Amplified luminescence proximity homogeneous assay法を用いた自己免疫性蕁麻疹の新規検査法の開発

概要

༤ኈㄽᩥ

Amplified luminescence proximity homogeneous assay ἲࢆ⏝࠸ࡓ
⮬ᕫච␿ᛶⶼ㯞⑈ࡢ᪂つ᳨ᰝἲࡢ㛤Ⓨ

ᗈᓥ኱Ꮫ኱Ꮫ㝔་ṑ⸆ಖ೺Ꮫ◊✲⛉ ་ṑ⸆Ꮫᑓᨷ
        ⑓㝔⸆๣Ꮫ◊✲ᐊ
ᖹᡂ 30 ᖺᗘධᏛ  ྂ㈡ ♸ᇶ
୺ᣦᑟᩍဨ  ᯇᑿ ⿱ᙲ

┠ḟ
ᗎㄽ............................................................................................................................................................. 1
ᮏㄽ............................................................................................................................................................. 6
➨ 1 ❶ ᢠ FcεRIα ⮬ᕫᢠయ࡟ᑐࡍࡿ AlphaCL ἲࡢᵓ⠏࠾ࡼࡧࡑࡢ᭷⏝ᛶࡢホ౯ .............................. 6
➨ 1 ⠇ ELISA ἲ࠾ࡼࡧ HRT ࡟ࡼࡿᢠ FcεRIα ⮬ᕫᢠయࡢ᳨ฟ....................................................... 7
1-1. ⥴ゝ ............................................................................................................................................. 7
1-2. ⤖ᯝ ............................................................................................................................................. 7
1-3. ⪃ᐹ ........................................................................................................................................... 11
➨ 2 ⠇ AlphaCL ἲ࡟ࡼࡿᢠ FcεRIα ⮬ᕫᢠయࡢ᳨ฟ᮲௳ࡢ᭱㐺໬ ................................................ 12
2-1. ⥴ゝ ........................................................................................................................................... 12
2-2. ⤖ᯝ ........................................................................................................................................... 12
2-3. ⪃ᐹ ........................................................................................................................................... 15
➨ 3 ⠇ AlphaCL ἲ࡟ࡼࡿ⾑Ύ୰ᢠ FcεRIα ⮬ᕫᢠయࡢ᳨ฟ ........................................................... 17
3-1. ⥴ゝ ........................................................................................................................................... 17
3-2. ⤖ᯝ ........................................................................................................................................... 17
3-3. ⪃ᐹ ........................................................................................................................................... 18
➨ 4 ⠇ ᑠᣓ ........................................................................................................................................ 20
➨ 2 ❶ ᢠ IgE ⮬ᕫᢠయ࡟ᑐࡍࡿ AlphaCL ἲࡢᵓ⠏࠾ࡼࡧࡑࡢ᭷⏝ᛶࡢホ౯ .................................. 22
➨ 1 ⠇ ELISA ἲ࠾ࡼࡧ HRT ࡟ࡼࡿᢠ IgE ⮬ᕫᢠయࡢ᳨ฟ .......................................................... 23
1-1. ⥴ゝ ........................................................................................................................................... 23
1-2. ⤖ᯝ ........................................................................................................................................... 23
1-3. ⪃ᐹ ........................................................................................................................................... 24
➨ 2 ⠇ AlphaCL ἲ࡟ࡼࡿᢠ IgE ⮬ᕫᢠయࡢ᳨ฟ᮲௳ࡢ᭱㐺໬ ...................................................... 25
2-1. ⥴ゝ ........................................................................................................................................... 25
2-2. ⤖ᯝ ........................................................................................................................................... 25
2-3. ⪃ᐹ ........................................................................................................................................... 28
➨ 3 ⠇ AlphaCL ἲ࡟ࡼࡿ⾑Ύ୰ᢠ IgE ⮬ᕫᢠయࡢ᳨ฟ ................................................................. 30
3-1. ⥴ゝ ........................................................................................................................................... 30
3-2. ⤖ᯝ ........................................................................................................................................... 30
3-3. ⪃ᐹ ........................................................................................................................................... 31
➨ 4 ⠇ ᑠᣓ ........................................................................................................................................ 33
⤖ㄽ........................................................................................................................................................... 35
ᐇ㦂ࡢ㒊 ................................................................................................................................................... 36
ㄽᩥ┠㘓 ................................................................................................................................................... 47
ཧ⪃ᩥ⊩ ................................................................................................................................................... 48
ㅰ㎡........................................................................................................................................................... 54

␎ㄒ୍ぴ

AAbs

autoantibodies

Abs

antibodies

aiCSU

autoimmune chronic spontaneous urticaria

Alpha

amplified luminescence proximity homogeneous assay

AlphaCL

Alpha involving cross-linking

ASST

autologous serum skin test

AU

absorbance unit

BAT

basophil activation test

CSU

chronic spontaneous urticaria

DNA

deoxyribonucleic acid

EDTA

ethylenediamine N, N, N’, N’ –tetraacetic acid

ELISA

enzyme linked immunosorbent assay

EPO

eosinophil peroxidase

EXiLE

IgE crosslinking-induced luciferase expression

FBS

fetal bovine serum

FcεRIα

high-affinity IgE receptor I α chain

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HMC

human mast cell

HPLC

high-performance liquid chromatography

HRP

horseradish peroxidase

HRT

histamine releasing test

HSA

human serum albumin

Ig

immunoglobulin

IU

international unit

mAb

monoclonal antibody

MBP

major basic protein

N.D.

not detected

nhIgE

native human IgE

N.P.

not performed

NF-AT

nuclear factor of activated T-cells

no.

number

nos.

numbers

O.D.

optical density

PBS

phosphate buffered saline

PBS-T

phosphate buffered saline Tween-20

pH

potential Hydrogen, power of Hydrogen

SD

standard deviation

Tween-20

polyoxyethylene (20) sorbitan monolaurate

UAS

urticaria activity score

v/v

volume per volume

w/v

weight per volume

ᗎㄽ

ⶼ㯞⑈ࡣࠊ⭾⑈ࡸᾋ⭘ࡀฟ⌧ࡍࡿ⓶⭵ࡢ⑌ᝈ࡛࠶ࡾࠊከࡃࡢሙྜࡣ⑛ࡳࢆక࠺ࠋⶼ㯞⑈࡟ྜే
ࡶࡋࡃࡣ༢⊂࡛㉳ࡇࡿࠊ⓶⭵ࢆ୰ᚰ࡜ࡋࡓ㝈ᒁᛶࡢᾋ⭘ࡣ⾑⟶ᛶᾋ⭘࡜࿧ࡤࢀࡿࠋⶼ㯞⑈ࡢⓎ⑕࡟
ࡣ⓶⭵࣐ࢫࢺ⣽⬊ࡢ⬺㢛⢏ࡀ㛵୚ࡍࡿ࡜⪃࠼ࡽࢀ࡚࠾ࡾࠊ⤌⧊㛫㝽࡟ᨺฟࡉࢀࡓࣄࢫࢱ࣑ࣥ࡞࡝ࡢ
໬Ꮫఏ㐩≀㉁ࡀ⓶⭵ᚤᑠ⾑⟶ࡸ⚄⤒࡟స⏝ࡋࠊ⭾⑈ࡸ⑛ࡳ࡞࡝ࡀច㉳ࡉࢀࡿࠋ
ⶼ㯞⑈ࡣࠊⓎ⑕ࡢᶵᗎࡸ⭾⑈ࡢㄏⓎᅉᏊࠊ⮫ᗋୖࡢ≉ᚩࡀከᒱ࡟ࢃࡓࡿࡇ࡜࠿ࡽࠊ」ᩘࡢ⑓ᆺ
࡟ศ㢮ࡉࢀࡿ (Table 1) [1,2]ࠋ≉࡟ immunoglobulin E (IgE) ᢠయࡢ㛵୚࡛㉳ࡇࡿ I ᆺ࢔ࣞࣝࢠ࣮ࡣࠊ
ⶼ㯞⑈ࡢ୺࡞Ⓨ⑕ᶵᗎ࡜ࡋ࡚▱ࡽࢀ࡚࠸ࡿࠋI ᆺ࢔ࣞࣝࢠ࣮࡛ࡣࠊ࣐ࢫࢺ⣽⬊ࡸዲሷᇶ⌫ࡢ⣽⬊⭷
ୖ࡟Ⓨ⌧ࡍࡿ㧗ぶ࿴ᛶ IgE ཷᐜయ α ࢧࣈࣘࢽࢵࢺ (FcεRIα) ࡟ IgE ᢠయࡀ⤖ྜࡋࠊࡑࡇ࡟ᢠཎࡀ 2
ࡘࡢ IgE ᢠయࢆᯫᶫࡍࡿࡼ࠺࡟⤖ྜࡍࡿࡇ࡜࡛⣽⬊ࡀάᛶ໬ࡉࢀࠊ⑕≧ࡀฟ⌧ࡍࡿ࡜⪃࠼ࡽࢀ࡚࠸
ࡿࠋ୍᪉ࠊI ᆺ࢔ࣞࣝࢠ࣮௨እ࡟࢔ࢫࣆࣜࣥ࡞࡝ࡢ⸆๣ࡢ౑⏝ࡸ㐠ື㈇Ⲵࠊᐮᬮᕪ࡞࡝ࢆཎᅉ࡜ࡍ
ࡿ่⃭ㄏⓎᆺࡢⶼ㯞⑈ࡸࠊ᫂ࡽ࠿࡞ཎᅉࡀศ࠿ࡽࡎ⑛ࡳࡸ⭾⑈ࡀ⧞ࡾ㏉ࡋฟ⌧ࡍࡿ≉Ⓨᛶࡢⶼ㯞⑈
࡞࡝ࡀ࠶ࡿ (Table 2) [1,2]ࠋ
Table 1. Subtypes of urticaria. [1,2]

1

Table 2. Factors associated with the pathogenesis of urticaria. [1,2]

≉Ⓨᛶⶼ㯞⑈ࡣࠊ඲ⶼ㯞⑈ᝈ⪅ࡢ⣙ 7 ๭ࢆ༨ࡵࠊࡑࡢ⑕≧ࡀ 6 㐌㛫௨ୖ⥆ࡃሙྜࡣ៏ᛶ≉Ⓨᛶ
ⶼ㯞⑈ (chronic spontaneous urticaria) ࡟ศ㢮ࡉࢀࡿ (Fig. 1) [2–4]ࠋ៏ᛶ≉Ⓨᛶⶼ㯞⑈ࡢⓎ⑕ᶵᗎࡣ
ᮍࡔ᏶඲࡟ࡣゎ᫂ࡉࢀ࡚࠸࡞࠸ࡀࠊⓎ⑕ࡢ୍ᅉ࡜ࡋ࡚⮬ᕫᢠయࡀ㛵୚ࡍࡿࡇ࡜ࡀ᫂࠿࡟ࡉࢀ࡚࠸ࡿ
[5–7]ࠋ

Fig. 1. Ratio of urticaria subtypes. [3]
2

⮬ᕫᢠయࢆ௓ࡋࡓ཯ᛂ࡟ࡼࡗ࡚ច㉳ࡉࢀࡿ៏ᛶ≉Ⓨᛶⶼ㯞⑈ࡣࠊ⮬ᕫච␿ᛶⶼ㯞⑈
(autoimmune chronic spontaneous urticaria: aiCSU) ࡜ᐃ⩏ࡉࢀࠊ⮬ᕫᢠయࡢࢡࣛࢫࡢ㐪࠸࡟ࡼࡾ I ᆺ࠾
ࡼࡧ IIb ᆺࡢ 2 ࡘࡢ࢚ࣥࢻࢱ࢖ࣉ࡟ศ㢮ࡉࢀࡿ [8]ࠋI ᆺᝈ⪅ࡢሙྜࠊ⮬ᕫᢠཎ࡟ᑐࡍࡿ IgE ⮬ᕫᢠ
యࡀཎᅉ࡜⪃࠼ࡽࢀ࡚࠾ࡾࠊ⮬ᕫᢠཎ࡜ࡋ࡚⏥≧⭢࣌ࣝ࢜࢟ࢩࢲ࣮ࢮ [9–12]ࠊ஧ᮏ㙐 DNA [12,13]ࠊ
࢖ࣥࢱ࣮ࣟ࢖࢟ࣥ-24 [11,14,15]ࠊ⤌⧊ᅉᏊ [16] ࠾ࡼࡧࢧ࢖ࣟࢢࣟࣈࣜࣥ [11] ࡞࡝ࡀᣲࡆࡽࢀࡿࠋ
୍᪉ࠊIIb ᆺᝈ⪅ࡢሙྜࡣࠊ࣐ࢫࢺ⣽⬊ࡸዲሷᇶ⌫ୖࡢ FcεRIα ࡸࡑࢀ࡟⤖ྜࡋࡓ IgE ࡟ᑐࡍࡿ IgG
⮬ᕫᢠయࡀཎᅉ࡜⪃࠼ࡽࢀࡿࠋ៏ᛶⶼ㯞⑈ᝈ⪅ࡢ࠺ࡕࠊ0–69%ࡢᝈ⪅ࡀ IgE ᢠయ࡟ᑐࡍࡿ IgG ⮬ᕫ
ᢠయࢆ᭷ࡋࠊ4–64%ࡢᝈ⪅ࡀ FcεRIα ࡟ᑐࡍࡿ IgG ⮬ᕫᢠయࢆ᭷ࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ [8]ࠋᢠ
IgE ⮬ᕫ IgG ᢠయࡣࠊ࣐ࢫࢺ⣽⬊ࡸዲሷᇶ⌫ୖࡢ FcεRIα ࡟⤖ྜࡋࡓ IgE ᢠయ࡜⤖ྜࡍࡿࡇ࡜࡛ 2 ศ
Ꮚ௨ୖࡢ FcεRIα ࢆᯫᶫࡋࠊ⬺㢛⢏ࢆ㉳ࡇࡍ (Fig. 2) [7,17–20]ࠋࡲࡓࠊᢠ FcεRIα ⮬ᕫ IgG ᢠయࡣࠊ
FcεRIα ࢆ┤᥋ᯫᶫࡍࡿࡇ࡜࡛ࠊ⬺㢛⢏ࢆᘬࡁ㉳ࡇࡍ (Fig. 2) [7,17–20]ࠋ

Fig. 2. Pathogenesis of type IIb aiCSU. ...

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