Serum Proteins and Secretory Component in Human Carious Dentin KEIJI OKAMURA, KUMIO TSUBAKIMOTO, KENICHI UOBE, KEN NISHIDA and MASAHIRO TSUTSUI Department of Oral Pathology, Osaka Dental University, Osaka, Japan By the use of the peroxidase-labeled antibody method, significant localization of IgG, IgA, albumin and transferrin was demonstrated in the deep lesion of 20 carious teeth, where the secretory component was absent. These serum proteins formed a distinct zone, surrounding the overlying, shallow lesion infected with bacteria. J Dent Res 58(3):1127-1133, March 1979
Introduction. Behavior of the serum proteins is accepted as one of the significant factors in modifying the carious process. Sumitani et al. 1 reported that 'y-globulin and albumin were found beneath a translucent zone in the human carious dentin. The purpose of our study was to demonstrate the localization of salivary proteins and serum proteins in human carious dentin by the use of the peroxidase-labeled antibody method, and also to identify the origin of these proteins, for the sake of obtaining more information about the reaction of dentin to its decay.
Materials and methods. Specimens of human dentin. -Human vital permanent teeth, five normal and twenty carious, ranging from 16-year-old to 40year-old, were extracted under the diagnosis of pulpitis or periodontitis, or for other reasons. Immediately after extraction, a burr hole was produced by the use of a dental drill at the height of the cervix of each tooth. The teeth were fixed in 4% paraformaldehyde/0.0 1 M phosphate buffer (pH 7.3) at 40C for 3 days, and divided at the height of the cervix into two segments. The coronal segments were washed with 0.01 M phosphate-buffered saline (PBS:
pH 7.1-7.2) at 40C for 24 hours, and decalcified with 10% ethylenediaminetetraacetic acid disodium salt (EDTA: pH 7.3) at 40C for 4-6 weeks. They were washed with PBS at 40C for 24 hours, and embedded in paraffin according to the method established by Sainte-Marie2 for immunofluorescent microscopy. Microtome sections, 7-10 pm, were serially made. Antisera.-The following commercially available antisera were used: (1) anti-human IgG goat serum (y-chain specific)*, (2) anti-human IgA goat serum (a-chain specific)*, (3) anti-human albumin goat serum*, (4) anti-human transferrin goat serum*, (5) anti-human secretory component goat serum*. The antisera to the human serum proteins were evaluated by the immunoelectrophoresis described by Grabar and Williams3 against human normal serum+. Monospecificity of each antiserum was confirmed. These antisera were also checked by Ouchterlony's double diffusion method4 against the human parotid saliva from a normal 28-year-old male. It was found that those against albumin and IgA reacted strongly with the saliva, while those against IgG and transferrin reacted only weakly. The antiserum to secretory component reacted with saliva, but not with the normal human serum, nor with the normal human dentin extract, prepared according to Jones and Leaver's method5. The potency of each antiserum was measured by Becker's method6. Specific antigens such as IgG*, albumin* and transferrint were commercially available for quantification, while IgA was prepared according to Vaerman et al.'s method7. Potency was found to be 1.4 mg antibody/
*Miles Laboratories, Inc., Elkhart, Ind. USA +Behringwerke AG, Marburg Lahn, W. Germany $AB KABI, Stockholm, Sweden 1127 Downloaded from jdr.sagepub.com at UCSF LIBRARY & CKM on April 5, 2015 For personal use only. No other uses without permission.
Received for publication November 15, 1977. Accepted for publication June 19, 1978.
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ml (to IgG), 1.0 mg antibody/ml (to IgA), 1.5 mg antibody/ml (to albumin) and 1.2 mg antibody/ml (to transferrin), respectively. Potency of the anti-human secretory component goat serum was not determined. Preparation of labeled antisera.-According to Nakane et al.'s method8, horseradish peroxidase (HRPO) was conjugated with the respective IgG fraction, which was separated from each anti-serum by the use of a DEAE-cellulosell column, and free HRPO was removed by the use of a Sephadex G-200* column. Solution of each labeled antibody was concentrated to 3 mg protein/ ml. Protein A. -Protein A, well-know for its specific reaction with the Fc part of IgG molecule, was also employed for the detection of human IgG in the tissue. In addition to protein A labeled with fluorescein isothiocyanate*, obtained commercially, HRPOlabeled protein A was prepared. Five mg of HRPO were treated with sodium periodate according to Nakane et al.'s method8. The activated HRPO was conjugated at room temperature for 3 hours with 5 mg of protein A*, being dissolved in 3 ml of 0.01 M carbonate buffer (pH 9.5), treated with 5 mg of NaBH4 at 40C for 3 hours, and dialyzed against PBS at 40C for 48 hours. The conjugate was applied upon an affinity column of CNBr-activated Sepharose 4B coupled with 20 mg of human IgG. After a small amount of unbound HRPO was removed by elution with PBS at 40C, the labeled protein A was obtained by elution at 40C with 0.1 M acetate buffer (pH 4.0) containing 1 M NaCl. Immediately, the solution of labeled protein A was dialyzed against PBS and concentrated at the same time to 3 mg protein/ml, by the use of Amicon Diaflo Cells: at 40C. Staining. - Following Sainte-Marie's procedure,2 the paraffin sections were deparaffinized, immersed in PBS at 40C for 30 minutes, and incubated with each one of the labeled antibodies or with the labeled protein A (0.3 mg protein/ml, respectively), in a moist chamber at room temperature for one hour. The sections were completely
I Sigma Chemical Co., St. Louis, Mo. USA IlWhatman, Ltd., Maidstone, Kent, England *Pharmacia Fine Chemicals AB, Uppsala, Sweden +Amicon Corporation, Lexington, Mass. USA
J Dent Res March 1 9 79
washed with PBS at 40C for 30 minutes, and stained according to Graham and Karnovsky's method9. Control study-Influence of the decalcifying process upon the antigenicity of human serum protein was examined. Human IgG dissolved in PBS (10 mg/ml) was dialyzed at 40C for 6 weeks against 10% EDTA as well as against PBS. According to Mancini et al.'s procedurel, immunochemical quantitation was carried out upon the remaining IgG after possible reduction with EDTA, and it was found that approximately 80% of the antigen remained after the EDTA treatment, whereas no reduction was observed in the case of PBS. The histochemical research technique was subjected to an immunochemical examination from various angles. In the first place, the dentinal sections were incubated with unlabeled goat y-globulin* (non-antibody: 10 mg/ml) at room temperature for one hour, prior to the incubation with labeled antibody (0.3 mg protein/ml) at room temperature for another hour. This was to ascertain absence of the non-specific inhibition of antigen-antibody reaction. Secondly, they were incubated with labeled goat y-globulin (non-antibody: 3 mg protein/ ml) at room temperature for one hour. This was to confirm no adsorption of globulin upon the sections. Third, they were incubated at room temperature for one hour with the labeled antibody solution (3 mg protein/ml) which had been absorbed by a column of CNBr-activated Sepharose 4B coupled with specific antigen. This was to confirm no adsorption of non-antibody globulin upon the sections. Finally, they were incubated with the unlabeled antiserum, diluted ten-fold with PBS, at room temperature for one hour, prior to the incubation with labeled antibody (0.3 mg protein/ml) at room temperature for another hour. This was done to ascertain complete inhibition of the specific antigen-antibody reaction.
Results. In five cases of normal dentin, no significant staining was observed following application of the respective labeled antibody to each serum protein or secretory component. *Miles Laboratories, Inc., Elkhart, Ind. USA
Downloaded from jdr.sagepub.com at UCSF LIBRARY & CKM on April 5, 2015 For personal use only. No other uses without permission.
Vol. S8 Nov. 3
SER UM AND SC IN CARIO US DENTIN
Fig. 1. -A section of the normal dentin, stained with labeled anti-lgG (Orig. mag. x 33). No notable staining is observed in the dentino-enainel junction.
The interglobular dentin and pre-dentin presented non-specific stainings in every section of all teeth examined. At the dentinoenamel junction, there was no marked staining in both sound and carious dentin (Fig. 1, 2a).
1129
In 20 cases of carious dentin, highly significant stainings in the form of a distinct zone (vide infra) were regularly demonstrated following application of the respective, labeled, antibody. Immunological specificity of the histological reaction was investigated in respect to serial sections of the carious dentin exhibiting such a highly significant staining. The staining reaction suffered from no change in intensity and distribution by the pre-incubation with unlabeled goat y-globulin (Fig. 2a); therefore, absence of the nonspecific inhibition of antigen-antibody reaction was ascertained. On the other hand, no notable staining was observed following incubation with labeled goat y-globulin (Fig. 2b), nor with the labeled antibody following absorption by a column of CNBractivated Sepharose 4B, coupled with the specific antigen; therefore, adsorption upon
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Introduction. Behavior of the serum proteins is accepted as one of the significant factors in modifying the carious process. Sumitani et al. 1 reported that 'y-globulin and albumin were found beneath a translucent zone in the human carious dentin. The purpose of our study was to demonstrate the localization of salivary proteins and serum proteins in human carious dentin by the use of the peroxidase-labeled antibody method, and also to identify the origin of these proteins, for the sake of obtaining more information about the reaction of dentin to its decay.
Materials and methods. Specimens of human dentin. -Human vital permanent teeth, five normal and twenty carious, ranging from 16-year-old to 40year-old, were extracted under the diagnosis of pulpitis or periodontitis, or for other reasons. Immediately after extraction, a burr hole was produced by the use of a dental drill at the height of the cervix of each tooth. The teeth were fixed in 4% paraformaldehyde/0.0 1 M phosphate buffer (pH 7.3) at 40C for 3 days, and divided at the height of the cervix into two segments. The coronal segments were washed with 0.01 M phosphate-buffered saline (PBS:
pH 7.1-7.2) at 40C for 24 hours, and decalcified with 10% ethylenediaminetetraacetic acid disodium salt (EDTA: pH 7.3) at 40C for 4-6 weeks. They were washed with PBS at 40C for 24 hours, and embedded in paraffin according to the method established by Sainte-Marie2 for immunofluorescent microscopy. Microtome sections, 7-10 pm, were serially made. Antisera.-The following commercially available antisera were used: (1) anti-human IgG goat serum (y-chain specific)*, (2) anti-human IgA goat serum (a-chain specific)*, (3) anti-human albumin goat serum*, (4) anti-human transferrin goat serum*, (5) anti-human secretory component goat serum*. The antisera to the human serum proteins were evaluated by the immunoelectrophoresis described by Grabar and Williams3 against human normal serum+. Monospecificity of each antiserum was confirmed. These antisera were also checked by Ouchterlony's double diffusion method4 against the human parotid saliva from a normal 28-year-old male. It was found that those against albumin and IgA reacted strongly with the saliva, while those against IgG and transferrin reacted only weakly. The antiserum to secretory component reacted with saliva, but not with the normal human serum, nor with the normal human dentin extract, prepared according to Jones and Leaver's method5. The potency of each antiserum was measured by Becker's method6. Specific antigens such as IgG*, albumin* and transferrint were commercially available for quantification, while IgA was prepared according to Vaerman et al.'s method7. Potency was found to be 1.4 mg antibody/
*Miles Laboratories, Inc., Elkhart, Ind. USA +Behringwerke AG, Marburg Lahn, W. Germany $AB KABI, Stockholm, Sweden 1127 Downloaded from jdr.sagepub.com at UCSF LIBRARY & CKM on April 5, 2015 For personal use only. No other uses without permission.
Received for publication November 15, 1977. Accepted for publication June 19, 1978.
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ml (to IgG), 1.0 mg antibody/ml (to IgA), 1.5 mg antibody/ml (to albumin) and 1.2 mg antibody/ml (to transferrin), respectively. Potency of the anti-human secretory component goat serum was not determined. Preparation of labeled antisera.-According to Nakane et al.'s method8, horseradish peroxidase (HRPO) was conjugated with the respective IgG fraction, which was separated from each anti-serum by the use of a DEAE-cellulosell column, and free HRPO was removed by the use of a Sephadex G-200* column. Solution of each labeled antibody was concentrated to 3 mg protein/ ml. Protein A. -Protein A, well-know for its specific reaction with the Fc part of IgG molecule, was also employed for the detection of human IgG in the tissue. In addition to protein A labeled with fluorescein isothiocyanate*, obtained commercially, HRPOlabeled protein A was prepared. Five mg of HRPO were treated with sodium periodate according to Nakane et al.'s method8. The activated HRPO was conjugated at room temperature for 3 hours with 5 mg of protein A*, being dissolved in 3 ml of 0.01 M carbonate buffer (pH 9.5), treated with 5 mg of NaBH4 at 40C for 3 hours, and dialyzed against PBS at 40C for 48 hours. The conjugate was applied upon an affinity column of CNBr-activated Sepharose 4B coupled with 20 mg of human IgG. After a small amount of unbound HRPO was removed by elution with PBS at 40C, the labeled protein A was obtained by elution at 40C with 0.1 M acetate buffer (pH 4.0) containing 1 M NaCl. Immediately, the solution of labeled protein A was dialyzed against PBS and concentrated at the same time to 3 mg protein/ml, by the use of Amicon Diaflo Cells: at 40C. Staining. - Following Sainte-Marie's procedure,2 the paraffin sections were deparaffinized, immersed in PBS at 40C for 30 minutes, and incubated with each one of the labeled antibodies or with the labeled protein A (0.3 mg protein/ml, respectively), in a moist chamber at room temperature for one hour. The sections were completely
I Sigma Chemical Co., St. Louis, Mo. USA IlWhatman, Ltd., Maidstone, Kent, England *Pharmacia Fine Chemicals AB, Uppsala, Sweden +Amicon Corporation, Lexington, Mass. USA
J Dent Res March 1 9 79
washed with PBS at 40C for 30 minutes, and stained according to Graham and Karnovsky's method9. Control study-Influence of the decalcifying process upon the antigenicity of human serum protein was examined. Human IgG dissolved in PBS (10 mg/ml) was dialyzed at 40C for 6 weeks against 10% EDTA as well as against PBS. According to Mancini et al.'s procedurel, immunochemical quantitation was carried out upon the remaining IgG after possible reduction with EDTA, and it was found that approximately 80% of the antigen remained after the EDTA treatment, whereas no reduction was observed in the case of PBS. The histochemical research technique was subjected to an immunochemical examination from various angles. In the first place, the dentinal sections were incubated with unlabeled goat y-globulin* (non-antibody: 10 mg/ml) at room temperature for one hour, prior to the incubation with labeled antibody (0.3 mg protein/ml) at room temperature for another hour. This was to ascertain absence of the non-specific inhibition of antigen-antibody reaction. Secondly, they were incubated with labeled goat y-globulin (non-antibody: 3 mg protein/ ml) at room temperature for one hour. This was to confirm no adsorption of globulin upon the sections. Third, they were incubated at room temperature for one hour with the labeled antibody solution (3 mg protein/ml) which had been absorbed by a column of CNBr-activated Sepharose 4B coupled with specific antigen. This was to confirm no adsorption of non-antibody globulin upon the sections. Finally, they were incubated with the unlabeled antiserum, diluted ten-fold with PBS, at room temperature for one hour, prior to the incubation with labeled antibody (0.3 mg protein/ml) at room temperature for another hour. This was done to ascertain complete inhibition of the specific antigen-antibody reaction.
Results. In five cases of normal dentin, no significant staining was observed following application of the respective labeled antibody to each serum protein or secretory component. *Miles Laboratories, Inc., Elkhart, Ind. USA
Downloaded from jdr.sagepub.com at UCSF LIBRARY & CKM on April 5, 2015 For personal use only. No other uses without permission.
Vol. S8 Nov. 3
SER UM AND SC IN CARIO US DENTIN
Fig. 1. -A section of the normal dentin, stained with labeled anti-lgG (Orig. mag. x 33). No notable staining is observed in the dentino-enainel junction.
The interglobular dentin and pre-dentin presented non-specific stainings in every section of all teeth examined. At the dentinoenamel junction, there was no marked staining in both sound and carious dentin (Fig. 1, 2a).
1129
In 20 cases of carious dentin, highly significant stainings in the form of a distinct zone (vide infra) were regularly demonstrated following application of the respective, labeled, antibody. Immunological specificity of the histological reaction was investigated in respect to serial sections of the carious dentin exhibiting such a highly significant staining. The staining reaction suffered from no change in intensity and distribution by the pre-incubation with unlabeled goat y-globulin (Fig. 2a); therefore, absence of the nonspecific inhibition of antigen-antibody reaction was ascertained. On the other hand, no notable staining was observed following incubation with labeled goat y-globulin (Fig. 2b), nor with the labeled antibody following absorption by a column of CNBractivated Sepharose 4B, coupled with the specific antigen; therefore, adsorption upon
am>-\ >9+9>s
