NL8302179A - IMMUNOCHEMICAL MEASUREMENT CARRIERS AND MEASUREMENT REAGENTS USING THESE CARRIERS. - Google Patents

Description

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Carriers for immunochemical measurement and measuring reagents using these carriers.

The present invention relates to immunochemical measurement carriers, the surface of which is coated with synthetic resin, and to immunochemical measurement reagents using these carriers.

In recent years, various immunochemical measurement reagents have been used as a means to measure concentrations of physiologically active substances such as peptide hormones, steroids, proteins, etc., as well as concentrations of drugs administered in vivo, which are administered in very small amounts in physiological samples such as serum, urine, etc. are present. Of these, reagents based on the enzyme immunoassay, radioimmunoassay, fluoro-inmuno assay, etc. are often used because they have high measurement sensitivity and excellent quantitative activity.

These reagents generally comprise (a) an insolubilized antibody or insolubilized antigen. obtained by binding an antibody or antigen corresponding to the substance to be measured to a carrier, and (b) a labeled antibody or labeled antigen obtained by labeling an antibody or antigen with a label agent such as an enzyme, etc. Measurement using such a reagent is carried out as follows: a substance to be measured and a labeled antibody or labeled antigen are reacted with an insolubilized antibody or insolubilized antigen, after which the label bound to the support (solid phase) and the unbound but present marker present in the liquid phase are separated. Thereafter, the activity of the marker in either the solid phase or the liquid phase is measured, thereby determining the amount or concentration of the substance to be measured. Therefore, the carrier should not non-specifically adsorb antigen, antibody or other components not involved in the immunological response.

On the other hand, attempts have been made to use containers as reagent carriers such as, for example, test tubes, etc. In other words, it has been expected that by binding an antigen or antibody to the surface of the inner wall of a container such as a test tube, etc., 8302179 i 2 and placing labeled antigen or labeled antibody in this container, a reagent is obtained which is convenient and stable for storage and allows for immediate measurement and thus can be conveniently used.

However, the storage stability of such a reagent has not been satisfactory to date and has shown the problem that after several months of storage the accuracy and sensitivity of the measurement decreases. The inventors of the present invention have conducted extensive research to clarify the cause of this problem and as a result they have found that the quality change of a reagent after storage can be attributed to the fact that the plastics used as carriers test tubes had small but influential gas permeability.

Although glass is a support material that is not permeable to gas, because glass adsorbs substances non-specifically, the use of glass as support is not suitable. The inventors have conducted further research to solve this problem and as a result have found that by coating the glass surface with a synthetic resin, non-specific adsorption can be prevented. The use of glass coated with a synthetic resin as a support for a reagent for immunochemical measurement has hitherto not been known.

The object of the present invention is to provide carriers for immunochemical measurement that prevent non-specific adsorption.

Another object of the present invention is to provide immunochemical measurement supports obtained by coating a glass surface with synthetic resin.

Another object of the present invention is to provide immunochemical measurement reagents which (a) an insolubilized antibody or insolubilized antigen obtained by binding the antibody or antigen to a glass support containing the. surface is coated with synthetic resin, (b) may comprise a labeled antibody or labeled antigen.

In the drawing, Fig. 1 is now a graph showing the standard curves of Example VII? and 8302179 * 3 Fig. 2 is a graph showing the standard curve of Example VIII.

Coating of a glass support can be carried out as follows: a material intended as a support, such as glass bead, a glass test tube, etc., is wetted with a synthetic resin solution or suspension to adhere said resin to the surface of the support. attach. Excess resin is removed if necessary and the wetted support material is then heat treated. The resin which is not coated on the support material 10 after the heat treatment is removed by washing. The thus-treated support is then attached to an antibody or antigen to obtain an insoluble antibody or insoluble antigen.

Various synthetic resins can be used as the coating agent. The synthetic resin can be classified into thermoplastic resins and thermosetting resins in accordance with the chemical structure and performance properties. The former resins are linerary polymers and become plastic and liquid on heating and solidify on cooling, which cycle is repeatable. The latter are resins such that their condensation products in early 20s have a linear structure but are large molecules; upon heating, they first soften and liquefy, but then undergo a cross-linking reaction between the molecules to form insoluble and fusible materials having a three-dimensional structure. Any of these types of synthetic resins can be used in the present invention.

As the thermoplastic resin, vinyl chloride resins, polystyrene resins, polypropylene resins, acrylic resins, fluorine resins, etc. can be used; as the thermosetting resin, silicone resins, phenolic resins, epoxy resins, etc. can be used. Of these, the silicone resins, polystyrene resins, acrylic resins and fluorine resins are particularly preferred.

As silicone resins there can be mentioned silicone oils with a linear structure, silicone rubbers with a partially cross-linked structure, silicone resins which are fully cross-linked and cured at the time of use, etc. Of these, the silicone oils are particularly preferred.

As polystyrene resins, there may be mentioned general purpose styrene resins obtained by mass homopolymerization of styrene; heat-resistant styrene resins, obtained by making a linear polymer with a higher degree of polymerization than the general-purpose styrene resins or by copolymerization with a monomer that imparts heat resistance, or by polymerization of α-methylstyrene; high impact styrene resins obtained by graft polymerization of styrene and butadiene, etc. Of these, the general purpose styrene resins are particularly preferred.

As acrylic resins, polymers of.

Acrylic acid esters and polymers of methacrylic acid esters, of which the polymers of acrylic acid esters are particularly preferred.

Polytetrafluoroethylene and polychlorotrifluoroethylene may be mentioned as fluorine resins, the polytetrafluoroethylene being particularly preferred.

These synthetic resins can be used as a solution in an organic solvent such as hexane, acetone, methyl chloride, chloroform, dichloroethane, trichlorethylene, etc., at a concentration of 1-30 w / w%, as a powder, as a paste, or as a suspension. When dissolved in an organic solvent, the coating is performed by contacting the surface of the support with the solution and then heat-treating the surface. On the other hand, a synthetic resin solution can be sprayed on the surface of the support and then the heat treatment carried out. The coating can also be carried out by using molten synthetic resin 25 'at a temperature above its melting point instead of using a solution in an organic solvent. Furthermore, it is also possible to make the synthetic resin into a paste which can be coated on the surface of the carrier, after which it is heated to realize a coating. For example, when a silicone resin is to be coated on a support material, the resin is dissolved in a solvent in a concentration of 1 - 30%, preferably 5 - 10%, the surface of the support is contacted with this solution, after which excess solution is removed, and heat treatment is performed for one hour at about 180 ° C. After cooling, the uncoated resin is removed by washing with trichlorethylene. A coated support is obtained after drying.

8302179 r ~ - «- # * 5%.

The shape of the carrier can be varied, and for example, various shapes such as beads, test tubes, drum beads, titre plates, ampoules, syringes, etc. can be used.

In order to bind antibody or antigen to the synthetic resin coated support, procedures such as those described in "Clinica Chimica Acta", 48, 15 (1973), "Journal of Immunology", 116, 1554 (1976), and Science, 158, 1570 (1967). For example, anti α-fetoprotein antibody is dissolved at a concentration of 1 mg / ml in glycine buffer pH 8.2, and this antibody solution is contacted with glass beads or a glass test tube coated with a silicone resin, followed by a reaction for 3 hours at 37 ° C. It is then washed with physiological saline followed by addition of phosphate buffer solution with a pH 7.0 containing 1% normal rabbit serum and allowed to stand overnight at 4 ° C to prepare an antibody-bound support.

As the labeling agent to be used in immunochemical measurement, enzymes (e.g., peroxidase, β-galactosidase, basic phosphatase, glucose oxidase, etc.), radioactive isotopes (e.g.

125 3 20 I, H, etc.), fluorescent materials (e.g. fluorescent isothiocyanate, tetramethylrhodamine isothiocyanate, etc.), etc. are used. When considering sensitivity, accuracy, simplicity, etc., the use of an enzyme is most preferred. The method of labeling the antibody or antigen with these markers is well known, and enzyme labeling can be performed, for example, by the method of Nakane, Kawaoi et al. ("J. Histochem. Cytochem.", 22, 1084 (1974)).

Using the above-obtained measuring reagent, composed of the insolubilized antibody or insolubilized antigen and the labeled antibody or labeled antigen, various physiologically active substances can be measured. For example, a test solution diluted to an appropriate concentration is added to a test tube carrying antibody bound thereto and the antigen-antibody reaction is performed. After the reaction, the test tube is washed, and labeled antibody is added to the test tube. This test tube is then washed and the amount of labeled antibody bound to the test tube is measured by suitable means. The amount of the substance to be measured is calculated by comparing the measured value with the standard curve, which is obtained in a similar manner. work at the standard of a known concentration -

Because conditions such as the amount of the sample, the concentration and amount of the labeled antibody used, the reaction temperature and time, etc. depend on the nature of the substance to be measured, the titre of the antibody used, the nature of the marker , etc., when the measurement is put into practice, optimal conditions are determined experimentally in each measuring system.

The immunochemical measurement reagents of the present invention include, as described above, a) an insoluble antibody or insoluble antigen, and b) a labeled antibody or labeled antigen, and if desired, the reagent may further comprise a buffer, a standard solution and, where an enzyme is used as a marker, an enzyme substrate contains a solution for dissolving the enzyme substrate, a reaction stopper, etc.

The buffer solution is used to dilute the sample to an appropriate concentration and also to maintain the site of the anti-no antibody reaction at an appropriate pH and ionic strength. As such a buffer solution, conventional and conventional buffer solutions, for example glycine buffered saline, phosphate buffered saline, borate buffered saline, etc. can be used in the immunochemical field. Furthermore, to enhance the reproducibility of the reaction, an appropriate amount of protein, for example 0.01-5%, preferably 0.5-2%, of bovine serum albumin (further referred to as BSA) may be present.

The labeled antibody or labeled antigen is obtained by labeling an antibody or antigen with an enzyme, a radioactive isotope or a fluorescent substance. By measuring the labeled substance that binds to or does not bind to the substance to be measured, which is bound to the solid phase by the antigen-antibody reaction, the physiologically active substance to be tested can be measured in the solution.

8302179

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Some or all of the components of the measuring reagent of the present invention may be in lyophilized form, and if so, an appropriate solvent for dissolving the lyophilized components may be combined with the reagent. Furthermore, the reagent of the present invention can be presented in the form of a measuring set by adding supplies such as test tubes, pipettes, etc. to facilitate its use.

Examples of the substances to be measured by the measuring reagents of the present invention include protein hormones such as human chorionic gonadotropin, insulin, human growth hormone, etc .; protein-containing substances such as α-fetoprotein (further referred to as AFP), type B hepatitis virus (HBs), immunoglobulin, an antigen-antibody complex, celluloplasmin, transferrin, etc., and haptics such as thyroxine, estradiol, progesterone, testosterone, phenytolne, phenobarbital, etc.

The measuring reagents of the present invention are free from ηση-specific adsorption by carriers, and therefore have not only high measuring accuracy, but also excellent stability which allows stable storage for a year or more.

The present invention is more particularly described by the following experimental example and the preparation and measurement examples.

Experimental Example Stability of AFP Measuring Reagent A conventional AFP measuring reagent using a polystyrene resin test tube as the support and an AFP measuring reagent according to the present invention using a glass test tube coated with silicone resin were prepared according to Example VI described below, and their storage stability was compared. They were stored at 4 ° C for 1 month, 3 months and 1 year. They were then tested for stability of their reactivities, using a blank solution (which did not contain AFP) and an AFP solution (80 ng / ml). Optical obsorbances of these solutions were measured by the reagents as will be described in Example VII, respectively. The differences in optical absorbance between the blank solution and the AFP solution were determined 8302179 8 and the results regarding the difference in absorbance for a storage period of 0 days taken as 100% are shown in Table A.

As shown in Table A, the reactivity (difference in the absorbance) in the conventional reagent after one year's storage is reduced to 60%, while practically no reactivity reduction was observed in the reagent of the present invention even after storage for a year. The decrease in reactivity naturally causes a decrease in the measurement accuracy and measurement sensitivity.

TABLE A

Storage period 0 1 month 3 months 1 year

Conventional reagent 100% 94% 82% 60%

Reagent according to the invention 100% 98% 98% 97%

Example I

Preparation of silicone resin coated test tubes

1 ml aliquot of a solution of a silicone resin diluted with n-hexane to 10% (Shin-Etsu Chemical Co., Ltd.

20 KC88) was placed in each of a series of glass test tubes (10 x 75 mm). After these test tubes were allowed to stand at room temperature for 10 min, the solution of the silicone resin in n-hexane was removed by aspiration. The test tubes were then heated to 180 ° C for 1 hour and allowed to cool. These test tubes were washed with 2 ml trichlorethylene, and dried to make silicone resin coated test tubes.

Example II

Preparation of silicone resin coated glass granules

One hundred glass beads (8 mm in diameter) were dipped in a 10% n-hexane solution of a silicone resin (Shin-Etsu Chemical Co., Ltd. KC88). After being allowed to stand at room temperature for 10 min, the granules were placed in a glass dish with tweezers, then heated at 180 ° C for one hour and allowed 8302179 to cool. They were then washed 5 times with 50 ml trichlorethylene to produce silicone resin coated glass beads.

9

Example III

5 Preparation of test tubes coated with polystyrene resin

1 ml aliquot of a polystyrene resin (Sanko Plastic Co., Ltd.), which had melted at 180 ° C, was placed in each of a series of glass test tubes (10 x 75 mm), removed quickly by suction and allowed to cool with polystyrene to manufacture coated test tubes.

Example IV

Preparation of acrylic resin coated test tubes

1 ml aliquot of an acrylic resin (Sanko Plastic Co., Ltd.), which had melted at 200 ° C, was placed in each of a series of glass test tubes (10 x 75 mm), removed quickly by suction and allowed to cool to manufacture acrylic resin coated test tubes. Example V

Preparation of Fluorine-Coated Test Tubes

A suspension of a fluorine resin (Asahi Glass Co., Ltd.) was sprayed on the inner walls of glass test tubes (10 x 75 mm), heated to 400 ° C, and allowed to cool to form fluorine resin coated test tubes. manufacture.

Example VI

Preparation of AFP measuring reagent a) Preparation of anti-AFP antibody 25 Extracted and purified AFP extracted from the ascites of a hepatoma patient according to the method of Nishi et al ("Cancer Res.", 30, 2707 (1970)). physiological saline at a concentration of 2 mg / ml, 0.5 ml of which was mixed with 0.5 ml of Freund's complete adjuvant. Rabbits were immunized with this mixture 5 or more times to obtain anti-AFP serum. This anti-serum was salted out twice with sodium sulfate and the globulin fraction was collected to prepare anti-AFP antibody.

b) Preparation of anti-AFP antibody-bound test tubes 8302179

To the in Examples I, III, IV and V respectively

Prepared coated test tubes became 0.5 ml of phosphate buffered saline

sing (further referred to as PBS), which contains 1 mg of the anti-AFP

10 contained antibody. A reaction was effected at 37 ° C for 3 hours, after which the test tubes were washed with PBS to prepare antibody-bound test tubes. c) Preparation of enzyme-labeled anti-AFP antibody 5 An enzyme-labeled antibody was prepared according to the method Nakane, Kawaoi et al ("J. Histochem. Cytochem.", 22, 1084 (1974)). 5 mg was dissolved horseradish peroxidase (further referred to as HRPO) in 1 ml 0.3M sodium hydrogen carbonate solution 0.1 ml of a 1% 2,4-dinitrofluorobenzene was added and the mixture was stirred at room temperature for 1 hour. solution 1 ml of 0.08 M sodium periodate solution was added and the solution was mixed at room temperature for 30 min, then 1 ml of a 0.16 M ethylene glycol solution was added and the mixture was continued for an hour at room temperature. Dialyzed overnight against 0.01 M carbonate buffer pH 9.5, then 1 ml of the anti-AFP antibody prepared in step (a) above and dissolved in 0.01 M carbonate buffer pH 9.5 at a concentration of 5 mg / ml, added and added reacted at room temperature for 3 hours, then 5 mg of sodium borohydride was added and the reaction was continued for an additional 3 hours at 4 ° C. After the reaction, the reaction mixture was dialyzed overnight against PBS, fractionated and purified using Sephadex G 200 to prepare HRPO-labeled anti-AFP antibody.

d) Preparation of AFP standard solution. AFP was extracted and purified from the ascites of a hepatoma patient according to the method of Nishi et al., supra. The AFP was dissolved in PBS containing 1% BSA and 0.1% Tween 20 at concentrations of 160, 80, 40, 20 and 10 ng / ml, respectively.

e) Preparation of AFP measuring reagents Using the anti-AFP antibody-bound test tubes and the HRPO-labeled anti-AFP antibody prepared in steps (b) and (c) above, AFP measuring reagents were prepared containing the following combinations had: 1) anti-AFP bound test house 35 2) HRPO-labeled anti-AFP antibody 3) AFP standard solution 8302179 11 4) enzyme substrate (o-phenylenediamins) 5) solution to dissolve the enzyme substrate (PBS containing 6 mM / 1 water dust peroxide) 6) reaction stopper (1N hydrochloric acid) 5 7) buffer solution (PBS containing 1% BSA).

Example VII Measurement of & FP

The AFP standard solutions with the respective concentrations of the AFP measuring reagent prepared in Example 6 described above were placed in the test tubes of the present invention in an amount of 0.1 ml each coated with various synthetic resins, and for comparison in test tubes other than the present invention, which were not coated with any synthetic resin. 0.4 ml of PBS containing 1% BSA was added to each test tube, then stirred and left to react for 2 hours. After completion of the reaction, each test tube was washed with distilled water. Then 0.5 ml of the HRPO-labeled anti-AFP prepared in Example VI (c) above and diluted 2000-fold with PBS containing 1% BSA was added for 2 hours at room temperature. reaction performed. After completion of the reaction, each test tube was washed with distilled water. To measure the HRPO activity, 0.5 ml of substrate solution (PBS) containing 6 mM / 1 hydrostipoxide and 20 mM / 1 o-phenylenediamine) was added to each test tube and the mixture was allowed to react at room temperature for 30 minutes while there was shielded from light. 2 ml of 1N hydrochloric acid were added and mixed, and the intensity of the developed color was measured at a wavelength of 492 nm. The results for the standard curves are given in Figure 1. When the test tubes other than those of the present invention were used, it could be observed that non-specific adsorption through the glass test tubes occurred as evidenced by the intensive color development even in the case of 0 nh / ml AFP and further from the fact that the gradient of the standard curve was small. In contrast, when the test tubes of the present invention were used, an accurate measurement was possible because hardly any color development was observed when AFP was absent and further the gradients of the standard curves 8302179 12

were great. example VIII Measurement of AFP

125

a) Preparation of I-AFP

20 µg AFP, extracted and purified from the ascites of a hepatoma patient according to the method of Nishi et al., 125 supra, 1 mCi of Na I and 250 µg chloramine T was reacted in 0.225 ml for 60 sec. 0.05 M phosphate buffer solution (pH 7.2), after which 600 µg sodium pyrosulfite was added thereto and allowed to react for 60 sec. After adding 5 mg of KI, the reaction mixture was subjected to gel filtration over Sephadex G-50, and the first fraction was collected to prepare 125 I-labeled AFP.

b) Measurement of AFP

0.1 ml of each AFP standard solution, 125 was added

15 in Example VI (d) and 0.1 ml of I-AFP prepared in Example VIII

(a) and diluted 20,000-fold with PBS containing 1% BSA to each test tube of the present invention prepared in Example VI

(b), and to any glass test tube other than the present invention that has not been coated with any synthetic resin. Then 0.3 ml PBS containing 1% BSA was added, stirred and reacted at 4 ° G for 18 hours. After the reaction, each test tube was washed with distilled water, and the radioactivity of each test tube was measured. The results for the standard curves are given in Figure 2.

As can be seen from Figure 2, test tubes of the present invention allowed accurate measurement due to the larger gradients of the standard curves.

8302179

Claims (24)

An immunochemical measurement support having a surface adapted to contact with a substance to be measured, which surface is coated with a synthetic resin. 2. Carrier as claimed in claim 1, wherein the carrier comprises a holder 5 and the surface comprises an inner wall of the holder. The carrier of claim 2, wherein the container is a test tube. The carrier according to claim 1, wherein the carrier comprises glass granules. Support according to claim 1, wherein the support is made of glass. Support according to claim 2, wherein the support is made of glass. 7. Carrier according to claim 3, wherein the carrier is made of glass. The carrier of claim 1, wherein the synthetic resin is selected from the group consisting of thermoplastic resins and thermosetting resins. The carrier of claim 1, wherein the synthetic resin 20 is selected from the group consisting of silicone resin, polystyrene resin, acrylic resin, and fluorine resin. The carrier of claim 2, wherein the synthetic resin is selected from the group consisting of silicone resin, polystyrene. resin, acrylic resin and fluorine resin. The carrier according to claim 3, wherein the synthetic resin is selected from the group consisting of silicone resin, polystyrene resin, acrylic resin and fluorine resin. 12. The carrier of claim 4, wherein the synthetic resin is selected from the group consisting of silicone resin, polystyrene resin, acrylic resin, and fluorine resin. The carrier according to claim 5, wherein the synthetic resin is selected from the group consisting of silicone resin, polystyrene resin, acrylic resin and fluorine resin. The carrier of claim 6, wherein the synthetic resin 8302179 is selected from the group consisting of silicone resin, polystyrene resin, acrylic resin, and fluorine resin. The carrier of claim 7, wherein the synthetic resin is selected from the group consisting of silicone resin, polystyrene resin, acrylic resin, and fluorine resin. 16. Reagent for immunochemical measurement, comprising a) insolubilized antibodies or insolubilized antigens, bound to a glass support surface, which surface is coated with synthetic resin, and b) labeled antibodies or labeled antigens. The reagent of claim 16, wherein the carrier comprises an interior wall surface of a container. The reagent of claim 17, wherein the container is a test tube. The reagent of claim 16, wherein the carrier comprises glass beads. Reagent according to claim 16, wherein the synthetic resin is selected from the group consisting of silicone resin, polystyrene resin, acrylic resin and fluorine resin. The reagent of claim 17, wherein the synthetic resin is selected from the group consisting of silicone resin, polystyrene resin, acrylic resin and fluorine resin. The reagent of claim 18, wherein the synthetic resin is selected from the group consisting of silicone resin, polystyrene resin, acrylic resin and fluorine resin. The reagent of claim 19, wherein the synthetic resin is selected from the group consisting of silicone resin, polystyrene resin, acrylic resin and fluorine resin. The reagent of claim 16, wherein at least one of the insolubilized antibodies or insolubilized antigens and said labeled antibodies or labeled antigens is in lyophilized form. 8302179