CA2356857A1 - Container for immunologic assay - Google Patents
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- CA2356857A1 CA2356857A1 CA002356857A CA2356857A CA2356857A1 CA 2356857 A1 CA2356857 A1 CA 2356857A1 CA 002356857 A CA002356857 A CA 002356857A CA 2356857 A CA2356857 A CA 2356857A CA 2356857 A1 CA2356857 A1 CA 2356857A1
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
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- Clinical Laboratory Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Automatic Analysis And Handling Materials Therefor (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Heterocyclic Compounds That Contain Two Or More Ring Oxygen Atoms (AREA)
Abstract
A container for an immunologic assay in which the saturation adsorption of molecules for use in the assay is 1x10-1 pmol/cm2 or smaller. It is free from non-specific adsorption causative of reagent loss, sensitivity decrease, and precision decrease.
Description
CA 02356857 2001-06-20 ,. " .,. . Li ~~s~as.
Description Container for Immunologic Assay Technical Field The present invention relates to a container used for storage, dilution, or reaction of a reagent and/or a test sample, in an immunoassay for detecting an antigen or an antibody through antigen-antibody reaction.
Background Art Conventional immunoassays employ a polystyrene- or polypropylene-made container for storage and dilution of a reagen c :.;r a sample which is ~ to be used. i~ow~ver; molecules contained in the reagent or the sample are non-specifically adsorbed onto such a container, and such adsorption necessarily causes loss of the reagent or the sample, as well as variation in concentration of a solution containing the reagent or the sample.
In recent years, in accordance wiith diversification of immunoassay methods, in most cases, naturally occurring , substances have been used after extraction and purification, particularly in the drug production deF>artment of drug manufactures. Generally speaking, such substances are obtained in very small amounts and thus. are quite expensive.
Therefore, reduction in the: amount of substance during storage or dilution, w~lich is caused by physical adsorption :, ~ ;,: _;; ,. ..~;:, onto a container, is not negligible.
When samples used for clinical diagnosis, such as serum and urine, are collected from patients, placed in a container, ar~d stored therein ur mil the _samples are sub.je cted .to ~,~;say, clinically important proteins contained in the samples, such as albumin, transferrin, and immunoglobulin, are adsorbed onto the container. Most containers uaed for clinical diagnosis, including syringes and cups used in the step of collecting a sample, tubes used in the step of storing the sample, and centrifugation tubes and test tubes used in the step o'f purifying, concentrating, or diluting the sample, are formed from polypropylene or polystyrene, and such a container is not subjected to surface -treatment. Therefore, when even a trace amour~t of proteins contained in thc: sample is adsorbed onto the container in each step, the concentration of the proteins is expeci~ed to vary greatly after all the steps have been performed, as compared with the concentration of the proteins at the time of collection of the sample.
In general, the price of a reagent: in immobilized form accounts for about 80~ the cost of a clinical test kit sold by a clinical test drug manufacturer. Therefore, when reduction in, the reagent due to adsorption onto a container is suppressed, production costs are greatly reduced.
In a solid phase method (a type of immunoassay method), assay is carried out by utilizing proteins immobilized onto the surface of a container for an immunoassay. Therefore; a CA 02356857 2001-06-20 ~ .. 'a~,~,.";"::-.
solid phase method employs a container subjected to "high adsorption treatment," in which, in order to increase the amount of a reagent which is to be immobilized onto the surface of the contai:nP~-, ~ ryd_ron~~ilic,-hydrophobic balance of the surface is regulated through introduction of a functional group such as a hydroxyl group, thereby increasing the saturation adsorption amount of the reagent.
In recent years, in order to shorten immunoassay time and to carry out immunoassay on a large scale, immunoassay methods making use of an automatic analyzer (robot) have been developed. Such methods have rapidly become prevalent, particularly in the drug production department of drug manufacturers .
When an assay is carried out by m~earis of a conventional solid phase method, a washing step for eliminating non-immobilized excess molecules is required. However, an automatic analyzer encounters difficulity in carrying out the washing step, in which fractional inje<a ion and suction of a washing solution are repeated. Therefore, a sequential addition method has been under developnnent as an immunoassay method suitable for an automatic analyzer, because such a .
method does not require separation ofra reacted substance and a non-reacted substance through a wasr~~.ng step.
In a sequential addition method, ~'.mmobilization of molecules is not carried out during reaction, and reaction is carried out in a solution. Therefore, 'when a container having a surface subjected to the aforementioned high adsorption treatment is used, unwanted adsorption of the molecules impedes reaction in the solution or lowers the reaction efficiency.
Tn rQC~~.t y ears , in accor~.~nce with progxess in __ ._ _ measurement techniques, evaluations through a fluorescence method or an emission method have been established, the method having high sensitivity as compared with absorbance assay by means of a conventional color:imetric method.
Therefore, in the future, unwanted adsorption of molecules onto a container is expected to induce problems in such ari assay'method having high sensitivity.
At the present time, a container used for such a method is provided without consideration of molecular adsorption;
i . a . , the coini:ainer is formed from polystyrene or polypropylene in consideration of only shapability, transparency, and low-temperature resistance, and the container is not subjected to surface t:reatment for suppressing adsorption of molecules. From the viewpoint of characteristics of the container, no ataempt has been made to solve problems such as loss of a reagent and reduction in sensitivity.
However, in order to control non-~;pecific adsorption of molecules onto *:he surface of a container for immunoassays, several tecl~:niques have hitherto been studied and carried out.
For example, a blocking method is most widely carried out, in which a container is coated with a protein inactive to a sample which is to be assayed. Since the method .... :.::..: '::, :,:. ..:. , . 'vx:iiwidl'iiat;.
basically utilizes non-specific adsorption of the protein onto the container, blocking effects may differ from container to container, and may depend. on the state of the protein. In addition, sinr..c the i~.~ct.i«e prc~tvein, is non-specifically adsorbed onto the container, the protein is easily detached from the container into a solution, and thus the container cannot be used for storing the solution.
Japanese Patent Application Laid-Open (xoxa.i) Nos. 6-174726 and 7-128336 disclose a technique in which such detachment of a protein is eliminated by chemically immobilizing the protein onto a container. However, thE: structure of the protein may vary in accordance with drying temperature, storage temperature, and storage time, and thus the container is not widely used in practice:. .
When the higher-order structure o:~ a protein adsorbed onto a container varies, the protein induces secondary adsorption. When a protein which is inactive in a. free state is adsorbed onto or chemically bound to a container, the protein cannot completely maintain its inactive state, due to alteration of the higher-order structure. Therefore, even when adsorption of another protein ontc> the container can be prevented, variance of the higher-order structure induces secondary adsorption between the proteins.
Secondary adsorption between proteins varies with types of proteins, 'and thus a protein suitable for blocking must be chosen every time a sample which is to be assayed is changed.
When a solution containing different proteins, such as serum, y. . .,..., ~...;.,:.. ~ ~i.. ."~ ~.~ ~~:~\
CA 02356857 2001-06-20 " "~
is used as a sample, no blocking protean can control adsorption of all the proteins contained in the sample.
Disclosure cf ':h.e Invention In view of the foregoing, the present inventors have performed extensive studies on characteristics of a container, and have found that when the saturation amount of molecules which are adsorbed onto the container, the molecules being used for an immunoassay, is controlled to a predetermined value or less, loss of a reagent or a sample is prevented during storage, dilution, and reaction, and the sample can be assayed at high sensitivity. The presE:nt invention has been accomplished on the basis of this finding.
~ccorciimgiy, the present invention provides a container for an immunoassay in which the saturation adsorption amount of molecules used for the assay is 1 x 10-1 pmol/cm2 or less .
Brief Description of Drawings Fig. 1 shows the concentration of proteins after bovine serum and albumin have been stored in i~he container for an immunoassay of the present invention air -80°C fo.r 48 hours ..
Fig. 2 shows reaction efficiency when an immunoassay is carried out in the container of the prEaent invention.
Best Mode for Carrying Out the Inventic>n In a conventional polystyrene- or polypropylene-made container for an immunoassay, the adsorption amount of ' 6 ~.4 - ~,~_~e_ molecules ( a . g . , proteins ) is about 1 --10 pmol,~cm? or. -~~rP
i.e., about 20-500 of molecules (e.g., proteins) used for an immunoassay are adsorbed onto the container, although the adsorption amount varies in accordance; with the concentration of a solution containing such molecule's and the contact area between the molecules and the container. G~Then the adsorbed molecules (20-50% of all the molecule;) are essential for reaction in the solution, reaction efficiency; i.e., assay sensitivity, is reduced by 20-50%. Meanwhile, when the adsorbed substance is such that it undergoes molecular structural changes due to adsorption t.o thereby cause unwanted reaction, considerable noise would result.
Therefore, a container onto which no molecules used for an immunoassay are adsorbed is most icLeal, but when the adsorption amount of molecules is substantially reduced to 1/10-1/100 with respect to the current level, satisfactory effects will be obtained.
Although the adsorption amount of molecules contained in a solution varies with the identity of the molecules, temperature; concentration of the solution, and the pH of the solvent, the container desirably meets the following conditions: the saturation adsorption amount of the molecules used in the immunoassav is 1 x 10-1 priiol/cm2 or less under the specific conditions ir~ terms of conc:entratiozn c-;f true solution, temperature, and pH of the,solvent under which the reaction and assay are carried out. In the case in which serum is used for an immunoassay, since serum is usually ~::,;
diluted up to i/10, tYve effer~. .~f the invention can be attained if the saturation adsorption amount of the molecules which participate in and/or affect the=_ assay, among a:11 molecules contained in the diluted serum, is always 1 x 10-1 pmol/cm2 or less at the diluted concentration of serum and under the specific conditions in terms of concentration of the solution, temperature, and pH of i~he solvent under which the reaction and assay are carr_Led out.
Similarly, when the container is used for storage and dilution of a reagent, the effect of the invention can be attained if the saturation adsorption amount of the molecules that undergo storage and dilution is always 1 x 10-1 pmol/cm2 or less under the specific conditions- in terms of concentration.nf..the.solnzti.on,, temberature, and pH of the solvent under which the reagent is removed from the storage container or dilution is carried out. In many cases, the reagent is stored in the containez: at a temperature as low as -80°C. However, adsorption of the molecules is an equilibrium reaction, and thus, it would be sufficient if the saturation adsorption amount of molecules is 1 x 10-1 pmol/cm2 or less under the specific conditions- in terms of concentration, temperature, and pH under which the reagent is removed from the container.
T~le saturation adsorption amount of the molecules is more preferably 1 x 10-2 pmol/cm2 or less, much more preferably h x 10-3 pmol/cm2 or less .
Examples of the molecules used iru an immunoassay ,..,.,. ~y:l" (~1:.
,.. ,k~~~,( -ie:rlude proteins ;e.g., enzymes, physiologically active proteins, and antibodies), nucleic acids, and physiologically active substances. Of these, proteins are particularly preferable. The saturation adsorption amount of the molecules can be measured by means of colloidal gold labeling immunoassay.
In the point that adsorption of a protein is prevented, the present invention exerts excellent: effects in addition to the aforementioned characteristic feature. Usually, when a protein is adsorbed onto a container, the structure of the protein is varied. Therefore, when an immunoassay is carried out, although a target protein is contained in a sample to be assayed, the protein may fail to be detected by an antibody, ~3~_,.e t~ variation in the structure of the protein. When a clinical test is carried out, in practice serum whose structure has been altered due to adsorption is assayed, even though serum must be assayed in the same state in which the serum is present in an organism. According to the present invention, since a protein is not adsorbed onto the container, the structure of the protein is not altered, and thus when a clinical test is carried out by use of the container, serum can be assayed in a state similar to that in which serum is present in an organism. Therefore, th~~ container of the present inVerition is very advantageoasly used as ~ container c far an immunoassay.
In a container for an immunoassay, the saturation adsorption amount of mclecules must be decreased at a portion ,.:.. .:".s~',, ,t, with t~:hich a reagent or a samp.lQ is brought into contact;
specifically, an inner surface of the container. Therefore, the molecular saturation adsorption amount at an inner surface of the container should be at least 1 x 10-1 pmol/cm2 or less.
In order to decrease the saturation adsorption amount of molecules at an inner surface of the container to ~. x 10-1 pmol/cm2 or less, preferably, at least. the inner surface is formed from a highly hydrophilic polymer or a highly hydrophobic polymer, or is coated with a highly hydrophilic polymer or a highly hydrophobic polymer. More preferably, at least the inner surface is coated with a highly hydrophilic polymer or a highly hydrophobic polyme r. Much more preferably, at least r.hP.i.nner surface is coated with a highly hydrophilic polymer. Particularly preferably, at least the inner surface is coated with an ultra-hydrophilic polymer.
Examples of highly hydrophobic polymers include fluorine-containing resins such as pol.ytetrafluoroethylene~
(PTFE) and 'silicon-containing resins. When the surface of the container is coated with a hydropr~obic polymer, the surface may be coated with the aforementioned hydrophobi c polymer, or the container may be fluoz~inated, thereby forming a fluorinated polymer fi~.m on the ,surf:ace thereof .
No particular limitation is impo~~ed on the highly hydrophilic polymerP so long as the polymer contains a hydrophilic group such as a carboxyl group or a hydroxyl ftiN.4ili'.
gro~zp. E~:.~-~J,yles of such a hydrophil is polymer include polymethacrylic acid, (meth)methacrylic acid-alkyl methacrylate copolymers, polyhydroxyalkyl rriethacrylates (e. g., polyhydroxyethyl methacrylate), hydroxyalkyl methacrylate-alkyl rnethacrylate copolymers, polyoxyalkylene-group-containing methacrylate polymer and copolymers containing the polymer, polyvinyl pyrrolidone, ethylene-vinyl alcohol copolymers, (2-methacryloyloxyethylphosphocholine) polymers (MPC) and copolymers containing the polymers (Seitai Zairyo, Vol. 9, No. 6, 1991), and phospholipid~polymer composites (Japanese Patent Application Laid-Open (kokai) Nos. 5-161491 and 6-46831). The container may be formed from such a hydrophilic polymer, or coated with the polymer.
Afvi.er. the container is formed from a suitable matFr.ial, such as polystyrene, a hydroxyl group or a carboxyl group may be introduced into the surface of the container, to thereby impart high hydrophilicity; i.e., low adsorbability, to the surface of the container. A surface exhibiting low adsorbability can be realized by means of surface modification. For example, when, in consideration of formability, the container is formed from a material which tends to induce non-specific adsorption, such as polystyrene or polypropylene, a carboxyl group, a carbonyl group, and/or a hydroxyl group may be introduced into the surface of the container through plasma exposure, to i:hereby impart low adsorbability to the surface. When, in consideration of transparency, the container is formed from polymethyl S
CA 02356857 2001-06-20 ",'Y~" ''...
methacrylate, a carboxyl group may be introd~:ccd into the surface of the container through partial hydrolysis of the surface by use of an alkali, to thereby impart low adsorbability to the surface.
When hydrophilicity is imparted t:o the inner surface of the container by use of a hydrophilic polymer, to thereby reduce the adsorption amount of molecules, the contact angle between the surface and water is preferably 30° or less (highly hydrophilic), more preferably 15° or less, much more preferably 1° or less (ultra-hydrophilic).
When, among the aforementioned hydrophilic polymers, there is employed a polyhydroxyalkyl m.ethacrylate, a polyoxy(C2-C9 alkylene-group-containing methacrylate) polymer or a copolymer containing the pol.j~mer.; _a. (2- . , methacryloyloxyethylphosphocholine) polymer or-a copolymer containing the polymer; a phospholipid~polymer composite; or polyvinyl pyrrolidone, the contact angle between the surface of the resultant container and water becomes 1° or less (i.e., the container is ultra-hydrophilic), and the saturation adsorption amount of proteins becomes 1 x 10'3 pmol/cm2 or less, which is particularly preferable.
The product form of the container of the present invention is not particularly limited, and the container may assume conventionally used product for:r~s, including a sample tube, a centrifugation tube, a multi-well plate, and a cuvette. However in order to carry Ollt storage, dilution, reaction, and assay of a sample in one container, the CA 02356857 2001-06-20 ,. .,... , ...
container prvferal~.~y assumes a form of multi-well plate.
Examples The present invention will next be described in more detail by way of Examples, which should not be construed as limiting the invention thereto.
(Example 1) A commercially available polypropylene-made 96-well plate (MS-3396P, product of Sumitomo E>akelite Co., Ltd.) was subjected to y-ray treatment at 70 kGy, to thereby generate a hydroxyl group on the surface of the ~>late. In the resultant plate, the saturation adsorption amount of proteins was 4.6 x 10-z pmol/cm2, and the contact angle between the surface and -~. ,,; Q ~. ~ -w s (Example 2) A commercially available polypropylene-made 96-well plate (MS-3396P, product of Sumitomo Bakelite Co., Ltd:) was coated with a commercially available fluorine-containing coating agent (Scotchguard, product of Sumitomo 3M Ltd.). In the resultant plate, the saturation adsorption amount of proteins was 2.7 x 10-2 pmol/cm2, and the contact angle between the surface and the water was 126°.
(Comparative Example 1) A cornme~cfally available polypsop~ylene-made 96-well plate (MS-3396P, product of Sumitorno Bakelite Co., Ltd.) was used as a comparative plate. In the plate, the saturation adsorption amount of proteins was 3.7 pmol/cm2, and the CA 02356857 2001-06-20 ~:7:
contact angle between the surface and the water was 92.~a., (Comparison of protein recovery percentage in containers usable as storage containers) For comparison of non-specific adsorption, solutions of an enzyme-labeled anti-bovine-albumin antibody (product of Cosmo Bio) were prepared (concentration of the antibody: 0.1 ng/mL, 1 ng/mL, 10 ng/mL, and 100 ng/mL, respectively); each solution was injected into 24 wells of each of the plates of Examples 1 and 2 and Comparative Example 1; the plate was stored at -80°C for 48 hours: and after storage time had elapsed, the concentration of the protein in each solution was measured by use of a substrate solution.
The results are shown in Fig. 1. The results show that ~he protein recovery percentage is high in.the plates Of Examples 1 and 2, as compared with the case of the plate of Comparative Example 1.
(Comparison of "in solution" reaction efficiency) In order to evaluate the efficiency of a reaction in a solution, the following test was carried out by using the plates of Examples 1 and 2 and Comparative Example 1 as reaction containers.
Rat albumin (product of Cosmo Bio) was dissolved in a phosphate buffer (Dulbecco PBS pH 7.4j to thereby prepare solutions (concentration of the albumin: l;? ng!mL,-~. ng/mL, and 0.1 ng/niL, respectively), and each solution was injected into four lines (i-:e., 32 wells) (100 pl per well) of each of the plates of Examples 1 and 2 and Comparative Example 1.
CA 02356857 2001-06-20 ... ... .,,.
Subsequently, a pnos~~~ra.:~~;e buffer (Dulbecco PBS pH 7.4) solution of a peroxidase-labeled anti-rat-albumin antibody (product of Cosmo Bio) (concentration of the antibody: 100 ng/mL, respectively) was injected into all the wells (100 ~.1 per well) of each plate.
After reaction had been carried cut in each well at 37°C
for 30 minutes, the solution in each well was transferred into a 96-well plate for ELISA in which an anti-rat-albumin antibody had been immobilized onto each well in advance, and then reaction was carried out again in each well at 37°C for 30 minutes.
After reaction was completed, a non-reacted peroxydase-labeled anti-rat-albumin antibody was washed with a washing solution (r7vl.?.~.e._~.co P~iS pH ?.,4 + 0,05% Tween 20) .
Subsequently, each plate was allowed to develop color by use of a commercially available chromophoric kit for peroxidase (ML-1120T, product of Sumitomo Bakelite Co., Ltd.), and then absorbance at 450 nm was measured using a plate reader.
The results are shown in Fig. 2. The results show that, in relation to the plate of Comparative Example 1, the absorbance is low when the concentration of the albumin is low; i.e., the reaction in the solution is impeded due to adsorption, and that, in relation to t:he plates of Examples 1 and 2, linearivy is obtained between t;he concentration of the ,;
albumin and~the absorbance when the albumin concentration is low; i.e., the antigen-antibody reaction in the solution is efficiently carried out.
CA 02356857 2001-06-20 ~. . .. ,..,....
(: E?'~ ~ J!~l~y.~ 1 C 3 ) ..
A commercially available polystyrene-made tube (Eiken tube for RIA No. 3, 70-12458) was coated with polyhydroxyethyl methacrylate (P-3932, product of SIGMA). In the resultant tube, the saturation ads>orption amount of proteins was 9.1 x 10-4 pmol/cm2, and the contact angle between the surface and the water was 0°.
(Example 4) Polytetrafluoroethylene was formE:d into a tube of the same inner diameter and volume as the tube of Example 1. In the resultant tube, the saturation adsorption amount of proteins was 7.2 x 10-3 pmol/cm2, and the contact angle between the surface and the water was 126°.
arr",~~arai-.ive Example 2) A commercially available polystyrene-made tube (Eiken tube for RIA No. 3, 70-12458) was used as a comparative tube.
In the tube, the saturation adsorption amount of proteins was 8.1 pmol/cm2, and the contact angle between the surface and the water was 85°.
(Comparison of assay sensitivity) In order to evaluate the assay sensitivity of a reaction in a solution, the following test was carried out by use of the tubes of Examples 3 and 4 and Comparative Example 2 as reac:Liom containers and an ELISA :ball as a carrier for E
reaction.
Phosphate buffer (pH 7.4) solutions of biotin hydrazide (product of Dojindo) were prepared in advance (concentration CA 02356857 2001-06-20 . _..... . ~ ;:~"r...
of biotin hydrazide: 0. 125 ~gi~uL, 0. a:'~~0 ~,g; mL, and 0. 500 ~g/mL, respectively). By use of the solutions, biotin hydrazide was immobilized onto ELISA ~>alls (amino-group-containing ball, product of Sumitomo Bakelite Co., Ltd.) through covalent bonding via glutarale'.ehyde, to thereby prepare ELISA balls having three different immobilization densities of biotin hydrazide.
A portion of each ELISA ball at which biotin hydrazide was not immobilized was subjected to blocking by use of skim milk so as to prevent adsorption.
Each of the above-prepared ELISA balls was placed into each of the tubes of Example 3, Example 4, and Comparative Example 2 (three tubes for each Example), a phosphate buffer (pH 7.4) solution of pPYoxydase-labeled av.idin,(product of Cappel) (concentration of avidin: 1 ~.g/mL) was injected into each tube (500 mL per tube), and reaction was carried out in each tube at room temperature for 30 minutes.
After the reaction was completed, non-reacted peroxydase-labeled avidin was washed with a washing solution (phosphate buffer pH 7.4 + 0.050 Tween 20). Subsequently, each ELISA ball was allowed to develop color by use of a commercially available chromophoric kit for peroxidase (ML-1120T, product of Sumitomo Bakelite Co., Ltd.), and then absorbanCe at 450 nm was measured by using a plate reader.
The results are shown in Table 1.; The results show that, in Examples 3 and--4, the absorbance varies linearly with respect to the density of biotin hydra:zide introduced onto ~.'~ ~ .a':~.K:
the surfac:=: ~~i she ELISA ball, and that, in Comparative Example 2, the absorbance does not vary with the different densities of biotin hydrazide.
In Examples 3 and 4, peroxydase-labeled avidin is reacted with only biotin hydrazide introduced onto the surface of the ELISA ball, and thus th.e absorbance is proportional to the density of biotin hydrazide. In contrast, in Comparative Example 2, peroxydase-labeled avidin remains in the tube due to adsorption, and the remaining avidin may act as a background, to thereby reduce assay sensitivity.
Table 1 Biotin-avidin reaction by use of ELISA ball ~g/mL Example 3 Example 4 Comparative Example 2 0.125 0.2 0.27 1.05 Om2 5- 0.54 0.62 1.03 - . : ..
0.5 0.97 0.91 ' 1.12 (Comparison of protein recovery percentage in containers usable as storage containers) For comparison of non-specific adsorption, solutions of an enzyme-labeled anti-bovine-albumin antibody (product of Cosmo Bio) were prepared (concentration of the antibody: 0.1 ng/mL, 1 ng/mL,.lO ng/mL, and 100 ng/mL, respectively); each solution was injected into 24 wells of each of the plate of Example 3, the plate of Example 4, and the plate of ComparativeExample 2; the plates were stored at -80°C for 48 hours; and after storage time had elapsed, the concentration of the protein in each solution was measured by use of a CA 02356857 2001-06-20 ,. ._ substrate solution.
The results are shown in Table 2. The results show that the protein recovery percentage is high in the plates of Examples 3 and 4, as compared with in the plate of Comparative Example 2.
Table 2 Protein concentration after storage (comparison of absorbance) ng/mL Example 3 Example 4 Comparative Example 2 0.1 0.24 0.26 0.01 1 0.41 0.33 0.07 0.76 0.66 0.07 100 1.02 0.82 ~
0.03 (Example 5) A 2.0 wt/volo methanol solution of polyhydroxyethyl methacryl.ate (P-3932, prcduct of SIGMA.) 2.5 mL) :~~as injected into a commercially available polystyrene-made tube (Eiken tube for RIA No. 3, 70-12458). Subsequently, the solution was removed from the tube, the tube was inverted so as to prevent the residual solution from remaining at the bottom, and the tube was dried at room temperature for 24 hours, and consequently the surface of the tube was coated with polyhydroxyethyl methacrylate. In the resultant tube, the saturation adsorption amount of proteins is 8.7 x 10-9 pmol/cm2, and the contact angle between the surface and water ,-is 0°.
(Example 6) A 0.5 wt/vol% ethanol. solution of an MPC polymer (2.5 rnL) was injected into a commercially available polystyrene-.. . . ... .,. ..., . ....... . ':,s'::: ~. .:yyVtwi,C3L4 made tube (Eik~n tubs. ;_or RIA No. 3, 70-12458), and the tube was allowed to stand at room temperature for 10 minutes.
Subsequently, the solution was removecL from the tube, the tube was inverted so as to prevent they residual solution from remaining at the bottom, and the tube was dried at room temperature overnight, and consequently the surface of the tube was coated with the MPC polymer. In the resultant tube, the saturation adsorption amount of proteins is 6.5 x 10-9 pmol/cm2, and the contact angle between the surface and water is 0° .
The MPC polymer was synthesized from an MPC-BMA (butyl methacrylate) copolymer (ratio of MPC to BMA = 3/7) which was prepared according to the procedure described in "Release of a dri~~:;, frc:m a hydw~c~e:l meml-hrane having a structure analogous to that of phospholipid," (Kobunshi Rc>nbunshu, 46, 591-595 (1989) ) .
(Comparative Example 3) A commercially available polystyrene-made tube (Eiken tube for RIA No. 3, 70-12458) was used in "as is" form as a comparative tube.
(Comparison of assay sensitivity) In order to evaluate the assay sensitivity of a reaction in a solution, the following test was carried out by use of the tubes of Examples 5 and 6 a.nd Comparative Examp~.e 3 as reaction containers, and an ELISP, ball (amino-group-containing ball, product of Sumitomo P~akelite Co., Ltd.) as a carrier for reaction.
..
,. ... ... ...: , .,u.
... .
~;
Phosphate buffer (pH 7.4) solutions of biotin luya.ra~zide (product of Dojindo) were prepared in advance (concentration of biotin hydrazide~ 0.125 ~.g/mL, 0.250 ~.g/mL, and 0.500 ~.g/mL). By use of the solutions, biotin hydrazide was immobilized onto ELISA balls through covalent bonding via glutaraldehyde, to thereby prepare ELI:SA balls having three different immobilization densities of biotin hydrazide.
A portion of each ELISA ball at which biotin hydrazide was not immobilized was subjected to ~>locking by use of skim milk so as to prevent adsorption.
Each of the above-prepared ELISA balls was placed into the tubes of Example 5, Example 6, and Comparative Example 3 (three tubes for each Example), a phosphate buffer (pH 7.4) solution of .peroxydase-labeled avidin l,~.r.odl.~,ct of ~a.ppell (concentration of avidin: 1 ~g/mL) was injected into each tube (S00 mL per tube), and reaction was carried out at room temperature for 30 minutes.
After the reaction was completed, non-reacted peroxydase-labeled avidin was washed with a washing solution (phosphate buffer pH 7.4 + 0.05% Tween 20). Subsequently, each ELISA ball was allowed to develop color by use of a .
commercially available chromophoric kit for peroxidase (ML-1120T, product of Sumitomo Bakelite Co., Ltd.), and then subjected to measurement of absorbance at 450 nm by use of a r plate reader.
The results are shown in Table 3. The results show that, in Examples 5 and 6, the absorbance varies linearly with CA 02356857 2001-06-20 . _,. . r ,';,~;;l.
respect to the density o~ biov:icv hydra.zide introduced onto the surface of the ELISA ball, and that, in Comparative Example 3, the absorbance does not vary with the different densities of biotin hydrazide.
In Examples 5 and 6, peroxydase-labeled avidin is reacted with only biotin hydrazide introduced onto the surface of the ELISA ball, and thus th.e absorbance is proportional to the density of biotin hydrazide. In contrast, in Comparative Example 3, peroxydase-labeled avidin remain s in the tube due to adsorption, and the remaining avidin may act as a background, to thereby lower assay sensitivity.
Table 3 Biotin-avidin reaction by use of ELISA ball ~g/mL Example 5 Example 6 Comparative Example 3 ~
~
0.125 0.22 .14 1.32 0.25 0.56 0.64 1.36 0.5 1.12 1.27 1.39 (Comparison of protein recovery percentage in containers usable as storage containers) For comparison of non-specific adsorption, solutions of an enzyme-labeled anti-bovine-albumin antibody (product of Cosmo Bio) were prepared (concentration of the antibody: 0.1 ng/mL, 1 ng/mL, 10 ng/mL; and 100 ng/mL); each solution was injected into 24 wells of each plate; the plates were stored at -80°C foz 48 hours; and after storage was completely the concentration of the protein in each solution was measured by use of a substrate solution.
... ;r~~rx.~;
m CA 0235685 7 2001-06-20 'lhe results are shown in Tr~ble 4. The results show that the protein recovery percentage is high in the plates of Examples 5 and 6, as compared with the plate of Comparative Example 3.
Table 4 Protein concentration after storage (comparison of absorbance) ng/mL Example 5 Example 6 Comparative Example 3 0.1 0.18 0.21 0.03 1 0.58. 0.78 0.02 1.01 1.36 0.03 100 1.63 1.87 0.05 Industrial Applicability In the container for an immunoassay of the present invention, the adsorption amount of molecules or serum used for tl~e assay is 1 x 10-1 pmol/cm2 or less, and !,-;h~:s _LUss oW a reagent, which is caused by adsorption, is prevented during storage or dilution of the reagent. Therefore, when the container is used for a liquid-phase reaction, an assay can be carried out at high sensitivity and high accuracy, since there is prevented decrease in reaction efficiency, which is caused by adsorption of molecules to be assayed, or impediment of reaction due to adsorption of unwanted molecules.
When the container is used for a clinical test employing serum, the test can be carriE=d out under conditions S
similar to those inside the body of an organism, since variation of the structure of serum components, which is caused by adsorption, does not occur in the container.
Description Container for Immunologic Assay Technical Field The present invention relates to a container used for storage, dilution, or reaction of a reagent and/or a test sample, in an immunoassay for detecting an antigen or an antibody through antigen-antibody reaction.
Background Art Conventional immunoassays employ a polystyrene- or polypropylene-made container for storage and dilution of a reagen c :.;r a sample which is ~ to be used. i~ow~ver; molecules contained in the reagent or the sample are non-specifically adsorbed onto such a container, and such adsorption necessarily causes loss of the reagent or the sample, as well as variation in concentration of a solution containing the reagent or the sample.
In recent years, in accordance wiith diversification of immunoassay methods, in most cases, naturally occurring , substances have been used after extraction and purification, particularly in the drug production deF>artment of drug manufactures. Generally speaking, such substances are obtained in very small amounts and thus. are quite expensive.
Therefore, reduction in the: amount of substance during storage or dilution, w~lich is caused by physical adsorption :, ~ ;,: _;; ,. ..~;:, onto a container, is not negligible.
When samples used for clinical diagnosis, such as serum and urine, are collected from patients, placed in a container, ar~d stored therein ur mil the _samples are sub.je cted .to ~,~;say, clinically important proteins contained in the samples, such as albumin, transferrin, and immunoglobulin, are adsorbed onto the container. Most containers uaed for clinical diagnosis, including syringes and cups used in the step of collecting a sample, tubes used in the step of storing the sample, and centrifugation tubes and test tubes used in the step o'f purifying, concentrating, or diluting the sample, are formed from polypropylene or polystyrene, and such a container is not subjected to surface -treatment. Therefore, when even a trace amour~t of proteins contained in thc: sample is adsorbed onto the container in each step, the concentration of the proteins is expeci~ed to vary greatly after all the steps have been performed, as compared with the concentration of the proteins at the time of collection of the sample.
In general, the price of a reagent: in immobilized form accounts for about 80~ the cost of a clinical test kit sold by a clinical test drug manufacturer. Therefore, when reduction in, the reagent due to adsorption onto a container is suppressed, production costs are greatly reduced.
In a solid phase method (a type of immunoassay method), assay is carried out by utilizing proteins immobilized onto the surface of a container for an immunoassay. Therefore; a CA 02356857 2001-06-20 ~ .. 'a~,~,.";"::-.
solid phase method employs a container subjected to "high adsorption treatment," in which, in order to increase the amount of a reagent which is to be immobilized onto the surface of the contai:nP~-, ~ ryd_ron~~ilic,-hydrophobic balance of the surface is regulated through introduction of a functional group such as a hydroxyl group, thereby increasing the saturation adsorption amount of the reagent.
In recent years, in order to shorten immunoassay time and to carry out immunoassay on a large scale, immunoassay methods making use of an automatic analyzer (robot) have been developed. Such methods have rapidly become prevalent, particularly in the drug production department of drug manufacturers .
When an assay is carried out by m~earis of a conventional solid phase method, a washing step for eliminating non-immobilized excess molecules is required. However, an automatic analyzer encounters difficulity in carrying out the washing step, in which fractional inje<a ion and suction of a washing solution are repeated. Therefore, a sequential addition method has been under developnnent as an immunoassay method suitable for an automatic analyzer, because such a .
method does not require separation ofra reacted substance and a non-reacted substance through a wasr~~.ng step.
In a sequential addition method, ~'.mmobilization of molecules is not carried out during reaction, and reaction is carried out in a solution. Therefore, 'when a container having a surface subjected to the aforementioned high adsorption treatment is used, unwanted adsorption of the molecules impedes reaction in the solution or lowers the reaction efficiency.
Tn rQC~~.t y ears , in accor~.~nce with progxess in __ ._ _ measurement techniques, evaluations through a fluorescence method or an emission method have been established, the method having high sensitivity as compared with absorbance assay by means of a conventional color:imetric method.
Therefore, in the future, unwanted adsorption of molecules onto a container is expected to induce problems in such ari assay'method having high sensitivity.
At the present time, a container used for such a method is provided without consideration of molecular adsorption;
i . a . , the coini:ainer is formed from polystyrene or polypropylene in consideration of only shapability, transparency, and low-temperature resistance, and the container is not subjected to surface t:reatment for suppressing adsorption of molecules. From the viewpoint of characteristics of the container, no ataempt has been made to solve problems such as loss of a reagent and reduction in sensitivity.
However, in order to control non-~;pecific adsorption of molecules onto *:he surface of a container for immunoassays, several tecl~:niques have hitherto been studied and carried out.
For example, a blocking method is most widely carried out, in which a container is coated with a protein inactive to a sample which is to be assayed. Since the method .... :.::..: '::, :,:. ..:. , . 'vx:iiwidl'iiat;.
basically utilizes non-specific adsorption of the protein onto the container, blocking effects may differ from container to container, and may depend. on the state of the protein. In addition, sinr..c the i~.~ct.i«e prc~tvein, is non-specifically adsorbed onto the container, the protein is easily detached from the container into a solution, and thus the container cannot be used for storing the solution.
Japanese Patent Application Laid-Open (xoxa.i) Nos. 6-174726 and 7-128336 disclose a technique in which such detachment of a protein is eliminated by chemically immobilizing the protein onto a container. However, thE: structure of the protein may vary in accordance with drying temperature, storage temperature, and storage time, and thus the container is not widely used in practice:. .
When the higher-order structure o:~ a protein adsorbed onto a container varies, the protein induces secondary adsorption. When a protein which is inactive in a. free state is adsorbed onto or chemically bound to a container, the protein cannot completely maintain its inactive state, due to alteration of the higher-order structure. Therefore, even when adsorption of another protein ontc> the container can be prevented, variance of the higher-order structure induces secondary adsorption between the proteins.
Secondary adsorption between proteins varies with types of proteins, 'and thus a protein suitable for blocking must be chosen every time a sample which is to be assayed is changed.
When a solution containing different proteins, such as serum, y. . .,..., ~...;.,:.. ~ ~i.. ."~ ~.~ ~~:~\
CA 02356857 2001-06-20 " "~
is used as a sample, no blocking protean can control adsorption of all the proteins contained in the sample.
Disclosure cf ':h.e Invention In view of the foregoing, the present inventors have performed extensive studies on characteristics of a container, and have found that when the saturation amount of molecules which are adsorbed onto the container, the molecules being used for an immunoassay, is controlled to a predetermined value or less, loss of a reagent or a sample is prevented during storage, dilution, and reaction, and the sample can be assayed at high sensitivity. The presE:nt invention has been accomplished on the basis of this finding.
~ccorciimgiy, the present invention provides a container for an immunoassay in which the saturation adsorption amount of molecules used for the assay is 1 x 10-1 pmol/cm2 or less .
Brief Description of Drawings Fig. 1 shows the concentration of proteins after bovine serum and albumin have been stored in i~he container for an immunoassay of the present invention air -80°C fo.r 48 hours ..
Fig. 2 shows reaction efficiency when an immunoassay is carried out in the container of the prEaent invention.
Best Mode for Carrying Out the Inventic>n In a conventional polystyrene- or polypropylene-made container for an immunoassay, the adsorption amount of ' 6 ~.4 - ~,~_~e_ molecules ( a . g . , proteins ) is about 1 --10 pmol,~cm? or. -~~rP
i.e., about 20-500 of molecules (e.g., proteins) used for an immunoassay are adsorbed onto the container, although the adsorption amount varies in accordance; with the concentration of a solution containing such molecule's and the contact area between the molecules and the container. G~Then the adsorbed molecules (20-50% of all the molecule;) are essential for reaction in the solution, reaction efficiency; i.e., assay sensitivity, is reduced by 20-50%. Meanwhile, when the adsorbed substance is such that it undergoes molecular structural changes due to adsorption t.o thereby cause unwanted reaction, considerable noise would result.
Therefore, a container onto which no molecules used for an immunoassay are adsorbed is most icLeal, but when the adsorption amount of molecules is substantially reduced to 1/10-1/100 with respect to the current level, satisfactory effects will be obtained.
Although the adsorption amount of molecules contained in a solution varies with the identity of the molecules, temperature; concentration of the solution, and the pH of the solvent, the container desirably meets the following conditions: the saturation adsorption amount of the molecules used in the immunoassav is 1 x 10-1 priiol/cm2 or less under the specific conditions ir~ terms of conc:entratiozn c-;f true solution, temperature, and pH of the,solvent under which the reaction and assay are carried out. In the case in which serum is used for an immunoassay, since serum is usually ~::,;
diluted up to i/10, tYve effer~. .~f the invention can be attained if the saturation adsorption amount of the molecules which participate in and/or affect the=_ assay, among a:11 molecules contained in the diluted serum, is always 1 x 10-1 pmol/cm2 or less at the diluted concentration of serum and under the specific conditions in terms of concentration of the solution, temperature, and pH of i~he solvent under which the reaction and assay are carr_Led out.
Similarly, when the container is used for storage and dilution of a reagent, the effect of the invention can be attained if the saturation adsorption amount of the molecules that undergo storage and dilution is always 1 x 10-1 pmol/cm2 or less under the specific conditions- in terms of concentration.nf..the.solnzti.on,, temberature, and pH of the solvent under which the reagent is removed from the storage container or dilution is carried out. In many cases, the reagent is stored in the containez: at a temperature as low as -80°C. However, adsorption of the molecules is an equilibrium reaction, and thus, it would be sufficient if the saturation adsorption amount of molecules is 1 x 10-1 pmol/cm2 or less under the specific conditions- in terms of concentration, temperature, and pH under which the reagent is removed from the container.
T~le saturation adsorption amount of the molecules is more preferably 1 x 10-2 pmol/cm2 or less, much more preferably h x 10-3 pmol/cm2 or less .
Examples of the molecules used iru an immunoassay ,..,.,. ~y:l" (~1:.
,.. ,k~~~,( -ie:rlude proteins ;e.g., enzymes, physiologically active proteins, and antibodies), nucleic acids, and physiologically active substances. Of these, proteins are particularly preferable. The saturation adsorption amount of the molecules can be measured by means of colloidal gold labeling immunoassay.
In the point that adsorption of a protein is prevented, the present invention exerts excellent: effects in addition to the aforementioned characteristic feature. Usually, when a protein is adsorbed onto a container, the structure of the protein is varied. Therefore, when an immunoassay is carried out, although a target protein is contained in a sample to be assayed, the protein may fail to be detected by an antibody, ~3~_,.e t~ variation in the structure of the protein. When a clinical test is carried out, in practice serum whose structure has been altered due to adsorption is assayed, even though serum must be assayed in the same state in which the serum is present in an organism. According to the present invention, since a protein is not adsorbed onto the container, the structure of the protein is not altered, and thus when a clinical test is carried out by use of the container, serum can be assayed in a state similar to that in which serum is present in an organism. Therefore, th~~ container of the present inVerition is very advantageoasly used as ~ container c far an immunoassay.
In a container for an immunoassay, the saturation adsorption amount of mclecules must be decreased at a portion ,.:.. .:".s~',, ,t, with t~:hich a reagent or a samp.lQ is brought into contact;
specifically, an inner surface of the container. Therefore, the molecular saturation adsorption amount at an inner surface of the container should be at least 1 x 10-1 pmol/cm2 or less.
In order to decrease the saturation adsorption amount of molecules at an inner surface of the container to ~. x 10-1 pmol/cm2 or less, preferably, at least. the inner surface is formed from a highly hydrophilic polymer or a highly hydrophobic polymer, or is coated with a highly hydrophilic polymer or a highly hydrophobic polymer. More preferably, at least the inner surface is coated with a highly hydrophilic polymer or a highly hydrophobic polyme r. Much more preferably, at least r.hP.i.nner surface is coated with a highly hydrophilic polymer. Particularly preferably, at least the inner surface is coated with an ultra-hydrophilic polymer.
Examples of highly hydrophobic polymers include fluorine-containing resins such as pol.ytetrafluoroethylene~
(PTFE) and 'silicon-containing resins. When the surface of the container is coated with a hydropr~obic polymer, the surface may be coated with the aforementioned hydrophobi c polymer, or the container may be fluoz~inated, thereby forming a fluorinated polymer fi~.m on the ,surf:ace thereof .
No particular limitation is impo~~ed on the highly hydrophilic polymerP so long as the polymer contains a hydrophilic group such as a carboxyl group or a hydroxyl ftiN.4ili'.
gro~zp. E~:.~-~J,yles of such a hydrophil is polymer include polymethacrylic acid, (meth)methacrylic acid-alkyl methacrylate copolymers, polyhydroxyalkyl rriethacrylates (e. g., polyhydroxyethyl methacrylate), hydroxyalkyl methacrylate-alkyl rnethacrylate copolymers, polyoxyalkylene-group-containing methacrylate polymer and copolymers containing the polymer, polyvinyl pyrrolidone, ethylene-vinyl alcohol copolymers, (2-methacryloyloxyethylphosphocholine) polymers (MPC) and copolymers containing the polymers (Seitai Zairyo, Vol. 9, No. 6, 1991), and phospholipid~polymer composites (Japanese Patent Application Laid-Open (kokai) Nos. 5-161491 and 6-46831). The container may be formed from such a hydrophilic polymer, or coated with the polymer.
Afvi.er. the container is formed from a suitable matFr.ial, such as polystyrene, a hydroxyl group or a carboxyl group may be introduced into the surface of the container, to thereby impart high hydrophilicity; i.e., low adsorbability, to the surface of the container. A surface exhibiting low adsorbability can be realized by means of surface modification. For example, when, in consideration of formability, the container is formed from a material which tends to induce non-specific adsorption, such as polystyrene or polypropylene, a carboxyl group, a carbonyl group, and/or a hydroxyl group may be introduced into the surface of the container through plasma exposure, to i:hereby impart low adsorbability to the surface. When, in consideration of transparency, the container is formed from polymethyl S
CA 02356857 2001-06-20 ",'Y~" ''...
methacrylate, a carboxyl group may be introd~:ccd into the surface of the container through partial hydrolysis of the surface by use of an alkali, to thereby impart low adsorbability to the surface.
When hydrophilicity is imparted t:o the inner surface of the container by use of a hydrophilic polymer, to thereby reduce the adsorption amount of molecules, the contact angle between the surface and water is preferably 30° or less (highly hydrophilic), more preferably 15° or less, much more preferably 1° or less (ultra-hydrophilic).
When, among the aforementioned hydrophilic polymers, there is employed a polyhydroxyalkyl m.ethacrylate, a polyoxy(C2-C9 alkylene-group-containing methacrylate) polymer or a copolymer containing the pol.j~mer.; _a. (2- . , methacryloyloxyethylphosphocholine) polymer or-a copolymer containing the polymer; a phospholipid~polymer composite; or polyvinyl pyrrolidone, the contact angle between the surface of the resultant container and water becomes 1° or less (i.e., the container is ultra-hydrophilic), and the saturation adsorption amount of proteins becomes 1 x 10'3 pmol/cm2 or less, which is particularly preferable.
The product form of the container of the present invention is not particularly limited, and the container may assume conventionally used product for:r~s, including a sample tube, a centrifugation tube, a multi-well plate, and a cuvette. However in order to carry Ollt storage, dilution, reaction, and assay of a sample in one container, the CA 02356857 2001-06-20 ,. .,... , ...
container prvferal~.~y assumes a form of multi-well plate.
Examples The present invention will next be described in more detail by way of Examples, which should not be construed as limiting the invention thereto.
(Example 1) A commercially available polypropylene-made 96-well plate (MS-3396P, product of Sumitomo E>akelite Co., Ltd.) was subjected to y-ray treatment at 70 kGy, to thereby generate a hydroxyl group on the surface of the ~>late. In the resultant plate, the saturation adsorption amount of proteins was 4.6 x 10-z pmol/cm2, and the contact angle between the surface and -~. ,,; Q ~. ~ -w s (Example 2) A commercially available polypropylene-made 96-well plate (MS-3396P, product of Sumitomo Bakelite Co., Ltd:) was coated with a commercially available fluorine-containing coating agent (Scotchguard, product of Sumitomo 3M Ltd.). In the resultant plate, the saturation adsorption amount of proteins was 2.7 x 10-2 pmol/cm2, and the contact angle between the surface and the water was 126°.
(Comparative Example 1) A cornme~cfally available polypsop~ylene-made 96-well plate (MS-3396P, product of Sumitorno Bakelite Co., Ltd.) was used as a comparative plate. In the plate, the saturation adsorption amount of proteins was 3.7 pmol/cm2, and the CA 02356857 2001-06-20 ~:7:
contact angle between the surface and the water was 92.~a., (Comparison of protein recovery percentage in containers usable as storage containers) For comparison of non-specific adsorption, solutions of an enzyme-labeled anti-bovine-albumin antibody (product of Cosmo Bio) were prepared (concentration of the antibody: 0.1 ng/mL, 1 ng/mL, 10 ng/mL, and 100 ng/mL, respectively); each solution was injected into 24 wells of each of the plates of Examples 1 and 2 and Comparative Example 1; the plate was stored at -80°C for 48 hours: and after storage time had elapsed, the concentration of the protein in each solution was measured by use of a substrate solution.
The results are shown in Fig. 1. The results show that ~he protein recovery percentage is high in.the plates Of Examples 1 and 2, as compared with the case of the plate of Comparative Example 1.
(Comparison of "in solution" reaction efficiency) In order to evaluate the efficiency of a reaction in a solution, the following test was carried out by using the plates of Examples 1 and 2 and Comparative Example 1 as reaction containers.
Rat albumin (product of Cosmo Bio) was dissolved in a phosphate buffer (Dulbecco PBS pH 7.4j to thereby prepare solutions (concentration of the albumin: l;? ng!mL,-~. ng/mL, and 0.1 ng/niL, respectively), and each solution was injected into four lines (i-:e., 32 wells) (100 pl per well) of each of the plates of Examples 1 and 2 and Comparative Example 1.
CA 02356857 2001-06-20 ... ... .,,.
Subsequently, a pnos~~~ra.:~~;e buffer (Dulbecco PBS pH 7.4) solution of a peroxidase-labeled anti-rat-albumin antibody (product of Cosmo Bio) (concentration of the antibody: 100 ng/mL, respectively) was injected into all the wells (100 ~.1 per well) of each plate.
After reaction had been carried cut in each well at 37°C
for 30 minutes, the solution in each well was transferred into a 96-well plate for ELISA in which an anti-rat-albumin antibody had been immobilized onto each well in advance, and then reaction was carried out again in each well at 37°C for 30 minutes.
After reaction was completed, a non-reacted peroxydase-labeled anti-rat-albumin antibody was washed with a washing solution (r7vl.?.~.e._~.co P~iS pH ?.,4 + 0,05% Tween 20) .
Subsequently, each plate was allowed to develop color by use of a commercially available chromophoric kit for peroxidase (ML-1120T, product of Sumitomo Bakelite Co., Ltd.), and then absorbance at 450 nm was measured using a plate reader.
The results are shown in Fig. 2. The results show that, in relation to the plate of Comparative Example 1, the absorbance is low when the concentration of the albumin is low; i.e., the reaction in the solution is impeded due to adsorption, and that, in relation to t:he plates of Examples 1 and 2, linearivy is obtained between t;he concentration of the ,;
albumin and~the absorbance when the albumin concentration is low; i.e., the antigen-antibody reaction in the solution is efficiently carried out.
CA 02356857 2001-06-20 ~. . .. ,..,....
(: E?'~ ~ J!~l~y.~ 1 C 3 ) ..
A commercially available polystyrene-made tube (Eiken tube for RIA No. 3, 70-12458) was coated with polyhydroxyethyl methacrylate (P-3932, product of SIGMA). In the resultant tube, the saturation ads>orption amount of proteins was 9.1 x 10-4 pmol/cm2, and the contact angle between the surface and the water was 0°.
(Example 4) Polytetrafluoroethylene was formE:d into a tube of the same inner diameter and volume as the tube of Example 1. In the resultant tube, the saturation adsorption amount of proteins was 7.2 x 10-3 pmol/cm2, and the contact angle between the surface and the water was 126°.
arr",~~arai-.ive Example 2) A commercially available polystyrene-made tube (Eiken tube for RIA No. 3, 70-12458) was used as a comparative tube.
In the tube, the saturation adsorption amount of proteins was 8.1 pmol/cm2, and the contact angle between the surface and the water was 85°.
(Comparison of assay sensitivity) In order to evaluate the assay sensitivity of a reaction in a solution, the following test was carried out by use of the tubes of Examples 3 and 4 and Comparative Example 2 as reac:Liom containers and an ELISA :ball as a carrier for E
reaction.
Phosphate buffer (pH 7.4) solutions of biotin hydrazide (product of Dojindo) were prepared in advance (concentration CA 02356857 2001-06-20 . _..... . ~ ;:~"r...
of biotin hydrazide: 0. 125 ~gi~uL, 0. a:'~~0 ~,g; mL, and 0. 500 ~g/mL, respectively). By use of the solutions, biotin hydrazide was immobilized onto ELISA ~>alls (amino-group-containing ball, product of Sumitomo Bakelite Co., Ltd.) through covalent bonding via glutarale'.ehyde, to thereby prepare ELISA balls having three different immobilization densities of biotin hydrazide.
A portion of each ELISA ball at which biotin hydrazide was not immobilized was subjected to blocking by use of skim milk so as to prevent adsorption.
Each of the above-prepared ELISA balls was placed into each of the tubes of Example 3, Example 4, and Comparative Example 2 (three tubes for each Example), a phosphate buffer (pH 7.4) solution of pPYoxydase-labeled av.idin,(product of Cappel) (concentration of avidin: 1 ~.g/mL) was injected into each tube (500 mL per tube), and reaction was carried out in each tube at room temperature for 30 minutes.
After the reaction was completed, non-reacted peroxydase-labeled avidin was washed with a washing solution (phosphate buffer pH 7.4 + 0.050 Tween 20). Subsequently, each ELISA ball was allowed to develop color by use of a commercially available chromophoric kit for peroxidase (ML-1120T, product of Sumitomo Bakelite Co., Ltd.), and then absorbanCe at 450 nm was measured by using a plate reader.
The results are shown in Table 1.; The results show that, in Examples 3 and--4, the absorbance varies linearly with respect to the density of biotin hydra:zide introduced onto ~.'~ ~ .a':~.K:
the surfac:=: ~~i she ELISA ball, and that, in Comparative Example 2, the absorbance does not vary with the different densities of biotin hydrazide.
In Examples 3 and 4, peroxydase-labeled avidin is reacted with only biotin hydrazide introduced onto the surface of the ELISA ball, and thus th.e absorbance is proportional to the density of biotin hydrazide. In contrast, in Comparative Example 2, peroxydase-labeled avidin remains in the tube due to adsorption, and the remaining avidin may act as a background, to thereby reduce assay sensitivity.
Table 1 Biotin-avidin reaction by use of ELISA ball ~g/mL Example 3 Example 4 Comparative Example 2 0.125 0.2 0.27 1.05 Om2 5- 0.54 0.62 1.03 - . : ..
0.5 0.97 0.91 ' 1.12 (Comparison of protein recovery percentage in containers usable as storage containers) For comparison of non-specific adsorption, solutions of an enzyme-labeled anti-bovine-albumin antibody (product of Cosmo Bio) were prepared (concentration of the antibody: 0.1 ng/mL, 1 ng/mL,.lO ng/mL, and 100 ng/mL, respectively); each solution was injected into 24 wells of each of the plate of Example 3, the plate of Example 4, and the plate of ComparativeExample 2; the plates were stored at -80°C for 48 hours; and after storage time had elapsed, the concentration of the protein in each solution was measured by use of a CA 02356857 2001-06-20 ,. ._ substrate solution.
The results are shown in Table 2. The results show that the protein recovery percentage is high in the plates of Examples 3 and 4, as compared with in the plate of Comparative Example 2.
Table 2 Protein concentration after storage (comparison of absorbance) ng/mL Example 3 Example 4 Comparative Example 2 0.1 0.24 0.26 0.01 1 0.41 0.33 0.07 0.76 0.66 0.07 100 1.02 0.82 ~
0.03 (Example 5) A 2.0 wt/volo methanol solution of polyhydroxyethyl methacryl.ate (P-3932, prcduct of SIGMA.) 2.5 mL) :~~as injected into a commercially available polystyrene-made tube (Eiken tube for RIA No. 3, 70-12458). Subsequently, the solution was removed from the tube, the tube was inverted so as to prevent the residual solution from remaining at the bottom, and the tube was dried at room temperature for 24 hours, and consequently the surface of the tube was coated with polyhydroxyethyl methacrylate. In the resultant tube, the saturation adsorption amount of proteins is 8.7 x 10-9 pmol/cm2, and the contact angle between the surface and water ,-is 0°.
(Example 6) A 0.5 wt/vol% ethanol. solution of an MPC polymer (2.5 rnL) was injected into a commercially available polystyrene-.. . . ... .,. ..., . ....... . ':,s'::: ~. .:yyVtwi,C3L4 made tube (Eik~n tubs. ;_or RIA No. 3, 70-12458), and the tube was allowed to stand at room temperature for 10 minutes.
Subsequently, the solution was removecL from the tube, the tube was inverted so as to prevent they residual solution from remaining at the bottom, and the tube was dried at room temperature overnight, and consequently the surface of the tube was coated with the MPC polymer. In the resultant tube, the saturation adsorption amount of proteins is 6.5 x 10-9 pmol/cm2, and the contact angle between the surface and water is 0° .
The MPC polymer was synthesized from an MPC-BMA (butyl methacrylate) copolymer (ratio of MPC to BMA = 3/7) which was prepared according to the procedure described in "Release of a dri~~:;, frc:m a hydw~c~e:l meml-hrane having a structure analogous to that of phospholipid," (Kobunshi Rc>nbunshu, 46, 591-595 (1989) ) .
(Comparative Example 3) A commercially available polystyrene-made tube (Eiken tube for RIA No. 3, 70-12458) was used in "as is" form as a comparative tube.
(Comparison of assay sensitivity) In order to evaluate the assay sensitivity of a reaction in a solution, the following test was carried out by use of the tubes of Examples 5 and 6 a.nd Comparative Examp~.e 3 as reaction containers, and an ELISP, ball (amino-group-containing ball, product of Sumitomo P~akelite Co., Ltd.) as a carrier for reaction.
..
,. ... ... ...: , .,u.
... .
~;
Phosphate buffer (pH 7.4) solutions of biotin luya.ra~zide (product of Dojindo) were prepared in advance (concentration of biotin hydrazide~ 0.125 ~.g/mL, 0.250 ~.g/mL, and 0.500 ~.g/mL). By use of the solutions, biotin hydrazide was immobilized onto ELISA balls through covalent bonding via glutaraldehyde, to thereby prepare ELI:SA balls having three different immobilization densities of biotin hydrazide.
A portion of each ELISA ball at which biotin hydrazide was not immobilized was subjected to ~>locking by use of skim milk so as to prevent adsorption.
Each of the above-prepared ELISA balls was placed into the tubes of Example 5, Example 6, and Comparative Example 3 (three tubes for each Example), a phosphate buffer (pH 7.4) solution of .peroxydase-labeled avidin l,~.r.odl.~,ct of ~a.ppell (concentration of avidin: 1 ~g/mL) was injected into each tube (S00 mL per tube), and reaction was carried out at room temperature for 30 minutes.
After the reaction was completed, non-reacted peroxydase-labeled avidin was washed with a washing solution (phosphate buffer pH 7.4 + 0.05% Tween 20). Subsequently, each ELISA ball was allowed to develop color by use of a .
commercially available chromophoric kit for peroxidase (ML-1120T, product of Sumitomo Bakelite Co., Ltd.), and then subjected to measurement of absorbance at 450 nm by use of a r plate reader.
The results are shown in Table 3. The results show that, in Examples 5 and 6, the absorbance varies linearly with CA 02356857 2001-06-20 . _,. . r ,';,~;;l.
respect to the density o~ biov:icv hydra.zide introduced onto the surface of the ELISA ball, and that, in Comparative Example 3, the absorbance does not vary with the different densities of biotin hydrazide.
In Examples 5 and 6, peroxydase-labeled avidin is reacted with only biotin hydrazide introduced onto the surface of the ELISA ball, and thus th.e absorbance is proportional to the density of biotin hydrazide. In contrast, in Comparative Example 3, peroxydase-labeled avidin remain s in the tube due to adsorption, and the remaining avidin may act as a background, to thereby lower assay sensitivity.
Table 3 Biotin-avidin reaction by use of ELISA ball ~g/mL Example 5 Example 6 Comparative Example 3 ~
~
0.125 0.22 .14 1.32 0.25 0.56 0.64 1.36 0.5 1.12 1.27 1.39 (Comparison of protein recovery percentage in containers usable as storage containers) For comparison of non-specific adsorption, solutions of an enzyme-labeled anti-bovine-albumin antibody (product of Cosmo Bio) were prepared (concentration of the antibody: 0.1 ng/mL, 1 ng/mL, 10 ng/mL; and 100 ng/mL); each solution was injected into 24 wells of each plate; the plates were stored at -80°C foz 48 hours; and after storage was completely the concentration of the protein in each solution was measured by use of a substrate solution.
... ;r~~rx.~;
m CA 0235685 7 2001-06-20 'lhe results are shown in Tr~ble 4. The results show that the protein recovery percentage is high in the plates of Examples 5 and 6, as compared with the plate of Comparative Example 3.
Table 4 Protein concentration after storage (comparison of absorbance) ng/mL Example 5 Example 6 Comparative Example 3 0.1 0.18 0.21 0.03 1 0.58. 0.78 0.02 1.01 1.36 0.03 100 1.63 1.87 0.05 Industrial Applicability In the container for an immunoassay of the present invention, the adsorption amount of molecules or serum used for tl~e assay is 1 x 10-1 pmol/cm2 or less, and !,-;h~:s _LUss oW a reagent, which is caused by adsorption, is prevented during storage or dilution of the reagent. Therefore, when the container is used for a liquid-phase reaction, an assay can be carried out at high sensitivity and high accuracy, since there is prevented decrease in reaction efficiency, which is caused by adsorption of molecules to be assayed, or impediment of reaction due to adsorption of unwanted molecules.
When the container is used for a clinical test employing serum, the test can be carriE=d out under conditions S
similar to those inside the body of an organism, since variation of the structure of serum components, which is caused by adsorption, does not occur in the container.
Claims (11)
1. A container for an immunoassay in which the saturation adsorption amount of molecules used for the assay is 1 x 10-1 pmol/cm2 or less.
2. A container for an immunoassay according to claim 1, wherein at least an inner surface of the container is formed from or coated with a highly hydrophilic polymer or a highly hydrophobic polymer.
3. A container for an immunoassay according to claim 1, wherein at least an inner surface of the container is formed from or coated with a highly hydrophilic polymer.
4. A container for an immunoassay according to claim 3, wherein the contact angle between the inner surface of the container and water is 30° or less.
5. A container for an immunoassay according to claim 3, wherein the contact angle between the inner surface of the container and water is 15° or less.
6. A container for an immunoassay according to claim 3, wherein the contact angle between the inner surface of the container and water is 1° or less.
7. A container for an immunoassay according to any one of claims 1 through 6, wherein the saturation adsorption amount of molecules used for the assay is 1 x 10-3 pmol/cm2 or less.
8. A container for an immunoassay according to any one of claims 3 through 7, wherein at least an inner surface of the container is coated with an ultra-hydrophilic polymer.
9. A container for an immunoassay according to claim 8, wherein the ultra-hydrophilic polymer is selected from among a polyhydroxyalkyl methacrylate, a polyoxy(C2-C4 alkylene-group-containing methacrylate) polymer or a copolymer containing the polymer, polyvinyl pyrrolidone, and a phopholipid-polymer composite.
10. A container for an immunoassay according to claim 8, wherein the ultra-hydrophilic polymer is a (2-methacryloyloxyethylphosphorylcholine) polymer or a copolymer containing the polymer.
11. (added) A container for an immunoassay according to any one of claims 2 through 8, wherein the highly hydrophilic polymer or the ultra-hydrophilic polymer is insoluble in water.
Applications Claiming Priority (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10/367404 | 1998-12-24 | ||
| JP36740498 | 1998-12-24 | ||
| JP5625399 | 1999-03-03 | ||
| JP11/56253 | 1999-03-03 | ||
| JP21209699 | 1999-07-27 | ||
| JP11/212096 | 1999-07-27 | ||
| PCT/JP1999/005979 WO2000039582A1 (en) | 1998-12-24 | 1999-10-28 | Container for immunologic assay |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2356857A1 true CA2356857A1 (en) | 2000-07-06 |
Family
ID=27295858
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002356857A Abandoned CA2356857A1 (en) | 1998-12-24 | 1999-10-28 | Container for immunologic assay |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP1152242B1 (en) |
| JP (1) | JP3681983B2 (en) |
| AT (1) | ATE314147T1 (en) |
| AU (1) | AU6366799A (en) |
| CA (1) | CA2356857A1 (en) |
| DE (1) | DE69929248T2 (en) |
| WO (1) | WO2000039582A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11421102B2 (en) * | 2015-11-24 | 2022-08-23 | Jsr Corporation | Method for manufacturing porous particles, porous particles, carrier, column, and method for separating target substance |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4774989B2 (en) * | 2003-06-25 | 2011-09-21 | 日油株式会社 | Embryoid body formation container and embryoid body formation method |
| DE10356752A1 (en) | 2003-12-04 | 2005-06-30 | Roche Diagnostics Gmbh | Coated test elements |
| WO2006040107A1 (en) * | 2004-10-12 | 2006-04-20 | Schott Ag | Multiplexed protein adsorption assay |
| JP2009050201A (en) * | 2007-08-27 | 2009-03-12 | Dainippon Printing Co Ltd | Culture tool for early embryo or the like |
| US10955410B2 (en) | 2016-05-24 | 2021-03-23 | Japanese Foundation For Cancer Research | Method of recovering extracellular vesicles and container for extracellular vesicles |
| WO2017217336A1 (en) | 2016-06-15 | 2017-12-21 | 日産化学工業株式会社 | Container for cryopreservation |
| JP7622432B2 (en) * | 2018-11-14 | 2025-01-28 | 日産化学株式会社 | Containers and methods for preserving, pretreating and analyzing biological materials |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BE789839A (en) * | 1971-10-08 | 1973-02-01 | Ceskoslovenska Akademie Ved | PROCESS FOR THE SURFICIAL TREATMENT OF ARTICLES |
| US4472357A (en) * | 1981-11-18 | 1984-09-18 | Medical Laboratory Automation, Inc. | Blood bank cuvette cassette and label therefor |
| FI833207A0 (en) * | 1983-09-08 | 1983-09-08 | Farmos Oy | REAKTIONSKAERL FOER IMMUNOLOGISKA BESTAEMNINGAR |
| JPS6091983A (en) * | 1983-10-25 | 1985-05-23 | Susumu Kogyo Kk | Membrane carrier for immobilizing protein and its preparation |
| JPH0419561A (en) * | 1990-05-14 | 1992-01-23 | Nippon Shokubai Co Ltd | Blocking agent for immunoassay |
| JPH0833472A (en) * | 1994-07-25 | 1996-02-06 | Terumo Corp | Cell reserver |
| JP3884510B2 (en) * | 1996-10-14 | 2007-02-21 | 日本油脂株式会社 | Method for stabilizing immobilized immunologically active substances during storage |
-
1999
- 1999-10-28 JP JP2000591430A patent/JP3681983B2/en not_active Expired - Fee Related
- 1999-10-28 AT AT99951118T patent/ATE314147T1/en not_active IP Right Cessation
- 1999-10-28 WO PCT/JP1999/005979 patent/WO2000039582A1/en active IP Right Grant
- 1999-10-28 CA CA002356857A patent/CA2356857A1/en not_active Abandoned
- 1999-10-28 DE DE69929248T patent/DE69929248T2/en not_active Expired - Lifetime
- 1999-10-28 AU AU63667/99A patent/AU6366799A/en not_active Abandoned
- 1999-10-28 EP EP99951118A patent/EP1152242B1/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11421102B2 (en) * | 2015-11-24 | 2022-08-23 | Jsr Corporation | Method for manufacturing porous particles, porous particles, carrier, column, and method for separating target substance |
Also Published As
| Publication number | Publication date |
|---|---|
| DE69929248D1 (en) | 2006-02-02 |
| AU6366799A (en) | 2000-07-31 |
| DE69929248T2 (en) | 2006-08-17 |
| ATE314147T1 (en) | 2006-01-15 |
| EP1152242B1 (en) | 2005-12-28 |
| EP1152242A1 (en) | 2001-11-07 |
| JP3681983B2 (en) | 2005-08-10 |
| EP1152242A4 (en) | 2002-03-06 |
| WO2000039582A1 (en) | 2000-07-06 |
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| FZDE | Discontinued |