JP5413863B2 - Test method and test reagent for fulminant type 1 diabetes - Google Patents

Description

  The present invention relates to a test method and a test reagent for fulminant type 1 diabetes (FT1DM).

Currently, AIP is diagnosed in accordance with the “Diagnostic criteria for autoimmune pancreatitis” published by the Japanese Pancreatic Society in 2002. According to this standard, it is a sufficient condition for diagnosis to recognize any one of hypergammaglobulinemia, hyperIgG, and autoantibodies, which are blood test items. further,
There is a report that the IgG4 subclass is specifically elevated among hyperIgG, and the anti-nuclear antibody and rheumatoid factor may be positive as autoantibodies.

Therefore, these γ-globulins, IgG, IgG4, antinuclear antibodies (SS
-A antibody and SS-B antibody) and rheumatoid factor must be measured, which is a problem that the cost is very high. In addition, these test items are elevated or positive in other diseases, and their diagnostic specificity is poor. Furthermore, even if this disease is diagnosed according to the above diagnostic criteria, it is very difficult to differentiate from pancreatic cancer, and there is a reality that unnecessary surgery is being performed, so a reliable differentiation method for both diseases is desired. ing.

Researchers have been searching for autoantibodies specific to AIP, but have found that there are activities of blood lactoferrin (LF) antibody and carbonic anhydrase II (CAII) antibody. However, the antibody positive rates in AIP are 73% and 53.8%, respectively, lacking in clinical sensitivity, lacking in method reliability and reproducibility, and are not suitable for diagnosis and differential diagnosis (Non-patent literature) 1, 2).

Furthermore, monitoring the progress of AIP treatment with a blood test is necessary to determine the dosage and timing, but for the same reason as described above, the problem is that the cost of testing is high. It has become.

On the other hand, the diagnosis of FT1DM is currently diagnosed according to the “diagnostic criteria for fulminant type 1 diabetes” presented by the Japan Diabetes Society Fulminant Diabetes Investigation Committee in 2004. According to this, as a specimen test, HbA1C, blood glucose level, urine or blood C-peptide are measured. Furthermore, as a reference finding, it is confirmed that autoantibodies such as GAD antibody and IA-2 antibody are negative, and an increase in blood pancreatic exocrine enzyme is observed.

  Therefore, the number of test items is also large in this disease, and the diagnosis is expensive and the test is nonspecific.

In addition, this disease often results in ketoacidosis within a few days after symptoms of hyperglycemia occur, and it is endangered if it is not diagnosed reliably and treatment is started immediately. Moreover, there are many cases that develop during pregnancy, and most of them cause fetal death. Therefore, it is desirable to measure the above test items even at the onset of normal diabetes to distinguish FT1DM from type 1 diabetes and type 2 diabetes, but there is no test marker for FT1DM for good exclusion diagnosis, and the number of test items Because there are many, the current situation is that no differential diagnosis is actually made.

N. Engl. J. Med. 2001, 344: 732-8 J. Gastroenterol. 2001, 36: 293-302

  The present invention has been made in view of the above-described problems of the prior art, and an object thereof is to provide a test method and test reagent for autoimmune pancreatitis (AIP) and fulminant type 1 diabetes (FT1DM). And

The present inventor has found that an antibody against HSP10, which is a kind of heat shock protein, is present in sera of patients with AIP and FT1DM and those who are highly likely to develop FT1DM, and have completed the present invention.

That is, the present invention provides the following.
(1) A method for examining autoimmune pancreatitis, comprising examining autoimmune pancreatitis by detecting an antibody immunologically reactive with HSP10 in a specimen.
(2) A method for testing fulminant type 1 diabetes, comprising detecting fulminant type 1 diabetes by detecting an antibody immunologically reactive with HSP10 in a sample.
In the test methods (1) to (2), it is preferable to detect the antibody by using an antigen that immunologically reacts with the antibody.

The present invention also includes an antigen that immunologically reacts with an antibody that immunologically reacts with HSP10.
Provided is a test reagent for use in the test methods (1) to (2) above.

  Diagnosis of autoimmune pancreatitis by measuring anti-HSP10 antibody is specific. Therefore, medical costs can be reduced by reducing examination costs by reducing the examination cost to one item, ensuring differential diagnosis with pancreatic cancer, and enabling unnecessary follow-up surgery and medication. Significant reduction of

The differentiation between fulminant type 1 diabetes and acute onset type 1 and type 2 diabetes is actually due to the lack of specific markers and the large number of test items for exclusion diagnosis as explained in the section of the prior art. Is not done. However, if the measurement according to the present invention is performed, a definitive diagnosis and a differential diagnosis can be performed with only one item of measurement. Moreover, if this measurement method is applied to a simple inspection method such as immunochromatography, fulminant type 1 diabetes can be diagnosed more quickly on the spot. In this way, it is possible to diagnose and save a patient who has been dying in the past (and the fetus of the patient) at an early stage, and to greatly reduce medical costs by reducing the cost of examination and becoming serious.

The measurement result of the reference example 2 is shown (electrophoresis photograph). (A) Western blot analysis, (b) ELISA. The measurement result of the presence rate of the autoantibody with respect to human HSP10 in the patient of various pancreatic diseases obtained in Example 1 is shown. AIP: autoimmune pancreatitis (before: before corticosteroid treatment, after: after corticosteroid treatment), CP: alcoholic chronic pancreatitis, PT: pancreatic tumor, N: healthy subject. In the figure, the dotted line indicates the average value of healthy individuals + 3SD. The measurement result of the presence rate of the autoantibody with respect to human HSP10 in various diabetic patients obtained in Example 2 is shown. FT1DM: Fulminant type 1, AT1DM: Acute type 1, T2DM: Type 2, N: Healthy subjects, FDR: First-degree relatives of FT1DM patients, Hashimoto: Hashimoto disease. In the figure, the dotted line indicates the average value of healthy individuals + 3SD. The time-dependent change of the anti- HSP10 antibody in the AIP patient and FT1DM patient obtained in Example 3 is shown. (A) AIP (the arrow in the figure indicates the start of corticosteroid treatment), (b) FT1DM. In the figure, the dotted line indicates the average value of healthy individuals + 3SD.

Embodiments of the present invention will be described below. First, the inspection method of the present invention will be described.
The test method of the present invention detects autoimmune pancreatitis (AIP) or fulminant type 1 diabetes by detecting an antibody that immunologically reacts with HSP10 (hereinafter also referred to as “anti-HSP10 antibody”) in a specimen. FT1DM).

The detection method is not limited as long as the anti-HSP10 antibody can be detected. Usually, an immunological method using an antigen or antibody that immunologically reacts with an anti-HSP10 antibody is used. Since an antigen is easier to prepare than an antibody, an immunological method using an antigen that immunologically reacts with an anti-HSP10 antibody is preferred. As an immunological method, the widely used ELISA
Law.

Any antigen may be used as long as it reacts immunologically with the anti-HSP10 antibody present in the specimen, and HSP10 molecules purified from humans or animals can be used. HSP10 molecules may be prepared by recombinant technology. For example, the base sequence encoding human HSP10 is known (GenBank accession number NM_002157. The encoded amino acid sequence is shown in SEQ ID NO: 1. It also immunologically reacts with the anti-HSP10 antibody present in the specimen. Insofar as a partial fragment of the HSP10 molecule can be used.

  The sample to be measured is usually serum, but is not particularly limited as long as it does not contain a substance that interferes with the reaction in the reaction process.

The detection conditions can be the same as in conventional immunological methods. That is, conditions such as the order of addition and amount of each reactant, reaction temperature, reaction time, etc. may be the same as in conventional immunological methods, for example, an antigen immobilized on a solid phase such as a bead, tube or plate, An example is a method in which serum is reacted to remove unbound IgG contained in the serum, and then an enzyme-labeled anti-IgG antibody is bound to the antibody bound to the antigen and detected by the reaction of the enzyme.

The amount of anti-HSP10 antibody is usually calculated as the measured value of the label. Alternatively, a calibration curve may be created using a standard sample containing a known concentration of anti-HSP10 antibody, and the concentration may be determined using the calibration curve.

  A positive determination can be made by comparing with the value of a healthy person. When the value is significantly higher than the value of healthy persons measured at the same time or measured in advance (for example, higher than 2SD, significantly higher by a statistical test), it can be determined as positive.

The rise in anti-HSP10 antibody measured by the test method of the present invention is specific to AIP and FT1DM.
According to the test method of the present invention, when AIP or FT1DM is suspected, AIP or FT1DM can be specifically tested by measuring one item.

  Next, the test reagent of the present invention will be described. The test reagent of the present invention is for carrying out the test method of the present invention, and contains an antigen that immunologically reacts with an anti-HSP10 antibody.

The antigen that immunologically reacts with the anti-HSP10 antibody is as described for the test method of the present invention.

Furthermore, the test reagent of the present invention may contain reagents necessary for immunological measurement, for example, a positive control, a buffer solution and the like, if necessary, and can also be provided as a kit.

  Each component of the test reagent of the present invention may be in a solution state or in a dry state such as a freeze-dried product. When it is in a dry state, a buffer solution for making it into a solution state before use may be further included in the test reagent of the present invention. The amount and form of each component are adjusted according to the conditions of the measurement method.

For example, in the case of an ELISA kit, a solid phase plate on which an antigen is immobilized, a washing solution, a specimen dilution solution,
A kit containing an enzyme-labeled anti-HSP10 antibody solution, a substrate solution, and a reaction stop solution can be obtained. In addition, the immunochromatography kit can be a kit including a reaction device and a developing solution.

  Hereinafter, the present invention will be described by way of examples. In Examples, “%” indicates wt% unless otherwise specified.

[Reference Example 1] Cloning of cDNA from human pancreas Human pancreatic cDNA library (λTriplEx2 human pancreatic large insert cDNA library, BD Bioscience Clontech) and E. coli XL-1 competent cells (BD Bioscience Clontech) were used. Plaques on the plate were transferred to a nitrocellulose membrane presoaked in 10 mM isopropyl-β-D-thiogalactoside (IPTG), washed with Tris buffered saline (TBST) containing 0.05% Tween 20, and washed with 1% bovine. Blocked with Tris buffered saline containing serum albumin. Membranes were incubated overnight at 4 ° C. with serum provided by chronic pancreatitis (AIP) patients (67 years old, male) (diluted 500-fold with TBST). Wash 4 times with TBST, goat horseradish peroxidase-conjugated anti-human IgG (American Qualex, diluted 2000-fold with TBST) and 30 at room temperature.
Reacted for minutes. After washing 4 times with TBST, a positive reaction was detected with 3,3′-diaminobenzidine.

By the above method, 2 × 10 ⁶ plaques obtained using the serum of an AIP patient were screened to obtain a positive clone.

The cDNA fragment of the positive clone was expressed as sense primer 5'-ATGGGGATCCGCAGGACAAGCGTTTAGA-3 '
(SEQ ID NO: 2) and antisense primer 5'-CTTCGAAT TCTCAGTCTACGTACTTTCC-3 '(
Amplified by PCR using SEQ ID NO: 3). The PCR product was digested with BamHI and EcoRI and ligated into pTrcHisB (Invitrogen). By sequencing the inserted cDNA and homology search, one of the 10 clones matched the sequence of human HSP10. Compared to the sequence of human HSP10 cloned by Monzini et al. (Biochim. Biophys. Acta. 1218: 478-480, 1994), it contained the full-length coding sequence.

[Reference Example 2] Western blot analysis and ELISA using antibody against human HSP10
As shown in Reference Example 1, IgG from AIP patients used for screening recognized HSP10. HSP10 tagged with a histidine tag was prepared using E. coli BL21. Specifically, after sequencing, the plasmid was transfected into E. coli BL-21 (Novagen), the recombinant protein was generated by induction with 1 mM IPTG and purified by His Bond column chromatography.

The prepared protein 0.1% SDS-15% polyacrylamide gel electrophoresis and transfer to a polyvinylidene difluoride (PVDF) membrane were performed according to the method described in Biochem. Biophys. Res. Commun. 190: 774-779, 2006. The modification was made as follows. Membranes were blocked with Tris-buffered saline (TBS) containing 2% BSA and 2% goat serum and incubated with AIP patient serum diluted 1000-fold with TBS containing 0.1% BSA for 1 hour at room temperature. The plate was washed 5 times with TBST and reacted with goat horseradish peroxidase-conjugated anti-human IgG (American Qualex, diluted 1: 2000 with 1% BSA) at room temperature for 30 minutes. A positive reaction was detected in the same manner as in Reference Example 1.

The results are shown in FIG. Patient serum (lane 1) clearly recognized the 14 kDa recombinant protein, but not healthy human serum (lanes 2 and 3). Immunostaining disappeared when the patient's serum was preincubated with the recombinant protein overnight at 4 ° C. (FIG. 1 (b), inset).

According to the ELISA described in Diabetes Care 24: 1661-7, 2001, the prepared protein was coated on a plate to construct an ELISA for detecting anti-HSP10 antibody in serum. Specifically, it is as follows. Microtiter plates (Coster 3590, Corning Inc.) were coated with 0.1 μg (50 μl) of recombinant human HSP10 at 4 ° C. overnight. Wash three times with phosphate buffered saline (PBST) containing 0.05% Tween 20 and add 200% 10% bovine serum albumin (BSA) solution in phosphate buffer.
Incubated with μl for 30 minutes. After washing with PBST, it was used for measurement. The measurement was performed in triplicate by diluting the patient's serum 200-fold with 1% BSA. The bound antibody was reacted with goat horseradish peroxidase-conjugated anti-human IgG (American Qualex, diluted 1: 2000 with 1% BSA) for 30 minutes at room temperature. After washing with PBST, the plate was incubated with 100 μl of 1-Step Slow TMB-ELISA (PIERCE) for 30 minutes. The reaction was stopped by adding 100 μl of 1 MH ₂ SO ₄ and the absorbance at 450 nm was measured.

  Compared to normal serum, patient serum produced a strong signal. This signal disappeared when the patient's serum (1000-fold diluted, 1 ml) was preincubated overnight at 4 ° C. with HSP10 (1 μg). The measurement result performed in triplicate is shown in FIG.

[Example 1] Presence of autoantibodies to human HSP10 in patients with pancreatic disease Various types of pancreatic disease patients (AIP 12 cases, alcoholic chronic pancreatitis 13 cases, pancreatic tumor [pancreatic cancer 2 cases, pancreas) The prevalence of autoantibodies against human HSP10 was measured in 6 cases of intravaginal papillary tumor (IPMT)] and healthy subjects (71 cases) (FIG. 2). When the average value of healthy subjects + 3SD was considered positive, 10 out of 12 AIP patients (before treatment) (95%) were positive for HSP10. However, other sera were all negative except that one healthy person became positive. In AIP patients treated with corticosteroids, 5 out of 8 (63%) were negative.

From these results, it was shown that this test method is a very effective method for the definitive diagnosis of AIP.

[Example 2] Prevalence of autoantibodies to human HSP10 in DM patients Using the ELISA of Reference Example 2, various types of DM patients (16 cases of fulminant type 1 DM, 40 cases of acute type 1 DM, 2
The presence of autoantibodies against human HSP10 was measured in 50 cases of type diabetes mellitus), 21 first-degree relatives of FT1DM patients, 54 cases of Hashimoto's disease patients, and 71 cases of healthy individuals (FIG. 3). Thirteen (81%) of 16 FT1DM patients were positive. However, it was detected infrequently in patients with acute onset type 1 DM and healthy individuals (10% and 2%, respectively). All patients with type 2 diabetes were negative. One patient (2%) was positive in patients with Hashimoto's disease, a typical organ-specific autoimmune disease. This antibody was positive in 1 case (5%) of first-degree relatives of FT1DM patients.

  From these results, it was shown that this test method is useful for the diagnosis of FT1DM.

[Example 3] Time course of anti-HSP10 antibody in AIP patients and FT1DM patients Using the above ELISA, the time course of anti-HSP10 antibodies in AIP patients and FT1DM patients was examined. The course was observed for 12 to 14 months for AIP patients (2 cases) and 7 to 10 weeks for FT1DM patients (2 cases). The measurement results performed in triplicate are shown in FIG. In AIP patients, they are positive from the onset, continue to be positive until corticosteroid treatment is started, and after starting corticosteroid treatment,
The antibody titer decreased or disappeared (FIG. 4 (a)). In FT1DM patients, the antibody titer was positive at the time of onset, but the antibody titer decreased with progress (FIG. 4 (b)).

  From the above results, it was shown that this inspection method is very effective for the follow-up of AIP and FT1DM.

Claims (3)

A method for testing fulminant type 1 diabetes, comprising detecting fulminant type 1 diabetes by detecting an antibody immunologically reactive with HSP10 in a sample. The test method according to claim 1, wherein the antibody is detected by using an antigen that immunologically reacts with the antibody. The test reagent for use in the test method according to claim 1, comprising an antigen that immunologically reacts with the antibody.