GB2125963A - Carriers for immunochemical measurement and measuring reagents utilizing said carriers - Google Patents

Abstract

A carrier for immunochemical measurement, the surface of which is coated with synthetic resin, and a reagent for immunochemical measurement utilizing said carrier are provided. The carrier of the reagent may be glass coated with synthetic resin. The carrier of the present invention does not show non-specific adsorption so that the reagent has sufficient reliability in measurement after storage for more than a year.

Description

SPECIFICATION Carriers for immunochemical measurement and measuring reagents utilizing said carriers The present invention relates to carriers for immunochemical measurement, the surface of which is coated with synthetic resin, and to reagents for immunochemical measurement utilizing said carriers.

In recent years, various reagents for immunochemical measurement have been employed as means for measuring concentrations of physiologically active substances, such as peptide hormones, steroids, proteins, etc., as well as concentrations of drugs administered in vivo, contained in very small quantities in physiological samples such as serum, urine, etc. Among these, reagents based on the enzymeimmunoassay, radioimmunoassay, fluoroimmunoassay, etc. are often employed because they have high measuring sensitivity and excellent quantitative efficacy.

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 labeling agent such as an enzyme, etc. Measurement using such a reagent is conducted as follows: A substance to be measured and a labeled antibody or labeled antigen are reacted with an insolubilized antibody or insolubilized antigen, and then the labeling agent bound to the carrier (solid phase) and the labeling agent not bound but present in the liquid phase are separated. Thereafter, the activity of the labeling agent either in the solid phase or in the liquid phase is measured, whereby the amount or concentration of the substance to be measured is determined.Therefore, the carrier must not non-specifically adsorb antigen, antibody or other components which do not participate in the immunological reaction.

On the other hand, it has been attempted to employ containers as carriers for reagents such as, for example, test tubes, etc. In other words, by binding an antigen or antibody to the inner wall surface of a container such as a test tube, etc., and placing labeled antigen or labeled antibody in this container, it has been expected to obtain a reagent which is suitable and stable for storage and permits instantaneous measurement and is thus conveniently used.

However, the storage stability of such a reagent has heretofore not been satisfactory and has had a problem that after several months' storage, the accuracy and sensitivity of measurement are decreased. The inventors of the present invention have studied extensively in order to clarify the cause of this problem and, as a result, they have discovered that the change in quality of the reagent after storage is attributable to the fact that the plastic test tubes used as carriers had small yet influential gas permeability.

Although glass is a carrier material having no gas permeability, because glass non-specifically adsorbs substances, the use of glass as a carrier is unsuitable. The inventors continued further study in order to solve this problem and, as a result, have discovered that by coating the glass surface with a synthetic resin, the non-specific adsorption can be prevented. The use of glass coated with a synthetic resin as a carrier for a reagent for immunochemical measurement has not heretofore been known.

SUMMARY OF THE INVENTION An object of the present invention is to provide carriers for immunochemical measurement which prevent non-specific adsorption.

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

A further object of the present invention is to provide reagents for immunochemical measurement which comprise (a) an insolubilized antibody or insolubilized antigen, obtained by binding the antibody or antigen to a glass carrier the surface of which is coated with synthetic resin, and (b) a labeled antibody or labeled antigen.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graph showing the standard curves in Example 7; and Fig. 2 is a graph showing the standard curves in Example 8.

DETAILED DESCRIPTION OF THE INVENTION Coating of a glass carrier may be effected as follows: A material intended for a carrier such as glass beads, a glass test tube, etc., is wetted with a synthetic resin solution or suspension to make said resin adhere onto the carrier surface. Excess resin is removed if necessary and the wetted carrier material is subsequently heat treated. The resin which has not been coated on the carrier material after heat treatment is removed by washing. The carrier thus treated is then bound with an antibody or antigen to obtain an insolubilized antibody or insolubilized antigen.

As the coating agent, various synthetic resins may be employed. The synthetic resin may be classified, according to the chemical structure and the working properties, into thermoplastic resins and thermosetting resins. The former resins are linear polymers and become softened and flowabie when heated and become solid when cooled, this cycle being repeatable. The latter are such resins that their condensation products in early stages are of a linear structure but are large molecules; when heated they become softened and flowable at first, but then undergo a cross-linking reaction between the molecules to form insoluble and infusible materials of three-dimensional structure. Either of these types of synthetic resins may be employed in the present invention.

As the thermoplastic resin, vinyl chloride resins, polystyrene resins, polypropylene resins, acrylic resins, fluorine resins, etc., may be employed; as the thermosetting resin, silicone resins, phenolic resins, epoxy resins, etc., may be employed. Especially preferred among these may be mentioned the silicone resins, polystyrene resins, acrylic resins and fluorine resins.

As the silicone resins, silicone oils having a linear structure, silicone rubbers having a partially crosslinked structure, silicone resins completely crosslinked and cured at the time of working, etc., may be mentioned. Among these, the silicone oils are especially preferred.

As the polystyrene resins, general-purpose styrene resins obtained by bulk homopolymerization of styrene; heat resistant styrene resins obtained by making a linear polymer having a higher degree of polymerization than the general-purpose styrene resins or by copolymerizing with a monomer which imparts heat resistance, or by polymerizing ar-methylstyrene; high impact styrene resins obtained by graft polymerization of styrene and butadiene, etc., may be mentioned. Among these, the generalpurpose styrene resins are especially preferred.

As the acrylic resins, polymers of acrylic acid esters and polymers of methacrylic acid esters may be mentioned, the polymers of acrylic acid esters being especially preferred.

As the fluorine resins, polytetrafluoroethylene and polychlorotrifluoroethylene may be mentioned, the polytetrafluoroethylene being especially preferred.

These synthetic resins may be employed as a solution in an organic solvent such as hexane, acetone, methyl chloride, chloroform, dichloroethane, trichloroethylene, etc., at a concentration of 1-30 w/w %, as a powder, as a paste, or as a suspention. When dissolved in an organic solvent, coating is effected by bringing the surface of the carrier into contact with said solution and subsequently subjecting the surface to a heat treatment. Alternatively, a synthetic resin solution may be sprayed over the surface of the carrier and thereafter the heat treatment may be carried out. Coating may also be effected by using molten synthetic resin at a temperature above its melting point instead of using a solution in an organic solvent.Further, it is also possible to make the synthetic resin into a paste to coat it on the surface of the carrier, and then to heat it to effect coating. For example, when a silicone resin is to be coated on a carrier material, the resin is dissolved in a solvent at a concentration of 130%, preferably 510%, the surface of the carrier is brought into contact with this solution, then excess solution is removed, and heat treatment is carried out at about 180"C for an hour. After cooling, the resin which has not been coated is removed by washing with trichloroethylene. After drying, a coated carrier is obtained.

The shape of the carrier may be varied, and, for example, various shapes such as beads, test tubes, drum-shaped beads, titer plates, ampules, syringes, etc., may be employed.

In order to bind antibody or antigen to the carrier coated with synthetic resin, procedures similar to those described in "Clinica Chimica Acta", 48, 15 (1973), "Journal of Immunology", 116, 1554 (1976), and "Science", 158, 1570 (1967) may be conducted. For example, anti afetoprotein antibody is dissolved at a concentration of 1 mg/ml in glycine buffer pH 8.2, and this antibody solution is brought into contact with glass beads or a glass test tube coated with a silicone resin, followed by a reaction at 370C for 3 hours. This is then washed with physiological saline and thereafter is added phosphate buffer solution of pH 7.0 containing 1% normal rabbit serum, allowed to stand at 40C overnight to prepare antibody-bound carrier.

As the labeling agent to be used in immunochemical measurement, enzymes (for example, peroxidase, 83-galactosidase, alkaline phosphatase, glucose oxidase, etc.), radioactive isotopes (for example '251, 3H, etc.), fluorescent substances (for example, fluorescein isothiocyanate, tetrnmethyirhodamine isothiocyanate, etc.), may be employed. When the sensitivity, accuracy, simplicity, etc., are taken into consideration, the use of an enzyme is most advantageous. The method for labeling the antibody or antigen with these labeling agents is generally known, and labeling with enzymes may be effected by, for example, 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 may be measured. For example, a solution to be tested which has been diluted to an appropriate concentration is added into a test tube carrying antibody bound thereto and the antigenantibody reaction is carried out. After the reaction, the test tube is washed, and labeled antibody is added to the test tube. Thereafter, this test tube is washed, and then the amount of the 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 to the standard curve obtained by similarly operating on the standard of a known concentration.

On practicing measurement, since conditions such as the amount of the specimen, the concentration and amount of the labeled antibody used, the reaction temperature and time, etc., can vary depending on the kind of the substance to be measured, the titer of the antibody used, the kind of the labeling agent, etc., optimum conditions are experimentally established in each measuring system.

The reagents for immunochemical measurement of the present invention comprise, as described above, a) an insolubilized antibody or insolubilized antigen and b) a labeled antibody or labeled antigen, and if necessary the reagent can further contain, for the convenience of measurement, a buffer, a standard solution, and, where an enzyme is employed as a labeling agent, an enzyme substrate, a solution for dissolving the enzyme substrate, a reaction stopper, etc.

The buffer solution is employed for diluting the specimen to an appropriate concentration and also for maintaining the site of the antigen-antibody reaction at an appropriate pH and ion strength. As such a buffer solution, buffer solutions conventionally and commonly employed in the immunochemical field, for example, glycine buffered saline, phosphate buffered saline, borate buffered saline, etc., may be employed. Further, in order to enhance reproducibility of the reaction, an appropriate amount of protein, for example, 0.01--59/c, preferably 0.52%, of bovine serum albumin (hereinafter referred to as BSA) may be contained.

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

Some or all constituents of the measuring reagent according to the present invention may be in lyophilized form and if such is the case, a suitable solvent for dissolving the lyophilized constituents may be attached to the reagent. Further, the reagent of the present invention may be provided as a measuring kit by supplementing accessories such as test tubes, pipettes, etc., in order to facilitate its use.

Examples of the substance to be measured by the measuring reagents according to the present invention include protein hormones such as human chorionic gonadotropin, insulin, human growth hormone, etc.; proteinaceous substances such as -fetoprotein (hereinafter referred to as AFP), Type B hepatitis virus (HBs), immunoglobulin, an antigen-antibody complex, celluloplasmin, transferrin, etc., and haptens such as thyroxin, estradiol, progesterone, testosterone, phenytoin, phenobarbital, etc.

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

The present invention is more particularly described by the following Experimental Example and Preparation and Measurement Examples.

EXPERIMENTAL EXAMPLE 1 Stability of AEP Measuring Reagent A conventional AFP measuring reagent utilizing a polystyrene resin test tube as a carrier and an AFP measuring reagent according to the present invention utilizing a glass test tube coated with silicone resin were prepared according to Example 6 described hereinbelow, and their storage stabilities were compared. They were stored at 40C for one month, 3 months and a year. Then they were examined for the stability of their reactivities using a blank solution (containing no AFP) and an AFP solution (80 ng/ml). Optical absorbances of these solutions were respectively measured by the reagents as will be described in Example 7.The differences in optical absorbance between the blank and AFP solution were determined and the results relative to the difference in the absorbance for a storage period of 0 day taken as 100% are given in Table 1.

As shown in Table 1, with the conventional reagent, the reactivity (difference in the absorbance) is reduced to 60% after a year's storage, whereas with the reagent of the present invention, practically no reduction in reactivity is observed even after a year's storage. The reduction in reactivity, of course, brings about a reduction in measuring accuracy and measuring sensitivity.

Table 1 Storing Period 0 1 month 3 months 1 year Conventional Reagent 100% 94% 82% 60% Reagent of the Invention 100% 98% 98% 97% EXAMPLE 1 Preparation of Silicone Resin Coated Test Tubes A solution of a silicone resin (Shin-Etsu Chemical Co., Ltd. KC88) diluted to 10% with n-hexane was allotted one ml each to glass test tubes (10 x 75 mm). After allowing these test tubes to stand at room temperature for 10 minutes, the solution of the silicone resin in n-hexane was removed by aspiration. Thereafter, the test tubes were heated at 1 800C for an hour and allowed to cool. These test tubes were washed with 2 ml of trichloroethylene, and dried to prepare silicone resin coated test tubes.

EXAMPLE 2 Preparation of Silicone Resin Coated Glass Beads 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 allowing them to stand at room temperature for 10 minutes, the beads were put into a glass dish with a pair of tweezers, then heated at 1 800C for an hour, and allowed to cool. They were then washed 5 times with 50 ml of trichloroethylene to prepare silicone resin coated glass beads.

EXAMPLE 3 Preparation of Polystyrene Resin Coated Test Tubes A polystyrene resin (Sanko Plastic Co., Ltd.) melted at 1 800C was allotted one ml each into glass test tubes (10 x 75 mm), rapidly removed by aspiration and allowed to cool to prepare polystyrene coated test tubes.

EXAMPLE 4 Preparation of Acrylic Resin Coated Test Tubes An acrylic resin (Sanko Plastic Co., Ltd.) melted at 2000C was allotted one ml each into glass test tubes (10 x 75 mm), rapidly removed by aspiration and allowed to cool to prepare acrylic resin coated test tubes.

EXAMPLE 5 Preparation of Fluorine Resin Coated Test Tubes A suspension of a fluorine resin (Asahi Glass Co., Ltd.) was sprayed over the inner walls of glass test tubes (10 x 75 mm), heated to 4000C, and allowed to cool to prepare fluorine resin coated test tubes.

EXAMPLE 6 Preparation of AFP Measuring Reagent a) Preparation of anti-AFP Antibody AFP extracted and purified from the ascites of a hepatoma patient according to the method of Nishi et al ("Cancer Res.", 30, 2707 (1970)) was dissolved in 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 5 times or more with this mixture to obtain anti-AFP serum. This anti-serum was salted out twice by sodium sulfate, and the globulin fraction was collected to prepare anti-AFP antibody.

b) Preparation of anti-AFP Antibody Bound Test Tubes To the coated test tubes prepared in Examples 1, 3, 4 and 5 respectively were added 0.5 ml of phosphate buffered saline (hereinafter referred to as PBS) containing 1 mg of the anti-AFP antibody.

Reaction was effected at 370C 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 An enzyme labeled antibody was prepared according to the method of Nakane, Kawaoi et al ("J.

Histochem. Cytochem.", 22, 1084 (1974)). 5 mg of horse-radish peroxidase (hereinafter referred to as HRPO) was dissolved in 1 ml of 0.3 M sodium hydrogen carbonate solution. 0.1 ml of 1% 2,4dinitrofluorobenzene was added thereto, and the mixture was stirred at room temperature for an hour.

To this solution was added 1 ml of 0.08 M sodium periodate solution and the solution was mixed at room temperature for 30 minutes, after which 1 ml of a 0.1 6 M ethylene glycol solution was added and the mixing was further continued at room temperature for an hour. The reaction mixture was dialyzed against 0.01 M carbonate buffer pH 9.5 overnight; then 1 ml of the anti-AFP antibody, prepared in (a) above and dissolved in 0.01 M carbonate buffer pH 9.5 at a concentration of 5 mg/ml, was added and reacted at room temperature for 3 hours, after which 5 mg of sodium borohydride was added and the reaction was continued at 40C for another 3 hours. After the reaction, the reaction mixture was dialyzed against PBS overnight, 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 respectively in (b) and (c) above, AFP measuring reagents having the following combinations were prepared: 1) Anti-AFP bound test tube 2) HRPO labeled anti-AFP antibody 3) AFP standard solution 4) Enzyme substrate (o-phenylenediamine) 5) Solution for dissolving the enzyme substrate (PBS containing 6 mM/I of hydrogen peroxide) 6) Reaction stopper(1 N hydrochloric acid) 7) Buffer solution (PBS containing 1% BSA).

EXAMPLE 7 Measurement of AFP The AFP standard solutions of the respective concentrations of the AFP measuring reagent prepared in Example 6 described above were taken up 0.1 ml each in the test tubes of the present invention which had been coated with various synthetic resins and, for comparison, in test tubes of other than the present invention which had not been coated with any synthetic resin. 0.4 ml of PBS containing 1% BSA was added to each test tube, and was then stirred and allowed to stand for 2 hours to effect a reaction. 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 6 (c) described above and diluted 2000-fold with PBS containing 1% S/o BSA, was added and reacted at room temperature for 2 hours. After completion of the reaction, each test tube was washed with distilled water. In order to measure the HRPO activity, 0.5 ml of substrate solution (PBS containing 6 mM/I of hydrogen peroxide and 20 mM/I of o-phenylenediamine) was added to each test tube and the mixture was allowed to react at room temperature while being shielded from light for 30 minutes. 2 ml of 1 N hydrochloric acid was 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.Where the test tubes of other than the present invention are used, it can be seen that non-specific adsorption by the glass test tubes is brought about as evidenced by the intense color development even in the case of 0 ng/mi of AFP and further by the fact that the gradient of the standard curve is small. On the contrary, where the test tubes of the present invention are used, since color development is hardly recognized when AFP is absent and further the gradients of the standard curves are great, accurate measurement is possible.

EXAMPLE 8 Measurement of AFP a) Preparation of 125l-AFP Twenty y9 of AFP extracted and purified from the ascites of a hepatoma patient according to the method of Nishi et al., supra, 1 mCi of Na1251 and 250 ,ug of chloramine T were reacted in 0.225 ml of 0.05 M phosphate buffer solution (pH 7.2) for 60 seconds, after which 600 y9 of sodium pyrosulfite was added thereto and allowed to react for 60 seconds. After adding 5 mg of KI, the reaction mixture was subjected to gel filtration on Sephadex G-50, and the first fraction was collected to prepare 1251 labeled AFP.

b) Measurement of AFP 0.1 ml of each AFP standard solution prepared in Example 6(d) and 0.1 ml of 125l-AFP prepared in Example 8(a) and diluted 20,000-fold with PBS containing 1% BSA were added to each test tube of the present invention prepared in Example 6(b) and to each glass test tube of other than the present invention which had not been coated with any synthetic resin. Thereafter, 0.3 ml of PBS containing 1% BSA was added, stirred and reacted at 40C 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 Fig. 2.

As seen from Fig. 2, test tubes of the present invention enable accurate measurement because of greater gradients of the standard curves.

Claims (26)

1. A carrier for immunochemical measurement having a surface adapted to contact a substance to be measured, said surface being coated with a synthetic resin.

2. A carrier according to Claim 1, in which said carrier comprises a container and said surface comprises an inner wall of said container.

3. A carrier according to Claim 2, wherein said container is a test tube.

4. A carrier according to Claim 1, wherein said carrier comprises glass beads.

5. A carrier according to Claim 1, wherein said carrier is made of glass.

6. A carrier according to Claim 2, wherein said carrier is made of glass.

7. A carrier according to Claim 3, wherein said carrier is made of glass.

8. A carrier according to Claim 1, wherein the synthetic resin is selected from the group consisting of thermoplastic resins and thermosetting resins.

9. A carrier according to Claim 1, wherein the synthetic resin is selected from the group consisting of silicone resin, polystyrene resin, acrylic resin and fluorine resin.

10. A carrier according to Claim 2, wherein the synthetic resin is selected from the group consisting of silicone resin, polystyrene resin, acrylic resin and fluorine resin.

11. A 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. A carrier according to Claim 4, wherein the synthetic resin is selected from the group consisting of silicone resin, polystyrene resin, acrylic resin and fluorine resin.

13. A 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.

14. A carrier according to Claim 6, wherein the synthetic resin is selected from the group consisting of silicone resin, polystyrene resin, acrylic resin and fluorine resin.

1 5. A carrier according to Claim 7, wherein the synthetic resin is selected from the group consisting of silicone resin, polystyrene resin, acrylic resin and fluorine resin.

1 6. A reagent for immunochemical measurement which comprises: a) insolubilized antibodies or insolubilized antigens bound to a glass carrier surface, said surface being coated with synthetic resin, and b) labeled antibodies or labeled antigens.

17. A reagent according to Claim 1 6, wherein said carrier comprises an inner wall surface of a container.

18. A reagent according to Claim 17, wherein said container is a test tube.

1 9. A reagent according to Claim 16, wherein said carrier comprises glass beads.

20. A 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.

21. A reagent according to Claim 17, wherein the synthetic resin is selected from the group consisting of silicone resin, polystyrene resin, acrylic resin and fluorine resin.

22. A reagent according to Claim 18, wherein the synthetic resin is selected from the group consisting of silicone resin, polystyrene resin, acrylic resin and fluorine resin.

23. A reagent according to Claim 19, wherein the synthetic resin is selected from the group consisting of silicone resin, polystyrene resin, acrylic resin and fluorine resin.

24. A reagent according to claim 1 6, wherein at least one of said insolubilized antibodies or insolubilized antigens and said antibodies or labeled antigens is in lyophilized form.

25. A carrier for immunochemical measurement as claimed in Claim 1 and substantially as described in any one of the specific examples hereinbefore set forth.

26. A reagent as claimed in Claim 1 6, and substantially as described in any one of the specific examples hereinbefore set forth.