JPH049262B2 - - Google Patents
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- Publication number
- JPH049262B2 JPH049262B2 JP9403882A JP9403882A JPH049262B2 JP H049262 B2 JPH049262 B2 JP H049262B2 JP 9403882 A JP9403882 A JP 9403882A JP 9403882 A JP9403882 A JP 9403882A JP H049262 B2 JPH049262 B2 JP H049262B2
- Authority
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- Prior art keywords
- crp
- solution
- serum
- sample
- absorbance
- Prior art date
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- 210000002966 serum Anatomy 0.000 claims description 30
- 238000000034 method Methods 0.000 claims description 18
- 238000002835 absorbance Methods 0.000 claims description 17
- 239000012086 standard solution Substances 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 9
- 238000011088 calibration curve Methods 0.000 claims description 8
- 239000012490 blank solution Substances 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 6
- 239000012224 working solution Substances 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 239000002504 physiological saline solution Substances 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 4
- 239000012085 test solution Substances 0.000 claims description 2
- 239000000523 sample Substances 0.000 description 12
- 238000005259 measurement Methods 0.000 description 8
- 238000004879 turbidimetry Methods 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000013024 dilution buffer Substances 0.000 description 4
- 238000000691 measurement method Methods 0.000 description 3
- 239000012723 sample buffer Substances 0.000 description 3
- 238000004062 sedimentation Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000760 immunoelectrophoresis Methods 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 235000004252 protein component Nutrition 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000008118 PEG 6000 Substances 0.000 description 1
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 210000003567 ascitic fluid Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 108010074605 gamma-Globulins Proteins 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 230000000409 histolytic effect Effects 0.000 description 1
- 230000000951 immunodiffusion Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- QYSGYZVSCZSLHT-UHFFFAOYSA-N octafluoropropane Chemical compound FC(F)(F)C(F)(F)C(F)(F)F QYSGYZVSCZSLHT-UHFFFAOYSA-N 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Description
本発明は血清CRPの簡易迅速定量法に係り、
殊に免疫比濁法を利用する定量法に係る。
周知のように、CRPは急性相反応性血漿蛋白
成分の一種であり、種々の炎症性及び組織崩壊性
疾患に際して出現する非特異的蛋白成分であつ
て、生体に上記疾患が生じた場合に６〜24時間以
内の短時間で増量しその回復に伴ない減量消失す
ると謂う特徴を有しており、従つてその検査は臨
床上不可欠とされている。
従来におけるCRP検査法としてはルーチン検
査法例えば毛細管法、ラテツクス凝集法、一元免
疫拡散法等が使用されて来たが、これら測定法で
はCRP値の変動を時間の経過と共に定量的に把
握することが不可能であつたり、測定感度、精度
や操作時間等の問題により特に低値域における
CRP値の変動の把握に難点がある等の欠陥があ
つた。
しかしながら最近では、検出方法の進歩により
血漿中の種々の微量蛋白が定量的に測定し得るよ
うになりつつある。この検出方法の代表例が比濁
法であり、既述のルーチン検査法に代つて普及し
つつある。
但し近年普及して来たこの比濁法はレーザー比
濁計等を用いているので高感度ではあるが、その
有用性については絶対的なものとは云えないのが
実情である。蓋し、機器が極めて高価であるのみ
ならず、測定精度や測定範囲に問題があり、又高
感度故に試薬や検体血清の澄明度に特別の注意を
払う必要があるのでその前処理が煩雑となる等の
欠点が存在するからであり、更にはレーザー比濁
法は脂濁血清、黄疸血清又は溶血血清を検体とす
る場合に不適当であり、又不活化しない血清では
可成りのバラツキの出ることが報告されているか
らである。
斯くて、本発明の目的は、免疫比濁法を利用す
るものではあるが、特殊にして高価な機器を必要
とせずに、安定且つ良好な精度にて実施し得る血
清CRPの比較的迅速な定量法を提供することで
ある。
本発明によれば、この目的は検体血清と抗血清
使用液との混合液である被験液Ｔと、検体血清と
検体ブランク緩衝液との混合液である検体ブラン
ク液SBと、生理食塩液と抗血清使用液との混合
液である試薬ブランク液RBと、生理食塩液と検
体ブランク緩衝液との混合液である緩衝液ブラン
ク液BBとを調製し、これらをそれぞれインキユ
ベートしてプラトーに達せしめた後に、それぞれ
の液の吸光度（Ａ、TASB、ARB及びABB）を測定
し、CRPによる検体血清の吸光度（ACRP）を式
ACRP＝AT−ASB−（ARB−ABB）として算定し、
一方上記と同様に、但し検体血清の代わりに各種
濃度のCRP標準液を用いて吸光度―CRP値の関
係を示す検量線を予め作成しておき、上記の算定
吸光度値に該当するCRP値を上記の検量線から
求めることにより達成される。
抗原―抗体複合体は光を消散し或は吸収する
が、同じ濁度（吸光度）は抗原過剰状態でも或い
は抗体過剰状態でも得られる可能性がある。免疫
比濁法による血清CRPの定量に際しては吸光度
測定を抗体過剰状態で行ない抗原過剰状態のもの
とは之を区別せねばならない。従つて、このため
には特異性が高く且つ高力価の抗血清が必要とさ
れる。
本発明方法に使用されるこの種の抗血清として
は例えば抗ヒトCRPヤギ血清から得られたγ―
グロプリン分画がある。
次に、試薬、標準液測定方法、結果等に関連し
て本発明方法を更に詳細に説明する。
ａ） 試薬及び標準液
１） 緩衝液
） 抗血清希釈緩衝液
HEPES bufferPH7.1
0.01M HEPES
0.1M NaCl，0.1％ NaN3
） 検体緩衝液
PEG6000 ３％
Tween20 0.2％
） 検体ブランク緩衝液
（抗血清希釈緩衝液を検体緩衝液で11倍に希釈
したもの）
２） 希釈液
CRP標準液及び検体の希釈用であつて、
生理食塩水をベースとする３％ウシアルブミ
ン液とヒトプール血清（CRP除去）との７
対３混液
３） 生理食塩液
４） CRP1次標準液
ガン患者のプール腹水からイオン交換クロマ
ト、ゲル過でCRPを純化したもの。その
純度はアクリルアミド電気泳動法、オクタロ
ニー法、免疫泳動法で確認され、このCRP
標準液の濃度は蛋白定量、アフイニテイーク
ロマトグラフイー及びSRID法により決定さ
れた。
５） CRP2次標準液
プール腹水から硫酸アンモニウム分画により
得た粗CRPを希釈液で一定の濃度に希釈し
たものであつて、その濃度は１次標準液を用
いSRID法により決定された。この２次標準
液は４℃に維持する場合には少なくとも６ケ
月間に亘り安定である。
６） 抗CRP血清
抗ヒトCRPヤギ血清から得たγ―グロブリ
ン分画を抗血清希釈緩衝液に溶解したもので
あつて、その特異性はオクタロニー法及び免
疫電気泳動法により確認された。
７） 抗血清原液
抗血清希釈緩衝液により２倍に希釈された抗
CRP血清。この原液は４℃に維持する場合
には少なくとも12ケ月間安定である。
８） 抗血清使用液
検体緩衝液により抗血清原液を11倍に希釈
し、ミリポアフイルタ（ボアサイズ0.45μm）
で過したもの。
ｂ） 測定法
下記表
The present invention relates to a simple and rapid method for quantifying serum CRP,
In particular, it relates to a quantitative method using immunoturbidimetry. As is well known, CRP is a type of acute phase reactive plasma protein component, and is a non-specific protein component that appears in various inflammatory and histolytic diseases. It has the characteristic that the weight increases in a short period of up to 24 hours, and the weight loss disappears as the weight recovers, so its examination is considered clinically essential. Conventional CRP testing methods have used routine testing methods such as the capillary tube method, latex agglutination method, and one-way immunodiffusion method, but these measurement methods do not allow quantitative understanding of changes in CRP values over time. Especially in the low value range, it may be impossible to
There were flaws, such as difficulty in understanding changes in CRP levels. However, recently, advances in detection methods have made it possible to quantitatively measure various trace proteins in plasma. A typical example of this detection method is turbidimetry, which is becoming popular in place of the routine testing methods described above. However, although this nephelometric method, which has become popular in recent years, is highly sensitive because it uses a laser nephelometer, etc., the reality is that its usefulness cannot be said to be absolute. Not only is the equipment extremely expensive, but there are also problems with measurement accuracy and measurement range, and because of its high sensitivity, special attention must be paid to the clarity of reagents and sample serum, making pretreatment complicated. Furthermore, laser turbidimetry is unsuitable when using lipid-turbid serum, icteric serum, or hemolyzed serum as a specimen, and there is considerable variation in serum values that are not inactivated. This is because it has been reported. Therefore, although the present invention utilizes immunoturbidimetry, it is a relatively rapid method for measuring serum CRP that can be performed stably and with good accuracy without the need for specialized and expensive equipment. The objective is to provide a quantitative method. According to the present invention, this purpose consists of a test solution T, which is a mixture of sample serum and an antiserum working solution, a sample blank solution SB, which is a mixture of sample serum and a sample blank buffer solution, and a physiological saline solution. Prepare a reagent blank solution RB, which is a mixture with the antiserum working solution, and a buffer blank solution BB, which is a mixture of physiological saline and sample blank buffer, and incubate each of these to reach a plateau. After that, the absorbance of each solution (A, T A SB , A RB and A BB ) is measured, and the absorbance of the sample serum due to CRP (A CRP ) is calculated using the formula A CRP = AT − A SB − (A RB − A BB ),
On the other hand, in the same way as above, however, a calibration curve showing the relationship between absorbance and CRP value is created in advance using CRP standard solutions of various concentrations instead of the sample serum, and the CRP value corresponding to the calculated absorbance value is calculated as above. This is achieved by determining from the calibration curve of Antigen-antibody complexes dissipate or absorb light, but the same turbidity (absorbance) can be obtained in antigen-rich or antibody-rich conditions. When quantifying serum CRP by immunoturbidimetry, it is necessary to measure absorbance in a state of excess antibody and to distinguish it from a state of excess antigen. Therefore, highly specific and high titer antisera are required for this purpose. Examples of this type of antiserum used in the method of the present invention include γ-
There is a glopurin fraction. Next, the method of the present invention will be explained in more detail in relation to reagents, standard solution measurement methods, results, etc. a) Reagents and standard solutions 1) Buffer solution) Antiserum dilution buffer HEPES bufferPH7.1 0.01M HEPES 0.1M NaCl, 0.1% NaN 3 ) Sample buffer PEG6000 3% Tween20 0.2%) Sample blank buffer (antiserum dilution Buffer solution diluted 11 times with sample buffer solution) 2) Dilution solution For diluting CRP standard solution and sample,
7 with saline-based 3% bovine albumin solution and human pool serum (CRP removed)
Mixture of 3 pairs 3) Physiological saline 4) CRP primary standard solution CRP purified from pooled ascitic fluid of cancer patients by ion exchange chromatography and gel filtration. Its purity was confirmed by acrylamide electrophoresis, Ouchterlony's method, and immunophoresis, and this CRP
The concentration of the standard solution was determined by protein quantification, affinity chromatography, and SRID method. 5) CRP secondary standard solution pool Crude CRP obtained from ascites by ammonium sulfate fractionation was diluted with a diluent to a certain concentration, and the concentration was determined by the SRID method using the primary standard solution. This secondary standard solution is stable for at least 6 months when maintained at 4°C. 6) Anti-CRP serum Anti-human CRP A γ-globulin fraction obtained from goat serum was dissolved in an antiserum dilution buffer, and its specificity was confirmed by the Ouchterlony method and immunoelectrophoresis. 7) Antiserum stock solution Antiserum diluted 2 times with antiserum dilution buffer
CRP serum. This stock solution is stable for at least 12 months when maintained at 4°C. 8) Antiserum working solution Dilute the antiserum stock solution 11 times with sample buffer and filter it through a Millipore filter (bore size 0.45 μm).
What I spent there. b) Measurement method Table below
【表】
液
[Table] Liquid
【表】
ク緩衝液
生理食塩液 − − 50μ 50μ
に見られる４種の液を調製し、各液を37℃でイ
ンキユベートした後に波長340nmでの吸光度を
測定し、検体血清の吸光度を式
ACRP＝AT−ASB−（ARB−ABB）
により算定し、一方上記と同様にして但し血清
の代りにCRP2次標準液を用いて操作して吸光
度−CRP濃度に関する検量線を作成し、上記
算定吸光度値に該当するCRP値を上記検量線
を利用して求めることにより測定する。
ｅ） 結果
） 経時変化
CRP2次標準液を用いそのCRP濃度が164
mg／(a)、63mg／(b)、15mg／(c)及び４
mg／(d)の場合の吸光度変化（AT−ASB）に
ついて、又ARB−ABB(e)について120分間に亘
り追究した処、反応は略々15分間で一定とな
り、その後は吸光度が変化しないことが判明
した（第１図参照）。これはインキユベート
時間が15分程度で充分なことを示している。
） 沈降素曲線
抗血清使用液ｆ及び希釈抗血清使用液ｇ
（検体ブランク緩衝液にて抗血清使用液を
１／２に希釈したもの）を用いて沈降素曲線
を追究した処、第２図に見られる通りであ
り、曲線は比較的緩やかであり、従つてこの
抗血清は免疫比濁法に使用するに当つて充分
な高力価を有し且つ測定範囲の汎いものであ
ることが判る。
） 検 線
CRP2次標準液を用いてそのCRP濃度が
8.0，29.5，65.0，114.5，164.0及び220.0mg／
のものを調製し、１日１回とし５日間に亘
り吸光度を測定し、その平均値をCRP値に
関してプロツトした処、第３図に示される通
りであり極めて安定した直線性が得られた。
尚CRP値が220mg／以上となると次第にスロ
ープを呈する。
） 測定値と算定値との相関性
CRP2次標準液を希釈してCRP値が７段階
の検体（即ちCRP値が220，163，122，62，
36，17及び９mg／のもの）を調製し、測定
値と算定値との関係につき５回の平均値をプ
ロツトした処、第４図に見られる通り安定し
た直線が得られた。
このことは、本方法がCRP値が220mg／
又はそれ以下の検体を良好な精度で測定し得
ることを示している。
） 干渉物質の影響
ビリルビン及びヘモグロビンを血清に添加
して測定したが、これら添加物質がCRP値
に及ぼす影響は第５図ａ及びｂに示されるよ
うに殆んど見られなかつた。
尚、脂濁血清、黄疸血清及び溶血血清に関
する本方法による測定値は次の各表に示され
る通りSRID法による測定値と類似した値を
示した（レーザー比濁法による測定値とも相
関関係の存在を示している。但し脂濁血清に
関してはレーザー比濁法では測定不能であつ
た）。
これらの事実は、レーザー比濁法で必要と
される検体血清の特別な前処理が、本発明で
は不要であることを如実に示している（表中
の数値の単位はmg／である）。[Table] Buffer saline − − 50μ 50μ
After incubating each solution at 37 °C , the absorbance at a wavelength of 340 nm was measured . ), and on the other hand, create a calibration curve for absorbance-CRP concentration by performing the same procedure as above, but using CRP secondary standard solution instead of serum, and calculate the CRP value corresponding to the above calculated absorbance value using the above calibration curve. It is measured by finding it using . e) Results) Change over time Using CRP secondary standard solution, the CRP concentration was 164
mg/(a), 63mg/(b), 15mg/(c) and 4
When we investigated the change in absorbance (A T - A SB ) in the case of mg/(d) and A RB - A BB (e) over a period of 120 minutes, we found that the reaction became constant in about 15 minutes, and after that the absorbance decreased. It was found that there was no change in (see Figure 1). This indicates that an incubation time of about 15 minutes is sufficient. ) Sedimentation curve Antiserum working solution f and diluted antiserum working solution g
(Antiserum solution diluted to 1/2 with sample blank buffer) was used to investigate the sedimentation curve, as shown in Figure 2, and the curve was relatively gentle. It can be seen that this antiserum has a sufficiently high titer for use in immunoturbidimetry and has a wide measurement range. ) Check line Check the CRP concentration using the CRP secondary standard solution.
8.0, 29.5, 65.0, 114.5, 164.0 and 220.0mg/
The absorbance was measured once a day for 5 days, and the average value was plotted against the CRP value. As shown in Figure 3, extremely stable linearity was obtained. Furthermore, when the CRP value exceeds 220mg/, a slope gradually appears. ) Correlation between measured values and calculated values Dilute the CRP secondary standard solution and prepare samples with CRP values in 7 levels (i.e. CRP values of 220, 163, 122, 62,
36, 17, and 9 mg/) were prepared, and the average value of five measurements was plotted for the relationship between the measured value and the calculated value, and a stable straight line was obtained as shown in Figure 4. This means that this method can reduce the CRP value to 220mg/
This shows that it is possible to measure samples with good accuracy. ) Effects of Interfering Substances Bilirubin and hemoglobin were added to serum for measurement, but as shown in Figures 5a and b, almost no effect of these added substances on CRP values was observed. The values measured using this method for lipid-turbid serum, icteric serum, and hemolytic serum showed values similar to those measured using the SRID method, as shown in the following tables (there is also a correlation with the measured values using laser turbidimetry). (However, it was not possible to measure lipid-turbid serum using laser turbidimetry). These facts clearly show that the present invention does not require special pretreatment of the sample serum required in laser turbidimetry (the units of numerical values in the table are mg/).
【表】【table】
【表】
叙上のように、本発明方法では測定値と計算値
とが極めて良好な直線的相関関係を呈するので、
機器として分光光度計さえあれば血清CRPの定
量測定が可能であり、これに検量線記憶、自動ブ
ランク補正、プリントアウト等の装置が付属して
いれば、その測定を迅速ならしめることができ、
更には測定自体の自動化が可能である。[Table] As mentioned above, in the method of the present invention, there is an extremely good linear correlation between the measured values and the calculated values.
Quantitative measurement of serum CRP is possible with just a spectrophotometer as an instrument, and if it is equipped with devices such as calibration curve storage, automatic blank correction, and printout, the measurement can be done quickly.
Furthermore, the measurement itself can be automated.
第１図はCRP濃度と吸光度との経時変化を示
すグラフ、第２図は抗血清液の沈降素曲線を示す
グラフ、第３図は検量線をその経日測定再現性と
共に示すグラフ、第４図は測定値と計算値との関
係を示すグラフ、第５図は干渉物質のCRP値に
及ぼす影響を示すグラフであつて、第５図ａ及び
第５図ｂは干渉物質としてそれぞれビリルビン及
びヘモグロビンを添加した場合を示したグラフで
ある。
Figure 1 is a graph showing the change in CRP concentration and absorbance over time, Figure 2 is a graph showing the sedimentation curve of the antiserum solution, Figure 3 is a graph showing the calibration curve along with its reproducibility over time, and Figure 4 is a graph showing the change in CRP concentration and absorbance over time. The figure is a graph showing the relationship between measured values and calculated values, and Figure 5 is a graph showing the influence of interfering substances on CRP values. It is a graph showing the case where is added.
Claims (1)
験液Ｔと、検体血清と検体ブランク緩衝液との混
合液である検体ブランク液SBと、生理食塩液と
抗血清使用液との混合液である試薬ブランク液
RBと、生理食塩液と検体ブランク緩衝液との混
合液である緩衝液ブランク液BBとを調製し、こ
れらをそれぞれインキユベートしてプラトーに達
せしめた後に、それぞれの液の吸光度（Ａ、TASB、
ARB及びABB）を測定し、CRPによる検体血清の
吸光度（ACRP）を式 ACRP＝AT−ASB−（ARB−ABB） として算定し、一方上記と同様に、但し検体血清
の代わりに各種濃度のCRP標準液を用いて吸光
度―CRP値の関係を示す検量線を予め作成して
おき、上記の算定吸光度値に該当するCRP値を
上記の検量線から求めることを特徴とする、免疫
比濁法による血清CRPの簡易迅速定量法。[Scope of Claims] 1. Test solution T, which is a mixture of sample serum and antiserum solution, sample blank solution SB, which is a mixture of sample serum and sample blank buffer solution, and physiological saline solution and antiserum. Reagent blank solution that is a mixture with the working solution
Prepare RB and buffer blank solution BB, which is a mixture of physiological saline and sample blank buffer solution, and incubate them to reach a plateau. Then, calculate the absorbance (A, T A SB ,
A RB and A BB ) were measured, and the absorbance of the sample serum by CRP (A CRP ) was calculated using the formula A CRP = AT − A SB − (A RB − A BB ), while as above, except that the sample serum A calibration curve showing the relationship between absorbance and CRP value is created in advance using CRP standard solutions of various concentrations instead of serum, and the CRP value corresponding to the above calculated absorbance value is determined from the above calibration curve. A simple and rapid method for quantifying serum CRP using immunoturbidimetry.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9403882A JPS58211659A (en) | 1982-06-03 | 1982-06-03 | Handy and quick assay of serum crp by immunological nephelometry |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9403882A JPS58211659A (en) | 1982-06-03 | 1982-06-03 | Handy and quick assay of serum crp by immunological nephelometry |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS58211659A JPS58211659A (en) | 1983-12-09 |
| JPH049262B2 true JPH049262B2 (en) | 1992-02-19 |
Family
ID=14099391
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP9403882A Granted JPS58211659A (en) | 1982-06-03 | 1982-06-03 | Handy and quick assay of serum crp by immunological nephelometry |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS58211659A (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6125061A (en) * | 1984-07-13 | 1986-02-03 | Nitsusui Seiyaku Kk | Improved assay of serum crp by immunonephelometry |
| JPS61243363A (en) * | 1985-04-22 | 1986-10-29 | Nitsusui Seiyaku Kk | Highly sensitive assay of crp |
| JP2607363B2 (en) * | 1986-02-20 | 1997-05-07 | 日水製薬 株式会社 | CRP measurement by immunoturbidimetry |
| EP0264866A3 (en) * | 1986-10-24 | 1990-12-27 | BEHRINGWERKE Aktiengesellschaft | Method for the determination of the presence of diagnostically relevant substances, particularly antibodies or antigens, by the "elisa" method using photometric evaluation |
| US6777198B2 (en) | 2000-10-11 | 2004-08-17 | Pharmacia Diagnostics Ab | Assay method and kit therefor |
| SE0003662D0 (en) * | 2000-10-11 | 2000-10-11 | Pharmacia Diagnostics Ab | Assay method and kit for that |
| US8865428B2 (en) | 2008-08-01 | 2014-10-21 | National University Corporation Okayama University | Protein production method, fusion protein, and antiserum |
-
1982
- 1982-06-03 JP JP9403882A patent/JPS58211659A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS58211659A (en) | 1983-12-09 |
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