JP2561170B2 - Method for measuring antigen-specific interleukin-2 reactivity of lymphocytes - Google Patents
Method for measuring antigen-specific interleukin-2 reactivity of lymphocytesInfo
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Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明はアレルギー性疾患における原因抗原の検索や
病勢の指標として有用なリンパ球の抗原特異的インター
ロイキン−２反応性を簡便かつ迅速に測定する方法に関
する。DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention conveniently and rapidly measures antigen-specific interleukin-2 reactivity of lymphocytes, which is useful as a search for causative antigens in allergic diseases and as an index of disease state. On how to do.
IgEを主体とする即時型（Ｉ型）アレルギー反応系あ
るいはＴ細胞から分泌されるサイトカインによって惹起
される遅延型（IV型）過敏反応系は、抗原刺激によって
活性化されたＴ細胞を介して行なわれる。すなわち、抗
原提示細胞により活性化されたＴ細胞は、インターロイ
キン−２（以下IL−２という）を吸収することによって
増殖し、IgE産生細胞の分化増殖を促進するIL4、IL5な
どのリンホカインを分泌する。また活性化Ｔ細胞は、Ig
Eを結合することによって脱顆粒をひきおこし即時型ア
レルギー反応の原因となる肥満細胞の増殖を促進するIL
3、IL4のリンホカインを分泌する。さらに、同様にして
活性化されたＴ細胞は遅延型過敏反応におけるヘルパー
Ｔ細胞として働き、遅延型過敏反応を誘導する。このよ
うにアレルゲンによりIL−２を介して活性化されたＴ細
胞は少なくともＩ型あるいはIV型アレルギー反応を促進
すると考えられている。The immediate type (I type) allergic reaction system mainly composed of IgE or the delayed type (IV type) hypersensitivity reaction system evoked by cytokines secreted from T cells is carried out through T cells activated by antigen stimulation. Be done. That is, T cells activated by antigen-presenting cells proliferate by absorbing interleukin-2 (hereinafter referred to as IL-2) and secrete lymphokines such as IL4 and IL5 that promote differentiation and proliferation of IgE-producing cells. To do. In addition, activated T cells are
IL promotes mast cell proliferation that causes degranulation by binding E and causes immediate allergic reaction
3, secretes IL4 lymphokine. Furthermore, similarly activated T cells act as helper T cells in the delayed hypersensitivity reaction and induce the delayed hypersensitivity reaction. Thus, T cells activated by IL-2 via allergen are considered to promote at least type I or type IV allergic reaction.
一方、臨床的に観ても、アレルギー患者のリンパ球の
IL−２反応性は抗原に特異的であることが知られてい
る。すなわち、例えば卵白アルブミンを原因抗原とする
患者のリンパ球は、卵白アルブミンで刺激した場合のみ
IL−２反応性が誘導され、他の抗原で刺激した場合には
IL−２反応性は誘導されない。On the other hand, clinically, the lymphocytes of allergic patients
IL-2 reactivity is known to be antigen specific. That is, for example, lymphocytes from patients whose ovalbumin is the causative antigen can only be stimulated with ovalbumin.
When IL-2 reactivity is induced and stimulated with other antigens,
IL-2 reactivity is not induced.
またリンパ球のIL−２反応性はアレルギー性患者の病
勢とも深い関わりを有している。すなわち、アレルギー
疾患患者が自然治癒していくに従ってその患者のリンパ
球のIL−２反応性が減衰していくことから、リンパ球の
IL−２反応性を測定することはアレルギー患者の病勢を
把握し、治療指針を企図するうえでも極めて重要である
〔臨床免疫,19（Suppl.12）,170（1987），アレルギー,
36,1075（1987）等〕。In addition, IL-2 reactivity of lymphocytes is closely related to the disease state of allergic patients. That is, as the patient with an allergic disease spontaneously heals, the IL-2 reactivity of the patient's lymphocytes attenuates.
Measurement of IL-2 reactivity is extremely important in understanding the disease state of allergic patients and in planning treatment guidelines [Clinical Immunity, 19 (Suppl.12), 170 (1987), Allergy,
36, 1075 (1987)].
このようにアレルギー性疾患における原因抗原の検索
や病勢の指標として有用なリンパ球のIL−２反応性の測
定法としては、被検リンパ球に抗原を反応させ、次いで
該反応液をIL−２を加えて培養し、これにトリパンブル
ーを加えて生細胞数を血算板を用いて算定する方法があ
った（トリパンブルー法）〔アレルギー,36,1075（198
7）等〕。As described above, a method for measuring IL-2 reactivity of lymphocytes, which is useful as a search for causative antigens in allergic diseases or as an index of disease state, is to react test lymphocytes with an antigen, and then to react the reaction solution with IL-2. There was a method in which trypan blue was added to this and cultured, and the viable cell count was calculated using a hemocytometer (trypan blue method) [Allergy, 36, 1075 (198
7) etc.].
しかしながら、従来のトリパンブルー法は顕微鏡下で
染色された細胞と非染色細胞を分別して生細胞数を算定
する方法であることから、その測定には長時間を要し、
操作が煩雑であり、かつ大量の検体を処理することがで
きないという欠点があった。However, since the conventional trypan blue method is a method of calculating the number of viable cells by separating stained cells and unstained cells under a microscope, the measurement requires a long time,
There are drawbacks that the operation is complicated and a large amount of specimens cannot be processed.
従って、簡便かつ迅速なリンパ球の抗原特異的IL−２
反応性の測定法の開発が望まれていた。Therefore, simple and rapid lymphocyte antigen-specific IL-2
It was desired to develop a method for measuring reactivity.
かかる実情において、本発明者は種々研究を重ねた結
果、リンパ球、抗原及びIL−２を一定時間反応させた後
の生細胞数を、蛍光色素であるヨウ化プロピジウムを利
用して蛍光色素量として定量すれば、リンパ球の抗原特
異的IL−２反応性が簡便、迅速かつ正確に測定できるこ
とを見出し、本発明を完成した。Under such circumstances, the present inventor has conducted various studies, and as a result, the number of viable cells after reacting lymphocytes, antigens and IL-2 for a certain period of time was measured using a fluorescent dye, propidium iodide, as the amount of the fluorescent dye. It was found that the antigen-specific IL-2 reactivity of lymphocytes can be simply, quickly and accurately measured by quantifying as, and completed the present invention.
すなわち、本発明は被検リンパ球に抗原を反応させ、
次いで該反応液にIL−２を加えて３〜４日間培養し、さ
らにヨウ化プロピジウム含有液を加えた後該培養液の蛍
光色素量を測定することを特徴とする、リンパ球の抗原
特異的IL−２反応性の測定法を提供するものである。That is, the present invention reacts a test lymphocyte with an antigen,
Next, IL-2 is added to the reaction solution, the mixture is cultured for 3 to 4 days, and further, a solution containing propidium iodide is added, and then the amount of the fluorescent dye in the culture solution is measured. It provides a method for measuring IL-2 reactivity.
本発明を実施するには、まず被検リンパ球に抗原を反
応させるが、用いられるリンパ球としてはヒト末梢血リ
ンパ球、特に単核球画分が好ましい。反応させる抗原と
しては、アレルギー性疾患患者の原因抗原が判明してい
る場合には、その原因抗原を用いればよい。一方、原因
抗原が不明の場合には、種々のアレルゲンが用いられ
る。通常用いられるアレルゲンとしては、卵白アルブミ
ン（OVA）、カゼイン、ダニ抗原、スギ花粉抗原、ブタ
クサ抗原、ハウスダスト等が挙げられる。被検リンパ球
と抗原との反応は、通常被検リンパ球をヒト保存AB血清
含有RPMI1640培地等の培地に加え、これに抗原を添加し
て37℃で５〜６日間培養することにより行なう。In carrying out the present invention, first, a test lymphocyte is reacted with an antigen, and as the lymphocyte used, human peripheral blood lymphocytes, particularly mononuclear cell fraction, are preferable. If the causative antigen of an allergic disease patient is known as the antigen to be reacted, the causative antigen may be used. On the other hand, when the causative antigen is unknown, various allergens are used. Commonly used allergens include ovalbumin (OVA), casein, mite antigen, cedar pollen antigen, ragweed antigen, house dust and the like. The reaction between the test lymphocytes and the antigen is usually carried out by adding the test lymphocytes to a medium such as human-preserved AB serum-containing RPMI1640 medium, adding the antigen thereto, and culturing at 37 ° C. for 5 to 6 days.
次に該反応液にIL−２を加えて３〜４日間培養する。
ここで用いられるIL−２としては、ヒトから抽出された
ものでもよいが遺伝子組み換えIL−２（ｒ−IL−２）を
用いてもよい。IL−２の添加量は、リンパ球に対するIL
−２の反応性がリンパ球２×105個当り0.1単位で平衡に
達するため、これ以上が好ましい。IL−２添加後の培養
は、リンパ球に対するIL−２の反応性が培養３日目にピ
ークとなるので、37℃３日間余とするのが好ましい。Next, IL-2 is added to the reaction solution and cultivated for 3 to 4 days.
IL-2 used here may be one extracted from human, but genetically modified IL-2 (r-IL-2) may also be used. The amount of IL-2 added is IL to lymphocytes.
Since the reactivity of −2 reaches equilibrium at 0.1 unit per 2 × 10 5 lymphocytes, more is preferable. In the culture after the addition of IL-2, the reactivity of IL-2 to lymphocytes reaches a peak on the third day of culture, and therefore, it is preferable that the culture is carried out at 37 ° C for 3 days or more.
次に培養液にヨウ化プロピジウム含有液を加えた後、
該培養液の蛍光色素量を測定する。本発明において用い
るヨウ化プロピジウムは、蛍光色素であり、２本鎖DNA
に特異的に結合し、１本鎖DNAには結合しないという特
性を有する。一方、生細胞のDNAの多くは２本鎖である
が、死細胞ではそのほとんどが１本鎖DNAとなってい
る。従って、２本鎖DNAに結合した蛍光色素量を測定す
れば、生細胞数を容易に定量できる。Next, after adding a liquid containing propidium iodide to the culture medium,
The amount of fluorescent dye in the culture solution is measured. Propidium iodide used in the present invention is a fluorescent dye and is a double-stranded DNA.
Has the property that it specifically binds to and does not bind to single-stranded DNA. On the other hand, most of live cell DNA is double-stranded, but most of dead cell DNA is single-stranded DNA. Therefore, the number of viable cells can be easily quantified by measuring the amount of the fluorescent dye bound to the double-stranded DNA.
ヨウ化プロピジウム含有液には、ヨウ化プロピジウム
以外に細胞膜破壊剤（例えばトリトン−Ｘ）及び色素
（例えばインク）を添加するのが望ましい。細胞膜破壊
剤は細胞膜を破壊してDNAをヨウ化プロピジウムと反応
しやすくするものであり、色素は未反応のヨウ化プロピ
ジウムの蛍光を減量せしめることによりバックグランド
となる蛍光量を低下させるものである。好ましいヨウ化
プロピジウム含有液は、ヨウ化プロビジウム約３〜15重
量％（好ましくは５〜10重量％）を含有し、トリトン−
Ｘ及びバックグランド蛍光量を５％以下とする量のイン
クを含有するEDTA水溶液である。In addition to propidium iodide, it is desirable to add a cell membrane disrupting agent (eg Triton-X) and a dye (eg ink) to the propidium iodide-containing liquid. The cell membrane disrupting agent disrupts the cell membrane and makes DNA easier to react with propidium iodide, and the dye reduces the fluorescence of unreacted propidium iodide to reduce the background fluorescence. . A preferred propidium iodide-containing liquid contains about 3 to 15% by weight (preferably 5 to 10% by weight) of probidium iodide, and Triton-
It is an EDTA aqueous solution containing X and an amount of ink that makes the background fluorescence amount 5% or less.
ヨウ化プロピジウム含有液を添加後、遠心（例えば10
00rpm、５分）して細胞沈渣をつくり、37℃、約30分間
静置後蛍光色素量を測定するのが好ましい。蛍光色素量
の測定には顕微光度計を用いるのが好ましい。After adding the solution containing propidium iodide, centrifuge (for example, 10
It is preferable to make a cell precipitate by applying (00 rpm, 5 minutes) and leave it at 37 ° C. for about 30 minutes, and then measure the amount of fluorescent dye. It is preferable to use a microphotometer for measuring the amount of fluorescent dye.
得られた蛍光色素量を例えば次式の如くしてIL−２非
添加群と比較することにより、IL−２反応性（SI）を算
出することができる。The IL-2 reactivity (SI) can be calculated by comparing the amount of the obtained fluorescent dye with the IL-2 non-added group, for example, as in the following formula.
〔作用及び発明の効果〕 本発明によれば、リンパ球のIL−２反応性を簡便な操
作で、迅速かつ正確に測定することができる。すなわ
ち、従来のトリパンブルー法においては生細胞数を顕微
鏡下に直接算定するため、煩雑であるばかりでなく熟練
者が長時間を要し、大量の検体を処理することは困難で
あったが、本発明方法では蛍光光学計を用い、通常の病
院等でも大量の検体を迅速に処理することができ、アレ
ルギー疾患の原因抗原の検索及び病勢の把握が容易とな
った。 [Action and Effect of the Invention] According to the present invention, IL-2 reactivity of lymphocytes can be measured quickly and accurately by a simple operation. That is, in the conventional trypan blue method, since the number of viable cells is directly calculated under a microscope, it is not only complicated but also requires a skilled person for a long time, and it was difficult to process a large amount of sample. In the method of the present invention, a large quantity of samples can be rapidly processed even in a normal hospital using a fluorescence optical meter, and it has become easy to search for the causative antigen of allergic disease and grasp the disease state.
次に実施例を挙げて本発明を詳細に説明する。 Next, the present invention will be described in detail with reference to examples.
実施例１ リンパ球の抗原特異的IL−２反応性の測定 ヒト末梢血より比重遠心法〔Scand.J.Clin.Lab.Inves
t.,21,51（1968）〕にて分離した単核細胞（１×106/m
l）に抗原〔ダニ抗原（Df,10μg/ml,鳥居薬品）〕を加
え、5mlの培養チューブ（Falcon）を用いて10％ヒトプ
ールAB血清加RPMI1640培地にて、10％CO2条件下5.5日間
培養した。培養後再度細胞数を２×105/150μ／穴に
調整した後、組み換えIL−２（武田薬品）0.1単位／穴
添加し、96穴平底マイクロプレートにて前記と同様の条
件で3.5日間培養した。次いでヨウ化プロピジウム含有
液50μ〔各種濃度のヨウ化プロピジウム液1.5μ
（4.9％EDTA中に1mg/mlのヨウ化プロピジウム含有液
（シグマ）を希釈して使用）、トリトンＸ−100（８％v
/v,和光純薬）1.5ml、各種濃度のインク（LEIZ社のdraw
ing inkを希釈して使用）0.1ml及びEDTA液（4.9％EDTA,
pH7.0,和光純薬）7.5mlより調製〕を添加し、遠心（100
0rpm,5分）し、37℃、30分間静置した後顕微光度計（ZE
ISS,MPM−03）を用いて各穴中の検体の蛍光を測定する
ことにより生細胞数を定量した。その結果を図１及び図
２に示す。その結果、生細胞数と蛍光色素量との間には
直線的正の相関が得られ、本発明方法が生細胞数を定量
するのに良好であることが判明した。また、測定光量を
一定にして、染色するヨウ化プロピジウム濃度とインク
濃度を変化させた時の（図１）、あるいは細胞を添加し
ない場合の測定蛍光色素量（バックグランド）を一定
（10）にした時の（図２）測定蛍光色素量と生細胞数と
の関係を検討したところ、ヨウ化プロピジウム濃度を原
液の２倍希釈（約７重量％）、インク濃度を1010倍希釈
したとき、測定値が一定となりまたその数値が大きかっ
た（図１及び図２）。また細胞を添加しない場合の測定
蛍光色素量（バックグランド）は全体の５％以下であっ
た。Example 1 Measurement of antigen-specific IL-2 reactivity of lymphocytes Specific gravity centrifugation [Scand.J.Clin.Lab.Inves] from human peripheral blood
t., 21, 51 (1968)] mononuclear cells (1 × 10 6 / m
l) to the antigen [Dani antigen (Df, 10 μg / ml, Torii Pharmaceutical)], and using a 5 ml culture tube (Falcon) in RPMI1640 medium with 10% human pool AB serum for 5.5 days under 10% CO 2 conditions. Cultured. After culturing, the cell number was adjusted again to 2 × 10 5 / 150μ / well, then 0.1 unit / well of recombinant IL-2 (Takeda Yakuhin) was added, and the cells were cultivated on a 96-well flat bottom microplate under the same conditions as above for 3.5 days. did. Next, propidium iodide-containing solution 50μ [propidium iodide solution of various concentrations 1.5μ
(Used by diluting a solution containing 1 mg / ml propidium iodide (Sigma) in 4.9% EDTA), Triton X-100 (8% v
/ v, Wako Pure Chemical Industries, Ltd. 1.5ml, ink of various concentrations (LEIZ draw
0.1 ml of diluted ing ink) and EDTA solution (4.9% EDTA,
pH 7.0, Wako Pure Chemical Industries, Ltd.) prepared from 7.5 ml], and centrifuged (100
After rotating at 0 rpm for 5 minutes at 37 ° C for 30 minutes, use a microphotometer (ZE
The number of viable cells was quantified by measuring the fluorescence of the sample in each well using ISS, MPM-03). The results are shown in FIGS. 1 and 2. As a result, a linear positive correlation was obtained between the number of viable cells and the amount of fluorescent dye, and it was found that the method of the present invention is good for quantifying the number of viable cells. In addition, the amount of fluorescent dye (background) measured when the concentration of propidium iodide and the ink for staining was changed (Fig. 1) or when cells were not added, was kept constant (10) by keeping the amount of measured light constant. When the relationship between the measured fluorescent dye amount and the number of viable cells was examined (Fig. 2), when the concentration of propidium iodide was diluted 2-fold (about 7% by weight) of the stock solution and the ink concentration was diluted 10 10- fold, The measured values were constant and the values were large (Figs. 1 and 2). The amount of fluorescent dye (background) measured when cells were not added was 5% or less of the whole.
なお、以下の実施例においてはヨウ化プロピジウム濃
度を原液の２倍希釈、インク濃度を1010倍希釈とした、
ヨウ化プロピジウム含有液を用いた。In the following examples, the propidium iodide concentration was diluted 2-fold from the stock solution, and the ink concentration was diluted 10 10- fold.
A liquid containing propidium iodide was used.
実施例２ リンパ球の抗原特異的IL−２反応性と添加IL−２濃度と
の関係 抗原としてダニ抗原（Df）の他に卵白アルブミン（OV
A,100μg/ml,シグマ）及びコンカナバリンＡ（ConA,5μ
g/ml,シグマ）を用い、添加IL−２濃度を0.0025〜1.0単
位／穴に変化させる以外は、実施例１と同様にして蛍光
を測定した。そして、次式に従い、IL−２反応性（SI）
を算出した。Example 2 Relationship between antigen-specific IL-2 reactivity of lymphocytes and added IL-2 concentration In addition to mite antigen (Df) as an antigen, ovalbumin (OV
A, 100 μg / ml, Sigma) and Concanavalin A (ConA, 5 μ
fluorescence was measured in the same manner as in Example 1 except that the concentration of added IL-2 was changed to 0.0025 to 1.0 unit / hole using g / ml (Sigma). Then, according to the following formula, IL-2 reactivity (SI)
Was calculated.
その結果、図３から明らかなようにIL−２濃度に応じ
て蛍光量が増加し、0.1単位／穴にてほぼ平衡状態に達
した。 As a result, as is clear from FIG. 3, the amount of fluorescence increased according to the IL-2 concentration, and the equilibrium state was reached at 0.1 unit / well.
よって、以下の実施例ではIL−２濃度を0.1単位／穴
として行なった。Therefore, in the following examples, the IL-2 concentration was 0.1 unit / hole.
実施例３ IL−２反応性とIL−２添加後の培養期間との関係 IL−２添加後の培養期間を変化させる以外は実施例１
と同様にしてIL−２反応性（SI）を測定した。Example 3 Relationship between IL-2 reactivity and culture period after addition of IL-2 Example 1 except that the culture period after addition of IL-2 was changed.
IL-2 reactivity (SI) was measured in the same manner as in.
その結果、IL−２添加後３日目において最大の染色色
素量（IL−２反応性）を示した（図４）。よってIL−２
反応性の評価はIL−２添加後３日目が適している。As a result, the maximum amount of dye (IL-2 reactivity) was shown on day 3 after the addition of IL-2 (Fig. 4). Therefore IL-2
The third day after addition of IL-2 is suitable for the evaluation of reactivity.
実施例４ アレルギー患者リンパ球におけるIL−２反応性の抗原特
異性の検討 各種アレルギー患者のリンパ球を用いて、実施例１と
同様にしてIL−２反応性（SI）を測定した。その結果を
表１及び図５に示す。なお、抗原としては、、ダニ抗原
（Df）、卵白アルブミン（OVA）、コンカナバリンＡ（C
onA）及びカゼイン（α−Casein,100μg/ml,シグマ）を
用いた。Example 4 Examination of Antigen Specificity of IL-2 Reactivity in Lymphocytes of Allergic Patients IL-2 reactivity (SI) was measured in the same manner as in Example 1 using lymphocytes of various allergic patients. The results are shown in Table 1 and FIG. The antigens are mite antigen (Df), ovalbumin (OVA), concanavalin A (C
onA) and casein (α-Casein, 100 μg / ml, Sigma) were used.
また、表１には抗原特異性を示すIgE RAST値も示し
た。IgE RAST値は、in vitroアレルゲン検索用キット、
RASTシオノギアイソトープ試薬を用いて測定した。In addition, Table 1 also shows IgE RAST values showing antigen specificity. IgE RAST value is a kit for in vitro allergen search,
It was measured using RAST Shionogi isotope reagent.
その結果、ConAで刺激した場合はSIが2.01±0.23（S
E）（ｎ＝10）のIL−２反応性が得られた。 As a result, SI was 2.01 ± 0.23 (S
E) (n = 10) IL-2 reactivity was obtained.
卵アレルギー患者末梢血リンパ球を用いてOVA刺激に
て誘導されるIL−２反応性（SI）は、1.91±0.14（ｎ＝
６）であった。ダニに感作されている小児気管支喘息児
末梢血リンパ球を用いてDf刺激により誘導されるIL−２
反応性（SI）は、1.69±0.10（ｎ＝６）であった。これ
に対し、健康人のリンパ球ではこれらのOVA,Dfで刺激し
た場合IL−２反応性は1.05±0.02（SE）（ｎ＝10）で反
応の誘導は認められなかった。またダニ抗原感作のすす
んでいない卵アレルギー患者の末梢血リンパ球をDfで刺
激した場合にもIL−２反応性は誘導されなかった。一方
卵に感作されていない気管支喘息小児ではOVAで刺激し
た場合IL−２反応性同様に誘導されなかった。また牛乳
により症状の出現する症例においてもα−caseinで刺激
した場合に得意的にIL−２反応性の誘導が認められた。
よって特異抗原刺激リンパ球において誘導されるIL−２
反応性は本発明方法で測定した場合、抗原特異的であ
る。IL-2 reactivity (SI) induced by OVA stimulation using peripheral blood lymphocytes of patients with egg allergy was 1.91 ± 0.14 (n =
6). IL-2 induced by Df stimulation using peripheral blood lymphocytes in children with bronchial asthma sensitized by ticks
The reactivity (SI) was 1.69 ± 0.10 (n = 6). On the other hand, IL-2 reactivity was 1.05 ± 0.02 (SE) (n = 10) when stimulated with these OVA and Df in healthy human lymphocytes, and no induction of response was observed. Also, IL-2 reactivity was not induced when Df stimulated peripheral blood lymphocytes of eggs allergic patients who were not sensitized with tick antigen. On the other hand, in children with bronchial asthma not sensitized to eggs, it was not induced as well as IL-2 reactivity when stimulated with OVA. In addition, even in the case where the symptoms appeared due to milk, the induction of IL-2 reactivity was found to be prominent when stimulated with α-casein.
Thus IL-2 is induced in specific antigen-stimulated lymphocytes
Reactivity is antigen-specific as measured by the method of the invention.
比較例 トリパンブルー法を用いたリンパ球のIL−２反応性の測
定 比重遠心法にて分離した単核細胞（１×106/ml）に10
0μg/mlのOVAあるいは10μg/ml Dfを添加し、5mlの培養
チューブを用いて10％ヒトプールAB血清加RPMI1640にて
５日間培養後、再度細胞数を２×105/0.2mlに調製した
のち、組み換えIL−２（武田）を添加し、96穴平底マイ
クロプレート（ヌンク）にて３日間培養後、測定直前に
１％トリパンブルー50μを添加し、よく撹拌後顕微鏡
下に生細胞数を血算板にて算定した（生細胞数の比率は
90％以上）。IL−２反応性の評価として、IL−２非添加
群との比率をSIとして求めた。Comparative Example Measurement of IL-2 reactivity of lymphocytes using trypan blue method Mononuclear cells (1 × 10 6 / ml) separated by specific gravity centrifugation were added to 10
After adding 0 μg / ml OVA or 10 μg / ml Df and culturing in a 5 ml culture tube in RPMI1640 with 10% human pool AB serum for 5 days, the cell number was adjusted to 2 × 10 5 /0.2 ml again. , Recombinant IL-2 (Takeda) was added, and after culturing for 3 days in a 96-well flat bottom microplate (Nunc), 1% trypan blue 50μ was added immediately before measurement, and after vigorous stirring, the viable cell number was measured under a microscope. Calculated using the abacus (the ratio of viable cells is
More than 90). As an evaluation of IL-2 reactivity, the ratio with the IL-2 non-added group was determined as SI.
同一の検体について、本発明方法（実施例１の方法）
とトリパンブルー法にてIL−２反応性を求めた結果を、
図６に示す。その結果、本発明方法とトリパンブルー法
とはその値がよく一致した。 The method of the present invention (method of Example 1) for the same specimen
And the result of determining IL-2 reactivity by the trypan blue method,
As shown in FIG. As a result, the values of the method of the present invention and the trypan blue method were in good agreement.
図１は、実施例１においてヨウ化プロピジウム（PI）及
びインク（ink）の濃度を変化させた場合の生細胞数と
蛍光色素量との関係を示す図面である。図２は実施例１
において、細胞を添加しない場合の測定蛍光色素量（バ
ックグランド）を一定（10）にしたときの生細胞数と蛍
光色素量との関係を示す図面である。図３は添加IL−２
濃度とIL−２反応性（SI）との関係を示す図面である。
図４はIL−２添加後の培養時間とIL−２反応性（SI）と
の関係を示す図面である。図５はアレルギー患者（●）
又は健常人（○）由来リンパ球のIL−２反応性（SI）を
示す図面である。図６は本発明方法とトリパンブルー法
との相関性を示す図面である。FIG. 1 is a diagram showing the relationship between the number of viable cells and the amount of fluorescent dye when the concentrations of propidium iodide (PI) and ink were changed in Example 1. FIG. 2 shows the first embodiment
3 is a drawing showing the relationship between the number of viable cells and the amount of fluorescent dye when the measured amount of fluorescent dye (background) is constant (10) when no cells are added. Figure 3 shows added IL-2
It is a figure which shows the relationship between a density | concentration and IL-2 reactivity (SI).
FIG. 4 is a drawing showing the relationship between the culture time after addition of IL-2 and IL-2 reactivity (SI). Figure 5 shows allergic patients (●)
Alternatively, it is a drawing showing the IL-2 reactivity (SI) of lymphocytes derived from a healthy person (◯). FIG. 6 is a drawing showing the correlation between the method of the present invention and the trypan blue method.
Claims (3)
反応液にインターロイキン−２を加えて３〜４日間培養
し、さらに細胞膜破壊剤及びヨウ化プロピジウム含有液
を加えた後該培養系の蛍光色素量を測定することを特徴
とする、リンパ球の抗原特異的インターロイキン−２反
応性の測定法。1. A test lymphocyte is reacted with an antigen, and then interleukin-2 is added to the reaction solution and the mixture is cultured for 3 to 4 days, and then a cell membrane disrupting agent and a propidium iodide-containing solution are added, and then the culture is performed. A method for measuring antigen-specific interleukin-2 reactivity of lymphocytes, which comprises measuring the amount of fluorescent dye in the system.
培養細胞を沈めたのち、得られた培養液を約30分間静置
した後蛍光色素量を測定するものである請求項１記載の
リンパ球の抗原特異的インターロイキン−２反応性の測
定法。2. A lymphocyte according to claim 1, which comprises adding a liquid containing propidium iodide and centrifuging to submerge the cultured cells, allowing the resulting culture to stand for about 30 minutes, and then measuring the amount of fluorescent dye. Of the method of measuring antigen-specific interleukin-2 reactivity of.
球２×105個当り0.1単位以上である請求項１記載のリン
パ球の抗原特異的インターロイキン−２反応性の測定
法。3. The method for measuring the antigen-specific interleukin-2 reactivity of lymphocytes according to claim 1, wherein the amount of interleukin-2 added is 0.1 unit or more per 2 × 10 5 lymphocytes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2186163A JP2561170B2 (en) | 1990-07-13 | 1990-07-13 | Method for measuring antigen-specific interleukin-2 reactivity of lymphocytes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2186163A JP2561170B2 (en) | 1990-07-13 | 1990-07-13 | Method for measuring antigen-specific interleukin-2 reactivity of lymphocytes |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0472563A JPH0472563A (en) | 1992-03-06 |
| JP2561170B2 true JP2561170B2 (en) | 1996-12-04 |
Family
ID=16183492
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2186163A Expired - Fee Related JP2561170B2 (en) | 1990-07-13 | 1990-07-13 | Method for measuring antigen-specific interleukin-2 reactivity of lymphocytes |
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| Country | Link |
|---|---|
| JP (1) | JP2561170B2 (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2214518A (en) * | 1988-01-28 | 1989-09-06 | Innofinance Altalanos Innovaci | Process and equipment for the rapid determination of the spermium cell count and/or living spermium count |
-
1990
- 1990-07-13 JP JP2186163A patent/JP2561170B2/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| アレルギー、３６［１２］（１９８７）野間他Ｐ．１０７５−１０８５ |
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