JP6249712B2 - Method of operating prognostic device for patients with non-paraneoplastic acute encephalitis - Google Patents
Method of operating prognostic device for patients with non-paraneoplastic acute encephalitis Download PDFInfo
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本発明は、非傍腫瘍性急性脳炎患者の予後診断装置の作動方法に関する。 The present invention relates to a method for operating a prognosis apparatus for patients with non-paraneoplastic acute encephalitis .
N-methyl-D-aspartate型グルタミン酸受容体抗体（NMDAR抗体）の測定方法には主として、イムノブロット法、cell-based assay法、ELISA法の３種類がある。 There are three main methods for measuring N-methyl-D-aspartate type glutamate receptor antibodies (NMDAR antibodies): immunoblotting, cell-based assay, and ELISA.
抗NMDAR抗体陽性脳炎は、2007年ペンシルヴェニア大学のDalmauらにより提唱された自己免疫性脳炎である。従来、cell-based assay法によるNMDAR抗体（Dalmau抗体）は、卵巣奇形腫のある抗NMDAR抗体陽性脳炎に特異的なマーカーと考えられていた。 Anti-NMDAR antibody-positive encephalitis is an autoimmune encephalitis proposed by Dalmau et al. At the University of Pennsylvania in 2007. Conventionally, NMDAR antibody (Dalmau antibody) by cell-based assay was considered to be a specific marker for anti-NMDAR antibody-positive encephalitis with ovarian teratoma.
しかしながら、今日では、NMDAR抗体は、奇形腫を合併しない脳炎、てんかん、Creutzfeldt-Jakob病、単純ヘルペス脳炎回復期、ミトコンドリア病等でも検出されるようになっている。 However, today, NMDAR antibodies are also detected in encephalitis, epilepsy, Creutzfeldt-Jakob disease, herpes simplex encephalitis recovery, mitochondrial disease, etc. that do not involve teratoma.
そこで、これらの症例の臨床特徴や予後の異なる神経疾患に共通して存在するNMDAR抗体の役割を理解するために、抗NMDAR抗体のIgGサブクラス測定方法が求められる。 Therefore, in order to understand the role of NMDAR antibodies that exist in common in clinical features and neurological diseases with different prognosis, a method for measuring the IgG subclass of anti-NMDAR antibodies is required.
IgGサブクラス測定方法には、免疫比ろう法（immunonephelometry: NIA）や免疫比濁法（immunoturbidimetry）等の比濁分析法（nephelometry）、一元放射状免疫拡散法（single radial immunodiffusionmethod: SRID）等の免疫拡散法、酵素抗体法（enzyme-linked immunosorbentassay: ELISA）等が開発されている。 IgG subclass measurement methods include immunodiffusion methods such as immunopeptometry (NIA), immunoturbidimetry (nephelometry), and single radial immunodiffusion method (SRID). And enzyme-linked immunosorbent assay (ELISA) have been developed.
このうち、ELISA法は比較的簡便で感度も良い方法であるため、ELISA法を用いた抗NMDAR抗体の各IgGサブクラスの濃度の定量方法が求められる（非特許文献１）。 Among these, since the ELISA method is a relatively simple method with good sensitivity, a method for quantifying the concentration of each IgG subclass of the anti-NMDAR antibody using the ELISA method is required (Non-patent Document 1).
本発明はかかる問題点に鑑みてなされたものであって、非傍腫瘍性急性脳炎患者の予後診断装置の作動方法を提供することを目的とする。 This invention is made | formed in view of this problem, Comprising: It aims at providing the operation | movement method of the prognosis apparatus of a non-paraneoplastic acute encephalitis patient .
本発明にかかる非傍腫瘍性急性脳炎患者の予後診断装置の作動方法は、非傍腫瘍性急性脳炎患者の血清又は髄液におけるGluN2B-NT2抗体のIgGサブクラス濃度を定量する手段と、定量されたIgGサブクラス濃度に基づいて前記非傍腫瘍性急性脳炎患者の予後を示す手段と、を有する、非傍腫瘍性急性脳炎患者の予後診断装置の作動方法において、前記IgGサブクラス濃度を定量する手段が、下記の工程a)〜i）を有し、
ａ）ELISAのプレートの検体用ウェルを、目的とする抗体の抗原を含有する溶液によりコーティングする工程；
ｂ）前記プレートの標準用ウェルを、希釈系列化されたIgG１〜IgG4によりコーティングする工程；
ｃ）プレートを、緩衝剤溶液及び非特異結合部位をブロックし得る溶液により洗浄する工程；
ｄ）プレートを、更に、洗浄緩衝剤により洗浄する工程；
ｅ）生物学的試料を前記検体用ウェルにおいて配置し、前記プレートをインキュベートする工程；
ｆ）プレートを洗浄緩衝剤により繰り返し洗浄する工程；
ｇ）標識二次抗体を含有する溶液を各ウェルに添加し、インキュベートする工程；
ｈ）プレートを洗浄緩衝剤により洗浄した後、発色基質を各ウェルに添加する工程；
ｉ）プレートに停止溶液を添加し、吸光度測定を行う工程；
前記予後を示す手段が、定量されたIgG1濃度が5ng/ml以上且つIgG3濃度が2ng/ml以上の場合に予後不良であると判定する工程を有する。
The method of operating the prognosis apparatus for patients with non-paraneoplastic acute encephalitis according to the present invention was quantified with means for quantifying the IgG subclass concentration of GluN2B-NT2 antibody in the serum or cerebrospinal fluid of patients with non-paraneoplastic acute encephalitis Means for determining the prognosis of the non-paraneoplastic acute encephalitis patient based on the IgG subclass concentration, and in the method of operating the prognosis device for a non-paraneoplastic acute encephalitis patient, the means for quantifying the IgG subclass concentration comprises: Comprising the following steps a) to i):
a) coating the sample well of the ELISA plate with a solution containing the antigen of the antibody of interest;
b) coating the standard wells of the plate with diluted serialized IgG1-IgG4;
c) washing the plate with a buffer solution and a solution capable of blocking non-specific binding sites;
d) further washing the plate with a wash buffer;
e) placing a biological sample in the specimen well and incubating the plate;
f) repeatedly washing the plate with wash buffer;
g) adding a solution containing the labeled secondary antibody to each well and incubating;
h) After the plate is washed with wash buffer, a chromogenic substrate is added to each well;
i) adding a stop solution to the plate and measuring absorbance;
The means for indicating a prognosis includes a step of determining that the prognosis is poor when the quantified IgG1 concentration is 5 ng / ml or more and the IgG3 concentration is 2 ng / ml or more.
本発明によれば、非傍腫瘍性急性脳炎患者の予後診断装置の作動方法が得られる。 ADVANTAGE OF THE INVENTION According to this invention, the operating method of the prognosis apparatus of a non-paraneoplastic acute encephalitis patient is obtained .
以下、添付の図面を参照して本発明の実施形態について具体的に説明するが、当該実施形態は本発明の原理の理解を容易にするためのものであり、本発明の範囲は、下記の実施形態に限られるものではなく、当業者が以下の実施形態の構成を適宜置換した他の実施形態も、本発明の範囲に含まれる。 Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. However, the embodiments are for facilitating understanding of the principle of the present invention, and the scope of the present invention is as follows. The present invention is not limited to the embodiments, and other embodiments in which those skilled in the art appropriately replace the configurations of the following embodiments are also included in the scope of the present invention.
本実施形態にかかる抗NMDAR抗体のIgGサブクラス濃度の定量方法は、下記工程を有することを特徴とする。
ａ）ELISAのプレートの検体用ウェルを、目的とする抗体の抗原を含有する溶液によりコーティングする工程；
ｂ）前記プレートの標準用ウェルを、希釈系列化されたIgG１〜4によりコーティングする工程；
ｃ）プレートを、緩衝剤溶液及び非特異結合部位をブロックし得る溶液により洗浄する工程；
ｄ）プレートを、更に、洗浄緩衝剤により洗浄する工程；
ｅ）生物学的試料を前記検体用ウェルにおいて配置し、前記プレートをインキュベートする工程；
ｆ）プレートを洗浄緩衝剤により繰り返し洗浄する工程；
ｇ）標識二次抗体を含有する溶液を各ウェルに添加し、インキュベートする工程；
ｈ）プレートを洗浄緩衝剤により洗浄した後、発色基質を各ウェルに添加する工程；
ｉ）プレートに停止溶液を添加し、吸光度測定を行う工程；
ELISAのプレートは、特に制限されるものではなく、市販の種々のELISAプレートを適用することができる。目的とする抗体の抗原を含有する溶液によるコーティングでは、プレートの一部のウェル（検体用ウェル）に抗原を吸着（コーティング）させる。目的とする抗体はNR2B-NT2抗体である。このコーティングは、通常、抗原を含む溶液をウェルに添加することにより行なう。市販のELISAプレートは通常96個のウェルを有するので、このうちの検体用ウェルに抗原を含む溶液を添加し、残りのウェルは溶液を添加しないでおく。
The method for quantifying the IgG subclass concentration of the anti-NMDAR antibody according to this embodiment has the following steps.
a) coating the sample well of the ELISA plate with a solution containing the antigen of the antibody of interest;
b) coating the standard wells of the plate with dilution serialized IgG1-4;
c) washing the plate with a buffer solution and a solution capable of blocking non-specific binding sites;
d) further washing the plate with a wash buffer;
e) placing a biological sample in the specimen well and incubating the plate;
f) repeatedly washing the plate with wash buffer;
g) adding a solution containing the labeled secondary antibody to each well and incubating;
h) After the plate is washed with wash buffer, a chromogenic substrate is added to each well;
i) adding a stop solution to the plate and measuring absorbance;
The ELISA plate is not particularly limited, and various commercially available ELISA plates can be applied. In coating with a solution containing the antigen of the target antibody, the antigen is adsorbed (coated) on a part of the plate (sample well). The antibody of interest is the NR2B-NT2 antibody. This coating is usually performed by adding a solution containing the antigen to the well. Since a commercially available ELISA plate usually has 96 wells, a solution containing the antigen is added to the sample well, and the remaining well is not added with the solution.
次に、プレートの標準用ウェルを、希釈系列化されたIgG１〜IgG4によりコーティングする。 Next, the standard wells of the plate are coated with IgG1 to IgG4 serialized in dilution.
次に、抗原がコーティングされたプレートを洗浄し、他の物質がプレートに吸着しないようにブロッキングを行なう。ブロッキングは、検体用ウェルと標準用ウェルの両方にブロッキング剤を含む溶液を添加することにより行なう。ブロッキング剤は、上記抗原と特異的な相互作用をしないものを用い、例えばPBSTに0.5%BSAを加えたものを用いる。 Next, the plate coated with the antigen is washed, and blocking is performed so that other substances are not adsorbed on the plate. Blocking is performed by adding a solution containing a blocking agent to both the sample well and the standard well. As the blocking agent, a blocking agent that does not specifically interact with the antigen is used, for example, PBST added with 0.5% BSA.
次に、プレートを、更に、洗浄緩衝剤により洗浄し、NR2B-NT2抗体の抗原と特異的に相互作用する免疫グロブリンを含む生物学的試料の溶液を検体用ウェルに添加し、抗原に結合させ、プレートを例えば37℃にて２時間インキュベートする。生物学的試料は特に限定されるものではないが、例えば血清又は髄液等である。 The plate is then further washed with a wash buffer and a solution of a biological sample containing immunoglobulin that specifically interacts with the antigen of the NR2B-NT2 antibody is added to the sample well to allow it to bind to the antigen. Incubate the plate, for example, at 37 ° C. for 2 hours. The biological sample is not particularly limited and is, for example, serum or cerebrospinal fluid.
次に、プレートを例えばPBSTで洗浄し、標識二次抗体を各ウェルに作用させ、プレートを例えば２時間の周囲温度でインキュベートする。 The plate is then washed, for example with PBST, a labeled secondary antibody is allowed to act on each well, and the plate is incubated, for example at ambient temperature for 2 hours.
次に、プレートを洗浄緩衝剤により洗浄した後、例えばTMB,H2O2等の発色基質を各ウェルに添加する。 Next, after the plate is washed with a washing buffer, a chromogenic substrate such as TMB or H 2 O 2 is added to each well.
次に、プレートに1M溶液のH2SO4等の停止溶液を添加し、吸光度測定を行う。吸光度測定は、例えば405nmで反応停止から30分以内に読み取る。 Next, a stop solution such as 1 M solution of H 2 SO 4 is added to the plate, and the absorbance is measured. Absorbance measurement is read, for example, at 405 nm within 30 minutes after stopping the reaction.
cell-based assay法によるNMDAR抗体（Dalmau抗体）又はELISAによるNR2B-NT2抗体陽性の非傍腫瘍性急性脳炎（抗NMDAR脳炎-NP）２１例の免疫治療前の髄液を用いて、NR2B-NT2抗体IgGサブクラスを定量した。疾病対照髄液としては、炎症を伴わない部分てんかん症例の髄液を使用した。後遺症の評価については、ADLはBarthel scoreで、てんかん発作、精神症状、認知機能、記憶機能、運動機能は下記表１に示す評価スコアを用いた。 NR2B-NT2 using cerebrospinal fluid before immunotherapy in 21 cases of NMDAR antibody (Dalmau antibody) by cell-based assay or NR2B-NT2 antibody-positive non-paraneoplastic acute encephalitis (anti-NMDAR encephalitis-NP) by ELISA The antibody IgG subclass was quantified. As the disease control cerebrospinal fluid, the cerebrospinal fluid of a case of partial epilepsy without inflammation was used. For the evaluation of sequelae, ADL was Barthel score, and the evaluation scores shown in Table 1 below were used for epileptic seizures, psychiatric symptoms, cognitive function, memory function, and motor function.
抗NMDAR抗体のうちのNR2B-NT2抗体のIgGサブクラス濃度を定量するELISAは、下記手順により行った。
１．Maxisorb plate(Nalge Nunc International,468667)をPBS300μlで５回洗浄した。
２．Maxisorb plateの検体用ウェルを、NR2B-NT2ペプチド(KERKWERVGKWKDK,100μg/ml)で、コーティングした（200μl well,４℃ 終夜）。
３．Maxisorb plateの標準用ウェルを、希釈系列化されたIgG1〜G4(フナコシ、ABD 5219-3004,5225-3004,5248-3004,5254-3004:1mg/ml)でコーティングした。
４．Maxisorb plateを、PBS 300μlで１回洗浄した。
５．Maxisorb plateを、0.5％ BSA in PBST(250μl)で２時間ブロッキングした。
６．Maxisorb plateを、PBST 300μlで１回洗浄した。
７．NR2B-NT2抗体陽性の非傍腫瘍性急性脳炎（抗NMDAR脳炎-NP）の免疫治療前の髄液(10％ BSA in PBSTで10倍希釈, 100μl)を検体用ウェルにおいて配置し、37℃、２時間インキュベートした。
８．Maxisorb plateを、PBST 200μlで５回洗浄した。
９．HRP conjugated anti-human IgG sabclass antibodies(Human IgG Subclass Screening Kit, フナコシ、CYGNUS,TECHNOROGIES ♯IM50)(1:100)の希釈液(PBSTで100倍希釈)を100μl/well入れて、２時間室温に置いた。
１０．Maxisorb plateを、PBST 200μlで５回洗浄した。
１１．TMB,H202を当量混和し(各6ml/枚)、100μl/well添加した。
１２．15分後に、1M H3PO4 100μl加えて反応を停止させた。
１３．30分以内に、450nmで吸光度測定を行った。
The ELISA for quantifying the IgG subclass concentration of the NR2B-NT2 antibody among the anti-NMDAR antibodies was performed according to the following procedure.
1. Maxisorb plate (Nalge Nunc International, 468667) was washed 5 times with 300 μl of PBS.
2. The sample well of the Maxisorb plate was coated with NR2B-NT2 peptide (KERKWERVGKWKDK, 100 μg / ml) (200 μl well, 4 ° C. overnight).
3. Standard wells of Maxisorb plate were coated with dilution serialized IgG1 to G4 (Funakoshi, ABD 5219-3004, 5225-3004, 5248-3004, 5254-3004: 1 mg / ml).
4). The Maxisorb plate was washed once with 300 μl of PBS.
5). The Maxisorb plate was blocked with 0.5% BSA in PBST (250 μl) for 2 hours.
6). The Maxisorb plate was washed once with 300 μl PBST.
7). Cerebrospinal fluid before immunotherapy of NR2B-NT2 antibody-positive non-paraneoplastic acute encephalitis (anti-NMDAR encephalitis-NP) was placed in the sample well and placed at 37 ° C, 10-fold diluted with 10% BSA in PBST. Incubated for 2 hours.
8). The Maxisorb plate was washed 5 times with 200 μl PBST.
9. Add 100 μl / well of HRP conjugated anti-human IgG sabclass antibodies (Human IgG Subclass Screening Kit, Funakoshi, CYGNUS, TECHNOROGIES # IM50) (1: 100) dilution (100 times diluted with PBST) for 2 hours at room temperature. It was.
10. The Maxisorb plate was washed 5 times with 200 μl PBST.
11. Equivalent amounts of TMB and H 2 0 2 were mixed (6 ml / plate each), and 100 μl / well was added.
12. After 15 minutes, 100 μl of 1M H 3 PO 4 was added to stop the reaction.
13. Absorbance measurement was performed at 450 nm within 30 minutes.
図１は、抗NMDAR脳炎-NPの髄液NR2B-NT2抗体のIgGサブクラスの測定結果を示す図である。図１に示されるように、IgGサブクラスごとの割合では、NR2B-NT2抗体IgG2、IgG4サブクラスが疾病対照髄液に比べて有意に増加し、IgG1サブクラスが有意に減少していた。 FIG. 1 is a graph showing the measurement results of IgG subclass of anti-NMDAR encephalitis-NP cerebrospinal fluid NR2B-NT2 antibody. As shown in FIG. 1, in the ratio for each IgG subclass, the NR2B-NT2 antibodies IgG2 and IgG4 subclass were significantly increased compared to the disease control cerebrospinal fluid, and the IgG1 subclass was significantly decreased.
図２は、抗NMDAR脳炎-NPの髄液NR2B-NT2抗体のIgGサブクラスの経時的変動を示す図である。図２に示されるように、抗NMDAR脳炎-NPの髄液では、脳炎発病後の経過で見ると、０〜５病日では、NR2B-NT2抗体のIgG1サブクラスは3/9、IgG2サブクラスは9/9、IgG3サブクラスは3/9、IgG4サブクラスは7/9例で、平均+2SDを超えていた。発病後１０〜２０病日では、NR2B-NT2抗体のIgG1サブクラスは4/6、IgG2サブクラスは5/6、IgG3サブクラスは4/6、IgG4サブクラスは4/6例で、平均+2SDを超えていた。以上より、発病期にはIgG2サブクラスが全例高値で、IgG4サブクラスも約80%の症例で高値であり、その後に低下する経過が推測された。IgG1、IgG3サブクラスは発病初期よりも１０〜２０病日に高値となる経過が推定された。 FIG. 2 is a graph showing the time course of IgG subclass of anti-NMDAR encephalitis-NP cerebrospinal fluid NR2B-NT2 antibody. As shown in FIG. 2, in the cerebrospinal fluid of anti-NMDAR encephalitis-NP, the NR2B-NT2 antibody IgG1 subclass is 3/9, and the IgG2 subclass is 9 on the 0th to 5th days after the onset of encephalitis. / 9, IgG3 subclass was 3/9, IgG4 subclass was 7/9 cases, exceeding the average + 2SD. On the 10th to 20th day after the onset of disease, the NR2B-NT2 antibody IgG1 subclass is 4/6, IgG2 subclass is 5/6, IgG3 subclass is 4/6, and IgG4 subclass is 4/6 cases, exceeding the average + 2SD It was. Based on the above, it was estimated that the IgG2 subclass was high in all cases and the IgG4 subclass was high in about 80% of cases during the onset stage, and subsequently decreased. It was estimated that IgG1 and IgG3 subclasses had a higher level 10 to 20 days after the onset of disease.
図３は、抗NMDAR脳炎-NPの髄液NR2B-NT2抗体のIgGサブクラスと急性期入院日数を示す図である。図３に示されるように、抗NMDAR脳炎-NPの髄液では、NR2B-NT2抗体IgG1サブクラスの濃度とIgG3サブクラスの濃度が急性期入院日数と有意に相関し、IgG1、IgG3サブクラス抗体の濃度が高いほど長期入院となり、重症化することが明らかになった。 FIG. 3 is a diagram showing IgG subclasses of anti-NMDAR encephalitis-NP cerebrospinal fluid NR2B-NT2 antibody and the number of days of acute hospitalization. As shown in FIG. 3, in the cerebrospinal fluid of anti-NMDAR encephalitis-NP, the concentrations of NR2B-NT2 antibody IgG1 subclass and IgG3 subclass were significantly correlated with the number of days of acute hospitalization, and the concentrations of IgG1 and IgG3 subclass antibodies It became clear that the higher the hospital, the longer the hospital stayed and the more serious it became.
図４は、抗NMDAR脳炎-NPの髄液NR2B-NT2抗体のIgG1サブクラスと予後を示す図である。図４に示されるように、抗NMDAR脳炎-NPの髄液では、NR2B-NT2抗体のIgG1サブクラス濃度はBarthelscoreと有意な相関が見られたが、他の予後とは相関が見られなかった。 FIG. 4 is a diagram showing the IgG1 subclass and prognosis of anti-NMDAR encephalitis-NP cerebrospinal fluid NR2B-NT2 antibody. As shown in FIG. 4, in the cerebrospinal fluid of anti-NMDAR encephalitis-NP, the IgG1 subclass concentration of NR2B-NT2 antibody was significantly correlated with Barthelscore, but was not correlated with other prognoses.
図５は、抗NMDAR脳炎-NPの髄液NR2B-NT2抗体のIgG2サブクラスと予後を示す図である。図５に示されるように、抗NMDAR脳炎-NPの髄液では、NR2B-NT2抗体のIgG2サブクラス濃度は予後と有意な相関が見られなかった。 FIG. 5 is a diagram showing the IgG2 subclass and prognosis of anti-NMDAR encephalitis-NP cerebrospinal fluid NR2B-NT2 antibody. As shown in FIG. 5, in the cerebrospinal fluid of anti-NMDAR encephalitis-NP, the IgG2 subclass concentration of NR2B-NT2 antibody was not significantly correlated with the prognosis.
図６は、抗NMDAR脳炎-NPの髄液NR2B-NT2抗体のIgG3サブクラスと予後を示す図である。図６に示されるように、抗NMDAR脳炎-NPの髄液では、NR2B-NT2抗体のIgG3サブクラス濃度はBarthelscore、てんかん発作(Epilepsy score)、認知機能(Cognitive score)、運動機能(Motor score)と有意な相関が見られた。 FIG. 6 is a diagram showing IgG3 subclass and prognosis of anti-NMDAR encephalitis-NP cerebrospinal fluid NR2B-NT2 antibody. As shown in FIG. 6, in the cerebrospinal fluid of anti-NMDAR encephalitis-NP, the IgG3 subclass concentration of NR2B-NT2 antibody is Barthelscore, epileptic seizure (Epilepsy score), cognitive function (Cognitive score), motor function (Motor score) and There was a significant correlation.
図７は、抗NMDAR脳炎-NPの髄液NR2B-NT2抗体のIgG4サブクラスと予後を示す図である。図７に示されるように、抗NMDAR脳炎-NPの髄液では、NR2B-NT2抗体のIgG4サブクラス濃度は予後と有意な相関が見られなかった。 FIG. 7 shows IgG4 subclass and prognosis of cerebrospinal fluid NR2B-NT2 antibody of anti-NMDAR encephalitis-NP. As shown in FIG. 7, in the cerebrospinal fluid of anti-NMDAR encephalitis-NP, the IgG4 subclass concentration of NR2B-NT2 antibody was not significantly correlated with the prognosis.
図８は、免疫介在性神経疾患の髄液NR2B-NT2抗体のIgGサブクラスの濃度比較を示す図である。なお、図において、NMDAR-E-NPは抗NMDAR脳炎-NPを示し、NMDAR-E-OTは抗NMDAR脳炎-OTを示し、CJDはCreutzfelt-Jakob病を示し、PE-Eは急性脳炎後部分てんかんを示し、Control PEは疾病対照部分てんかんを示す。図８に示されるように、NMDAR抗体の一つであるNR2B-NT2抗体のIgG1サブクラス濃度は、抗NMDAR脳炎-NP、抗NMDAR脳炎-OT、Creutzfelt-Jakob病、急性脳炎後部分てんかん等の種々の疾患で対照に比べて高値となった。NR2B-NT2抗体のIgG2サブクラスは精神症状を主症状とする抗NMDAR脳炎-NP、Creutzfelt-Jakob病等で対照に比べて高値となった。 FIG. 8 shows a comparison of IgG subclass concentrations of cerebrospinal fluid NR2B-NT2 antibody in immune-mediated neurological diseases. In the figure, NMDAR-E-NP indicates anti-NMDAR encephalitis-NP, NMDAR-E-OT indicates anti-NMDAR encephalitis-OT, CJD indicates Creutzfelt-Jakob disease, and PE-E indicates the part after acute encephalitis Indicates epilepsy, Control PE indicates disease control partial epilepsy. As shown in FIG. 8, the IgG1 subclass concentration of NR2B-NT2 antibody, which is one of NMDAR antibodies, is various, such as anti-NMDAR encephalitis-NP, anti-NMDAR encephalitis-OT, Creutzfelt-Jakob disease, and partial epilepsy after acute encephalitis. It was higher than the control in the disease. The IgG2 subclass of NR2B-NT2 antibody was higher than the control in anti-NMDAR encephalitis-NP, Creutzfelt-Jakob disease, etc. whose main symptoms are psychiatric symptoms.
以上、上述の実施例に示したように、NMDAR抗体の一つであるNR2B-NT2抗体のIgG1〜4サブクラスをELISAで定量する方法を確立した。 As described above, as shown in the above-mentioned Examples, a method for quantifying the IgG1 to 4 subclass of NR2B-NT2 antibody, which is one of NMDAR antibodies, by ELISA has been established.
図２に示したように、抗NMDAR脳炎-NPでは、発病初期にNR2B-NT2抗体のIgG1〜4サブクラスのうち、補体活性化に関与しないIgG2サブクラスが著しく増加し、またIgG4サブクラスも増加していた。そのためこれらのIgGサブクラスが発病初期の辺縁系症状に関与していると推測した。IgG2サブクラス抗体はNMDARの内在化を起こし、NMDAR拮抗作用で精神症状をもたらすと推測した。 As shown in FIG. 2, in anti-NMDAR encephalitis-NP, among IgG1 to 4 subclasses of NR2B-NT2 antibody, IgG2 subclass not involved in complement activation is markedly increased in the early stage of disease, and IgG4 subclass is also increased. It was. Therefore, it was speculated that these IgG subclasses are involved in limbic symptoms in the early stages of onset. It was speculated that IgG2 subclass antibodies caused internalization of NMDAR and caused psychiatric symptoms by NMDAR antagonism.
図２に示したように、抗NMDAR脳炎-NPでは、発病後１０〜２０日でNR2B-NT2抗体のIgG1〜4サブクラスのうち、補体活性化に関与するIgG1、IgG3サブクラスが増加する症例があった。 As shown in FIG. 2, in anti-NMDAR encephalitis-NP, among IgG1 to 4 subclasses of NR2B-NT2 antibody, IgG1 and IgG3 subclasses involved in complement activation increase 10 to 20 days after onset of disease. there were.
図３に示したように、抗NMDAR脳炎-NPでは、NR2B-NT2抗体のIgG1〜4サブクラスのうち、補体活性化に関与するIgG1、IgG3サブクラスと急性期入院日数（重症度）とが相関し、補体介在性の神経障害が重症化と関連し、予後が不良となる可能性が示唆された。 As shown in FIG. 3, among anti-NMDAR encephalitis-NP, among IgG1 to 4 subclasses of NR2B-NT2 antibody, IgG1 and IgG3 subclasses involved in complement activation correlate with acute hospitalization days (severity) This suggests that complement-mediated neuropathy is associated with increased severity and a poor prognosis.
図６に示したように、抗NMDAR脳炎-NPでは、NR2B-NT2抗体のIgG1サブクラス濃度はBarthelscoreと相関し、NR2B-NT2抗体のIgG3サブクラス濃度は、Barthel score、てんかん発作、認知機能、運動機能と有意な相関が見られ、補体介在性の神経障害がてんかん発作、認知機能、運動機能等の予後を規定している可能性が示唆された。 As shown in FIG. 6, in anti-NMDAR encephalitis-NP, the IgG1 subclass concentration of NR2B-NT2 antibody is correlated with Barthelscore, and the IgG3 subclass concentration of NR2B-NT2 antibody is Barthel score, epileptic seizure, cognitive function, motor function A significant correlation was found, suggesting that complement-mediated neuropathy may define the prognosis of epileptic seizures, cognitive function, motor function, etc.
抗NMDAR抗体のIgGサブクラス濃度の定量に利用できる。 It can be used for quantification of IgG subclass concentration of anti-NMDAR antibody.
Claims (2)
定量されたIgGサブクラス濃度に基づいて前記非傍腫瘍性急性脳炎患者の予後を示す手段と、を有する、非傍腫瘍性急性脳炎患者の予後診断装置の作動方法において、
前記IgGサブクラス濃度を定量する手段が、下記の工程a)〜i）を有し、
ａ）ELISAのプレートの検体用ウェルを、目的とする抗体の抗原を含有する溶液によりコーティングする工程；
ｂ）前記プレートの標準用ウェルを、希釈系列化されたIgG１〜IgG4によりコーティングする工程；
ｃ）プレートを、緩衝剤溶液及び非特異結合部位をブロックし得る溶液により洗浄する工程；
ｄ）プレートを、更に、洗浄緩衝剤により洗浄する工程；
ｅ）生物学的試料を前記検体用ウェルにおいて配置し、前記プレートをインキュベートする工程；
ｆ）プレートを洗浄緩衝剤により繰り返し洗浄する工程；
ｇ）標識二次抗体を含有する溶液を各ウェルに添加し、インキュベートする工程；
ｈ）プレートを洗浄緩衝剤により洗浄した後、発色基質を各ウェルに添加する工程；
ｉ）プレートに停止溶液を添加し、吸光度測定を行う工程；
前記予後を示す手段が、定量されたIgG1濃度が5ng/ml以上且つIgG3濃度が2ng/ml以上の場合に予後不良であると判定する工程を有する、
ことを特徴とする非傍腫瘍性急性脳炎患者の予後診断装置の作動方法。 Means for quantifying the IgG subclass concentration of GluN2B-NT2 antibody in serum or cerebrospinal fluid of patients with non-paraneoplastic acute encephalitis;
Means for indicating the prognosis of the non-paraneoplastic acute encephalitis patient based on the quantified IgG subclass concentration, and a method for operating the prognosis device for the non-paraneoplastic acute encephalitis patient,
The means for quantifying the IgG subclass concentration comprises the following steps a) to i):
a) coating the sample well of the ELISA plate with a solution containing the antigen of the antibody of interest;
b) coating the standard wells of the plate with diluted serialized IgG1-IgG4;
c) washing the plate with a buffer solution and a solution capable of blocking non-specific binding sites;
d) further washing the plate with a wash buffer;
e) placing a biological sample in the specimen well and incubating the plate;
f) repeatedly washing the plate with wash buffer;
g) adding a solution containing the labeled secondary antibody to each well and incubating;
h) After the plate is washed with wash buffer, a chromogenic substrate is added to each well;
i) adding a stop solution to the plate and measuring absorbance;
The means for indicating the prognosis comprises a step of determining that the prognosis is poor when the quantified IgG1 concentration is 5 ng / ml or more and the IgG3 concentration is 2 ng / ml or more,
A method for operating a prognosis apparatus for a patient with non-paraneoplastic acute encephalitis, characterized by the above.
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