) The radio-immuno assay was performed essentially according to Catt & Tregear (1967). Polysterene tubes (AB Labassco, Goteborg, Sweden) were coated with 0.5 ml of an optimal concentration of antigen (the supernatant of the sonicated plaque) by incubation with gentle shaking for 3 hours at 37°C, Before use the tuhes were rinsed 3 times with PBS (pH = 7,2) containing 0,05 % Tween 20, Test serum 0.5 ml in serial dilutions in PBS containing 0.5 % Tween, was incubated in fhe tuhes. for 6 hours at room temperature, after which the tubes were rinsed anew, Ammonium-precipifated radio iodinated anti-dog serum (0,5 ml) was then added (0.16 mg protein/ml) to the tubes atid the tuhes were incubated ior 16 hours at room temperature. After rinsing all the tubes, counting was performed in a Tri-Carb liquid Scintillation Spectrometer® (Packard Instrument Co, Inc, Illinois, USA), Background levels were determined by using PBS instead of test serum. All serum dilutions were tested in duplicate. The antibody level in serum dilution 1 : 10 was expressed as per cent of the counts per minutes (cpm) of a standard immune serum. The Tween 20 was included in all huffers
RYLANDER
used in the antibody assay, except when adsorbing the antigen onto the tubes, to lower the unspecific adsorption to the plastic surface and thus decrease the background radioactivity level. Inhibition test The specificity of the antigen-antibody reactions was tested by pre-incuhation for 3 hours at 37°C in antisera diluted 50-fold with different concentration of antigen (0,8100 /ig/ml). Inhibition tests were also performed hy adding the same amounts of bovine serum albumin (BSA). Results
The counts ohtained in the IRMA proved proportional to the concentration (0,08-0.33 mg/ml) of the labelled anti-dog immunoglobulin serum used. However, comparable results were obtained for antibody quantities. A concentration of 0,16 mg/ml was therefore used In the assay. Optimal antigen concentration for coating of the tubes Coating of the tuhes with different antigen concentrations gave a biphasic count pattern (Table 1), The optimal concentration of antigen solution for the used tubes was found to be about 5 X lO"^ mg/ml. This concentration was then used throtighotit the study. The specificity of the IRMA The immunological specificity of the antigen-antibody reaction was tested by inhibition experiments. As illustrated in Fig. 1, preincuhation with dental plaque antigen significantly inhibited the binding of the antibodies in immune serum to antigen coated tubes. But the slope of the inhibition curve was not very steep. In contrast, preincubation of the immune serum with BSA did not significantly affect the reaction he-
S E R U M
A N T I B O D I E S
T O
P L A Q U E
A N T I G E N
227
Table 1
Radioactivity in tubes coated with different antigen concentrations tested with a serum diluted 1 :100. Antigen concentration mg/ml
CPM X 10=
5 X 10-'
5 X 10-'
5 X 10-*
5 X 10-'
0
14.5
17.9
7.1
3.2
2.1
tween antibodies in antiserum and dental plaque antigen. The reproducibility of the radio-immuno assay The variation between duplicate samples in the assay was estimated using the student's T-test. From 100 determinations the deviation between duplicates was found to be 17.8 % + 4.6 (p < 0.01) of the mean value. To test the reproducibility of different antigen coatings, 3 sera were tested on 2 different occasions. The differences between the assays were of the same magnitude as between duplicates i. e. 13.0 % ± 6.6 (p < 0.01). The antihody response in immunized dogs The typical pattern of the serum titration
curves is illustrated in Fig. 2. All the sera shown are from one of the dogs and were tested on a single occasion. The immunizing procedure raised the antibody levels in the animals in relation to the immunization schedule (Fig. 3), All the animals responded to the immunizations with a rise in antibody level. The increase varied between 2 and 5 fold the initial values. Later during the observation period the antibody level sometimes fell. In all dogs the final antibody determinations were higher than the initial ones (Fig. 3). Discussion
It is established from the present observations that subcutaneous immunization of
ANTiSEftUM CSJJTION
'BOVINE SERUMALBUMSN CONCENTRATION Cf
n ^ / m l
Immunoradiometnc assay for quantification of serum antibodies to dental plaque antigen in immunized dogs STAFFAN AHLSTEDT AND HARALD RYLANDER
Department of Immunology, Institute of Medical Microbiology and Department of Periodontology, Faculty of Odontology, University of Goteborg, Gbteborg, Sweden An immunoradiometric assay (IRMA) was used for quantifying dog serum antibodies to antigens from dental plaque collected from full-grown dogs. The antigens were adsorbed onto the inner surface of plastic tubes and then incubated with dog-anti-plaque serum. I'^I-labelled anti-dog plasma-immunoglobulin was used for quantification of the specific antibodies. Four 10 months old Beagle dogs in excellent gingival health were immunized for 10 weeks with ultrasonicated dog dental plaque. The anibody levels in antisera sampled 6, 8, 10 and 11 weeks after the first antigen injection were 2 to 5 times as high as those recorded before the immunizing period. Tbe variability of the assay as judged from the difference between duplicate samples was found to be 18 % ± 4 (p < 0.01) of the mean value and the variability between the same serum ran on different test occasions 13 % + 7 (p < 0,01). The specificity of the antigen-antibody reaction in the immuno assay was tested by inhibition experiments, Preincubation of the antisera with dental plaque antigen significantly inhibited the antigenantibody reaction in .tbe IRMA, while bovine serum albumin did not, (Received for publication December 10, 1974; accepted Apr. 4,1975}
° Specific antibodies formed locally in the dento-gingival region or present io serum against dentai plaque antigens have been suggested to be of importance for the development of tbe gingival inflammation (Berglund 1971), The tissue alterations have been regarded as an immunopathological reaction to dental plaque antigens (Mergenhagen et al, 1970, Berglund 1971), Thus Rizzo & Mitchell (1966), Ranney & Zander (1970), McDougall (1974) found that application of antigen onto the gingiva of
hyperimmunized animals induced inflammatory tissue alterations similar to the lesion seen in naturaiiy occurring gingivitis induced by accumulation of dental plaque (Theilade et al, 1966, Attstrom & Egelberg 1967, Lindhe, Hamp & Loe 1973), A chronic gingivai lesion is characterized by a subepithelial zone of infiltrating leukocytes, the majority of which can be morphologically classified as cells belonging to the lymphocyte line (Zachrisson 1968, Schroeder, Munzel-Pedrazzoli 1973, Page & Schroeder 1974), Since these cells have the capacity to produce immunoglobulins
SERUM
A N T I B O D I E S
(Brandzaeg 1973) the histoiogicai picture thus supports the hypothesis that immunopathological reactions might contribute to the destruction of the periodontal tissue, i, e, through antigen-antibody complexes and complement activation. This hypothesis is supported also by recent demonstration of antibodies to plaque microorganisms from diseased human gingival cells as well as from sertim (Berglund 1971, Mergenhagen et al, 1970), This paper concerns a) a method (immuno-radio-metric-assay, IRMA) for quantifying serum antibodies to dental plaque antigens and b) estimation of the levels of serum antibodies to dental plaque antigens during experimental immunization of Beagle dogs with such antigen. Materials and Methods
Antigens Dental plaque was collected from full-grown Beagle dogs. The animals had for several years been fed a standardized diet (Hamp, Lindhe & Loe 1973) and had accumulated abundant plaque on their tooth surfaces. The gingiva showed clinical sigos of severe chronic inflammation. Following plaque sampling the different plaques were pooled and suspended in phosphate buffer saline, PBS (0,14 M NaCl, 0,01 M phosphate: pH 7,2), The pooled plaque antigen was dispensed using a Whirlimixer® (Fisons, England) at maximal speed for about 2 minutes. The suspension was then ultrasonicated (20 khz) in a cell disrupter, ModeU B-12 Soaifier® (Branson Sonic Power Company, USA) for 15 minutes in 2 minute intervals
Abbreviations used in this paper: BSA = bovine serum albumin, CPM = counts per minute, FCA = Freund's complete adjuvant, IRMA = Immunoradiometric assay, PBS = phosphate buffered saline.
TO
PLAOUE
ANTIGEN
225
with cooling in ice-water (Lehner, Wilton & Ward 1970), After centrifugation at 12,000 g for one hour at +4°C the supernatant was sterilized by filtering through a 0,45 pm Millipore filter. The protein content of the supernatant was determined according to Lowry et al, (1951) with thyrosine as a standard and adjusted to 0,5 mg protein/ml by PBS, This solution was stored at —20°C until used. The pellet was suspended in 0,5 % formalin over night and then washed three times in PBS at pH 7,2, Also this suspension was stored at —20°C until used, Antisera Four Beagle dogs, about 10 months old, and in excellent gingival health as evaluated by clinical and histoiogicai inspection (Lindhe, Hamp & Loe 1973), were immunized with dental plaque antigen. The antigen used for immunization consisted of 2,0 mg protein (Lowrj' et al, 1951) from ultrasonicated plaque substances and 50 mg pellet in 4 ml PBS, Together with an equal volume of Freund's complete adjuvant (FCA) the antigen extract was injected stibcixtaneotisly close to lymph nodes in four different sites in the back of the animal. Immunization was repeated 3, 6, 8 and 10 weeks later without FCA, In order to study the antibody response to the plaque antigen, antisera from the dogs were sampled before and 6, 8, 10 and 11 weeks after the initial injection. Labelling of anti-dog serum with '^'Iodine The j-globulin fraction of 2 ml anti-dog plasma protein serum (Behringewerke, Marburg, Germany) was prepared by precipitation of the serum with an equal volume of saturated ammonium-sulphate. The precipitate was suspended in 2 ml PBS (pH = 7,6) and dialysed for 24 bours against the same PBS, The antiserum was labelled essentially according to Hunter & Greenwood (1962). Thtis, 2 mCi of Nai^si was added to 2 ml of the dialysed antiserum (16,4 mg/ml) and
226
AHLSTEDT
AND
25 /ji chloramine T solution (8 mg/ml in PBS pH = 7,6) was added for iodination. The reaction was allowed to continue for 4 minutes, after which it was terminated by addition of 100 ^1 sodium metabisulphite (4,8 mg/ml in PBS pH = 7.6), The labelled protein was dialysed free from unreacted 1251 after addition of 100 ^1 KI (20 mg/ml) in PBS pH = 7,6 as carrier and then stored at -20°C utitil used in the IRMA, The activity of all radio-iodinated anti-dog sera were checked by coating polystyrene tubes with ammonium-sulphate precipitated dog y-globulin, followed by incubation with the labelled anti-dog serum as described helow. Immunoradiometric Assay (IRMA) The radio-immuno assay was performed essentially according to Catt & Tregear (1967). Polysterene tubes (AB Labassco, Goteborg, Sweden) were coated with 0.5 ml of an optimal concentration of antigen (the supernatant of the sonicated plaque) by incubation with gentle shaking for 3 hours at 37°C, Before use the tuhes were rinsed 3 times with PBS (pH = 7,2) containing 0,05 % Tween 20, Test serum 0.5 ml in serial dilutions in PBS containing 0.5 % Tween, was incubated in fhe tuhes. for 6 hours at room temperature, after which the tubes were rinsed anew, Ammonium-precipifated radio iodinated anti-dog serum (0,5 ml) was then added (0.16 mg protein/ml) to the tubes atid the tuhes were incubated ior 16 hours at room temperature. After rinsing all the tubes, counting was performed in a Tri-Carb liquid Scintillation Spectrometer® (Packard Instrument Co, Inc, Illinois, USA), Background levels were determined by using PBS instead of test serum. All serum dilutions were tested in duplicate. The antibody level in serum dilution 1 : 10 was expressed as per cent of the counts per minutes (cpm) of a standard immune serum. The Tween 20 was included in all huffers
RYLANDER
used in the antibody assay, except when adsorbing the antigen onto the tubes, to lower the unspecific adsorption to the plastic surface and thus decrease the background radioactivity level. Inhibition test The specificity of the antigen-antibody reactions was tested by pre-incuhation for 3 hours at 37°C in antisera diluted 50-fold with different concentration of antigen (0,8100 /ig/ml). Inhibition tests were also performed hy adding the same amounts of bovine serum albumin (BSA). Results
The counts ohtained in the IRMA proved proportional to the concentration (0,08-0.33 mg/ml) of the labelled anti-dog immunoglobulin serum used. However, comparable results were obtained for antibody quantities. A concentration of 0,16 mg/ml was therefore used In the assay. Optimal antigen concentration for coating of the tubes Coating of the tuhes with different antigen concentrations gave a biphasic count pattern (Table 1), The optimal concentration of antigen solution for the used tubes was found to be about 5 X lO"^ mg/ml. This concentration was then used throtighotit the study. The specificity of the IRMA The immunological specificity of the antigen-antibody reaction was tested by inhibition experiments. As illustrated in Fig. 1, preincuhation with dental plaque antigen significantly inhibited the binding of the antibodies in immune serum to antigen coated tubes. But the slope of the inhibition curve was not very steep. In contrast, preincubation of the immune serum with BSA did not significantly affect the reaction he-
S E R U M
A N T I B O D I E S
T O
P L A Q U E
A N T I G E N
227
Table 1
Radioactivity in tubes coated with different antigen concentrations tested with a serum diluted 1 :100. Antigen concentration mg/ml
CPM X 10=
5 X 10-'
5 X 10-'
5 X 10-*
5 X 10-'
0
14.5
17.9
7.1
3.2
2.1
tween antibodies in antiserum and dental plaque antigen. The reproducibility of the radio-immuno assay The variation between duplicate samples in the assay was estimated using the student's T-test. From 100 determinations the deviation between duplicates was found to be 17.8 % + 4.6 (p < 0.01) of the mean value. To test the reproducibility of different antigen coatings, 3 sera were tested on 2 different occasions. The differences between the assays were of the same magnitude as between duplicates i. e. 13.0 % ± 6.6 (p < 0.01). The antihody response in immunized dogs The typical pattern of the serum titration
curves is illustrated in Fig. 2. All the sera shown are from one of the dogs and were tested on a single occasion. The immunizing procedure raised the antibody levels in the animals in relation to the immunization schedule (Fig. 3), All the animals responded to the immunizations with a rise in antibody level. The increase varied between 2 and 5 fold the initial values. Later during the observation period the antibody level sometimes fell. In all dogs the final antibody determinations were higher than the initial ones (Fig. 3). Discussion
It is established from the present observations that subcutaneous immunization of
ANTiSEftUM CSJJTION
'BOVINE SERUMALBUMSN CONCENTRATION Cf
n ^ / m l
