GB1572220A - Creation of Inventions and Patents with Artificial Intelligence

GB1572220A – Immunochemical process of measuring physiologically active substances
– Google Patents

GB1572220A – Immunochemical process of measuring physiologically active substances
– Google Patents
Immunochemical process of measuring physiologically active substances

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Info

Publication number
GB1572220A

GB1572220A
GB40344/77A
GB4034477A
GB1572220A
GB 1572220 A
GB1572220 A
GB 1572220A
GB 40344/77 A
GB40344/77 A
GB 40344/77A
GB 4034477 A
GB4034477 A
GB 4034477A
GB 1572220 A
GB1572220 A
GB 1572220A
Authority
GB
United Kingdom
Prior art keywords
substance
antibody
measured
bindable
antigen
Prior art date
1976-10-07
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.)

Expired

Application number
GB40344/77A
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

Mochida Pharmaceutical Co Ltd

Original Assignee
Mochida Pharmaceutical Co Ltd
Priority date (The priority date 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 date listed.)
1976-10-07
Filing date
1977-09-28
Publication date
1980-07-30

1976-10-07
Priority claimed from JP51120622A
external-priority
patent/JPS607229B2/en

1976-10-07
Priority claimed from JP12062176A
external-priority
patent/JPS5347518A/en

1977-09-28
Application filed by Mochida Pharmaceutical Co Ltd
filed
Critical
Mochida Pharmaceutical Co Ltd

1980-07-30
Publication of GB1572220A
publication
Critical
patent/GB1572220A/en

Status
Expired
legal-status
Critical
Current

Links

Espacenet

Global Dossier

Discuss

238000000034
method
Methods

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title
claims
description
58

230000000984
immunochemical effect
Effects

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title
claims
description
6

239000013543
active substance
Substances

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title
description
2

239000000126
substance
Substances

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claims
description
77

238000006243
chemical reaction
Methods

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claims
description
34

230000000694
effects
Effects

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claims
description
21

238000002372
labelling
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claims
description
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239000003795
chemical substances by application
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claims
description
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reaction mixture
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claims
description
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mixture
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description
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description
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description
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material
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description
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antigenic effect
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description
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processed proteins & peptides
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description
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processed proteins & peptides
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claims
description
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amines
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claims
description
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glycans
Chemical class

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claims
description
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isolation
Methods

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claims
description
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description
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description
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polysaccharide
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insulin
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antigens
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antigens
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solution
Substances

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insulin
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measurement
Methods

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dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride
Chemical compound

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phosphate buffered saline
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Methods

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testing method
Methods

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Polysorbate 20
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polyoxyethylene sorbitan monolaurate
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polyoxyethylene sorbitan monolaurate
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suspension
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physiological saline solution
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cellulose
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Hydrogen peroxide
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dilution
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substrate
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Polyethylene
Substances

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enzyme
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incubation
Methods

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polyethylene
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Carbon
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description
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Sodium Carbonate
Chemical compound

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0.000
description
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radioimmunoassay
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separation method
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test solution
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Glycine
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fluid
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mesalamine
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Ethylene glycol
Chemical compound

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Human genes

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Phosphoric Monoester Hydrolases
Proteins

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Sodium Sulfate
Chemical compound

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Sodium hydroxide
Chemical compound

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centrifugation
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immunoglobulin
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proteins and genes
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proteins and genes
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receptors
Human genes

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receptors
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sodium carbonate
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sodium sulfate
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sodium sulphate
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testis
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Alkaline Phosphatase
Human genes

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Hydrogen
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Streptococcus agalactiae IgA FC receptor
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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

G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

G01N33/54306—Solid-phase reaction mechanisms

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

G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic

Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC

Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

Y10S435/00—Chemistry: molecular biology and microbiology

Y10S435/971—Capture of complex after antigen-antibody reaction

Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC

Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

Y10S436/00—Chemistry: analytical and immunological testing

Y10S436/815—Test for named compound or class of compounds

Y10S436/817—Steroids or hormones

Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC

Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

Y10S436/00—Chemistry: analytical and immunological testing

Y10S436/815—Test for named compound or class of compounds

Y10S436/817—Steroids or hormones

Y10S436/818—Human chorionic gonadotropin

Description

PATENT SPECIFICATION ( 11) 1 572 220
o ( 21) Application No 40344/77 ( 22) Filed 28 Sep 1977 ( 19) > ( 31) Convention Application No’s 51/120621 ( 32) Filed 7 Oct 1976 i /, 51/120622 CA ( 33) Japan (JP) C ( 44) Complete Specification Published 30 Jul1980 I t, ( 51) INT CL 3 G 01 N 33/54 /E V-1 C 07 G 7/00 C 12 N 11/00 ( 52) Index at Acceptance G 1 B BA CB C 3 H 202 203 220 242 H 1 HX 1 HX 2 ( 54) IMMUNOCHEMICAL PROCESS FOR MEASURING PHYSIOLOGICALLY ACTIVE SUBSTANCES ( 71) We, MOCHIDA SEIYAKU KABUSHIKI KAISHA, a Corporation duly organized and existing under the laws of Japan, having its principal office at 7 banchi, 1-chome, Yotsuya, Shinjuku-ku, Tokyo, Japan do hereby declare the invention for which we pray that a patent may be granted to us and the method by which it is to be performed to be
particularly described in and by the following statement: 5
Substances such as insulin, chorionic gonadotropin, growth hormone, fetoprotein and immunoglobulin having antigenicity hereinafter referred to as antigenic substances bind to the antibodies against these substances very specifically and very sensitively Taking advantage of these features, numerous processes have been developed to measure the antigenic substances or their antibodies and many immunochemical measuring processes are already 10 in practical use To mention some examples, there are, immunodiffusion methods, in which the antigen and the antibody are caused to react with each other in agar gel; agglutination reactions and agglutination inhibition reaction methods, in which blood cells or fine particles like polystyrene latex are utilized as carrier of antigen or antibody; radioimmunoassay (RIA), in which radioisotopes are employed to label the antigen or antibody; emzyme 15 immunoassay (EIA), in which enzymes are employed for the same purpose; and fluorescence immunoassay, in which fluorescent materials are employed for same purpose.
Meanwhich there is a competitive protein binding assay, in which a receptor protein or binding protein, i e, a protein which specifically binds to the substance to be measured is utilized instead of the antibody in the immunochemical process 20 These processes with respective characters are finding wide applications Among others, RIA and EIA, which are far superior in the sensitivity of measurement and quantitative precision, are widely used, the substances measurable by them ranging from high molecular substances such as protein hormones, virus, immunoglobulins to low molecular ones such as peptides, steroids, synthetic medicines 25 The principle, which is common to RIA and EIA, is characterized by two methods; competitive method and sandwich method.
The present specification concerns a case of the antigen as the substance to be measured and the antibody as the substance specifically bindable with said antigen Not being confined to this case alone, however, the present invention is applicable equally to a case of the 30 antibody as the substance to measured and of the antigen as the bindable substance with said antibody; to an antigen-antibody system; and even to a physiologically active substance-receptor protein system.
Next, referring to a drawing, the competitive method and the sandwich method in RIA and in EIA are to be described 35 As schematically illustrated in Fig 1, the competitive method works such that an unknown amount of unlabeled antigen 1 and a given amount of labeled antigen 2 which is labeled by labeling agent are caused to react competitively with a given amount of the corresponding antibody 3 In this case, the unlabeled antigen 1 and the labeled antigen 2 bind to the antibody 3 in proportion to their respective amounts and thereby the amount of 40 the labeled antigen 2 to bind to the antibody 3 is reversely proportional to the amount of the unlabeled antigen 1 Next, by appropriate way the bound labelled antigen 2 a and the free labeled antigen 2 are separated and the activity of the labeling agent in either of the two is measured In the meantime a dilution series of reference substance, whose concentration is known, is prepared and in the same way described above the activity of labeling 45 2 1,572,220 2 agent in each dilution is measured A standard curve obtained by plotting the measured activities is utilized for determining the amount of the substance to be measured.
In the competitive method, the antigen is measured by causing the antigen to be measured and the labeled antigen to compete one with another for binding to the antibody If the antibody used is one insolubilized, the antigen can be measured in the same way; moreover, 5 the antibody too can be measured by causing the antibody to be measured and the insolubilized antibody to compete one with another for binding to the labeled antigen U S Patents No 3,654,090 and No 3,850,752 disclose the processes based on this principle In these processes, the labeled substance directly binds to the insolubilized antibody, and this binding is competed by the binding of the substance to be measured to the insolubilized anti 10 body and/or to labeled substance Thus the amount of the labeled substance to bind to the insolubilized antibody is in reverse proportion to the amount of the substance to be measured.
Next the sandwich method works, as schematically illustrated in Fig 2, such that an unknown amount of unlabeled antigen 1 to be measured is caused to react with the insol 15 ubilized antibody 4, i e, an antibody to the antigen which has been insolubilized Both come to bind to each other, yielding an antigen-antibody complex 5 (the first reaction) The complex 5 is separated from the reaction mixture and caused to react with a given amount of the labeled antibody 6 in which an antibody to antigen to be measured is bound to a labeling agent (the second reaction) The labeled antibody 6 binds to said complex 5, but a 20 part of it which exceeds the bindability of the insoluble complex 5 remains in free state in the solution without binding to the insoluble complex 5 Next the bound labeled antibody 6 a and the free labeled antibody 6 are separated and the activity of labeling agent in either of them is measured In the meantime a dilution series of reference substance, whose concentration is known, is prepared and in the same way described above the activity of 25 labeling agent in each dilution is measured A standard curve obtained by plotting the measured activities is utilized for determining the amount of the substance to be measured.
In the sandwich method, since the labelled substance does not directly bind to the insolubilized antibody but does so through the substance to be measured, the amount of the labeled substance to bind to the insolubilized antibody will be proportional to the amount of 30 the substance to be measured.
U.S Patent No 3,791,932 discloses a process based on this principle.
Whereas in the competitive method of change in the activity of the labeling agent corresponding to increase or decrease of the amount of the antigen to be measured is little and accordingly with a mild slope of the standard curve the sensitivity of measurement is 35 relatively low, in the sandwich method a change in the amount of the antigen to be measured directly corresponds to increase or decrease in the activity of the labeling agent and accordingly with a steep slope of the standard curve the sensitivity of measurement is high.
In the conventional sandwich method, however, in which after the first reaction between the antigen to be measured and the insolubilized antibody the antigenantibody complex 40 yielded has to be separated from the reaction mixture, the upper limit of measurable amount of antigen is 102-103 times the lower limit Meanwhile, the upper limit of the amount of antigen to be measured may often amount to 105 _ 106 times the lower limit For instance the blood level of a fetoprotein is in normal humans less than 1 x 101 ng/ml, but in a patient with primary hepatoma it may reach even 1 x 106 ng/ml; in the latter case the 45 specimens at several levels of dilution have to be prepared so that they can be made measurable by the process, whereby repeated dilution is likely to lower the precision of measurement.
According to the present invention there is provided an immunochemical measuring process which comprises; 50 firstly reacting a bindable substance to be measured with either (a) an insolubilized substance which specifically binds the substance to be measured, or (b) with a labelled substance which is bindable to said bindable substance, and secondly reacting the resultant product without isolation with the substance (a) or (b) not utilized in said first reaction, and finally separating the labelled substance which has been 55 bound to the insolubilized substance and the bindable substance from the labelled substance which has not been so bound, and measuring the activity of either of said separated substances.
In one aspect of the invention the bindable substance to be measured is reacted with an insolubilised substance specifically binding the bindable substance and thereafter reacting 60 the mixture so produced with said labelled substance.
In another aspect of the invention said bindable substance is reacted with said labelled substance and the resulting reaction mixture is then reacted with said insolubilised substance In a modification said bindable substance is reacted simultaneously with said labelled substance and said insolubilised substance 65 3 1,572,2203 Following is a description with reference to the accompanying drawings and by way of example only of methods of carrying the invention into effect.
In the drawings:
Figure 1 is a pattern illustrating the principle of the competitive method of the prior art.
Figure 2 is a pattern illustrating the principle of the sandwich method of the prior art 5
Figure 3 is a pattern illustrating the principle of the measuring process according to the present invention.
Figure 4 shows a standard curve for AFP measurement in the Example 1.
Figure 5 shows the standard curves for AFP measurement in the Example 3 ( A) and Comparative case 1 (B) 10 Figure 6 shows the results of radioactivity measurement in the Example 4.
Figure 7 shows the results of absorbancy measurement in the Example 5.
Figure 8 shows a standard curve for insulin measurezent in the Example 6.
Figure 9 shows a standard curve for h CG measurement in the Example 7.
Referring to Figure 2 and 3, the principle of the process according to the present inven 15 tion is to be described.
First an unknown amount of the antigen 1 to be measured and a given amount of the insolubilized antibody 4 to said antigen are to be caused to react one with another Thereby if the amount of said antigen 1 is smaller than that of said insolubilized antibody 4, the unknown amount of said antigen 1 should be measured in accordance with the principle of 20 the sandwich method illustrated in Fig 2 (without separating the antigenantibody complex from the reaction mixture).
Next if the amount of said antigen 1 is larger than that of said insolubilized antibody 4, as shown in Fig 3, a given amount of a labeled antibody 6 is caused to react with the mixture of them in the presence of a free antigen 1 in addition to the antigenantibody complex 5 25 and in consequence a competition to bind to the labeled antibody 6 takes place between the antigen-antibody complex 5 and said free antigen 1 Thereby the more the free antigen 1, the less becomes the amount of the labeled antibody 6 which binds to the antigen-antibody complex 5 Thus the activity of the labeling agent which has bound to the antigen-antibody complex will decrease in reverse proportion to an increase in the amount of the antigen 30 Namely in the present invention the appropriate principle can be selected depending on the quantitative relation between antigen and antibody When the amount of the antigen to be measured is smaller than that of insolubilized antibody in the reaction system, the principle of the sandwich method works; and when vice versa, the principle of the competitive method does In a case of measuring the activity of the labeling agent which has bound 35 to the antigen-antibody complex, the standard curve makes an inverted Vletter with an increase in the amount of the antigen to be measured; and in the case of measuring the activity of the labeling agent which has not bound to it, the standard curve makes a V-letter with an increase in the amount of the antigen to be measured.
In the present invention unlike in the U S patent No 3,654,090, even when working on 40 the principle of the competitive method the labeled substance binds to the insolubilized antibody through the substance to be measured just as in the sandwich method Thus with selection of the binding mode it is possible to combine the sandwich method with the competitive method and accordingly to enlarge the range of the measurable amount of the substance to be measured 45 Moreover in the present invention which is characterized by no separation of the antigen-antibody complex from the reaction mixture it is possible to reverse the order of the first and second reactions or to execute them simultaneously Namely, the antigen to be measured and the labeled antibody are caused to react together to form an antigenantibody complex in the first reaction; and without separation of said complex from the 50 reaction mixture said complex is continuously caused to react with the insolubilized antibody in the second reaction; or the first and the second reaction take place by causing the antigen to be measured to react with a preliminaryily obtained mixture of the labeled antibody and the insolubilized antibody Next the complex of labeled antibody and the antigen to be measured and the insolubilized antibody is separated from the reaction 55 mixture and then the activity of the labeling agent in said complex or that of the labeling agent remaining in the reaction mixture is measured.
According to this process, the reaction between the antigen to be measured and the labeled antibody takes place in liquid-liquid phase Therefore with the reaction fully promoted, a greater amount of the labeled antibody binds to the antigen, resulting in a steep 60 enough slope of the standard curve and in an increased sensitivity of measurement.
For the purpose of separating the labeled antibody which has bound to the antigenantibody complex from the labeled antibody which has not, usually an insolubilized antibody, i e, an antibody which has been insolubilized as the result of binding to a carrier such as polysaccharides (for instance, cellulose, agarose, dextran) or plastics (for instance, polys 65 1,572,220 A 1,572,220 4 tyrene, polyethylene, polypropylene, polyvinylchloride, polyacetal, acrylonitrilebutadiene-styrene copolymer) is used.
As the labeling agent the following are available; radioisotopes (for instance, 125 j 1311, 3 H, ‘t); enzymes (for instance, horseradish peroxidase, alkaline phosphatase, /3 a D-galactosidase, glucose oxidase, glucoamylase); and fluorescent materials (for instance, 5 fluorescein isothiocyanate, rhodamine) They have respective characters, but considering the sensitivity, the precision and the convenience of measurement, enzymes are found the most advantageous.
The present invention is executed usually as follows.
An antigen solution with an appropriate concentration is added to a suspension of the 10 insolubilized antibody for reaction To the reaction mixture is added a solution of the labeled antibody and after the reacti the solid phase is centrifugally separated Following a well washing, the activity of the labeling agent contained is measured When for instance the labeling agent is an enzyme, the well-washed solid phase is added to the solution of substrate against said enzyme and after the reaction the activity of the enzyme is measured 15 Plotting the measured value over the standard curve obtained by using a reference material with known concentration, the amount of antigen in the test solution is determined.
In the execution of measurement, the conditions such as volume of test solution, the concentration and the volume of the reagent to be used and the reaction temperature and time, which depend on the kind of substance to be measured, the titer of antibody, the kind 20 and activity of the labeling agent, should be experimentally optimized for each measurement.
The activity measurement of the labeling agent may be done either in the solid phase or liquid phase after centrifugal separation In the present invention, in which after the reaction between the test solution and the insolubilized antibody the antigenantibody complex 25 is not separated from the reaction mixture, the activity measurement of the liquid phase is likely to be hindered by presence of impurities in the test solution Therefore when the labeling agent is an enzyme, it would be advantageous to measure the activity of solid phase.
The measurable substances by the present invention include high molecular substances, 30 for instance, human chorionic gonadotropin, growth hormone, insulin, glucagon, adrenocorticotropic hormone, thyroid stimulating hormone, immunoglobulin E, a-fetoprotein, hepatitis B antigen, human placental lactogen and their antibody; and low molecular ones, for instance, steroids such as testosterone, estriol, progesterone, corticosterone, aldosterone; thyroid hormones such as thyroxine, triiodothyronin; physiologically 35 active peptides such as bradykinin, gastrin, angiotensin, thyroid hormonereleasing hormone, luteinizing hormone-releasing hormone; physiologically active amines such as epinephrine, norepinephrine, histamine, serotonin; prostaglandin.
The present invention possesses the following advantages over the conventional measuring process 40 The present invention, as mentioned above, is a combination of the sandwich principle and the competitive principle and accordingly it is applicable over a wide range of concentration of substance to be measured This means that when the amount of substance to be measured present in the test solution is unpredictable because of wide range of the amount of the substance to be measured, there is no need of preparing several dilutions to fit into 45 the conventional measurable range; the operation is simplified; there is little error due to dilution and the precision of measurement can be improved Whereas in the conventional sandwich method the separation of the reaction product has to be done two times, in the present invention only one time of separation suffices, thereby simplifying the operation, decreasing a loss of the sample through the operation and improving the precision of 50 measurement.
Further, in the present invention, in which after reaction between antigen and antibody the reaction is allowed to go on without separating the antigen which has not bound to the antibody, even the antigen which has not fully reacted with the antibody in short time can well bind to the antibody Thus with the amount of the antigen to bind to the antibody 55 increased, the process according to the present invention can have the sensitivity of measurement which is improved over that of conventional processes.
Furthermore, in the present invention, if the antigen to be measured and the labeled antibody are first caused to react together as the first reaction, the reaction between the antigen t 9 be measured and the labeled antibody will take place in liquid-liquid phase Thus 60 with the reaction fully promoted, more of the labeled antibody can bind to the antigen to be measured, resulting in the slope of the standard curve becoming steep enough and the sensitivity of measurement being improved.
The present invention is illustrated by the following examples.
Example 1 Measurement of a-fetoprotein 65 A 1,572,220 a) Preparation of standard a-fetoprotein solutions a-fetoprotein (AFP) extracted from abdominal ascites of patient with primary hepatoma and purified by S Nishi and others’ method (S Nishi, Cancer Res, 30, 25072513 ( 1970)) was dissolved in PBS (Phosphate buffered saline) containing 0 5 % Tween 20 and 1 % (bovine serum albumin) to concentrations of 101, 102, 103, 104, 105, 106, and 107 ng/ml 5 b) Preparation of anti-AFP antibody The purified AFP in a) was dissolved to a concentration of 2 mg/ml in physiological saline and 0 5 ml of the solution was mixed with 0 5 ml of Freund’s complete adjuvant and the mixture was injected more than 5 times into a rabbit, and therefrom anti-AFP antiserum was obtained This antiserum was salted out by sodium sulfate and thus anti-AFP 10 antibody globulin was obtained.
c) Preparation of anti-AFP antibody coupled cellulose.
Eight g of cellulose powder (Merck Chemical) was added to 320 ml of 2 5 % cyanogen bromide; adjusted to p H 10 ’11 with 1 N sodium hydroxide; stirred for two minutes to 15 react; passed through a glass filter; and washed with 0 1 M sodium bicarbonate, producing 15 activated cellulose The activated cellulose obtained was suspended in 32 ml of 0 1 M sodium bicarbonate To this suspension was added 8 mg of said anti-AFP antibody and reacted for 22 hours at 4 C under stirring After this reaction, the antiAFP antibody coupled cellulose was washed with 8 M urea 0 2 M glycine mixture (p H 7 0) and suspended to a concentration of 10 % in PBS containing 0 5 %Tween 20 and 1 % BSA 20 d) Preparation of anti-HRP (horseradish peroxidase) was dissolved in 1 ml of 0 3 M sodium bicarbonate; mixed with 0 1 ml of 1 % 2 4-dinitrofluorobenzene; and stirred for one hour at room temperature To the solution obtained was added 1 ml of 0 08 M sodium periodate, followed by stirring for 30 minutes at room temperature; after adding 1 0 ml of 0 16 M ethyleneglycol solution the mixture was stirred for one hour at room temperature 25 After dialysis overnight against 0 01 M sodium carbonate buffer (p H 9 5), to the dialyzate was added 1 0 ml of anti-AFP antibody solution i e, the anti-AFP antibody prepared in b) was dissolved to a concentration of 5 mg/ml in O O 1 M sodium carbonate buffer (p H 9 5).
After the reaction for three hours at room temperature, 5 mg of sodium borohydride was added to the reaction mixture followed by incubation for three more hours at 4 C After 30 dialysis overnight against PBS (p H 7 2), anti-AFP antibody-HRP conjugate (hereinafter referred to anti-AFP-HRP) was purified through fractionation by Sephadex G-200.
e) Measurement of AFP In a test tube 0 1 ml of the standard AFP solution of each concentration prepared in a) was put and to this, 0 4 ml of the anti-AFP antibody coupled cellulose suspension in c) was 35 added and the suspension was reacted for 60 minutes at room temperature Following the reaction, 0 1 ml of anti-AFP-HRP obtained in d) was added to the reaction mixture and was reacted for 120 minutes at room temperature After the reaction, the solid phase was centrifugally separated; washed two items with a physiological saline containing 0 005 % Tween 20; and then to the washed solid phase was added to 3 ml of a substrate solution 40 containing 60 mg/dl of 5-aminosalicylic acid and 1 ml/dl of 0 3 % hydrogen peroxide and the enzyme-substrate mixture was kept standing for 60 minutes at room temperature The reaction was stopped and after centrifugation, the absorbancy of the supernatant fluid was measured at the wave length of 500 nm The standard curve obtained thereby is illustrated in Fig4 45 Next, examples of measurement using this curve are described.
Example 2 Measurement of AFP in a healthy person’s serum and in a patient’s serum.
In accordance with the procedure in Example 1, the amount of AFP in serum was measured on healthy persons, pregnant women and patients with liver disease.
In test tubes 0 05 ml and 0 1 ml of specimens were put and to them was added 0 4 ml of 50 the anti-AFP antibody coupled cellulose obtained in Example 1-c) and then the tubes were incubated for 60 minutes at room temperature Subsequently the absorbancy at 500 nm was measured in the same way as in Example 1-e).
The absorbancy (OD O 05) of 0 05 ml of the specimen was compared with the (OD 01) of 55 0.1 ml of the specimen In the case of OD 005 < OD O 01, the rising gradient side of the standard curve, and in the case of OD O 05 > OD O o 1, the falling gradient side of it were used to estimate the AFP concentration in serum, the results being summarized in Table 1.
1,572,220 Table 1
Case No Subject AFP content 1 Healthy less than 10 ng/ml 5 2, 3,, 4 Hepatitis 82 ng/ml ” less than 10 ng/ml 6 Liver Cirrhosis 45 ng/ml 10 7 Pregnant 120 ng/ml 8 ” 53 ng/ml 9 ” 220 ng/ml ” 260 ng/ml 11 Liver cancer 8180 ng/ml 15 12 ” 410 ng/ml 13 ” 165 ng/ml 14 ” 6262 ng/ml Example 3 Measurement of AFP 20 Standard AFP solutions were prepared by dissolving the AFP purified in Example 1-a) in PBS containing 1 % BSA and Tween 20 to concentrations of 100, 80, 60, 40 and 20 ng/ml.
In a test tube 0 1 ml of each of these solutions was put and to this was added 0 4 ml of anti-AFP-HRP obtained in Example 1-d) and the test tubes were incubated for 60 minutes at room temperature After the incubation, 0 1 ml of the anti-AFP antibody coupled 25 cellulose suspension obtained in Example 1-c) was added to each reaction mixture and the test tubes were incubated for 120 minutes at room temperature After the incubation the solid phase was centrifugally separated; washed two times with a physiological saline containing 0 005 % Tween 20; and 3 ml of a substrate solution containing 60 mg/dl of 5-aminosalicylic acid and 1 ml/dl of 0 3 % hydrogen peroxide was added to the washed solid 30 phase and the test tubes were incubated for 60 minutes at room temperature Thereafter the absorbancy at 500 nm was measured The standard curve obtained thereby is illustrated in Fig 5 lAl Compared with the one for conventional process, this curve has a slope two times steeper and it can sensitively detect a change in the AFP concentration as a change in the absorbancy 35 Comparative case 1 Measurement of AFP In test tubes 0 1 ml of each of the AFP standard solutions in Example 3 was put and to these was added 0 4 ml of the anti-AFP antibody coupled cellulose suspension in Example 1-c and the tubes were incubated for 60 minutes at room temperature After the incubation the solid phase was centrifugally separated; washed with a physiological saline containing 40 0.005 % Tween 20; and suspended in PBS containing 0 5 % BSA Then to this suspension was added 0 4 ml of anti-AFP-HRP in Example 1-d) and the tubes were incubated for 120 minutes at room temperature After the incubation, the solid phase was centrifugally separated; washed with a physiological saline containing 0 005 % Tween 20; and then added to 3 ml of a substrate solution containing 60 mg/dl of 5-aminosalicylic acid and 1 ml/dl of 0 3 % 45 of 0 3 % hydrogen peroxide and the tubes were incubated for 60 minutes at room temperature Then the absorbancy at 500 nm was measured, the standard curve thereby being illustrated in Fig lBl.
Example 4 Measurement of AFP a) Preparation of standard AFP solutions 50 In accordance with the procedure in Example 1-a), standard solutions containing a purified AFP in concentrations of 320, 160, 80,40, 20, 10, 5 and 0 ng/ml were prepared.
b) Labeling of anti-AFP antibody with radioactive iodine The anti-AFP antibody in Example 1-b) was labeled with ” 3 ‘I In a small test tube 0 005 ml of 2 m Ci Na 131 I was added to 0 025 ml of 0 5 M phosphate buffer Next, 0 025 ml of 55 anti-AFP antibody solution and 0 02 ml of chloramine T were put and mixed together In seconds after this, the reaction was stopped by adding 0 1 ml of sodium pyrosulfite solution Through fractionation by a column of Sephadex G-50, 13 I-labeled anti-AFP antibody was obtained.
c) Measurement of AFP 60 In a counting test tube 0 1 ml of a standard AFP solution with each concentration prepared in a) and 0 4 ml of the anti-AFP antibody coupled cellulose suspension obtained in Example 1-c) were added and the tubes were incubated for 60 minutes at room temperature Next, O 1 ml of said 13 I-labeled anti-AFP antibody in b) was added and the tubes were incubated for 120 minutes at room temperature Then the solid phase wascentrifugally 65 A r separated; washed with a physiological saline containing 0 005 % Tween 20; and then the radioactivity was measured, some of the results being given in Fig 6.
Example 5 Measurement of insulin a) Preparation of standard insulin solutions Bovine crystal insulin (Sigma chemical) was dissolved in PBS containing 0 1 % BSA to 5 concentration of 160, 80, 40, 20, 10 and 0 /zl U/ml.
b) Preparation of anti-insulin antibody Bovine crystal insulin suspended in physiological saline; thereafter dissolved by adding 0.1 N hydrochloric acid drop by drop and adjusted to a concentration of 2 mg/ml The insulin solution was mixed with activated charcoal powder (Wako Pure Chemical) at a rate 10 of 10 mg to 1 ml of said insulin solution, thereby causing insulin to be adsorbed on activated charcoal The insulin adsorbed charcoal was centrifugally separated By adding 0 5 ml of physiological saline to 10 mg of this charcoal, the insulin adsorbed activated charcoal suspension was obtained A guinea pig was injected every other week a mixture of 0 25 ml of this suspension and 0 25 ml of Freund’s complete adjuvant and the injection was repe 15 ated 10 times One week after the final injection, the blood was collected from the animal’s carotid, thereby producing a guinea pig anti-insulin serum The antiserum thus obtained was salted out two times with sodium sulfate and the anti-insulin antibody was obtained.
c) Preparation of anti-insulin antibody-alkaline phosphatase conjugate.
Three tenth ml of alkaline phosphatase (Grade I) (Boehringer mannheim) solution ( 5 20 mg/ml) was centrifuged and, after removed of the supernatant fluid, it was dissolved in 0 1 ml of the anti-insulin antibody in b) After dialysis overnight against PBS, it was mixed with 0.01 ml of 4 2 % glutaraldehyde and the mixture was incubated for 2 hours at room temperature Then, the volume of the solution was adjusted to 1 ml with addition of PBS After dialysis overnight against PBS, dialysate was fractionated by a Sepharose 6 B column 25 chromatography, thereby yielding an anti-insulin antibody-alkaline phosphatase conjugate.
d) Preparation of anti-insulin antibody-sensitized polyethylene stick.
The anti-insulin antibody in b) was dissolved to a concentration of 10 / g/ml in a glycine buffer (p H 8 2) Into 10 ml of this antibody solution was immersed 100 pieces of polyethylene stick, 3 mm in diameter and 8 mm in length, and the mixture was incubated 30 for three hours at 37 C The sticks were then washed with physiological saline and immersed in PBS containing 1 % normal guinea pig serum After kept standing this solution overnight at 4 C, anti-insulin antibody-sensitized polyethylene sticks were obtained.
e) Measurement of insulin One tenth ml of standard insulin solution of each concentration obtained in a) was put in 35 a test tube and was mixed with 0 6 ml of PBS containing 0 5 % BSA and 0 5 % Tween 20.
Then the anti-insulin antibody-sensitized polyethylene sticks prepared in d) were put, three pieces of stick to each test tube, and the tubes were incubated for one hour at room temperature Next, 0 1 ml of the anti-insulin antibody-alkaline phosphatase conjugate solution in c) was added and the tubes were incubated overnight at 4 C After the incubation, 40 polyethylene sticks, which had been washed with a physiological saline containing 0 005 % Tween 20, were put in 3 ml of a substrate solution (pnitrophenylphosphate 1 mg/ml, magnesium chloride 1 m M: sodium carbonate buffer p H 9 8) After the reaction for one hour at room temperature, 0 3 ml of 1 N Na OH was added and the absorbancy of 400 nm was measured, some of the results being given in Fig7 45 Example 6 Measurement of insulin a) Preparation of standard insulin solutions Bovine crystal insulin was dissolved in PBS (p H 6 4) containing 0 1 % BSA to concentrations of 104, 103, 320, 80 and 20,u U/ml.
b) Preparation of anti-insulin antibody-HRP conjugate 50 In accordance with the procedure in Example 1-d), reaction was made between 5 mg of the anti-insulin antibody prepared in Example 5-b) and 5 mg of HRP, producing an antiinsulin antibody-HRP conjugate.
c) Measurement of insulin In a test tube 0 1 ml of the standard insulin solution in a), 0 3 ml of PBS containing 0 5 % 55 BSA and 0 5 %Tween 20 and 0 1 ml of the anti-insulin antibody-HRP conjugate in b) were put and the tubes were incubated for one hour at room temperature Next, 0 5 ml of PBS containing 0 5 % BSA and 0 5 % Tween 20 and three pieces of anti-insulin antibodysensitized polyethylene stick in Example 5-d) were added together and the tubes were incubated overnight at 4 C After the incubation, these sticks were washed with a 60 physiological saline containing 0 005 % Tween 20 and thereto was added 3 ml of a substrate solution ( 5-amino-salicylic acid 60,g/dl, 0 3 % hydrogen peroxide 1 ml/dl) and the tubes were incubated for 60 minutes at room temperature, after which the absorbancy at 500 nm was measured The standard curve obtained thereby is illustrated in Fig 8.
Example 7 Measurement of human chorionic gonadotropin (h CG) 65 1,572,220 8 1,572,220 8 a) Preparation of standard h CG solutions Standard h CG specified in Japanese pharmacopoeia was dissolved in PBS containing 0.1 % BSA to concentrations of 30,000, 3,000, 300, 30, 3, and O IU/1.
b) Preparation of rat’s testis gonadotropin receptor Tunica albuginea of two testes of a 7 ’10 weeks old rat was torn off and thereto was 5 added a small amount of PBS The whole thing was homogenized in a whirling blender for about 30 seconds The suspension obtained was filtered through a piece of gauze and the filtrate was centrifuged to separate for 10 minutes at 3,000 rpm The precipitate obtained was resuspended in 10 ml of PBS per one testis 10 c) Preparation of anti-h CG antibody.
Commercial h CG was purified by using the anion exchange resin and the gel filtration with Sephadex Using the product obtained as the antigen, a rabbit was immunized in the same way as in Example 1-b) Normal human serum and urine were added to the obtained antiserum; the antibody to the components of serum and urine was eliminated through 15 absorption; and after salting out with sodium sulfate, an anti-h CG antibody was obtained.
d) Preparation of anti-h CG antibody HRP conjugate.
In accordance with the procedure in Example 1-d), 5 mg of HRP and 5 mg of the anti-h CG antibody in c) were bound, yielding an anti-h CG antibody-HRP conjugate e) Measurement of h CG In test tube, 0 4 ml of PBS containing 0 1 % BSA, 0 1 ml of the standard h CG solution in 20 a) and 0 1 ml of the receptor suspension in b) were added and the tubes were incubated for minutes at 37 C Next 0 1 ml of the anti-h CG antibody-HRP conjugate solution was added and the tubes were incubated for 60 minutes at 37 C After centrifugation for 10 minutes at 3,000 rpm, the supernatant fluid was removed; the precipitated receptor was 5 washed with PBS: and 3 ml of the substrate solution in Example l-e) was added and the tubes were incubated for one hour at room temperature.
After the reaction was stopped, centrifugation was done and then the absorbancy of the supernatant fluid was measured, some of the results being given in Fig 9.
The words “Tween”, “Sephadex”, and “Sepharose” used herein are Registered Trade 30 Marks.

Claims (6)

WHAT WE CLAIM IS:

1 An immunochemical measuring process which comprises; firstly reacting a bindable substance to be measured with either (a) an insolubilized substance which specifically binds the substance to be measured, or (b) with a labelled substance which is bindable to said bindable substance, and secondly reacting the resultant product without isolation with the substance (a) or (b) not utilized in said first reaction, and finally separating the labelled substance which has been bound to the insolubilized substance and the bindable substance from the labelled substance which has not been so bound, and measuring the activity of either of said separated substances.

2 A process as claimed in claim 1 wherein the bindable substance to be measured is reacted with an insolubilised substance specifically binding the bindable substance and thereafter reacting the mixture so produced with said labelled substance.

3 A process as claimed in claim 1 wherein said bindable substance is reacted with said labelled substance and the resulting reaction mixture is then reacted with said insolubilised 45 substance.

4 A modification of the process as claimed in claim 1 wherein said first and second reaction steps are effected simultaneously.

A process as claimed in any preceding claim wherein the bindable substance to be measured is one or more of antigenic substances, haptens, antibodies and physiologically 50 active peptides and amines.

6 For the Applicants F.J CLEVELAND & COMPANY 40-43, Chancery Lane, London WC 2.
Printed for Her Majest’, Statunery Office bl Croydon Printing Company Limited Croydon Surrey 1980.
Published bh The Patent Office 25 Southampton Buildings, London WC 2 A IAY from sshich copies may be obtained.
6 A process as claimed in any preceding claim wherein the insolubilized substance is a bindable substance insolubilized by binding to a carrier.
7 A process as claimed in claim 6 wherein the carrier is a polysaccharide or a plastics material 55 8 A process as claimed in any preceding claim wherein the labelling agent is radioisotope, enzyme or fluorescent material.
9 A process as claimed in claim 1 and substantially as described in any one of the specific examples hereinbefore set forth.

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US
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patent/FR2367286A1/en
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Also Published As

Publication number
Publication date

NL178193B
(en)

1985-09-02

NL7710980A
(en)

1978-04-11

NL178193C
(en)

1986-02-03

US4248965A
(en)

1981-02-03

FR2367286B1
(en)

1982-09-17

DE2744836A1
(en)

1978-04-13

FR2367286A1
(en)

1978-05-05

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