JP6310631B2 - Periodontal pathogen plasma or serum antibody titer kit - Google Patents

Description

  The present invention relates to a periodontal pathogen plasma or serum antibody titer test kit, more specifically a periodontal pathogen plasma or serum antibody titer kit comprising a specific periodontal pathogen antigen protein suitable for automated testing by an instrument, the kit The present invention relates to a modified polypeptide used in the present invention, and a method for measuring antibody titers against periodontal pathogens in blood samples.

Periodontal disease is a bacterial infection that develops when oral bacteria infect periodontal tissues.
Diagnosis of periodontal disease in the clinical practice of the dentistry is performed by comprehensively combining the results of clinical tests such as clinical symptoms, intraoral photographs, X-ray images or periodontal tissue examinations. Among these examinations, intraoral photographs and X-ray examinations visually assess morphological changes in periodontal tissues, and periodontal examinations include plaque adhesion, periodontal pocket depth, and probing. It measures and evaluates various clinical items such as bleeding and tooth mobility. Therefore, these examinations require complicated operations, and a high level of skill is required of the operator in order to accurately diagnose the periodontal pathology of the patient.

  That is, depending on the case, the test result may differ depending on the skill level of the operator, and thus the diagnosis for the patient may differ. Moreover, in the dental clinical examination as described above, the periodontal disease is evaluated not at the “infection” level of periodontal pathogens but at the level of “morphological change” of the periodontal tissue, even though the periodontal disease is a bacterial infection. It is an objective periodontal that has been examined by the subjectivity of the surgeon, so far has been appropriate from a bacteriological and immunological viewpoint, and does not produce a difference in the test result depending on the skill of the surgeon. There was a need for disease testing.

  Under such circumstances, a periodontal disease inspection system using a serum antibody titer against periodontal pathogens as an index of periodontal disease inspection is performed (Non-Patent Document 1). This periodontal disease test system detects and quantifies "specific antibodies" against periodontal pathogens from a small amount of blood collected and separated from the fingertips of patients, thereby infecting periodontal pathogens and the severity of periodontal diseases. (Inflammation state) is evaluated, and the pathological condition of periodontal disease can be objectively and uniformly evaluated by adopting an immunological technique.

In this periodontal disease test system, plasma is separated from blood collected from the fingertips, a sample of the plasma is mailed to a test institution, and the IgG antibody titer against periodontal pathogens is measured by the test institution to evaluate the importance of periodontal disease. After assessing the severity, each patient is notified of the results of the periodontal disease test (periodontal pathogen plasma antibody titer test). Therefore, it becomes possible to support periodontal disease examinations at general practitioners or at home, and by collecting and analyzing examination data at once, it is possible to correlate with a disease state using a huge amount of data.
On the other hand, in such a periodontal disease inspection system, since a large amount of samples are handled, it is required to automatically perform high-speed processing.

In such a test system, the more types of antibodies to the antigen to be tested in the sample (blood), the higher the correlation between the test result and periodontal disease, and the more accurately test for periodontal disease. Is possible. In humans infected with periodontal disease, the types of periodontal pathogen antigens recognized due to periodontal pathogens and individual differences between humans are different. The greater the number, the more accurately the periodontal disease can be examined.
However, when attempting to measure the antibody titers of various antibodies, it is difficult to process the sample at high speed, which is not suitable for automated inspection using an instrument.

  The antigen currently used for antibody titer measurement is a solution prepared by crushing periodontal pathogens such as Porphyromonas gingivalis, including various bacterial proteins (LPS, membrane lipids) ). Therefore, it has been difficult to automatically process a large number of samples at high speed using an instrument.

Eiko Kudo, Journal of Okayama Dental Society, 28, No. 1 (2009) 1-14

  The object of the present invention is to cover a wide range of periodontals of patients with various immunotypes covering various antigen-antibody reactions caused by changing antigenicity of periodontal pathogens and various immunoreactivity of test subjects. Periodontal pathogen plasma or serum antibody titer test kit that can test disease with high accuracy and can be processed at high speed with automated equipment, periodontal pathogen antigen protein that can be suitably used for the kit, and An object of the present invention is to provide a method for examining periodontal pathogen plasma or serum antibody titer of a blood sample using the kit.

Under these problems, the present inventors examined selection of periodontal pathogen proteins capable of accurately determining periodontal disease while covering various antigen-antibody reactions even with a small number of types. As a result, specific combinations of periodontopathic proteins react specifically with plasma antibodies in blood samples isolated from periodontal disease patients, and various antigen-antibody reactions can be achieved by selectively using these bacterial protein combinations. The present inventors have found that a blood sample can be examined with high accuracy and can be processed at high speed with an automated apparatus, and the present invention has been completed.
That is, the present invention
[1]
A modified polypeptide having the amino acid sequence of SEQ ID NO: 141;
[2]
The modified polypeptide according to the above [1], comprising the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 142;
[3]
A modified polypeptide having the amino acid sequence of SEQ ID NO: 143;
[4]
The modified polypeptide according to the above [3], comprising an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 144;
[5]
A modified polypeptide having the amino acid sequence of SEQ ID NO: 145;
[6]
The modified polypeptide according to the above [5], comprising an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 146;
[7]
A modified polypeptide having the amino acid sequence of SEQ ID NO: 147;
[8]
The modified polypeptide according to the above [7], comprising an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 148;
[9]
SEQ ID NOs: 1, 3, 5, 9, 15, 17, 19, 23, 31, 35, 37, 41, 43, 47, 141, 143, 145, 147, 151, 153 and 155 Periodontal pathogen plasma or serum antibody titer test kit comprising a polypeptide having one or more amino acid sequences;
[10]
The polypeptide is one or more selected from the group consisting of SEQ ID NOs: 1, 3, 5, 9, 15, 17, 19, 31, 37, 41, 43, 141, 143, 147, 151, 153 and 155 Periodontal pathogen plasma or serum antibody titer test kit according to [9] above, which is a polypeptide having two or more amino acid sequences and a polypeptide having the amino acid sequence of SEQ ID NO: 145;
[11]
The polypeptide is based on SEQ ID NOs: 2, 4, 6, 10, 16, 18, 20, 24, 32, 36, 38, 42, 44, 48, 142, 144, 146, 148, 152, 154 and 156. The periodontal pathogen plasma or serum antibody titer test kit according to [9] above, which is a polypeptide encoded by a polynucleotide sequence selected from the group consisting of:
[12]
A method for measuring an antibody titer against periodontal pathogens in a blood sample comprising contacting a blood sample separated from a human body with a periodontal pathogen antigen polypeptide, wherein the periodontal pathogen antigen polypeptide is SEQ ID NO: One, selected from the group consisting of 1, 3, 5, 9, 15, 17, 19, 23, 31, 35, 37, 41, 43, 47, 141, 143, 145, 147, 151, 153 and 155 The method comprising a polypeptide having two or more amino acid sequences;
[13]
A method for measuring an antibody titer against periodontal pathogens in a blood sample comprising contacting a blood sample separated from a human body with a periodontal pathogen antigen polypeptide, wherein the periodontal pathogen antigen polypeptide is SEQ ID NO: It has one or more amino acid sequences selected from the group consisting of 1, 3, 5, 9, 15, 17, 19, 31, 37, 41, 43, 141, 143, 147, 151, 153 and 155 A polypeptide and a polypeptide having the amino acid sequence of SEQ ID NO: 145;
[14]
The polypeptide is based on SEQ ID NOs: 2, 4, 6, 10, 16, 18, 20, 24, 32, 36, 38, 42, 44, 48, 142, 144, 146, 148, 152, 154 and 156. The method according to [12] or [13] above, which is a polypeptide encoded by a polynucleotide sequence selected from the group consisting of:
[15]
A typing kit for a Porphyromonas gingivalis strain comprising a polypeptide having one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 151, 153 and 155;
[16]
The typing kit according to [15], wherein the polypeptide is a polypeptide encoded by one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs: 152, 154 and 156;
[17]
A polypeptide having the amino acid sequence of SEQ ID NO: 151;
[18]
The polypeptide according to [17] above, comprising the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 152;
[19]
A polypeptide having the amino acid sequence of SEQ ID NO: 153;
[20]
The polypeptide according to [19] above, comprising the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 154;
[21]
A polypeptide having the amino acid sequence of SEQ ID NO: 155;
[22]
The polypeptide according to [21] above, comprising the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 156.

In a first aspect, the present invention provides a periodontal pathogen plasma or serum antibody titer test kit containing a specific antigen protein.
The periodontal pathogen plasma or serum antibody titer test kit of the present invention reacts with a small amount of blood separated from a human body to measure the IgG antibody titer against periodontal pathogens in a blood sample (plasma or serum). To test for peripathogenic infections,
(1) A lancet to cause a small amount of bleeding by damaging the subject body (2) A capillary for collecting the blood (3) A bottle containing a solution containing a specific periodontal pathogen antigen protein (4) Cylinder for separating the plasma from the blood by compressing the inside of the bottle. (5) A cap that seals the bottle. By mixing the blood collected by the capillary with the solution in the bottle, the periodontium is contained in the blood sample. When IgG antibodies against pathogenic bacteria are present, an antigen-antibody reaction with the antigen protein in the bottle is performed, and infection by periodontal pathogens is examined by measuring immunoprecipitation,
(1) Immobilize on a 96-well plate to immobilize the antigen protein,
(2) Add a blood sample (plasma or serum) to the 96-well plate to perform the antigen-antibody reaction,
(3) After washing the 96-well plate, an anti-human IgG secondary antibody was added to cause an antigen-antibody reaction,
(4) After washing the 96-well plate, one that is used in an ELISA method to detect the signal by performing color development or luminescence reaction due to the presence of a specifically bound anti-human IgG secondary antibody,
(1) Immobilizing the antigen protein on the filter (such as biotinylation)
(2) A blood sample (plasma or serum) is added to the filter, and the antigen-antibody reaction is performed in the filter.
(3) After washing the filter, add an anti-human IgG secondary antibody to perform the antigen-antibody reaction,
(4) There are those used in the antigen-immobilized filter method in which after the filter is washed, color development or luminescence reaction is performed due to the presence of the specifically bound anti-human IgG secondary antibody to detect the signal.

The feature in this aspect of the present invention resides in the antigenic protein of the periodontal pathogen Porphyromonas gingivalis contained in the test kit, specifically reacts with an antibody against periodontal pathogens possessed by a periodontal disease patient, and It reacts with antibodies possessed by a wide range of periodontal disease patients.
That is, the periodontal pathogen antigen protein contained in the periodontal pathogen plasma or serum antibody titer test kit of the present invention is SEQ ID NO: 1, 3, 5, 9, 15, 17, 19, 23, 31, 35 of the sequence listing. , 37, 41, 43, 47, 141, 143, 145, 147, 151, 153 and 155, a polypeptide having one or two or more amino acid sequences, and a kind of these antigenic proteins Alternatively, by using a combination of two or more kinds, it is possible to examine the antibody titer and type of antibodies possessed by a periodontal disease patient and to examine the degree of infection and the type of infection of periodontal pathogens. Moreover, although there are individual differences in the antigenic proteins of periodontal pathogens and the immunotypes of individual periodontal disease patients (types of antibodies against periodontal pathogens), a wide range of use can be made by combining the antigenic proteins of the present invention. It can cover patients with immune type periodontal disease. In addition, periodontal pathogen SU63 strain, which is a risk factor of cardiovascular or cerebrovascular disease, can be detected.
In addition, the periodontal pathogen antigen protein used in the present invention is preferably SEQ ID NO: 2, 4, 6, 10, 16, 18, 20, 24, 32, 36, 38, 42, 44, 48 in the sequence listing. , 142, 144, 146, 148, 152, 154 and 156, a polypeptide encoded by one or more nucleotide sequences selected from the group consisting of.
Furthermore, preferably, the periodontal pathogen antigen protein contained in the periodontal pathogen plasma or serum antibody titer test kit of the present invention is SEQ ID NO: 3, 5, 15, 19, 31, 41, 141, 143, A polypeptide having one or more amino acid sequences selected from the group consisting of 145, 147, 151, 153 and 155, preferably SEQ ID NOs: 4, 6, 16, 20, 32, 42, 142 , 144, 146, 148, 152, 154 and 156 are polypeptides encoded by one or more polynucleotide sequences selected from the group consisting of:

In a second aspect, the present invention provides a modified polypeptide for use in the aforementioned periodontal pathogen bacteria plasma or serum antibody titer test kit.
The present inventors have found that a periodontal pathogen antigen protein suitable for an antibody titer test kit has a protease activity despite the fact that some antigen proteins react with a wide range of periodontal disease patients. It was found that the test was hindered by the self-digesting action and the degradation action of other antigenic proteins. Therefore, the present inventors produced a modified polypeptide that lost the protease activity while maintaining the antigenicity of these antigenic proteins by a genetic engineering technique.

  That is, the modified polypeptide used in the periodontal pathogen plasma or serum antibody titer test kit of the present invention is a polypeptide having the amino acid sequence of SEQ ID NO: 63, 65, 67, 141, 143, 145 or 147, No. 51 modified polypeptide in which the cysteine residue at position 477 or 488 of the wild-type polypeptide has been deleted or replaced with another amino acid residue, preferably an alanine residue (SEQ ID NO: : 63 and 65) and a modified polypeptide in which the cysteine residue at position 471 of the wild type polypeptide of SEQ ID NO: 57 has been deleted or replaced with another amino acid residue, preferably with an alanine residue (SEQ ID NO: 67). The amino acid sequences of SEQ ID NOs: 141 and 143 are about half of the amino acid sequences on the N-terminal side and C-terminal side of the mutated Lys-gingipain having the amino acid sequence of SEQ ID NO: 63, respectively. The amino acid sequences of 145 and 147 are about half of the amino acid sequences on the N-terminal side and C-terminal side of the mutated arginine-specific cysteine proteinase RgpA having the amino acid sequence of SEQ ID NO: 67, respectively.

  In the third aspect, the present invention provides a method for measuring antibody titer against periodontal pathogens in a blood sample, which comprises contacting a blood sample isolated from a human body with a periodontal pathogen antigen polypeptide, and is used in the method. The blood sample is preferably blood, serum or plasma collected from finger capillaries or veins, and the periodontal pathogen antigen polypeptide is SEQ ID NO: 1, 3, 5, 9, 15, 17, 19, 23, 31. , 35, 37, 41, 43, 47, 141, 143, 145, 147, 151, 153 and 155, a polypeptide having one or more amino acid sequences selected from the group consisting of. Preferably, the periodontal pathogen antigen polypeptide is from the group consisting of SEQ ID NOs: 1, 3, 5, 9, 15, 17, 19, 31, 37, 41, 43, 141, 143, 147, 151, 153 and 155 A polypeptide having one or more selected amino acid sequences and a polypeptide having the amino acid sequence of SEQ ID NO: 145.

The method according to this aspect of the present invention can be preferably performed using the periodontal pathogen plasma or serum antibody titer test kit of the first aspect, and preferably performed using the periodontal pathogen plasma antibody titer test. can do.
Furthermore, the progress (severity) of periodontal disease can be examined based on the antibody titer.

  In a fourth embodiment, the present invention provides a typing kit for Porphyromonas gingivalis strain, which kit is a Porphyromonas gingivalis strain, particularly FDC381 strain, present in a blood sample isolated from the human body. The strain SU63 can be typed to test which strain is present in the sample. The kit comprises a polypeptide having one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 151, 153 and 155, preferably the polypeptide is from the group consisting of SEQ ID NOs: 152 and 156 It is encoded by one or two selected polynucleotides.

ADVANTAGE OF THE INVENTION According to this invention, the periodontal disease test kit which can test | inspect periodontal disease at high speed and with high precision can be provided. Moreover, the periodontal disease test kit which can test | inspect a periodontal disease objectively without depending on the skill level of a practitioner technique can be provided.
In addition, with the spread of the periodontal disease test kit of the present invention, it is possible to examine periodontal disease by a unified method in dental clinics nationwide, and by creating a database of the obtained measurement results, Can develop or revise disease criteria and treatment guidelines.
Furthermore, the periodontal disease test kit of the present invention can detect a risk factor of cardiovascular or cerebrovascular disease.

It is a figure which shows the antigen antibody reaction with respect to the human serum of a periodontal pathogen antigen protein. A: Antigen-antibody reaction against normal serum, B and C: Antigen-antibody reaction against periodontal disease patient serum. FIG. 2 is an SDS-PAGE electrophoretogram of Porphyromonas gingivalis FDC381 strain antigen protein roughly purified using an antibody column. Lane A: antigen protein roughly purified from serum column of healthy subject, lane B: antigen protein roughly purified from column 1 of patient serum, lane C: antigen protein roughly purified from patient serum 2 It is a figure which shows the antigen antibody reaction of the crudely purified FDC381 strain antigen protein and serum. Lane A: antigen protein roughly purified from a healthy subject serum column, lane B: antigen protein roughly purified from one patient serum column, lane C: antigen protein roughly purified from two patient serum columns. It is an SDS-PAGE electrophoretic diagram of Porphyromonas gingivalis SU63 strain antigen protein roughly purified using an antibody column. Lane A: antigen protein roughly purified from a healthy subject serum column, lane B: antigen protein roughly purified from one patient serum column, lane C: antigen protein roughly purified from two patient serum columns. It is a figure which shows the antigen antibody reaction of the roughly purified SU63 strain antigen protein and serum. Lane A: antigen protein roughly purified from a healthy subject serum column, lane B: antigen protein roughly purified from one patient serum column, lane C: antigen protein roughly purified from two patient serum columns. It is a figure which shows the Mascot search result of an antigen protein. It is a figure which shows the gene information and selection result of the identified antigen protein. It is a figure which shows the antigen antibody reaction of serum and a synthetic antigen protein. It is a figure which shows the antigen antibody reaction signal value of serum and a synthetic antigen protein. It is an SDS-PAGE electrophoretic diagram showing the stability of a synthetic antigen protein. It is a figure which shows the antigen antibody reaction signal value of healthy subject serum and synthetic antigen protein. It is a figure which shows the antigen antibody reaction signal value of various patient sera and antigen protein. It is the figure which summarized the antigen antibody reaction of various patient sera and antigen protein. It is a figure which shows stability and antigenicity of a modified polypeptide. It is a figure which shows the antigen antibody reaction of serum and a synthetic antigen protein. It is a figure which shows the antigen antibody reaction signal value of serum and a synthetic antigen protein. It is the figure which summarized the antigen antibody reaction of various serum and an antigen protein. It is a figure which shows the antigen antibody reaction signal average value and S / N value of a healthy subject and patient serum, and an antigen protein. It is a figure which shows the ROC curve and AUC value which were calculated | required about the signal value of each serum with respect to various antigen proteins. It is a figure which shows the antigen antibody reaction of serum and modified antigen protein. It is a figure which shows the antigen antibody reaction of serum and various antigen proteins. It is a figure which shows the antigen antibody reaction signal value of serum and a synthetic antigen protein. It is a figure which shows the sensitivity and specificity of various antigen proteins calculated | required based on various cut-off values. It is a figure which shows the ROC curve and AUC value which were calculated | required about the signal value of each serum with respect to various antigen proteins. It is a figure which shows the antigen antibody reaction of serum and various antigen proteins (SU63 strain | stump | stock). It is the figure which summarized the antigen antibody reaction of serum and antigen protein (SU63 strain).

  Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples.

  Throughout this example, Porphyromonas gingivalis FDC381 strain and SU63 strain were used as periodontal pathogens. FDC381 strain is sold by Sumisho Pharma International Co., Ltd., and SU63 strain is available as type II or type IV pili holding strain.

Preparation of antibody column When humans are infected with periodontal pathogens, the human body produces various antibodies against the antigens of periodontal pathogens by immune reaction. The antigen recognized by the antibody produced here varies depending on the immune response of the individual.
Therefore, it was investigated which antigen is the target of the antibody from the composition of the antigen preparation solution of periodontal pathogens, and sera for purifying more antigens were selected based on the results.

Preparation of antigen protein Antigen preparation solution obtained by ultrasonically crushing Porphyromonas gingivalis (FDC381 and SU63 strains) and collecting the ultracentrifugated supernatant fraction (Special Immunology Institute, equivalent to 200 μg protein) To this, phosphate buffered saline (PBS) was added to make 270 μl. Trichloroacetic acid was added to this, and it left still in ice, Then, it centrifuged at low temperature and the supernatant was removed. The precipitate was washed with ice-cold ethanol, centrifuged again at a low temperature, and the supernatant was removed. Further, the above operation was performed twice. After air drying, 120 μl of PBS containing 0.06% sodium dodecyl sulfate (SDS) was added to dissolve the precipitate, and an antigen protein solution was prepared for each strain.

Quantification of the antigen protein 25 μl of the prepared standard (concentration: 1000, 500, 250, 125, 62.5 or 31.25 ng / μl), 25 μl of the antigen protein solution diluted with PBS, and 25 μl of PBS as a control were added to each well of the 96-well plate, Then, 200 μl of protein working solution (Thermo scientific, Reagent A: B = mixed at a dose of 50: 1) was added to each well. Next, the reaction solution was stirred with a shaker, incubated at 37 ° C. for 30 minutes in a thermo-hygrostat, and then the absorbance at 577 nm was measured using a plate reader (Intermed, NJ2000) to quantify the collected antigen protein.

SDS-PAGE electrophoresis Quantitative gingivalis (FDC381 strain and SU63 strain) antigen proteins were each prepared to a protein concentration of 400 ng / μl using a sample buffer (Invitrogen). The prepared sample was heat-denatured and subjected to SDS-PAGE electrophoresis (5 μl of protein solution).

The gel after SDS-PAGE electrophoresis was blotted onto a polyvinylidene fluoride membrane (PVDF membrane) using an iBlot drive blotting system (Invitrogen).
After transferring to the PVDF membrane, the migrated layout (molecular weight marker, FDC381 strain, SU63 strain) was cut out as one set. The cut out set of slits was subjected to Ponceau staining to confirm protein blotting. The remaining slits were placed in a falcon tube and immersed in a blocking solution (Tris-buffered saline (hereinafter referred to as “TBS”) containing 3% skim milk and 0.1% Tween 20).

Antigen-antibody reaction using serum After blocking, the blocking solution was removed, and then the serum reaction solution (20 ml TBS with 3% skim milk plus 8 μl serum collected from healthy or periodontal patients) And shaken at room temperature. Next, the slit was washed with TBS added with 0.05% Tween20. After washing, horseradish peroxidase-conjugated goat anti-human IgG antibody reaction solution diluted 5000 times (a solution obtained by adding anti-human secondary antibody (CHEMICON) to TBS with 3% skim milk) was added and shaken at room temperature. Next, the slit was washed with TBS added with 0.05% Tween20. After washing, the slit was immersed in TBS containing 0.61 mg / mL 4-methoxy-1-naphthol and 0.018% aqueous hydrogen peroxide to confirm color development, washed with purified water, and dried.

The reaction results of the antigen protein and human serum are shown in FIG.
In the antigen-antibody reaction with the healthy subject serum, almost no signal was observed (FIG. 1A: healthy subject subject serum).
On the other hand, in the antigen-antibody reaction with periodontal disease sera, clear signals were observed, and the patterns varied (FIGS. 1B and 1C). The various signals observed in the antigen-antibody reaction with patient sera could be roughly divided into two. That is, it was divided into a signal showing a clear band (FIG. 1B) and a signal showing a smear spreading throughout (FIG. 1C). Various antigen-antibody reaction patterns were formed due to differences in size and specificity to strains.

And the signal of the size shown below was strongly observed specifically for the sera of many patients.
46kDa (antigen protein of FDC381 and SU63)
25-37kDa (antigen protein of FDC381 strain and SU63 strain)
100-110kDa (antigen protein of FDC381 strain and SU63 strain)
57 kDa (antigenic protein of SU63 strain)
150-250kDa (antigen protein of SU63 strain)

  Looking at the results of antigen-antibody reactions using periodontal disease sera, two types of signal patterns (clear bands and smears spreading throughout) were observed, and various antigen antibodies depending on their combination (size / strain) A reaction pattern was formed. That is, it was shown that antibodies produced by persons infected with Gingivalis bacteria are various and recognized as antigenic proteins in some sera but may not be recognized at all in other sera. This is consistent with reports that antibodies contained in various periodontal disease sera have various proteins as antigens. Therefore, it was shown that a variety of test antigen proteins are required to measure the infection of Gingivalis by antibody titer.

In addition, when an antigen protein common to FDC381 strain and SU63 strain is used for the test, it is considered difficult to identify the strain. On the other hand, if only an antigen protein specific for a strain is used for the test, if the test shows a positive result, infection by the strain is suspected.
The antigen-antibody response patterns observed in patient sera varied, but no significant differences were observed in the patterns of first-patient (FV) and maintenance patients (SPT). Based on this, an antigen protein showing a strong signal in the patient serum was identified and used as a candidate antigen for use in the test.

  Bands showing strong signals for many patient sera include antigenic protein groups of 46 kDa, 25-37 kDa, 100-110 kDa, 57 kDa and 150-250 kDa as shown in the results. Therefore, the presence or absence of antigenicity of these protein groups against the sera of various patients was examined. The results are shown in Table 1.

  As can be seen from Table 1, by using a combination of these five antigenic protein groups, it is possible to confirm the presence or absence of antibodies against gingivalis in all patient sera, and a wide range of patients with periodontal disease can be examined. It was shown that it can be covered.

Selection of sera used for immunoaffinity column preparation Next, sera used for column preparation were selected in order to purify these five antigen protein groups using immunoaffinity columns. At that time, in consideration of the antigen-antibody reaction pattern of the patient serum and the measured value of the plasma antibody titer, two types of patient serum pools were used for the production of the antibody column. That is, the antigen pools of 46kDa (FDC381 and SU63 strains) and 57kDa (SU63 strain) antigen pools of No.7350, No.6921 and No.6870 serum pools showing a clear band in the target antigen and high plasma antibody titers. Was used to prepare an antibody column for purification. On the other hand, the serum pools of No.7107, No.7523 and No.6980, which show a smear band as a whole but a strong signal to the target antigen and high plasma antibody titers, are 25-37 kDa (FDC381 and SU63 strains) , 100-110 kDa (FDC381 strain and SU63 strain) and 150-250 kDa (SU63 strain) antigen proteins were used to prepare antibody columns. For comparison, the NAI, TOM, and KOB serum pools of healthy subjects were used to prepare antibody columns for purifying antigen proteins of healthy subjects.

  On the other hand, five antigen protein groups (46 kDa, 25-37 kDa, 100-110 kDa, 57 kDa, and 150-250 kDa) were selected as antigen proteins used for the plasma antibody titer test. In order to purify these five antigen protein groups using an immunoaffinity column, three types of columns were prepared. That is, column A: healthy serum column (NAI, TOM and KOB serum ligand), column B: clear band purification column (No. 7350, No. 6921 and No. 6870 serum ligand) and column C: smear band purification Columns (No. 7107, No. 7523 and No. 6980 serum ligands) were prepared.

Purification of antigenic protein using immunoaffinity column In order to identify the selected antigenic protein, it is necessary to purify the antigenic protein from the antigen preparation. Therefore, an antibody column was prepared using the selected serum, and the antigen protein was purified.

Immunoaffinity columns are written by Masato Okada and Kaoru Miyazaki, “Protein Experiment Note (Volume 2)”, Yodosha, pp.131-136 (1990) and Seishi Takatsu et al., “Antibody Experiment Manual for Protein Research”, It was produced by the following method based on Yodosha, pp.53-61 (2005).
Periodontal disease patient serum or healthy subject serum 1.5ml (each serum 500μl x 3 samples), antibody binding buffer (50mM tartrate buffer solution, 3M NaCl, pH9.0) 2.5ml, sodium chloride 0.26g is added and mixed, An antibody reaction solution was prepared.

On the other hand, Protein G Sepharose (GE Healthcare) was added to an Econopack column (BIO-RAD), washed with ultrapure water, and further washed with an antibody binding buffer. The whole amount of the prepared antibody reaction solution was added to the column, and the column was sealed, followed by stirring using a rotator. After stirring, the antibody reaction solution was removed, and the column was washed with an antibody binding buffer (50 mM tartrate buffer solution, 3M NaCl, pH 9.0).
Dissolve 100 mg of the crosslinker BS3 (PIERCE) in 6.8 ml of the crosslinker solution (0.2 M triethanolamine-HCl, pH 8.0) and distribute it to three columns (a normal serum column and two patient serum columns). After pouring, the mixture was stirred at room temperature using a rotator.

  After removing the crosslinker solution from the column, the column was washed with a blocking solution (0.2 M ethanolamine-HCl, pH 8.0). After sealing the column, a blocking solution was added and stirred at room temperature using a rotator. Thereafter, the blocking solution was removed, the column was washed with an elution solution (0.1 M glycine-HCl, pH 2.8), then washed with 50 mM Tris-HCl (pH 7.5), and then added with 50 mM Tris-HCl (pH 7.5). Stored (immunoaffinity columns A, B and C).

Preparation of antigen protein sample PBS was added to an antigen preparation solution (Special Immunology Research Institute, equivalent to 200 μg protein) of Gingivalis (FDC381 strain and SU63 strain) to prepare 270 μl. Trichloroacetic acid was added thereto, and the mixture was allowed to stand in ice and centrifuged at a low temperature to remove the supernatant. Further, ice-cold ethanol was added, the precipitate was washed, and then centrifuged again at a low temperature to remove the supernatant. The above operation was performed three times.
After air drying, 200 μl of PBS supplemented with 0.06% SDS was added to dissolve the precipitate, and the protein solution for each strain was collected. Antigen protein samples were prepared for each strain by adding the same amount of PBS (containing 0.01% Brij-35 and 0.2% CHAPS) to the combined protein solution to dissolve the precipitate.

Antigen protein quantification 25 μl of prepared standards (concentrations: 1000, 500, 250, 125, 62.5 and 31.25 ng / μl), 25 μl of antigen protein sample diluted in PBS, and 25 μl of PBS as a control were added to each well of a 96-well plate. 200 μl of protein working solution (Thermo scientific, Reagent A: B = mixed at a 50: 1 dose) was added to each well. Next, the reaction solution was stirred with a shaker and incubated at 37 ° C. for 30 minutes in a thermo-hygrostat, and then the absorbance at 577 nm was measured using a plate reader (Intermed, NJ2000) to quantify the antigenic protein in the sample. .

Purification of antigenic proteins using immunoaffinity columns
About 133 μg / 1.5 ml of antigen protein sample was applied to each immunoaffinity column (A, B and C) equilibrated with PBS (sample buffer composition; 0.03% SDS, 0.005% Brij-35 and 0.2% CHAPS ( PBS containing Dojindo Laboratories). After stirring at room temperature using a rotator, the flow-through was collected and stored as a sample. In addition, a wash buffer (0.005% Brij-35, 0.1% CHAPS, 20 mM Tris-HCl and 500 mM NaCl, pH 7.5) was added, and the column was washed by stirring at room temperature using a rotator. After stirring, the flow-through was collected and stored as a sample. This operation was performed three times.
After washing the column with ultrapure water, 5 ml of elution buffer (0.05% trifluoroacetic acid) was added twice, and the eluted protein solution was recovered and lyophilized.

SDS-PAGE electrophoresis Antigen protein samples, flow-through of each process, and a part of the eluted protein were collected and prepared to be a sample buffer containing 5% final concentration of mercaptoethanol.
The prepared sample was heat denatured and subjected to SDS-PAGE electrophoresis (10 μl for CBB staining, 5 μl for antigen-antibody reaction).

The gel after SDS-PAGE electrophoresis was washed with distilled water, immersed in a CBB staining solution, and stirred at room temperature. Then, it washed with distilled water until the band was clearly observable.
Further, the gel after SDS-PAGE electrophoresis was blotted on a PVDF membrane using an iBlot drive blotting system (Invitrogen).
After transferring to the PVDF membrane, the layout was cut out as one set and immersed in a blocking solution (TBS with 3% skim milk and 0.1% Tween20).

Antigen-antibody reaction using serum After blocking, the blocking solution was removed, and then a serum reaction solution (a solution obtained by adding 8 μl of serum to 20 ml of TBS containing 3% skim milk) was added and shaken at room temperature. The set of sera added is described below.
Set A (Healthy Serum): NAI, TOM, KOB
Set B (Patient serum 1): No.7350, No.6921
Set C (Patient serum 2): No.7107, No.7523, No.6980
Wash the slit with TBS containing 0.05% Tween20 and add 5000-fold diluted horseradish peroxidase-conjugated sheep anti-human secondary antibody reaction solution (TBS containing 3% skim milk plus human secondary antibody (CHEMICON)) And stirred at room temperature.
Next, the slit was washed with TBS containing 0.05% Tween 20, and the slit was immersed in a coloring solution (TBS containing 0.61 mg / ml 4-methoxy-1-naphthol and 0.018% hydrogen peroxide solution) to confirm color development. , Washed with purified water and dried.

Purification of FDC381 strain antigen protein FIG. 2 shows an SDS-PAGE electropherogram of the antigen protein purified from the column.
When flow-through after the antigen protein sample was applied to each immunoaffinity column was confirmed, no significant difference was observed between the healthy serum column and the patient serum column. However, when the purified protein was observed, the CBB staining signal was clearly stronger when purified from the patient serum column (Fig. 2, lane A) than when purified from the healthy serum column (Fig. 2). , Lanes B and C). The band around 50 kDa in particular showed a significantly strong signal in the patient serum column. A band slightly larger than 25 kDa also showed a strong signal in patient serum (FIG. 2, lanes B and C).

Purification of SU63 strain antigen protein FIG. 4 shows an SDS-PAGE electrophoretogram of the antigen protein purified from the column.
Observing the purified protein after subjecting the antigen protein sample to each immunoaffinity column is not as clear as in the case of the FDC381 strain, but compared to the one purified from the healthy serum column (Figure 4, Lane A). ) Apparently the CBB staining signal of antigen protein purified from patient serum column was strong (Figure 4, lanes B and C). A particularly significant difference was observed in a band slightly larger than 25 kDa and a band slightly larger than 50 kDa. The strong signal observed in the purification of FDC381 strain tended to show a strong signal in the purification of SU63 strain (FIG. 4).

Antigen-antibody reaction of antigen protein
The results of the antigen-antibody reaction of the antigen proteins of FDC381 strain and SU63 strain subjected to SDS-PAGE electrophoresis are shown in FIGS. 3 and 5, respectively.
Serum set A (serum for healthy subjects) reacted to high molecular weight proteins but hardly reacted to proteins of 150 kDa or less to antigen proteins eluted from both healthy and patient serum columns (Fig. 3 and Figure 5).
On the other hand, serum set B (patient serum 1; the serum group used for preparation of column B) reacted strongly with the antigen protein eluted from the patient serum column (FIGS. 3 and 5; lane B and “patient serum 1”) C). A stronger signal was observed with the protein eluted from the patient serum column (column B).
Serum set C (patient serum 2; serogroup used to prepare column C) also reacted strongly with the protein eluted from the patient serum column as in serum set B (Fig. 3 and Fig. 5, "Patient Serum 2" Lanes B and C). On the other hand, serum set C also reacted relatively with the protein eluted from the serum column of healthy subjects (FIGS. 3 and 5, lane A of “patient serum 2”). A stronger signal was observed for the protein eluted from the patient serum column (column C).

When the protein eluted from the immunoaffinity column was observed, the protein eluted from the patient serum column was clearly more than the protein eluted from the healthy subject serum column. This is presumably because protein G sepharose in the patient serum column is bound with a large amount of antibodies against gingivalis bacteria, and can be purified by binding more with gingivalis antigen protein in the purification process.
Therefore, antigen protein candidates were selected by comparing proteins purified using the healthy subject serum column and the patient serum column.

  As a result of comparing the purified antigen proteins, the antigen proteins containing the common antigens (46 kDa, 25-37 kDa and 100-110 kDa) shown as candidates in Table 1 were obtained from the healthy serum column in the protein purified by the patient serum column. It was found to be contained more than the purified protein. Therefore, it is possible to select the antigen protein candidate that can be used in the test kit by clarifying the structure of the entire purified protein and comparing the two, so that each purified protein is composed. All proteins were identified by mass spectrometry.

Mass spectrometry The antigen protein roughly purified as described above was separated by SDS-PAGE electrophoresis, and CBB-stained eluted protein bands (columns A, B, and C) were cut out together for 3 lanes each. . The cut gel was put in a falcon tube, 200 μl of ultrapure water was added, and the following mass analysis was performed.

Preparation of Sample for Mass Spectrometry The prepared sample was subjected to protein digestion with ProGest (Genomic Solutions) workstation as described below, reduced with dithiothreitol at 60 ° C., and then cooled to room temperature. It was then alkylated with iodoacetamide. The mixture was incubated at 37 ° C. for 4 hours in the presence of trypsin, and the reaction was stopped by adding formic acid. The supernatant after stopping the reaction was used as a sample for analysis.

LC / MS / MS
About the prepared sample, nano LC / MS / MS analysis was performed using ThermoFisher LTQ Orbitrap XL.
30 μl of hydrolyzate was applied to a 5 mm × 75 μm ID C12 column (Jupiter Proteo, Phenomenex) vented column. Gradient elution was performed at 300 nl / min on a 15 cm × 75 μm ID C12 column.
MS / MS was analyzed with a mass spectrometer operated in data-dependent mode, six most abundant ions. Orbitrap MS scans were performed at 60000 FWHM resolution.
MS / MS data was searched using a local copy of Mascot (www.matrixscience.com).
The LC / MS / MS search parameters were set as follows.
Search type: MS / MS ion search Classification: All bacteria or all species Enzyme: Trypsin Default modification: Carbamide methyl Variable modification: Oxidation, acetylation, pyroglutamylation, deamidation Mass value: Monoisotopic Protein mass: Restriction No Peptide mass tolerance: ± 10ppm (Orbitrap)
Fragment mass tolerance: ± 0.5 Dalton (LTQ)
Maximum number of miscuts: 2

SCAFFOLD
Samples were processed within the Scaffold algorithm (www.proteomesoftware.com) using DAT files created by Mascot. The LTQ Orbitrap XL data parameter identified proteins that matched two or more peptides.
The results are shown in FIG.

FDC381 strain As a result of Mascot search for all bacteria (A to C: A: healthy serum column, B: patient serum column 1, C: patient serum column 2), a total of 28 types were analyzed. The protein was identified (Figure 6 left). Of the identified proteins, Nos. 9, 17 and 18 were part of the IgG antibody. In addition, 15 of 28 proteins were identified only in the protein group purified from the patient serum columns (B and C). On the other hand, the other 10 proteins were observed in the protein group purified from the healthy subject serum column (A), but in the protein group purified from the patient serum column (B and C), the spectrum count Was expensive.

As a result of Mascot search for all bacteria, the total of 28 proteins were identified (Fig. 6, right). Of the identified proteins, Nos. 8 and 12 were part of the IgG antibody. In addition, 20 of 28 proteins were identified only in the protein group purified from patient serum columns (B and C). On the other hand, five types of proteins were also observed in the healthy subject serum column (A) purified protein group, but in the patient serum column (B and C) purified protein group, the spectrum count was high. Moreover, No. 22 was identified only in the healthy subject serum column (A).

  Looking at the results obtained this time, in both FDC381 and SU63 strains, the protein group eluted from the patient serum column was clearly more in both type and amount than the protein group eluted from the healthy subject serum column. This suggests that the antigen protein retained in the antibody of the patient serum was purified by the immunoaffinity column method. Among the identified proteins, proteins already reported as antigens were included. From this, it was considered that the protein obtained by purification using the immunoaffinity column method is highly likely to be an antigenic protein.

Synthetic protein selection
The proteins observed in the two antigen protein identifications were arranged, and the proteins that were actually synthesized were selected.
For the identified proteins, the proteins identified in both strains based on the accession number are described as the same line, and the genetic information is examined to determine whether the function is known or unknown, or whether the antigenicity is known or unknown. (Fig. 7).
When the protein identified from the healthy subject column clearly shows a higher spectral count as to whether it is specific to the patient or not, the spectral count of the protein identified from the healthy subject column and the patient column is not much different △ Obviously, a protein with a high spectral count identified from the patient column was marked as ○ (primary selection). In addition, in order to examine whether it is specific for Gingivalis, proteins with high homology were examined based on the base sequence. A protein having a slight homology with other bacterial species was indicated by Δ, a protein having a high homology with other bacterial species was indicated by ×, and a protein having a low homology and specific for Gingivalis was designated by ○ (secondary selection).

  As a result of arranging the proteins identified by the two mass spectrometry, a total of 37 proteins were identified as antigen protein candidates. No. 3, 4, 6, 9, 10, 15, 16, 19, 24, 26, 37, 32 and 37 are the proteins that satisfy all the requirements as a result of amino acid sequence duplication, primary selection and secondary selection. A total of 13 proteins.

  We also considered selecting patients-specific and gingivalis-specific proteins by primary and secondary selection, but selecting candidate proteins at this stage may result in the loss of usable antigenic proteins. There is. For example, even a protein that is not selected as non-specific to a patient is a protein that can be used as an antigen for testing if the antibody titer against the patient is sufficiently higher than the antibody titer against the healthy subject. Therefore, it was considered that the evaluation of antigenicity in the actually synthesized protein can eliminate the leakage of antigen protein candidates.

  Therefore, protein synthesis was carried out using a total of 31 proteins excluding No. 1, 17, 18, 25, 30 and 31 that had overlapping amino acid sequences from a total of 37 proteins identified by mass spectrometry as candidates.

Protein synthesis and antigenicity evaluation of synthetic proteins To evaluate whether 31 candidate proteins actually show antigenicity, protein synthesis is performed, and then the synthesized proteins are analyzed using normal and patient sera. Antigenicity was evaluated.

  Obtain the gene information of each target protein from the database (antigen protein information), amplify the target gene from the genomic DNA of Gindivarius strain using the synthesized primer pairs shown in SEQ ID NOs: 71 to 132 in the sequence listing, and gateway system (Invitrogen) was used to clone into plasmid DNA (pDONR vector).

Subsequently, the pDONR vector DNA into which the gene was cloned was treated with a restriction enzyme and ligated to a protein expression vector (Cell Free Science: pEu vector) treated with the restriction enzyme. The ligation product was introduced into E. coli by transformation. Subsequently, clones into which the gene was introduced were selected.
Plasmid DNA was collected from the selected clones and subjected to sequence analysis. For clones in which no significant mutation was found from the results of sequence analysis, a large amount of plasmid was prepared, the protein was synthesized by a wheat germ cell-free protein synthesis system, and purified using a GST tag.

  We aimed for in vitro protein synthesis for all 31 genes, but No.33 could not be cloned into a protein expression vector, and No.5 could not be synthesized or had a very high synthesis amount. Since the results were small, the remaining 29 proteins were evaluated for antigenicity. Dot blot analysis was performed to evaluate the antigenicity of the synthetic protein.

Dot blot
29 antigen proteins were subjected to dot blot. The amount of antigen protein was adjusted to 50 ng, and four sets of dot blots were prepared for each antigen protein. However, since proteins No. 32 and No. 35 could not be quantified, 4 μl of the synthetic protein solution was applied to the dot blot. After dot blotting, it was immersed in a blocking solution (TBS solution containing skim milk (3%) and Tween20 (0.1%)).
As a primary antibody, 8 μl of healthy subject serum pool mixed with 3 μl of serum from NAI, TOM and KOB for healthy subjects, patient serum pool 1 mixed with 4 μl of serum from periodontal disease patients No. 7350 and 6921 Add 8 μl and 8 μl of patient serum pool 2 mixed with 3 μl of serum from periodontal disease patients No. 7107, 7523 and 6980 to 20 ml of TBS containing 3% skim milk, respectively. A dot blot slit was added to allow antigen-antibody reaction.
Subsequently, the slit was washed with TBS containing Tween 20 (0.05%). After washing, a horseradish peroxidase-conjugated goat anti-human IgG antibody reaction solution diluted 5000 times (a solution with TBS containing skim milk (3%) attached with an anti-human secondary antibody (CHEMICON)) was added and shaken at room temperature. Subsequently, the slit was washed with TBS containing Tween 20 (0.05%). After washing, the slit was immersed in TBS containing 4-methoxy-1-naphthol (0.61 mg / ml) and hydrogen peroxide (0.018%) to confirm color development, washed with purified water, and dried.
The layout of the synthetic protein attached to the dot blot and the results of dot blot analysis are shown in FIG.

As a result of dot blot analysis, spots No. 2, 6, 10, 26, 29, 32 and 35 were colored in the reaction with the serum pool of healthy subjects. On the other hand, in the reaction with patient serum pool 1, coloring of spots No.2, 3, 4, 6, 9, 10, 11, 13, 19, 21, 22, 24, 26, 28, 32 and 35 was observed. In the reaction with patient serum pool 2, spots No. 2, 3, 4, 6, 10, 11, 19, 24, 26, 32 and 35 were colored.
As a result of visual confirmation, color development in the patient serum pool was higher in all spots than in the healthy subject serum pool. In the patient serum pool, spot coloration that was not observed in the healthy subject serum pool could be confirmed.

Detection of Signal Value Next, the results of dot blot analysis were quantified by the following method.
1. An image of the membrane was taken using ImageQuant LAS4000 (GE Healthcare). After focusing, an image was taken under the following conditions.
・ Image capture conditions
Exposure Type: Precision
Exposure Time: 1/100 Sec
Sensitivity / Resolution: Standard
2. Using ImageQuant TL (GE Healthcare), the signal value of the dot of the captured image was determined.
・ Signal value capture conditions
In the Array analysis mode, the signal value was digitized in accordance with the dot spot size and arrangement. In addition, the background setting set a part close to the spot.
3. After digitization, the signal values of each spot were arranged as a table. Moreover, the signal value which showed the highest signal value among the signal values of the spot (blank) which did not mount an antigen protein was made into the reference | standard, and the signal value below it was processed as noise. The quantified signal value is shown in FIG.

  As a result of quantifying the spot signal, almost the same result as that visually confirmed was obtained. However, the signal value of No. 28 in the patient serum pool 1 was so low that it was judged as noise. On the other hand, for No. 34, a signal value was detected although a spot could hardly be visually confirmed. In addition, the signal value of No. 19 in the patient serum pool 2 was low enough to be judged as noise.

Summing up these results, among the 29 proteins synthesized, the proteins that did not react with the serum of healthy subjects but reacted only with the patient serum were No. 3, 4, 9, 11, 13, 19, 21, 22, There were 10 proteins of 24 and 28. On the other hand, proteins with higher color development in the serum of patients than in healthy subjects are Nos. 2, 3, 4, 6, 9, 10, 11, 13, 19, 21, 22, 24, 26, 28, 32 and 35. There were 16 types of proteins.
From these results, the proteins of Nos. 2, 3, 4, 6, 9, 10, 11, 13, 19, 21, 22, 24, 26, 28, 32 and 35 are included in the antibody titer test kit of the present invention. It was shown to be suitable as an antigen protein to be used.

Synthetic Protein Stability For the synthesized antigenic proteins, in order to confirm whether these proteins could actually be used in the antibody titer test kit, SDS-PAGE was performed to examine the state of the synthetic protein.
Samples were prepared to be 50 ng / 10 μl sample buffer (+ DTT). Since the proteins No. 5, 32 and 35 had no quantitative values, 4 μl of the synthetic protein solution and 6 μl of the sample buffer (+ DTT) were mixed to prepare a sample.
The prepared sample was heat denatured, and a sample of 10 μl in total was applied and subjected to SDS-PAGE electrophoresis. Subsequently, the gel after the electrophoresis was washed with distilled water and then washed with distilled water until CBB staining and a band could be clearly observed. The results are shown in FIG.

For proteins other than Nos. 5, 32 and 35, it was confirmed that proteins of the desired size were synthesized. On the other hand, no bands were observed for the No. 5 protein, and multiple bands were observed for the No. 32 and 35 proteins.
As for No. 5, the synthesis of messenger RNA was confirmed, but the protein synthesis was not confirmed. Therefore, this protein was expected to be a very unstable protein or a protein that was difficult to synthesize.
On the other hand, the proteins of No. 32 and No. 35 are known to be proteases, and it was thought that the synthetic protein also has protease activity and thus self-digests. When a part of the antigen protein has protease activity as described above, it causes degradation of other antigen proteins contained in the antibody titer test kit of the present invention, and the protein stability is poor, so it is considered that the protein cannot be used for the test. .

  Therefore, it was found that the No. 5 protein cannot be used because it is difficult to synthesize, and the No. 32 and 35 proteins have protease activity and cannot be used as they are.

Antigen-antibody reaction with patient sera As can be seen from Table 1, it is conceivable that the proteins used as antigens by antibodies in individual sera are different. Therefore, in order to select an antigen protein highly reactive with many patient sera, the antigen-antibody reaction with various patient sera was examined for the protein in which antigenicity was observed.
In this experiment, 16 types of antigen proteins that were highly colored in patient serum than in healthy subject serum were subjected to dot blot. The amount of protein attached to the dot blot was 50 ng. For No. 32 and 35, since the protein concentration was unknown, 4 μl of the synthetic protein solution was applied. In addition, each antigen protein was reacted with individual normal serum to set a reference value.

Detection of signal value An image of the membrane was taken using ImageQuant LAS4000 (GE Healthcare). After focusing, an image was taken under the following conditions.
・ Image capture conditions
Exposure Type: Precision
Exposure Time: 1/100 Sec
Sensitivity / Resolution: Standard
2. Next, the signal value of the dot of the photographed image was obtained using ImageQuant TL (GE Healthcare).
・ Signal value capture conditions
In the array analysis mode, the dot spot sizes were matched, and the signal values were digitized as the arrangement of 3 columns × 8 rows with respect to 2 columns × 8 rows (2 × 8).
3. After digitization, from the signal value of each spot, the middle row was used as the background close to the spot, and the difference was arranged in a table as the signal value of each spot. In addition, the signal value of the spot which cannot be confirmed visually was processed as undetectable.
4). NAI, TOM, and KOB signal values were determined as normal criteria. Among these, the signal values of the spots that could be visually confirmed were compared, and the highest signal value among the three samples was used as the serum standard for healthy subjects (FIG. 11). The reference value was set to 0 for spots where color development could not be confirmed in any specimen. Spots that showed a signal value higher than the signal value were marked with a circle (Figure 12 and Figure 13).

As a result, it was confirmed that the number of colored spots was clearly larger than that of normal serum and that the signal value was higher than that of normal serum. As expected, some patients had proteins that were not antigens.
Here, it was shown that periodontal disease of all patients can be determined by using antigen protein No. 32 or 35 that has been found to react with all patient antibodies. By selecting and using a combination of two or more of the selected proteins in consideration of sex and various immunoreactivity of the test target, it is possible to test for a wide range of periodontal pathogens and periodontal diseases in the test target Become.
The color signal value reflects the antibody titer against periodontal pathogen protein in the patient's plasma or serum. Therefore, although the progress (severity) of periodontal disease can be examined based on the obtained signal value, a wide range of teeth can be selected by appropriately selecting one or more of the selected proteins. It becomes possible to test periodontal disease progression by periodontal pathogens and test target.

Comparison of signal values between serum of healthy subjects and patient sera Antigen proteins whose signal values were higher than those of healthy subjects were sorted out. It showed a signal value higher than that of the standard for the sera of sera, and was found to be particularly suitable as an antigen protein used in the antibody titer test kit of the present invention (FIGS. 12 and 13). In addition, each of the antigen proteins No. 2, 3 and 26 showed a signal value higher than the standard for the serum of 19, 16 and 12 out of the 23 examined, and the coverage rate of the test object was It was found to be relatively high (FIGS. 12 and 13).

Thus, the antigenic proteins of No. 32 and No. 35 have high signal values and high coverage, and were found to be suitable as antigens for use in antibody titer test kits. Not available.
On the other hand, in order to use a protein other than No. 32 and 35 as an antigen of an antibody titer test kit, it is necessary to combine a plurality of types of antigen proteins. For example, it was confirmed that the coverage could be increased to 100% by combining antigen proteins No. 2 and No. 3.

As described above, the antigen proteins No. 32 and No. 35 have excellent properties but are proteases, and therefore cannot be used as test antigens.
Therefore, a modified polypeptide in which the protease activity was lost was prepared in a state where the antigenicity of these antigen proteins remained.

The amino acid sequences of the No. 32 and 35 proteins were analyzed using the Genetyx homology search tool, and the two cysteine residues confirmed for each protein were each replaced with an alanine residue by the following method. The amino acid sequences of the two modified polypeptides (No. 32A and 32B) prepared for No. 32 are shown in SEQ ID NOs: 63 and 65, and the polynucleotide sequences encoding them are shown in SEQ ID NOs: 64 and 66, respectively. The amino acid sequences of the two modified polypeptides (No. 35A and 35B) prepared for No. 35 are shown in SEQ ID NOs: 67 and 69, and the polynucleotide sequences encoding them are shown in SEQ ID NOs: 68 and 70, respectively. .
Preparation of protein expression plasmid / primer synthesis A primer pair shown in SEQ ID NO: in the following sequence listing including a mutation introduction site was synthesized.
Modified polypeptide No.32A
Forward primer SEQ ID NO: 133
Reverse primer SEQ ID NO: 134
Modified polypeptide No.32B
Forward primer SEQ ID NO: 135
Reverse primer SEQ ID NO: 136
Modified polypeptide No. 35A
Forward primer SEQ ID NO: 137
Reverse primer SEQ ID NO: 138
Modified polypeptide No. 35B
Forward primer SEQ ID NO: 139
Reverse primer SEQ ID NO: 140
・ Primer phosphorylation: T4 Polynucleotide Kinase (Toyobo)
Composition of reaction solution 20μl Preparation synthetic primer (50μM) 14μl
10 × Protruding End Kinase Buffer 2μl
10 mM ATP 2 μl
T4 Polynucleotide Kinase (5-20U / μl) 2μl

Reaction composition
After holding at 37 ° C. for 60 min and then at 95 ° C. for 5 min, 50 μl of DW was added (10 pmol / μl primer DNA).
・ Inverse PCR: Prime STAR MAX (Takara)
Preparation of reaction solution 50μl
Takara PrimeSTAR MAX Premix (2 ×) 25μl
Forward primer (10 pmol/μl) 1.5μl
Reverse primer (10 pmol/μl) 1.5μl
Template DNA (No.32 or 35 antigen protein plasmid DNA: 10ng / μl) 1μl
Sterile water 21 μl
Reaction conditions
After 30 seconds of treatment at 98 ° C, 30 cycles of 98 ° C for 10 seconds, 55 ° C for 5 seconds, and 72 ° C for 50 seconds were performed.

After purifying the PCR product using the QIAGEN kit, DpnI treatment was performed to degrade Template DNA (plasmid DNA).
The PCR product was purified using the QIAGEN kit with the following composition.
・ DpnI-treated purified PCR product 30μl
NEB4 5μl
BSA 5μl
DpnI (20unit / μL) 0.5μl
Sterile water 9.5μl
37 ° C, 1 hour

Purification of restriction enzyme treated product: PCI treatment was performed to purify the DNA product.
・ Ligation purified DNA 5μl
Ligation Mighty Mix 5μl
The ligation product was introduced into E. coli by transformation at 16 ° C. for 1 hour.
Sequence analysis was performed on plasmid DNA for which gene transfer was confirmed.
A large amount of a plasmid into which the target mutation was introduced was prepared, a protein was synthesized by a wheat germ cell-free protein synthesis system, and purified using a GST tag.

Confirmation of synthetic protein
SDS-PAGE
A sample was prepared by mixing 4 μl of the synthetic protein solution and 6 μl of the sample buffer (+ DTT). The prepared sample was heat denatured, a total amount of 10 μl of sample was applied, and subjected to SDS-PAGE electrophoresis.
CBB staining
The gel after SDS-PAGE was washed with distilled water, immersed in a CBB staining solution, and then stirred.
Washed with distilled water until the band could be clearly observed.
Dot Blot Four types of modified polypeptides (No. 32A, 32B, 35A and 35B) prepared were subjected to dot blot. 4 μl of 12.5 ng / μl of antigen protein to be dot blotted was applied to make 50 ng. Three sets of dot blots were performed. The dot blot was blocked by immersing in TBS with 5% skim milk overnight and allowed to react with the following serum solution at room temperature.
Serum solution (primary antibody)
A: NAI, TOM and KOB sera mixed with 3 μl each 8 μl mixed with 20 ml TBS containing 3% skim milk
B: Mix 8 μl of 4 μl of No.7350 and 6921 serum with 20 ml of TBS containing 3% skim milk
C: 3 μl each of No. 7107, 7523 and 6980 sera mixed 8 μl mixed with 20 ml TBS containing 3% skim milk 140 mL 1 × TBS
Subsequently, the slit was washed with TBS containing Tween 20 (0.05%). After washing, 20 ml of a horseradish peroxidase-conjugated goat anti-human IgG antibody reaction solution diluted 5000 times (a solution obtained by adding anti-human secondary antibody (CHEMICON) to TBS containing skim milk (3%)) was added and shaken at room temperature. Subsequently, the slit was washed with TBS containing Tween 20 (0.05%). After washing, the slit was immersed in TBS containing 4-methoxy-1-naphthol (0.61 mg / ml) and hydrogen peroxide (0.018%) to confirm color development, washed with purified water, and dried.

The results are shown in FIG.
For No. 32, protease activity was suppressed in both modified polypeptides. On the other hand, for No. 35, the protease activity was not completely suppressed in No. 35B, whereas the protease activity was suppressed in No. 35A. Further, as a result of examining the antigenicity, it was confirmed that any modified polypeptide maintained the antigenicity.

Test method and dot blot analysis As in the previous experiment, 16 antigen proteins were dot-blotted on the sera of 10 healthy individuals and 20 periodontal disease patients (however, No.32 and No.35). For the antigen protein of No. 32A and No. 35A, the obtained modified polypeptides were used). The amount of protein applied to the dot blot was 50 ng (however, the antigen concentration of No. 4 was 37 ng because the protein concentration was low and the protein solution was insufficient during the test, so serum numbers H9, H10, P10) , P20 not blotted).
After dot blotting, it was immersed in a blocking solution (TBS solution containing skim milk (5%)).
As a primary antibody solution, use a solution in which 8 μL of individual healthy subject serum (H1 to H10) or periodontal disease patient serum (P1 to P20) is mixed with 20 mL of TBS containing 3% skim milk. And the antigen-antibody reaction was performed at room temperature for 2 hours.
Subsequently, the slit was washed with TBS containing Tween 20 (0.05%). After washing, add 20 mL of a horseradish peroxidase-conjugated goat anti-human IgG antibody reaction solution (a solution of TBS containing skim milk (3%) to which anti-human secondary antibody (MILLIPORE) is added) diluted 5000 times, and antigen at room temperature for 1 hour. An antibody reaction was performed.
Subsequently, the slit was washed with TBS containing Tween 20 (0.05%). After washing, the slit was immersed in TBS containing 4-methoxy-1-naphthol (0.61 mg / mL) and hydrogen peroxide (0.018%) to confirm color development, washed with purified water, and dried. The results are shown in FIG.

・ Signal value detection An image of the membrane was taken using ImageQuant LAS4000 (GE Healthcare). After focusing, an image was taken under the following conditions.
-Image capture conditions-
Exposure Type: Precision
Exposure Time: 1/100 Sec
Sensitivity / Resolution: Standard
2. Next, the signal value of the dot of the photographed image was obtained using ImageQuant TL (GE Healthcare).
-Signal value capture conditions-
In the array analysis mode, a circular cursor was placed so as to surround the entire spot of dots, and the arrangement was matched as a spot layout of 2 columns × 8 rows, and the signal value was digitized.
For the background setting, Spot Edge Average mode was set, and the signal value of the spot was reflected to the background around the spot.

・ Signal value analysis For the signal values of each serum for each antigen protein, the mean signal values of the healthy and patient serum groups were calculated (FIGS. 16 and 17). As a result, it was found that the antigen proteins No. 13, 21, 22, and 28 did not react with any serum or the reaction rate was low.
Next, the Signal / Noise ratio was determined with the signal value of the patient serum group as the Signal value and the signal value of the healthy subject serum group as the Noise value. In addition, since the calculation value cannot be calculated for the healthy subject serum group whose signal average value is zero, it is indicated as Noise: 0 (FIG. 18). As a result, the No. 2, 6, 10 and 26 antigen proteins have high signal mean but low S / N ratio and are not suitable for testing periodontal disease in a wide range of patients with various immunotypes. It was shown that. On the other hand, the antigen proteins No. 3, 4, 9, 11, 19, 24, 32A and 35A were considered to have a high S / N ratio and high specificity for patient serum.
2. With respect to the signal value of each serum for each antigen protein, the ROC (Receiver Operating Characteristic) curve and the area under the ROC curve (Area) were calculated using Excel Statistics 2010 software (FIG. 19).

Here, the ROC curve is a graph showing how the positive rate (sensitivity) and false positive rate (1-specificity) change when the boundary value (cutoff value) is changed. Considering an ideal test, a state where both sensitivity and specificity are 1.0, that is, when it is at the upper left point, is an ideal state. Therefore, the graph that changes so that the ROC curve is close to the upper left corner is considered to have high diagnostic and predictive ability of the examination. Therefore, by measuring the area under the ROC curve, it is possible to judge the predictability and diagnostic ability of the test.
In general, the predictive ability / diagnostic ability based on the value of AUC is determined as follows.
AUC 0.9−1.0 High accuracy
AUC 0.7−0.9 Moderate accuracy
AUC 0.5−0.7 Low accuracy

  From the results of FIG. 19, it was found that the antigen proteins No. 3, 4, 9, 11, 19, 24, 32A and 35A have diagnostic ability / prediction ability of AUC 0.6 or more.

Production of highly expressed antigenic protein Test method No.32N, 32C, 35N and 35C protein synthesis
Based on the nucleotide sequences of No. 32A and No. 35A, the target gene sequence was amplified using the synthesized primer pairs shown in the following primer list so that the nucleotide size was about half.
Preparation of protein expression plasmid and primer synthesis Primer pairs shown in SEQ ID NOs:
Modified polypeptide No.32N
Forward primer SEQ ID NO: 157
Reverse primer SEQ ID NO: 158
Modified polypeptide No.32C
Forward primer SEQ ID NO: 159
Reverse primer SEQ ID NO: 160
Modified polypeptide No.35N
Forward primer SEQ ID NO: 161
Reverse primer SEQ ID NO: 162
Modified polypeptide No.35C
Forward primer SEQ ID NO: 163
Reverse primer SEQ ID NO: 164

Subsequently, the amplified DNA product was treated with a restriction enzyme, and ligated so that the reading frame matched with a protein expression vector (Cell Free Science: pEu vector) treated with the restriction enzyme. The ligation product was introduced into E. coli by transformation. Subsequently, clones into which the gene was introduced were selected.
Plasmid DNA was collected from the selected clones and subjected to sequence analysis. The amino acid sequences of the two modified polypeptides (No. 32N and 32C) prepared for No. 32A are shown in SEQ ID NOs: 141 and 143, and the polynucleotide sequences encoding them are shown in SEQ ID NOs: 142 and 144, respectively. In addition, the amino acid sequences of two types of modified polypeptides (No. 35N and 35C) prepared for No. 35A are shown in SEQ ID NOs: 145 and 147, and the polynucleotide sequences encoding them are shown in SEQ ID NOs: 146 and 148, respectively. .
For clones in which no significant mutation was found from the results of sequence analysis, a large amount of plasmid was prepared, the protein was synthesized by a wheat germ cell-free protein synthesis system, and purified using a GST tag.
We succeeded in synthesizing four types of proteins, N-terminal and C-terminal of No.32A and N-terminal and C-terminal of No.35A, and performed dot blot analysis.

・ Dot blot analysis
Four antigenic proteins (No. 32N, 32C, 35N and 35C) were dot blotted. The amount of protein attached to the dot blot was 10 ng and 200 ng.
After dot blotting, it was immersed in a blocking solution (TBS solution containing skim milk (5%)).
Normal antibody pool serum (H1 to H10), periodontal disease patient pool serum 1 (P1 to P10) or periodontal disease patient pool serum 2 (P11 to P20) 4 μL as primary antibody solution, 10 mL of TBS containing 3% skim milk Using the mixed solution, the slit after blocking was immersed in this, and antigen-antibody reaction was performed at room temperature for 2 hours.
Subsequently, the slit was washed with TBS containing Tween 20 (0.05%). After washing, add 10 mL of horseradish peroxidase-conjugated goat anti-human IgG antibody reaction solution (a solution of TBS containing skim milk (3%) plus anti-human secondary antibody (MILLIPORE)) diluted 5000 times, and antigen at room temperature for 1 hour An antibody reaction was performed.
Subsequently, the slit was washed with TBS containing Tween 20 (0.05%). After washing, the slit was immersed in TBS containing 4-methoxy-1-naphthol (0.61 mg / mL) and hydrogen peroxide (0.018%) to confirm color development, washed with purified water, and dried. The results are shown in FIG.
As a result, both the antigenic proteins of No. 32A and 35A differed in antigenicity between the N-terminal and C-terminal fragments, and the C-terminal side showed reactivity with normal serum, but the N-terminal side showed periodontal disease. Specific reactivity was shown in patient sera.

Selection of antigen protein with high test accuracy Test method and dot blot analysis
Ten antigen proteins were dot blotted. The amount of protein attached to the dot blot was 200 ng. However, No. 32C and 35C were 10 ng because of their high sensitivity.
After dot blotting, it was immersed in a blocking solution (TBS solution containing skim milk (5%)).
As a primary antibody solution, use 8 μL of individual healthy subject serum (H1 to H10) or periodontal disease patient serum (P1 to P20) mixed with 10 mL of TBS containing 3% skim milk. And the antigen-antibody reaction was performed at room temperature for 2 hours.
Subsequently, the slit was washed with TBS containing Tween 20 (0.05%). After washing, add 10 mL of horseradish peroxidase-conjugated goat anti-human IgG antibody reaction solution (a solution of TBS containing skim milk (3%) plus anti-human secondary antibody (MILLIPORE)) diluted 5000 times, and antigen at room temperature for 1 hour An antibody reaction was performed.
Subsequently, the slit was washed with TBS containing Tween 20 (0.05%). After washing, the slit was immersed in TBS containing 4-methoxy-1-naphthol (0.61 mg / mL) and hydrogen peroxide (0.018%) to confirm color development, washed with purified water, and dried. The results are shown in FIG.

・ Signal value detection The membrane image was scanned using CanoScan 9000F (Canon). After placing the membrane on the scanning board, the image was scanned under the following conditions.
-Image capture conditions-
-Input settings Document type: Paper / Photo color mode: Grayscale-Output settings Output resolution: 300 dpi
Output size: Free size and image settings Image preparation: Automatic Other items: Off

Next, the captured image was trimmed into each slit using ArcSoft PhotoStudio 6 software and converted into a Tiff file.
2. Next, the signal value of the dot of the photographed image was obtained using ImageQuant TL (GE Healthcare).
-Signal value capture conditions-
In the array analysis mode, a circular cursor was placed so as to surround the entire spot of dots, and the arrangement was matched as a spot layout of 2 columns × 6 rows, and the signal value was digitized.
For the background setting, Spot Edge Average mode was set, and the signal value of the spot was reflected to the background around the spot. The results are shown in FIG.

・ Signal value analysis The mean value, SD value, maximum value in the group, second value, mean value + 2SD value, mean value + 3SD value were determined from the signal values in the healthy subject serum group. With these maximum values, second values, average + 2SD, and average + 3SD as boundary values (cut-off values), true positive, false negative, false positive, and true negative were determined from the test results for each antigen protein.
Then, sensitivity and specificity were determined from the results.
2. The ROC curve and AUC were calculated | required using the Excel statistics 2010 software about the signal value of each serum with respect to each antigen protein.

Test results Based on the sensitivity and specificity results based on the cut-off value obtained from the healthy serum group (Figure 23), the second value of the healthy serum is the cut-off value. It was found that sensitivity 0.85 and specificity 0.9 could be achieved.
The results of ROC curve and AUC for each antigen protein (Fig. 24) are low accuracy tests for No. 3 and No. 9 antigen proteins, but Moderate Accuracy tests for other antigen proteins. Turned out to be able to provide. In particular, it has been found that the No. 35N antigen protein can provide a high accuracy test.
Regarding the No. 3 and No. 9 antigen proteins, the AUC value obtained in this test with better antigen-antibody reaction conditions was lower than the AUC value obtained in the previous test. It was considered that one of the causes was that protein denaturation progressed due to repeated freeze-thawing of the antigen protein.

Antigen protein that recognizes Porphyromonas gingivalis strain SU63 Test method / Verification of homology between strains
Based on the amino acid sequence of each protein of the FDC381 strain, a search was performed using blastp of BLAST (Basic Local Alignment Search Tool) on the NCBI site.
From the search results, homology with proteins in Porphyromonas gingivalis strains W83, ATCC33277 and TDC60 registered in the database was confirmed.
-Gene cloning and protein synthesis from the SU63 strain Amplify the target gene from the genomic DNA derived from the Gindivarius strain SU63 using the primer pair indicated in the sequence number of the following sequence listing by referring to the gene information of the selected protein from the database did.
Su63 strain No.15 antigen protein (No.15Su)
Forward primer SEQ ID NO: 165
Reverse primer SEQ ID NO: 166
Su63 strain No.16 antigen protein (No.16Su)
Forward primer SEQ ID NO: 167
Reverse primer SEQ ID NO: 168
Su63 strain No.34 antigen protein (No.34Su)
Forward primer SEQ ID NO: 169
Reverse primer SEQ ID NO: 170
Su63 strain No.37 antigen protein (No.37Su)
Forward primer SEQ ID NO: 171
Reverse primer SEQ ID NO: 172

Subsequently, the amplified DNA product was treated with a restriction enzyme and ligated to a protein expression vector (Cell Free Science: pEu vector) treated with the restriction enzyme. The ligation product was introduced into E. coli by transformation. Subsequently, clones into which the gene was introduced were selected.
Plasmid DNA was collected from the selected clones and subjected to sequence analysis. The amino acid sequences of the antigen proteins No. 15Su, No. 16Su, No. 34Su and No. 37Su are respectively shown in SEQ ID NOs: 149, 151, 153 and 155, and the polynucleotide sequences encoding them are respectively shown in SEQ ID NOs: 150 and 152. , 154 and 156. As a result, the polynucleotide sequence encoding the No.15Su antigen protein is one base different from the corresponding polynucleotide sequence of the W83 strain, and the polynucleotide sequences encoding the No.16Su antigen protein are those of the TDC60 strain and ATCC33277 strain. It was found that the sequence of the polynucleotide encoding No.34Su antigen protein is highly homologous to the polynucleotide sequence of ragA (A011 / 9 strain).

For clones in which no significant mutation was found from the results of sequence analysis, a large amount of plasmid was prepared, the protein was synthesized by a wheat germ cell-free protein synthesis system, and purified using a GST tag.
As a result, four types of proteins No. 15, 16, 34 and 37 derived from the SU63 strain were successfully synthesized, and dot blot analysis was performed on them.

・ Dot blot analysis
Eight antigen proteins (FDC381 strain: No.15, 16, 34 and 37, SU63 strain: No.15Su, 16Su, 34Su and 37Su) were dot-blotted. The amount of protein attached to the dot blot was 50 ng.
After dot blotting, it was immersed in a blocking solution (TBS solution containing skim milk (5%)).
As a primary antibody solution, use a solution in which 4 μL of individual healthy subject serum (H1 to H10) or periodontal disease patient serum (P1 to P20) is mixed with 10 mL of TBS containing 3% skim milk. And the antigen-antibody reaction was performed at room temperature for 2 hours.
Subsequently, the slit was washed with TBS containing Tween 20 (0.05%). After washing, add 10 mL of horseradish peroxidase-conjugated goat anti-human IgG antibody reaction solution (a solution of TBS containing skim milk (3%) plus anti-human secondary antibody (MILLIPORE)) diluted 5000 times, and antigen at room temperature for 1 hour An antibody reaction was performed.
Subsequently, the slit was washed with TBS containing Tween 20 (0.05%). After washing, the slit was immersed in TBS containing 4-methoxy-1-naphthol (0.61 mg / mL) and hydrogen peroxide (0.018%) to confirm color development, washed with purified water, and dried. The results are shown in FIGS. 24 and 25.
As is clear from these figures, the No. 15 antigen protein was antigenic to the patient serum P19 with both FDC381 and SU63 antigen proteins. On the other hand, for the antigen proteins No. 16, 34 and 37, either one of the FDC381 strain or SU63 strain antigen protein showed antigenicity against either healthy subject serum or patient serum.
Therefore, it was suggested that the No. 16, 34, and 37 antigen proteins can distinguish and grasp the infection of FDC381 strain and SU63 strain.

Test results As a result of examining the homology between strains, the homology between each protein in the FDC381 strain and the protein on the database was as follows, and it was confirmed that the homology was low between strains.
No. W83 stock ATCC33277 stock TDC60 stock
15 387/387 (100%) 202/398 (51%) 213/402 (53%)
16 553/553 (100%) 242/566 (43%) 243/566 (43%)
34 1015/1016 (99%) 737/1039 (71%) 719/1022 (70%)
37 500/500 (100%) 249/511 (49%) 249/511 (49

As a result of sequence analysis, the homology of each protein derived from the FDC381 strain and the SU63 strain was as follows.
No.15: 99.7%
No.16: 41.4%
No.34: 65.2%
No.37: 47.3%

  The periodontal pathogen plasma or serum antibody titer test kit of the present invention is suitably used for a periodontal pathogen plasma or serum antibody titer test system that automatically and rapidly processes a large amount of samples.

SEQ ID NO: 1 is the amino acid sequence of a conserved hypothetical protein having a zinc carboxypeptidase domain.
SEQ ID NO: 2 is a nucleotide sequence encoding a conserved hypothetical protein having a zinc carboxypeptidase domain.
SEQ ID NO: 3 is the amino acid sequence of hypothetical protein PG1881.
SEQ ID NO: 4 is the base sequence encoding hypothetical protein PG1881.
SEQ ID NO: 5 is the amino acid sequence of hypothetical protein PGN_0291.
SEQ ID NO: 6 is the base sequence encoding hypothetical protein PGN_0291.
SEQ ID NO: 7 is the amino acid sequence of hypothetical protein PG0491.
SEQ ID NO: 8 is the base sequence encoding hypothetical protein PG0491.
SEQ ID NO: 9 is the amino acid sequence of hypothetical protein PGN_1611.
SEQ ID NO: 10 is the base sequence encoding hypothetical protein PGN_1611.
SEQ ID NO: 11 is the amino acid sequence of hypothetical protein PGN_0477.
SEQ ID NO: 12 is the base sequence encoding hypothetical protein PGN_0477.
SEQ ID NO: 13 is the amino acid sequence of hypothetical protein PGN_0860.
SEQ ID NO: 14 is the base sequence encoding hypothetical protein PGN_0860.
SEQ ID NO: 15 is the amino acid sequence of the 53 kDa major outer membrane protein.
SEQ ID NO: 16 is a nucleotide sequence encoding a 53 kDa major outer membrane protein.
SEQ ID NO: 17 is the amino acid sequence of the 35 kDa hemin binding protein.
SEQ ID NO: 18 is a nucleotide sequence encoding a 35 kDa hemin binding protein.
SEQ ID NO: 19 is the amino acid sequence of heme-binding protein FetB.
SEQ ID NO: 20 is the base sequence encoding heme-binding protein FetB.
SEQ ID NO: 21 is the amino acid sequence of NAD-dependent glutamate dehydrogenase.
SEQ ID NO: 22 is the base sequence encoding NAD-dependent glutamate dehydrogenase.
SEQ ID NO: 23 is the amino acid sequence of phosphoserine aminotransferase.
SEQ ID NO: 24 is a nucleotide sequence encoding phosphoserine aminotransferase.
SEQ ID NO: 25 is the amino acid sequence of TonB binding receptor Tlr.
SEQ ID NO: 26 is the base sequence encoding TonB binding receptor Tlr.
SEQ ID NO: 27 is the amino acid sequence of fimbrillin (FDC381 strain).
SEQ ID NO: 28 is the base sequence encoding fimbrillin.
SEQ ID NO: 29 is the amino acid sequence of the minor component FimE (FDC381 strain).
SEQ ID NO: 30 is a base sequence encoding the trace component FimE.
SEQ ID NO: 31 is the amino acid sequence of HmuY ′.
SEQ ID NO: 32 is the base sequence encoding HmuY ′.
SEQ ID NO: 33 is the amino acid sequence of the M24 family peptidase.
SEQ ID NO: 34 is the base sequence encoding M24 family peptidase.
SEQ ID NO: 35 is the amino acid sequence of glyceraldehyde-3-phosphate dehydrogenase type 1.
SEQ ID NO: 36 is the base sequence encoding glyceraldehyde-3-phosphate dehydrogenase type 1.
SEQ ID NO: 37 is the amino acid sequence of ferritin.
SEQ ID NO: 38 is a nucleotide sequence encoding ferritin.
SEQ ID NO: 39 is the amino acid sequence of serine hydroxymethyltransferase.
SEQ ID NO: 40 is the base sequence encoding serine hydroxymethyltransferase.
SEQ ID NO: 41 is the amino acid sequence of outer membrane lipoprotein Omp28.
SEQ ID NO: 42 is the base sequence encoding outer membrane lipoprotein Omp28.
SEQ ID NO: 43 is the amino acid sequence of a promising lysyl endopeptidase precursor.
SEQ ID NO: 44 is a nucleotide sequence encoding a promising lysyl endopeptidase precursor.
SEQ ID NO: 45 is the amino acid sequence of the quinone family NAD (P) dehydrogenase.
SEQ ID NO: 46 is the base sequence encoding quinone family NAD (P) dehydrogenase.
SEQ ID NO: 47 is the amino acid sequence of DNA-binding protein from starved cell Dps.
SEQ ID NO: 48 is the base sequence encoding DNA-binding protein from starved cells Dps.
SEQ ID NO: 49 is the amino acid sequence of the immunoreactive 42 kDa antigen PG33.
SEQ ID NO: 50 is the base sequence encoding immunoreactive 42 kDa antigen PG33.
SEQ ID NO: 51 is the amino acid sequence of Lys-gingipain.
SEQ ID NO: 52 is the base sequence encoding Lys-gingipain.
SEQ ID NO: 53 is the amino acid sequence of peptidyl arginine deiminase.
SEQ ID NO: 54 is the base sequence encoding peptidylarginine deiminase.
SEQ ID NO: 55 is the amino acid sequence of the ragA protein (FDC381 strain).
SEQ ID NO: 56 is the base sequence encoding ragA protein.
SEQ ID NO: 57 is the amino acid sequence of arginine-specific cysteine proteinase RgpA.
SEQ ID NO: 58 is the base sequence encoding arginine-specific cysteine proteinase RgpA.
SEQ ID NO: 59 is the amino acid sequence of outer membrane protein 41 precursor.
SEQ ID NO: 60 is the base sequence encoding outer membrane protein 41 precursor.
SEQ ID NO: 61 is the amino acid sequence of lipoprotein RagB (FDC381 strain).
SEQ ID NO: 62 is the base sequence encoding lipoprotein RagB.
SEQ ID NO: 63 is the amino acid sequence of mutated Lys-gingipain.
SEQ ID NO: 64 is the base sequence encoding the mutated Lys-gingipain.
SEQ ID NO: 65 is the amino acid sequence of mutated Lys-gingipain.
SEQ ID NO: 66 is the base sequence encoding mutated Lys-gingipain.
SEQ ID NO: 67 is the amino acid sequence of the mutated arginine-specific cysteine proteinase RgpA.
SEQ ID NO: 68 is the base sequence encoding the mutated arginine-specific cysteine proteinase RgpA.
SEQ ID NO: 69 is the amino acid sequence of the mutated arginine-specific cysteine proteinase RgpA.
SEQ ID NO: 70 is the base sequence encoding the mutated arginine-specific cysteine proteinase RgpA.
SEQ ID NO: 71 is a forward primer used for PCR amplification of a polynucleotide encoding a conserved hypothetical protein having a zinc carboxypeptidase domain.
SEQ ID NO: 72 is a reverse primer used for PCR amplification of a polynucleotide encoding a conserved hypothetical protein having a zinc carboxypeptidase domain.
SEQ ID NO: 73 is a forward primer used for PCR amplification of a polynucleotide encoding hypothetical protein PG1881.
SEQ ID NO: 74 is a reverse primer used for PCR amplification of a polynucleotide encoding hypothetical protein PG1881.
SEQ ID NO: 75 is a forward primer used for PCR amplification of a polynucleotide encoding the hypothetical protein PGN_0291.
SEQ ID NO: 76 is a reverse primer used for PCR amplification of a polynucleotide encoding the hypothetical protein PGN_0291.
SEQ ID NO: 77 is a forward primer used for PCR amplification of a polynucleotide encoding hypothetical protein PG0491.
SEQ ID NO: 78 is a reverse primer used for PCR amplification of a polynucleotide encoding hypothetical protein PG0491.
SEQ ID NO: 79 is a forward primer used for PCR amplification of a polynucleotide encoding the hypothetical protein PGN_1611.
SEQ ID NO: 80 is a reverse primer used for PCR amplification of a polynucleotide encoding the hypothetical protein PGN_1611.
SEQ ID NO: 81 is a forward primer used for PCR amplification of a polynucleotide encoding the hypothetical protein PGN_0477.
SEQ ID NO: 82 is a reverse primer used for PCR amplification of a polynucleotide encoding the hypothetical protein PGN_0477.
SEQ ID NO: 83 is a forward primer used for PCR amplification of a polynucleotide encoding the hypothetical protein PGN_0860.
SEQ ID NO: 84 is a reverse primer used for PCR amplification of a polynucleotide encoding the hypothetical protein PGN_0860.
SEQ ID NO: 85 is a forward primer used for PCR amplification of a polynucleotide encoding a 53 kDa major outer membrane protein.
SEQ ID NO: 86 is a reverse primer used for PCR amplification of a polynucleotide encoding a 53 kDa major outer membrane protein.
SEQ ID NO: 87 is a forward primer used for PCR amplification of a polynucleotide encoding a 35 kDa hemin binding protein.
SEQ ID NO: 88 is a reverse primer used for PCR amplification of a polynucleotide encoding a 35 kDa hemin binding protein.
SEQ ID NO: 89 is a forward primer used for PCR amplification of a polynucleotide encoding the heme binding protein FetB.
SEQ ID NO: 90 is a reverse primer used for PCR amplification of a polynucleotide encoding the heme binding protein FetB.
SEQ ID NO: 91 is a forward primer used for PCR amplification of a polynucleotide encoding NAD-dependent glutamate dehydrogenase.
SEQ ID NO: 92 is a reverse primer used for PCR amplification of a polynucleotide encoding NAD-dependent glutamate dehydrogenase.
SEQ ID NO: 93 is a forward primer used for PCR amplification of a polynucleotide encoding phosphoserine aminotransferase.
SEQ ID NO: 94 is a reverse primer used for PCR amplification of a polynucleotide encoding phosphoserine aminotransferase.
SEQ ID NO: 95 is a forward primer used for PCR amplification of a polynucleotide encoding TonB binding receptor Tlr.
SEQ ID NO: 96 is a reverse primer used for PCR amplification of a polynucleotide encoding the TonB binding receptor Tlr.
SEQ ID NO: 97 is a forward primer used for PCR amplification of a polynucleotide encoding fimbrillin.
SEQ ID NO: 98 is a reverse primer used for PCR amplification of a polynucleotide encoding fimbrillin.
SEQ ID NO: 99 is a forward primer used for PCR amplification of a polynucleotide encoding the trace component FimE.
SEQ ID NO: 100 is a reverse primer used for PCR amplification of a polynucleotide encoding the trace component FimE.
SEQ ID NO: 101 is a forward primer used for PCR amplification of a polynucleotide encoding HmuY ′.
SEQ ID NO: 102 is a reverse primer used for PCR amplification of a polynucleotide encoding HmuY ′.
SEQ ID NO: 103 is a forward primer used for PCR amplification of a polynucleotide encoding M24 family peptidase.
SEQ ID NO: 104 is a reverse primer used for PCR amplification of a polynucleotide encoding M24 family peptidase.
SEQ ID NO: 105 is a forward primer used for PCR amplification of a polynucleotide encoding glyceraldehyde-3-phosphate dehydrogenase type 1.
SEQ ID NO: 106 is a reverse primer used for PCR amplification of a polynucleotide encoding glyceraldehyde-3-phosphate dehydrogenase type 1.
SEQ ID NO: 107 is a forward primer used for PCR amplification of a polynucleotide encoding ferritin.
SEQ ID NO: 108 is a reverse primer used for PCR amplification of a polynucleotide encoding ferritin.
SEQ ID NO: 109 is a forward primer used for PCR amplification of a polynucleotide encoding serine hydroxymethyltransferase.
SEQ ID NO: 110 is a reverse primer used for PCR amplification of a polynucleotide encoding serine hydroxymethyltransferase.
SEQ ID NO: 111 is a forward primer used for PCR amplification of a polynucleotide encoding outer membrane lipoprotein Omp28.
SEQ ID NO: 112 is a reverse primer used for PCR amplification of a polynucleotide encoding outer membrane lipoprotein Omp28.
SEQ ID NO: 113 is a forward primer used for PCR amplification of a polynucleotide encoding a promising lysyl endopeptidase precursor.
SEQ ID NO: 114 is a reverse primer used for PCR amplification of a polynucleotide encoding a promising lysyl endopeptidase precursor.
SEQ ID NO: 115 is a forward primer used for PCR amplification of a polynucleotide encoding quinone family NAD (P) dehydrogenase.
SEQ ID NO: 116 is a reverse primer used for PCR amplification of a polynucleotide encoding quinone family NAD (P) dehydrogenase.
SEQ ID NO: 117 is a forward primer used for PCR amplification of a polynucleotide encoding a DNA-binding protein from starved cells Dps.
SEQ ID NO: 118 is a reverse primer used for PCR amplification of a polynucleotide encoding a DNA-binding protein from starved cells Dps.
SEQ ID NO: 119 is a forward primer used for PCR amplification of a polynucleotide encoding the immunoreactive 42 kDa antigen PG33.
SEQ ID NO: 120 is a reverse primer used for PCR amplification of a polynucleotide encoding the immunoreactive 42 kDa antigen PG33.
SEQ ID NO: 121 is a forward primer used for PCR amplification of a polynucleotide encoding Lys-gingipain.
SEQ ID NO: 122 is a reverse primer used for PCR amplification of a polynucleotide encoding Lys-gingipain.
SEQ ID NO: 123 is a forward primer used for PCR amplification of a polynucleotide encoding peptidylarginine deiminase.
SEQ ID NO: 124 is a reverse primer used for PCR amplification of a polynucleotide encoding peptidylarginine deiminase.
SEQ ID NO: 125 is a forward primer used for PCR amplification of a polynucleotide encoding ragA protein.
SEQ ID NO: 126 is a reverse primer used for PCR amplification of a polynucleotide encoding ragA protein.
SEQ ID NO: 127 is a forward primer used for PCR amplification of a polynucleotide encoding arginine-specific cysteine proteinase RgpA.
SEQ ID NO: 128 is a reverse primer used for PCR amplification of a polynucleotide encoding arginine-specific cysteine proteinase RgpA.
SEQ ID NO: 129 is a forward primer used for PCR amplification of a polynucleotide encoding outer membrane protein 41 precursor.
SEQ ID NO: 130 is a reverse primer used for PCR amplification of a polynucleotide encoding outer membrane protein 41 precursor.
SEQ ID NO: 131 is a forward primer used for PCR amplification of a polynucleotide encoding lipoprotein RagB.
SEQ ID NO: 132 is a reverse primer used for PCR amplification of a polynucleotide encoding lipoprotein RagB.
SEQ ID NO: 133 is a forward primer used for PCR amplification of a polynucleotide encoding the mutated Lys-gingipain.
SEQ ID NO: 134 is a reverse primer used for PCR amplification of a polynucleotide encoding the mutated Lys-gingipain.
SEQ ID NO: 135 is a forward primer used for PCR amplification of a polynucleotide encoding the mutated Lys-gingipain.
SEQ ID NO: 136 is a reverse primer used for PCR amplification of a polynucleotide encoding a mutated Lys-gingipain.
SEQ ID NO: 137 is a forward primer used for PCR amplification of a polynucleotide encoding a mutated arginine-specific cysteine proteinase RgpA.
SEQ ID NO: 138 is a reverse primer used for PCR amplification of a polynucleotide encoding the mutated arginine-specific cysteine proteinase RgpA.
SEQ ID NO: 139 is a forward primer used for PCR amplification of a polynucleotide encoding a mutated arginine-specific cysteine proteinase RgpA.
SEQ ID NO: 140 is a reverse primer used for PCR amplification of a polynucleotide encoding a mutated arginine-specific cysteine proteinase RgpA.
SEQ ID NO: 141 is the amino acid sequence of about half of the N-terminal side of the mutated Lys-gingipain.
SEQ ID NO: 142 is the base sequence encoding about half of the N-terminal side of the mutated Lys-gingipain.
SEQ ID NO: 143 is the amino acid sequence of about half of the C-terminal side of the mutated Lys-gingipain.
SEQ ID NO: 144 is the base sequence encoding about half of the C-terminal side of the mutated Lys-gingipain.
SEQ ID NO: 145 is the amino acid sequence of about half of the N-terminal side of the mutated arginine-specific cysteine proteinase RgpA.
SEQ ID NO: 146 is a nucleotide sequence encoding about half of the N-terminal side of the mutated arginine-specific cysteine proteinase RgpA.
SEQ ID NO: 147 is the amino acid sequence of about half of the C-terminal side of the mutated arginine-specific cysteine proteinase RgpA.
SEQ ID NO: 148 is a nucleotide sequence encoding about half of the C-terminal side of the mutated arginine-specific cysteine proteinase RgpA.
SEQ ID NO: 149 is the amino acid sequence of fimbrillin (SU63 strain).
SEQ ID NO: 150 is the base sequence encoding fimbrillin (SU63 strain).
SEQ ID NO: 151 is the amino acid sequence of the minor component FimE (SU63 strain).
SEQ ID NO: 152 is the base sequence (SU63 strain) encoding the trace component FimE.
SEQ ID NO: 153 is the amino acid sequence of the ragA protein (SU63 strain).
SEQ ID NO: 154 is the base sequence (SU63 strain) encoding ragA.
SEQ ID NO: 155 is the amino acid sequence of lipoprotein RagB (SU63 strain).
SEQ ID NO: 156 is the base sequence (SU63 strain) encoding lipoprotein RagB.
SEQ ID NO: 157 is a forward primer used for PCR amplification of a polynucleotide encoding about half of the N-terminal side of the mutated Lys-gingipain.
SEQ ID NO: 158 is a reverse primer used for PCR amplification of a polynucleotide encoding about half of the N-terminal side of the mutated Lys-gingipain.
SEQ ID NO: 159 is a forward primer used for PCR amplification of a polynucleotide encoding about half of the C-terminal side of the mutated Lys-gingipain.
SEQ ID NO: 160 is a reverse primer used for PCR amplification of a polynucleotide encoding about half of the C-terminal side of the mutated Lys-gingipain.
SEQ ID NO: 161 is a forward primer used for PCR amplification of a polynucleotide encoding about half of the N-terminal side of the mutated arginine-specific cysteine proteinase RgpA.
SEQ ID NO: 162 is a reverse primer used for PCR amplification of a polynucleotide encoding about half of the N-terminal side of the mutated arginine-specific cysteine proteinase RgpA.
SEQ ID NO: 163 is a forward primer used for PCR amplification of a polynucleotide encoding about half of the C-terminal side of the mutated arginine-specific cysteine proteinase RgpA.
SEQ ID NO: 164 is a reverse primer used for PCR amplification of a polynucleotide encoding about half of the C-terminal side of the mutated arginine-specific cysteine proteinase RgpA.
SEQ ID NO: 165 is a forward primer used for PCR amplification of a polynucleotide encoding fibrin of the SU63 strain.
SEQ ID NO: 166 is a reverse primer used for PCR amplification of the polynucleotide encoding fibrin of the SU63 strain.
SEQ ID NO: 167 is a forward primer used for PCR amplification of a polynucleotide encoding the trace component FimE of the SU63 strain.
SEQ ID NO: 168 is a reverse primer used for PCR amplification of a polynucleotide encoding the trace component FimE of the SU63 strain.
SEQ ID NO: 169 is a forward primer used for PCR amplification of a polynucleotide encoding the ragA protein of the SU63 strain.
SEQ ID NO: 170 is a reverse primer used for PCR amplification of a polynucleotide encoding ragA protein of SU63 strain.
SEQ ID NO: 171 is a forward primer used for PCR amplification of a polynucleotide encoding lipoprotein RagB of SU63 strain.
SEQ ID NO: 172 is a reverse primer used for PCR amplification of a polynucleotide encoding lipoprotein RagB of SU63 strain.

Claims (6)

A typing kit for distinguishing between Porphyromonas gingivalis SU63 strain and FDC381 strain comprising a polypeptide having one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 151 and 155.   The typing kit according to claim 1, wherein the polypeptide is a polypeptide encoded by one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs: 152 and 156.   A polypeptide having the amino acid sequence of SEQ ID NO: 151.   The polypeptide according to claim 3, which consists of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 152.   A polypeptide having the amino acid sequence of SEQ ID NO: 155.   The polypeptide according to claim 5, which consists of an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 156.