JPH01126554A - Reagent for immunological measurement - Google Patents
Reagent for immunological measurementInfo
- Publication number
- JPH01126554A JPH01126554A JP28310787A JP28310787A JPH01126554A JP H01126554 A JPH01126554 A JP H01126554A JP 28310787 A JP28310787 A JP 28310787A JP 28310787 A JP28310787 A JP 28310787A JP H01126554 A JPH01126554 A JP H01126554A
- Authority
- JP
- Japan
- Prior art keywords
- antibody
- solid phase
- amount
- solution
- layer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000003153 chemical reaction reagent Substances 0.000 title claims description 14
- 238000005259 measurement Methods 0.000 title description 16
- 230000001900 immune effect Effects 0.000 title 1
- 239000007790 solid phase Substances 0.000 claims abstract description 32
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims abstract description 15
- 108010039918 Polylysine Proteins 0.000 claims abstract description 13
- 229920000656 polylysine Polymers 0.000 claims abstract description 13
- 239000000427 antigen Substances 0.000 claims abstract description 10
- 102000036639 antigens Human genes 0.000 claims abstract description 10
- 108091007433 antigens Proteins 0.000 claims abstract description 10
- 229910052588 hydroxylapatite Inorganic materials 0.000 claims abstract description 7
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims abstract description 7
- 238000003018 immunoassay Methods 0.000 claims description 13
- 229940027941 immunoglobulin g Drugs 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 18
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000004132 cross linking Methods 0.000 abstract description 2
- 238000000034 method Methods 0.000 description 23
- 239000000243 solution Substances 0.000 description 23
- 230000000052 comparative effect Effects 0.000 description 21
- 238000010586 diagram Methods 0.000 description 21
- 239000004793 Polystyrene Substances 0.000 description 16
- 229920002223 polystyrene Polymers 0.000 description 16
- 108090000790 Enzymes Proteins 0.000 description 11
- 102000004190 Enzymes Human genes 0.000 description 11
- 229940088598 enzyme Drugs 0.000 description 11
- 238000001179 sorption measurement Methods 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 239000012153 distilled water Substances 0.000 description 7
- 238000011156 evaluation Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000002860 competitive effect Effects 0.000 description 3
- 238000011017 operating method Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- SVOAXTTVWPHOQW-UHFFFAOYSA-N 2-acetylsulfanylbutanedioic acid Chemical compound CC(=O)SC(C(O)=O)CC(O)=O SVOAXTTVWPHOQW-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 108010015776 Glucose oxidase Proteins 0.000 description 2
- 239000004366 Glucose oxidase Substances 0.000 description 2
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 239000008351 acetate buffer Substances 0.000 description 2
- XECAHXYUAAWDEL-UHFFFAOYSA-N acrylonitrile butadiene styrene Chemical compound C=CC=C.C=CC#N.C=CC1=CC=CC=C1 XECAHXYUAAWDEL-UHFFFAOYSA-N 0.000 description 2
- 229920000122 acrylonitrile butadiene styrene Polymers 0.000 description 2
- 239000004676 acrylonitrile butadiene styrene Substances 0.000 description 2
- 238000003452 antibody preparation method Methods 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 229940116332 glucose oxidase Drugs 0.000 description 2
- 235000019420 glucose oxidase Nutrition 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- -1 potassium ferricyanide Chemical compound 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- PVGATNRYUYNBHO-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-(2,5-dioxopyrrol-1-yl)butanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCN1C(=O)C=CC1=O PVGATNRYUYNBHO-UHFFFAOYSA-N 0.000 description 1
- HCILGDWKPMTTSN-UHFFFAOYSA-N 2-(2,5-dioxopyrrolidin-1-yl)-4-(2,5-dioxopyrrol-1-yl)butanoic acid Chemical compound O=C1CCC(=O)N1C(C(=O)O)CCN1C(=O)C=CC1=O HCILGDWKPMTTSN-UHFFFAOYSA-N 0.000 description 1
- OBYNJKLOYWCXEP-UHFFFAOYSA-N 2-[3-(dimethylamino)-6-dimethylazaniumylidenexanthen-9-yl]-4-isothiocyanatobenzoate Chemical compound C=12C=CC(=[N+](C)C)C=C2OC2=CC(N(C)C)=CC=C2C=1C1=CC(N=C=S)=CC=C1C([O-])=O OBYNJKLOYWCXEP-UHFFFAOYSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 1
- NBSCHQHZLSJFNQ-GASJEMHNSA-N D-Glucose 6-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@H]1O NBSCHQHZLSJFNQ-GASJEMHNSA-N 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- VFRROHXSMXFLSN-UHFFFAOYSA-N Glc6P Natural products OP(=O)(O)OCC(O)C(O)C(O)C(O)C=O VFRROHXSMXFLSN-UHFFFAOYSA-N 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 239000012506 Sephacryl® Substances 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000002494 anti-cea effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229920006026 co-polymeric resin Polymers 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000010324 immunological assay Methods 0.000 description 1
- PZLXYMQOCNYUIO-UHFFFAOYSA-N lithium;hydrochloride Chemical compound [Li].Cl PZLXYMQOCNYUIO-UHFFFAOYSA-N 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000036963 noncompetitive effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
【発明の詳細な説明】
Ａ産業上の利用分野
本発明は、固相を用いた免疫測定試薬であり、固相にハ
イドロキシアバクイト（Ｃａ１゜（ＰＯａ） ｓ （Ｏ
Ｈ） ｘ）を使い、ポリリジン層とグルタルアルデヒド
層と抗体または抗原層を順次形成して、固相への抗体結
合量及び抗体結合力を増加させ、測定範囲の拡大、検出
限界の感度の上昇、及び再現性の向上を得た免疫測定試
薬に関するものである。DETAILED DESCRIPTION OF THE INVENTION A. Industrial Application Field The present invention is an immunoassay reagent using a solid phase, in which hydroxyabacterium (Ca1°(POa) s (O
H) Using x), sequentially form a polylysine layer, a glutaraldehyde layer, and an antibody or antigen layer to increase the amount of antibody binding to the solid phase and the antibody binding strength, expanding the measurement range and increasing the sensitivity of the detection limit. , and an immunoassay reagent with improved reproducibility.
Ｂ発明の概要
本発明は、ハイドロキシアバタイ）・を固相として、そ
の固相上に、ポリリジン層とグルタルアルデヒド層と抗
体または抗原層を順次形成したことからなる免疫測定試
薬に関するものである。B. Summary of the Invention The present invention relates to an immunoassay reagent comprising a solid phase of hydroxyabatai, on which a polylysine layer, a glutaraldehyde layer, and an antibody or antigen layer are sequentially formed.
Ｃ従来の技術
固相を用いる免疫学的測定法には、大きく分けて、競合
法と非競合法に分類される。前者の代表例は、第１抗体
固相法、後者ンよ、サンドイッチ測定法がある。C. Conventional Technology Immunoassay methods using solid phases are broadly classified into competitive methods and non-competitive methods. A typical example of the former is the first antibody solid phase method, and the latter is the sandwich measurement method.
このサンドイツチ法は、固相に抗体を吸着させ、そこに
抗原をトラップさせる。次に酵素標識抗体をその抗原に
結合させ、結合した酵素標識抗体量から抗原の量を測定
する方法である。In this sandwich method, antibodies are adsorbed onto a solid phase and antigens are trapped there. Next, an enzyme-labeled antibody is bound to the antigen, and the amount of the antigen is measured from the amount of bound enzyme-labeled antibody.
上記の原理のため、サンドイツチ法の検出感度や上昇さ
せるためにｌ、？ 、固相への抗体結合量及び抗体結合
力が、大きく影響してくる。Due to the above principle, is it possible to increase the detection sensitivity of the Sanderch method? , the amount of antibody bound to the solid phase and the antibody binding strength have a large influence.
従来、固相の材料として１よポリスチレン、ガラス、ア
クリルニトリル−ブタジェン−スチレン共重合樹脂（略
してＡＢＳ）などが多く用いられた。Conventionally, polystyrene, glass, acrylonitrile-butadiene-styrene copolymer resin (abbreviated as ABS), etc. have often been used as materials for the solid phase.
これらの固相への抗体結合方法は、多くは物理的に吸着
させる方法であった。しかし、この方法では、固相の抗
体結合量が少ない、固相の抗体結合力が弱い、測定値の
バラツキが大きい、非特異的吸着が大きいなどの問題点
があった。Most of the methods for binding antibodies to these solid phases involve physical adsorption. However, this method has problems such as a small amount of antibody bound to the solid phase, weak antibody binding strength of the solid phase, large variations in measured values, and large nonspecific adsorption.
以前、特願昭６０−４５７４２で本発明者らが出願した
「免疫学的な測定試薬の調整法とこれによって得た試薬
」では、上記の問題点を解決するために、固相をアミノ
酸処理した後、二官能性アルデヒド処理し、抗体結合さ
せたものであり、その効果は上記問題点を解決するに至
った。Previously, in the patent application No. 60-45742 entitled "Method for preparing immunological assay reagents and reagents obtained thereby," the solid phase was treated with amino acids in order to solve the above problems. After that, it was treated with a bifunctional aldehyde and bound to an antibody, which has the effect of solving the above problems.
Ｄ発明が解決しようとする問題点
しかし、免疫測定試薬としての検出限界、測定範囲及び
再現性のより一層の向上が望まれていた。D. Problems to be Solved by the Invention However, it has been desired to further improve the detection limit, measurement range, and reproducibility of the immunoassay reagent.
本発明は、かかる問題点を解決するためになされたもの
で、固相の抗体結合量が多く、固相の抗体結合力も強く
、測定値のバラツキが小さい、非特異的吸着の少ない免
疫測定試薬を得ろことはもちろんの事として、免疫測定
試薬としての測定範囲の拡大、検出限界の感度上昇及び
再現性のより一層の向上を成し得た免疫測定試薬を得る
ことを目的とする。The present invention has been made to solve these problems, and is an immunoassay reagent that has a large amount of antibody binding on the solid phase, strong antibody binding force on the solid phase, small variation in measured values, and low nonspecific adsorption. The object of the present invention is, of course, to obtain an immunoassay reagent that can expand the measurement range, increase the sensitivity of the detection limit, and further improve reproducibility.
Ｅ問題点を解決するための手段
この発明に係わる免疫測定試薬では、ボリリジノ層とグ
ルタルアルデヒド層と抗体または抗原層を順次形成した
、生体親和性の高いハイドロキシアパタイトを使い、そ
のハイドロキシアバタイ１−を固相としたものである。E Means for Solving Problems The immunoassay reagent according to the present invention uses hydroxyapatite with high biocompatibility, in which a boriridino layer, a glutaraldehyde layer, and an antibody or antigen layer are sequentially formed, and the hydroxyapatite 1- is used as a solid phase.
Ｆ作用
生体親和性の高いハイドロキレアパタイトｅ固相に使う
ことにより、固相とポリリジンの結合力と結合量が増加
する。それにより、架橋するグルタルアルデヒド量が増
加し、抗体結合量及び抗体結合力が増加する。F action By using hydroxyleapatite e solid phase, which has high biocompatibility, the binding force and amount of binding between the solid phase and polylysine increases. As a result, the amount of crosslinking glutaraldehyde increases, and the amount of antibody binding and antibody binding strength increase.
Ｇ実施例
（１１”’ｉｃ Ｅ Ａ （ガン胎児性抗原）を用いた
抗体結合量の評価
実施例１゜
第１図は本発明の一実施例を示す免疫測定試薬の調整方
法図である。Example G (Evaluation of antibody binding amount using 11"'ic EA (carcinoembryonic antigen) Example 1) FIG. 1 is a diagram showing a method for preparing an immunoassay reagent according to an embodiment of the present invention.
調整方法は、ハイドロキシアパタイト（Ｃｉ、、（ＰＯ
４） Ｇ （０［［） ２） （以下、ＨＡｐボールと
略す）をＡ液（０，１ｍｇ／−ポリリジンを含む０．１
５ｍｏｌ／ ｌホウ酸緩衝液（、［１８，５） ）中に
室温で、１晩浸漬してポリリジン処理を行った。蒸留水
で洗浄し、５％グルタルアルデヒド水溶液に、３０℃で
２時間浸漬してグルタルアルデヒド処理を行った。蒸留
水で洗浄し、Ｂ ２１２ （０，１ｍｇ／ｍｌ免疫グロ
ブリンＧ（ＩｇＧと略す）、０．１ｎａｏｌ／ｌ”）
ン酸緩衝液（ｐＦ［７，５１） ４℃で１＠浸漬した。The adjustment method is hydroxyapatite (Ci, (PO
4) G (0[[) 2) (hereinafter abbreviated as HAp ball) was mixed with solution A (0.1 mg/- containing polylysine).
Polylysine treatment was performed by immersing it in 5 mol/l borate buffer (, [18,5)) at room temperature overnight. The sample was washed with distilled water and immersed in a 5% aqueous glutaraldehyde solution at 30° C. for 2 hours to perform a glutaraldehyde treatment. Wash with distilled water and add B 212 (0.1 mg/ml immunoglobulin G (abbreviated as IgG), 0.1 naol/l”).
It was immersed in acid buffer (pF[7,51) at 4°C.
さらにＢ液で洗浄した後、Ｃ液（０，Ｏ１ｍｏｌ／ｌリ
ン酸緩衝液（ｐ［（７，０）、０．１ｍｏｌ／４１ Ｎ
ａＣｌ、 ０．ＩＸ牛血清アルブミン（Ｂ Ｓ Ａと略
す）、０．１にＮａｐ、混合液）で洗浄した後、Ｃ液に
浸漬し、４℃で保存した。 本実施例では、ＩｇＧを抗
ガン胎児性抗原（抗ＣＥＡと略すｌ−ＩｇＧとして被覆
ＨＡｐボールを調整した。After further washing with solution B, solution C (0, O1 mol/l phosphate buffer (p[(7,0), 0.1 mol/41 N
aCl, 0. After washing with IX bovine serum albumin (abbreviated as BSA), 0.1% Nap, mixed solution), it was immersed in solution C and stored at 4°C. In this example, coated HAp balls were prepared using IgG as anti-carcinoembryonic antigen (anti-CEA, abbreviated as 1-IgG).
第２図は競合反応による抗体結合量の測定操作図であり
、第１図の方法で調整された抗ＣＥＡ−ＩｇＧ被覆ＨＡ
ｐボールを１”Ｉ−ＣＥ Ａ溶液（約２ｘｌＯ’Ｃｐ耐
］カウント・パー ξニフフ］）１００μ ｌ、 Ｃ
ＥＡ溶液 （０〜１１０００ｎ／ｍ１）１００μｌ 、
Ｃ）ｌｉｌＯＱμｌの混合溶液で１晩反応させ、洗浄
後、γ−ウェルカウンターで計測した。Figure 2 is a diagram showing the procedure for measuring the amount of antibody binding by competitive reaction, and shows the anti-CEA-IgG-coated HA prepared by the method shown in Figure 1.
Add the p-ball to 100 μl of 1" I-CE A solution (approximately 2xlO'Cp resistance]), C
EA solution (0-11000n/ml) 100μl,
C) Reaction was carried out overnight with a mixed solution of lilOQμl, and after washing, measurement was performed using a γ-well counter.
第１図で調整した抗ＣＥＡ−ＩｇＧ被覆ＨＡｐボールを
第２図の通り、”’ｉｃ Ｅ Ａにより、抗体結合量を
測定した。結果は、第３図に示す。The amount of antibody bound to the anti-CEA-IgG-coated HAp balls prepared in FIG. 1 was measured by ``ic EA as shown in FIG. 2. The results are shown in FIG. 3.
比較例１゜
第１図と同様の方法で、抗ＣＥＡ−ＩｇＧ被覆ポリスチ
レンボールを調整し、第２図と同様の方法で、抗体結合
量を測定した。Comparative Example 1 An anti-CEA-IgG coated polystyrene ball was prepared in the same manner as in FIG. 1, and the amount of antibody bound was measured in the same manner as in FIG.
結果は、第３図に示す。The results are shown in FIG.
第３図は、実施例１と比較例１の結果であり、ＨＡｐボ
ールとポリスチレンボールの抗体結合量変化図である。FIG. 3 shows the results of Example 1 and Comparative Example 1, and is a diagram of changes in the amount of antibody binding between HAp balls and polystyrene balls.
図において（ａ）は実施例１、（ｂ）は比較例１の結果
を示している。In the figure, (a) shows the results of Example 1, and (b) shows the results of Comparative Example 1.
図より、抗体結合量はＨＡｐボールの方がポリスチレン
ボールより相対的に大であることが判明した。From the figure, it was found that the amount of antibody bound to the HAp ball was relatively larger than that of the polystyrene ball.
これは、ＨＡｐがポリスチレンに比べて生体親和性が強
いためである。This is because HAp has stronger biocompatibility than polystyrene.
（２）酵素標識抗体の固相への非特異的吸着の評価実施
例２゜
第４図は酵素免疫測定（Ｅｎｚｙｍｅ Ｉ＋ａ＋ｕｎｏ
人５ｓａｙ ：ＥＩＡと略す）を行う時のＩｇＧ−ＣＯ
Ｄ（グルコースオキシダーゼ）標識抗体ＰＩ整方法図で
あり、第５図は非特異的吸着の評価方法の操作図である
。(2) Evaluation Example 2 of non-specific adsorption of enzyme-labeled antibodies to solid phase Figure 4 shows enzyme immunoassay (Enzyme I+a+uno
IgG-CO when performing human 5say (abbreviated as EIA)
FIG. 5 is a diagram of a D (glucose oxidase)-labeled antibody PI preparation method, and FIG. 5 is an operational diagram of a method for evaluating nonspecific adsorption.
ＩｇＧ−ＣＯＤ標識抗体調整方法は、まずＧＯＤ約３ｍ
ｇ７０．３−を４倍量の０．１ｍｏｌ／ｊリン酸緩衝液
（ｐＨ７，０）に溶かし、ＧＯＤ： ＧＭＢＳ（ザクシ
ンイミジル−４−マレイミドブチレイト）＝１：５Ｇの
比で添加し、それをＯ，１ｍｏｌ／ ｊリン酸ｍ衝液（
ｐＨ６，０）で平衡化したセファデックスＧ ２５ （
ＩＸ３０カラム）を用いて１２ｍｊ／ｈｒで脱塩し、１
ｒｎｌずつ分取し、マレイミドＣＯＤを得た。次に、Ｉ
ｇＧに２＋ｎｔ’の０．１ｍｏｌ／ｌ’リン酸緩衝液（
ｐ［（６，０） ５ｍ・ｍｏｌ／ｊ ＥＤＴＡを加え
、Ｓ−アセチルメルカプトこはく酸を（Ｓ−アセチルメ
ルカプトこはく酸： Ｉ ｇＧ ＝ ３００： １
の比で）レメチルフォルマイドに溶解し添加する。室温
で３０分間攪拌後、０．１ｍｏｌ／ｊ ｈリス−塩酸（
ｐＨ７，Ｏ） ０．１−１Ｏ，１ｍｏｌ／ ｌ ＥＤＴ
Ａ （ｐｌ［７，０） ０．０２−０２−１ｌ／Ｉヒド
ロキシジアミン水溶液（ｐＨ７，０） ０．１ｍｊを各
々加え、３０℃４分間反応させた。Ｄ液で平衡化したセ
ファデックｘ Ｇ ２５ （１ｘ３０カラム）を用いて
、１２ｍｊ／ｈｒの速度で脱塩し、１ｍｌずつ分取し、
水冷中コロンオンバッグで濃縮し、ＳＨ−ＩｇＧを得た
。得られたマレイミドＣＯＤとＳＨ−ＩｇＧを等モル混
和し、３０℃１時間静置後、４℃で１晩静置した。０．
１ｍｏｌ／ｌリン酸緩衝液（ｐＨ６，５）、５ ｍｆｆ
Ｉｏｌ／１ＥＤＴ人で平衡化したセファクリル３００
（ＩＸ９０カラム）に、６ｍ１／ｈｒで上記試料を溶出
し、１ｒｎｌずつ分取し、ＩｇＧ−ＣＯＤ標識抗体を得
た。これを０．１％ＮａＮ、、０．１％ＢＳＡとなるよ
うに添加し、４℃で保存する。The IgG-COD labeled antibody preparation method begins with GOD of approximately 3m
g70.3- was dissolved in 4 times the volume of 0.1 mol/j phosphate buffer (pH 7.0), added at a ratio of GOD: GMBS (succinimidyl-4-maleimidobutyrate) = 1:5G, and O, 1 mol/j phosphoric acid solution (
Sephadex G 25 (pH 6,0) equilibrated with
IX30 column) at 12 mj/hr, and
rnl was fractionated to obtain maleimide COD. Next, I
gG with 2+nt' in 0.1 mol/l' phosphate buffer (
p[(6,0) 5 mmol/j Add EDTA and add S-acetylmercaptosuccinic acid (S-acetylmercaptosuccinic acid: IgG = 300: 1
) in remethylformide and add. After stirring at room temperature for 30 minutes, 0.1 mol/j h lithium-hydrochloric acid (
pH7, O) 0.1-1O, 1mol/l EDT
A (pl [7,0) 0.02-02-1 l/I hydroxydiamine aqueous solution (pH 7,0) 0.1 mj was added to each, and the mixture was reacted at 30°C for 4 minutes. Desalting was carried out at a rate of 12 mj/hr using Sephadec
It was concentrated in a colon-on bag while cooling with water to obtain SH-IgG. The obtained maleimide COD and SH-IgG were mixed in equimolar amounts, left at 30°C for 1 hour, and then left at 4°C overnight. 0.
1 mol/l phosphate buffer (pH 6,5), 5 mff
Sephacryl 300 equilibrated with Iol/1EDT humans
(IX90 column), the above sample was eluted at 6 ml/hr, and 1 rnl was fractionated to obtain an IgG-COD labeled antibody. Add this to 0.1% NaN and 0.1% BSA and store at 4°C.
非特異的吸着は、第５図に示すように、第１図で得られ
た抗ＣＥＡ−ＩｇＧ被覆ＨＡｐボールを、Ｃ液で希釈し
た抗ＣＥＡ−ＩｇＧ−ＧＯＤ標識抗体０、１ｒｎｌとＣ
液０．２ｔｎｌ’に室温で１晩静置し、蒸留水で洗浄後
、０．５ｍｏｌ／ ｌグルコース、０． Ｏ１ｍｏｌ／
ｌ酢酸緩衝液（ｐＨ５，１） ０．３ｍｌを加え、３
７℃２時間静置した。As shown in Fig. 5, non-specific adsorption was performed using the anti-CEA-IgG-coated HAp balls obtained in Fig. 1 with 0 and 1 rnl of anti-CEA-IgG-GOD-labeled antibodies diluted with C solution.
The solution was left standing overnight at room temperature in 0.2 tnl', washed with distilled water, and then treated with 0.5 mol/l glucose and 0.5 mol/l glucose. O1mol/
Add 0.3 ml of l acetate buffer (pH 5,1) and
It was left standing at 7°C for 2 hours.
０、１ｍｊサンプリングし、２ｘｌＯ−’ｍｏｌ／ ｌ
ルミノール、０．２ｍｏｌ／ｊ炭酸緩衝液（ｐｌ［９，
８） ０．５ｍｊ 、 ６Ｘ１０−’ｍｏｌ／ｌフェリ
シアン化カリ水溶液０．５ｍＮを各添加し、１５秒待ち
、１６〜４５秒間の発光量を算出することによって求め
た。0, 1 mj sampled, 2xlO-'mol/l
Luminol, 0.2 mol/j carbonate buffer (pl[9,
8) Each addition of 0.5 mN of 0.5 mj, 6 x 10-' mol/l potassium ferricyanide aqueous solution, waiting for 15 seconds, and calculating the luminescence amount for 16 to 45 seconds, was performed.
比較例２゜
第１図と同様に調整した抗ＣＥＡ−ＩｇＧ被覆ポリスチ
レンボールを用いて、第５図の様にポリスチレンボール
の非特異的吸着量を求めた。Comparative Example 2 Using anti-CEA-IgG coated polystyrene balls prepared in the same manner as shown in Fig. 1, the amount of non-specific adsorption of the polystyrene balls was determined as shown in Fig. 5.
実施例２と比較例２で、酵素標識抗体の固相への非特異
的吸着を比較した。なお、評価法は、添加ＩｇＧ−ＧＯ
Ｄ標識抗体に対するＩｇＧ−ＣＯＤ標識抗体の固相への
吸着量の割合（％）とした。結果を表１に示す。In Example 2 and Comparative Example 2, nonspecific adsorption of an enzyme-labeled antibody to a solid phase was compared. In addition, the evaluation method is based on added IgG-GO
It was expressed as the ratio (%) of the amount of IgG-COD labeled antibody adsorbed on the solid phase to the D labeled antibody. The results are shown in Table 1.
ｉ Ｉ ＨＡ ｐボールとポリスチレンボールにおける
非特異的吸着の比較
サンドイッチ測定法の感度を左右する主な要因の１っで
ある酵素標識抗体の固相への非特異的吸着は両者に差が
ないという結果を得た。これは抗体を固相へ吸着後、牛
血清アルブミンによるブロックがＨＡ ｐの場合でも、
ポリスチレンと同程度に有効であるためである。i I HA Comparison of nonspecific adsorption between p-balls and polystyrene balls It is said that there is no difference between the two in terms of nonspecific adsorption of enzyme-labeled antibodies to the solid phase, which is one of the main factors that influences the sensitivity of the sandwich assay method. Got the results. This is true even if HAp is blocked by bovine serum albumin after adsorbing the antibody to the solid phase.
This is because it is as effective as polystyrene.
（３）検出限界、測定範囲の評価 第６図は測定範囲および検出限界の比較操作図である。(3) Evaluation of detection limit and measurement range FIG. 6 is a comparison diagram of measurement range and detection limit.
以下にその操作を示す。第１図の操作で得られた抗ＣＥ
Ａ−ＩｇＧ被覆ＨＡｐボールおよび抗ＣＥＡ−１ｇＧ被
覆ポリスチレンボールをＣＥＡ標準液（Ｃ液で希釈）０
．１ｍｌとＣ液０．２艷に室温で６時間静置し、蒸留水
で洗浄後、Ｃ液で希釈した抗ＣＥＡ−１，ｇＧ−ＣＯＤ
標識抗体０．１−とＣ液０．２ｍＪに室温で１晩静置し
、蒸留水で洗浄後、０，５ｍｏｌ／ｌグルコース、０．
０１ｍｏｌ／ｌ酢酸緩衝液（ｐＨ５，１） ０．３ｍｌ
を加え、３７℃２時間静置した。０．１−サンプリング
し、２Ｘ１０−’ｌ１ｏｔ／ １ルミノール、０．２ｍ
ｏｌ／ｊ炭酸緩衝液（ｐ［１９，８） ０．５ｍｔｌ、
６Ｘ１０−”ｍｏｌ／ ｌフェリシアン化カリ水溶液０
．５ｍｌを各添加し、１５秒待ち、１６〜４５秒間の発
光量を算出する。The operation is shown below. Anti-CE obtained by the procedure shown in Figure 1
A-IgG coated HAp balls and anti-CEA-1gG coated polystyrene balls were mixed with CEA standard solution (diluted with C solution) 0
．． Anti-CEA-1, gG-COD diluted with 1 ml of C solution and 0.2 mL of C solution at room temperature for 6 hours, washed with distilled water, and diluted with C solution.
The labeled antibody 0.1- and C solution 0.2 mJ were allowed to stand overnight at room temperature, washed with distilled water, and then 0.5 mol/l glucose and 0.5 mol/l glucose were added.
0.3 ml of 01 mol/l acetate buffer (pH 5,1)
was added and allowed to stand at 37°C for 2 hours. 0.1-sampled, 2X10-'l1ot/1 Luminol, 0.2 m
ol/j carbonate buffer (p[19,8) 0.5mtl,
6X10-”mol/l potassium ferricyanide aqueous solution 0
．． Add 5 ml each, wait 15 seconds, and calculate the amount of luminescence for 16 to 45 seconds.
実施例２と比較例２について検出限界および測定範囲を
比較した。第７図は、その結果であり、ＣＥＡ濃度−発
光量変化図である。図において、（ｃ）は実施例２、（
ｄ）は比較例２の結果を示している。The detection limits and measurement ranges of Example 2 and Comparative Example 2 were compared. FIG. 7 shows the results and is a CEA concentration-emission amount change diagram. In the figure, (c) is Example 2, (
d) shows the results of Comparative Example 2.
図よりＨＡｐの方が、ポリスチレンよりＣＥＡの検出限
界、測定範囲いずれも優れていることが判明した。The figure shows that HAp is superior to polystyrene in both the detection limit and measurement range of CEA.
これは、ＨＡｐの抗体結合量が、大であることと、固相
への抗体結合の際、抗体の免疫活性が失われにくいこと
の２点が挙げられる。また、ＨＡｐとポリスチレンの測
定上限が同値なのは、添加した酵素標識抗体量が制限因
子になっているためと考えろ。This is due to two reasons: the amount of antibody binding to HAp is large, and the immunoactivity of the antibody is less likely to be lost when the antibody is bound to the solid phase. Also, the reason that the upper limits of measurement for HAp and polystyrene are the same is considered to be because the amount of enzyme-labeled antibody added is the limiting factor.
（４）検出限界における再現性の評価
実施例２と比較例２で検出限界における日内変動と日間
変動を比較した。結果を表２に示す。(4) Evaluation of reproducibility in detection limits In Example 2 and Comparative Example 2, intra-day and day-to-day variations in detection limits were compared. The results are shown in Table 2.
表２ ＨＡ ｐボールとポリスチレンボールの検出にお
ける再現性の比較
口内変動 測定回数ｎ ＝ ８
日間変動 測定回数ｎ ＝ ６
表中（）は、ＣＥＡｇ度を示す。Table 2 Comparison of reproducibility in detection of HA p balls and polystyrene balls Intraoral variation Number of measurements n = 8 Daily variation Number of measurements n = 6 In the table, () indicates the degree of CEAg.
検出限界でのＨＡｐボールの再現性が、ポリスチレンボ
ールよりも優れている。これは、ＨＡｐボールの抗体結
合量が多く、比較する固相の個体差が、ポリスチレンボ
ールよりも小さいためである。The reproducibility of HAp balls at the detection limit is better than polystyrene balls. This is because the amount of antibody bound to the HAp ball is large, and the individual differences in the solid phase to be compared are smaller than that of the polystyrene ball.
（５）固相の抗体結合ｍ整法の評価 比較例３゜ 第８図は、比較例３を示す操作方法図である。(5) Evaluation of solid phase antibody binding method Comparative example 3゜ FIG. 8 is an operating method diagram showing Comparative Example 3.
調整方法は、Ｂ液に４℃で１晩浸漬し、さらにＢ液で洗
浄した後、Ｃ液で３回洗浄した後、Ｃ液に浸漬し、４℃
で保存する。The adjustment method is to soak in liquid B overnight at 4°C, wash with liquid B, wash three times with liquid C, then soak in liquid C at 4°C.
Save with .
比較例４゜ 第９図は、比較例４を示す操作方法図である。Comparative example 4゜ FIG. 9 is an operating method diagram showing Comparative Example 4.
調整方法は、ＨＡｐボールをＡ液中に室温で、１晩浸漬
してポリリジン処理を行った。蒸留水で洗浄し、Ｂ液で
４℃で１晩浸漬した。さらにＢ液で洗浄した後、Ｃ液で
３回洗浄した後、Ｃ液に浸漬し、４℃で保存する。The preparation method was to immerse the HAp ball in liquid A at room temperature overnight to perform polylysine treatment. It was washed with distilled water and immersed in solution B at 4°C overnight. Further, after washing with B solution and three times with C solution, it is immersed in C solution and stored at 4°C.
比較例５゜ 第１０図は、比較例５を示す操作方法図である。Comparative example 5゜ FIG. 10 is an operation method diagram showing Comparative Example 5.
調整方法は、ＨＡｐボールを５％グルタルアルデヒド水
溶液に、３０℃で２時間浸漬してグルタルアルデヒド処
理を行った。蒸留水で洗浄し、Ｂ液４℃で１晩浸漬した
。さらにＢ液で洗浄した後、Ｃ液で３回洗浄した後、Ｃ
液に浸漬し、４℃で保存する。The preparation method was to perform glutaraldehyde treatment by immersing the HAp ball in a 5% aqueous glutaraldehyde solution at 30° C. for 2 hours. It was washed with distilled water and immersed in Solution B at 4°C overnight. After further washing with B solution and 3 times with C solution, C
Immerse in liquid and store at 4°C.
実施例２と比較例３，４，５について測定範囲及び検出
限界を第６図の方法で比較した。第１１図はその結果で
、処理条件の比較図である。The measurement range and detection limit of Example 2 and Comparative Examples 3, 4, and 5 were compared using the method shown in FIG. FIG. 11 shows the results and is a comparison diagram of the processing conditions.
図において、（ｅ）は比較例３、（ｆ）は比較例４、（
ｇ）は比較例５の結果を示している。In the figure, (e) is Comparative Example 3, (f) is Comparative Example 4, (
g) shows the results of Comparative Example 5.
図より、第１図の方法で、ＷｒＪ整したＨＡｐの方がＣ
ＥＡの検出限界′及び測定範囲のいずれにおいても優れ
ていることが判明した。From the figure, it can be seen that HAp with WrJ adjusted using the method shown in Figure 1 has a higher C.
It was found that the EA was excellent in both the detection limit' and measurement range.
第１図の調整法はボールをポリリジンで被覆後、グルタ
ルアルデヒドで架橋されるため、固相が安定する。この
様な安定した固相に結合した抗体により、抗体結合量及
び抗体結合力が増加した。In the preparation method shown in FIG. 1, the ball is coated with polylysine and then crosslinked with glutaraldehyde, so that the solid phase is stabilized. Antibody bound to such a stable solid phase increased the amount of antibody bound and the antibody binding strength.
今回は抗体量を検出するための標識物を酵素（ＧＯＤ）
の場合のみ記したが、酵素のみならず蛍光発光物質、放
射性同位元素でも同様な結果を得ている。以下に標識物
の例を示す。酵素（グルコースオキシダーゼ、西洋ワサ
ビペルオキシダーゼ、β−Ｄ−ガラクトシグーゼ、グル
コース６リン酸、脱水素酵素、アルカリホスファターゼ
等）、発光物ｆｆ１（フルオレセインイソチオシアネ−
１−１テトラメチルローダミンイソチオシアネート等）
、化学発光物質（アミノブチルエチルイソルミノール、
アミノブチルエチルイソルミノール、アミノペンチルエ
チルイソルミノール、アミノヘキシルエチルイソルミノ
ール等）、放射性同位元素（３Ｈ１”４Ｃ，”Ｐ、１２
’ｌｊ 、１３１ ■等）Ｈ発明の効果
ポリリジン層とグルタルアルデヒド層と抗体または抗原
層を順次形成した、生体親和性の高いへイドロキシアパ
タイトを固相としたので、ポリリジンの被覆量が上昇し
、グルタルアルデヒド処理によって、ポリリジンの一部
のアミノ基が互いにグルタルアルデヒドで、架橋される
ため同相が安定する。この様な安定した固相に結合した
抗体により、抗体結合量及び抗体結合力が増加する。This time, we will use an enzyme (GOD) as a label to detect the amount of antibodies.
Although only the case is described, similar results have been obtained not only with enzymes but also with fluorescent substances and radioactive isotopes. Examples of labeled objects are shown below. Enzymes (glucose oxidase, horseradish peroxidase, β-D-galactosigase, glucose 6-phosphate, dehydrogenase, alkaline phosphatase, etc.), luminescent substance ff1 (fluorescein isothiocyane-
1-1 tetramethylrhodamine isothiocyanate, etc.)
, chemiluminescent substances (aminobutylethylisoluminol,
aminobutylethylisoluminol, aminopentylethylisoluminol, aminohexylethylisoluminol, etc.), radioactive isotopes (3H1"4C,"P, 12
'lj, 131 ■, etc.) H Effects of the Invention Since the solid phase is hydroxyapatite, which has high biocompatibility and has a polylysine layer, a glutaraldehyde layer, and an antibody or antigen layer formed in sequence, the amount of polylysine covered increases. By treatment with glutaraldehyde, some amino groups of polylysine are crosslinked with each other with glutaraldehyde, so that the same phase becomes stable. Antibody bound to such a stable solid phase increases the amount of antibody binding and the antibody binding strength.
そのため、従来よりも測定範囲が拡大し、検出限界が上
昇し、再現性が向上するという効果がある。Therefore, the measurement range is expanded, the detection limit is increased, and the reproducibility is improved compared to the conventional method.
第１図（よ本発明の一実施例を示す免疫測定試薬の調整
方法図、第２図は競合反応による抗体結合量の測定操作
図、第３図はＨＡ ｐボールとポリスチレンボールの抗
体結合量変化図、第４図はＩｇＧ−ＧＯ［）標識抗体調
整方法図、第５図は非特異的吸着の評価方法の操作図、
第６図は測定範囲および検出限界の比較操作図、第７図
はＣＥＡ濃度−発光量変化図、第８図は比較例３を示す
操作方法図、第９図は比較例４を示す操作方法図、第１
０図は比較例５を示す操作方法図、第１１図はその結果
で、処理条件の比較図である。Figure 1 is a diagram of a method for preparing an immunoassay reagent showing an embodiment of the present invention, Figure 2 is a diagram of the procedure for measuring the amount of antibody binding by competitive reaction, and Figure 3 is a diagram of the amount of antibody binding between HA p balls and polystyrene balls. Figure 4 is a diagram of the IgG-GO[) labeled antibody preparation method, Figure 5 is a diagram of the method for evaluating non-specific adsorption,
Fig. 6 is a comparison operation diagram of measurement range and detection limit, Fig. 7 is a CEA concentration-light emission change diagram, Fig. 8 is an operation method diagram showing Comparative Example 3, and Fig. 9 is an operation method showing Comparative Example 4. Figure, 1st
FIG. 0 is an operating method diagram showing Comparative Example 5, and FIG. 11 is a comparison diagram of processing conditions, showing the results.
Claims (2)
は抗原層を順次形成したハイドロキシアパタイトを固相
としたことを特徴とする免疫測定用試薬。(1) An immunoassay reagent characterized in that the solid phase is hydroxyapatite in which a polylysine layer, a glutaraldehyde layer, and an antibody or antigen layer are sequentially formed.
Ｇとしたことを特徴とする特許請求の範囲第１項記載の
免疫測定用試薬。(2) The immunoassay reagent according to claim 1, wherein the antibody is anti-carcinoembryonic antigen-immunoglobulin G.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP28310787A JPH01126554A (en) | 1987-11-11 | 1987-11-11 | Reagent for immunological measurement |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP28310787A JPH01126554A (en) | 1987-11-11 | 1987-11-11 | Reagent for immunological measurement |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01126554A true JPH01126554A (en) | 1989-05-18 |
Family
ID=17661310
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP28310787A Pending JPH01126554A (en) | 1987-11-11 | 1987-11-11 | Reagent for immunological measurement |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01126554A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5540995A (en) * | 1993-10-05 | 1996-07-30 | Asahi Kogaku Kogyo Kabushiki Kaisha | Granular polymer composite and production process thereof as well as diagnostic agent |
| GB2307552A (en) * | 1995-11-21 | 1997-05-28 | Asahi Optical Co Ltd | Evaluating an immunoassay test sheet by immunoassay |
-
1987
- 1987-11-11 JP JP28310787A patent/JPH01126554A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5540995A (en) * | 1993-10-05 | 1996-07-30 | Asahi Kogaku Kogyo Kabushiki Kaisha | Granular polymer composite and production process thereof as well as diagnostic agent |
| US5897953A (en) * | 1993-10-05 | 1999-04-27 | Asahi Kogaku Kogyo Kabushiki Kaisha | Granular polymer composite and production process thereof as well as diagnostic agent |
| GB2307552A (en) * | 1995-11-21 | 1997-05-28 | Asahi Optical Co Ltd | Evaluating an immunoassay test sheet by immunoassay |
| GB2307552B (en) * | 1995-11-21 | 2000-02-23 | Asahi Optical Co Ltd | Method of evaluating a testing element for antigens or antibodies |
| US6040196A (en) * | 1995-11-21 | 2000-03-21 | Asahi Kogaku Kogyo Kabushiki Kaisha | Method of evaluating a testing element for antigens or antibodies |
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