JP4353856B2 - Immunological reagent - Google Patents

Description

  The present invention relates to a reagent for latex agglutination reaction, and more particularly to a reagent for latex agglutination reaction having good dispersibility and excellent stability.

  Measuring an antigen or antibody in a biological sample is one of the extremely important means for diagnosing a disease. Among immunological measurement methods, an insoluble suspendable particulate carrier is sensitized with an antibody (or antigen) against an antigen (or antibody) in a sample, and the antigen (or antibody) in the sample and the particulate carrier Unlike RIA and EIA, the latex agglutination method, which measures the degree of turbidity change due to agglutination, can be measured with an automatic analyzer widely used in hospitals and examination centers. It is useful from the viewpoints of reducing the amount of reaction system, improving sensitivity, shortening reaction time, and the like.

  Various reagents for latex agglutination reaction are on the market, but conventional latex agglutination reaction reagents naturally aggregate antibody-sensitized latex particles during storage even if no sample is present. It is known. The occurrence of such agglomeration has the problem that aggregation due to antigen-antibody reaction cannot be measured correctly because agglutination occurs in the absence of a measurement sample, and sensitivity becomes unstable due to long-term storage. there were.

As a method for suppressing such natural aggregation, a method of adding sucrose or the like (see Patent Document 1) is known. However, when a large amount of sucrose is added in order to obtain a sufficient inhibitory effect, the viscosity increases, the antigen-antibody reaction is suppressed, and the sensitivity is lowered.
JP 54-26327 A

  Accordingly, an object of the present invention is to provide a reagent for latex agglutination reaction that does not cause spontaneous aggregation, has good dispersibility, and has a stable sensitivity.

As a result of intensive studies in view of such circumstances, the present inventors have used a specific buffer as a suspension of protein-immobilized latex particles, and set the concentration to 3 to 18 mM, thereby storing it for a long time. However, the present inventors have found that a natural agglutination of the latex particles does not occur and a latex agglutination reagent having a stable sensitivity can be obtained, and the present invention has been completed.
In the present specification, “protein-immobilized latex particles” refers to particles in which antigens, antibodies, antibody digests such as Fab, F (ab ′) _{2 and the} like are coated with latex by a conventional method.
That is, according to the present invention, the protein-immobilized latex particles are composed of 3-18 mM phosphoric acid, 2-amino-2-hydroxymethyl-1,3-propanediol, 2- (N-morpholino) ethanesulfonic acid, 3- (N- Morpholino) -2-hydroxypropanesulfonic acid, N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid, 3- (N-morpholino) propanesulfonic acid, bis (2-hydroxyethyl) iminotris (hydroxy A buffer selected from methyl) methane, N- (2-acetamido) iminodiacetic acid, N- (2-acetamido) -2-aminoethanesulfonic acid and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid A reagent for latex agglutination reaction suspended in a liquid is provided.

  The present invention also provides a method for preventing spontaneous aggregation of latex particles during storage, characterized by suspending protein-immobilized latex particles in a buffer solution of 3 to 18 mM.

The latex agglutination reagent of the present invention suppresses the occurrence of spontaneous aggregation, has good dispersibility, and provides stable sensitivity. The reagent for latex agglutination reaction of the present invention is useful for finding a disease, grasping a disease state, treating it, and the like.
Further, according to the method for preventing spontaneous aggregation of the present invention, it is possible to prevent spontaneous aggregation of protein-fixed latex particles even when stored for a long period of time.

  The present applicant has found that the latex particles do not spontaneously aggregate even when stored for a long time by suspending the protein-immobilized latex particles in a buffer solution of 3 to 18 mM. In this specification, “suspension” means that protein-immobilized latex particles are dispersed in a buffer solution, and “suspension” means that protein-immobilized latex particles are dispersed. A buffer solution.

  The buffer solution that can be used in the method for preventing spontaneous aggregation of the protein-fixed latex particles of the present invention is not particularly limited, but phosphate, 2-amino-2-hydroxymethyl-1,3-propanediol (Tris), or Good's buffer solution. In particular, Good's buffer is preferred. Good buffers include 2- (N-morpholino) ethanesulfonic acid (MES), 3- (N-morpholino) -2-hydroxypropanesulfonic acid (MOPSO), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), 3- (N-morpholino) propanesulfonic acid (MOPS), bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), N- (2-acetamide) ) Iminodiacetic acid (ADA), N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) or N-tris ( Hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) is preferred. These buffers may be used alone or in combination of two or more. The concentration of the buffer solution is 3 to 18 mM from the viewpoint of suppressing spontaneous aggregation, and 3 to 10 mM is particularly preferable. This concentration is the final buffer concentration before reacting the sample with the protein-immobilized latex particle suspension.

  The pH of the protein-immobilized latex particle suspension is preferably 4 to 10, particularly 6 to 9.

  The latex particles of protein-fixed latex particles are not particularly limited as long as they are fine particle carriers generally used in immunological aggregation reactions and aggregation inhibition reactions using latex aggregation reactions, but are industrially mass-produced. Organic fine particles that can be used are preferred. Examples of such organic fine particles include homopolymers or copolymers of vinyl monomers such as styrene, vinyl chloride, acrylonitrile, vinyl acetate, acrylic acid ester, and methacrylic acid ester; styrene-butadiene copolymer, methyl Examples thereof include butadiene-based copolymers such as methacrylate-butadiene copolymers. In addition, reactive organic fine particles in which a functional group such as a carboxyl group, a primary amino group, a carbamoyl group, a hydroxyl group, or an aldehyde group is bonded to such organic fine particles can also be preferably used. Among the latex particles described above, polystyrene latex particles such as polystyrene and styrene-butadiene copolymer are preferred from the viewpoint that they have excellent antigen or antibody adsorptivity and can stably maintain biological activity for a long period of time.

  The shape of the latex particles is not particularly limited, but the average particle size is desirably large enough to allow the aggregates resulting from the aggregation reaction between the protein on the latex particle surface and the substance to be measured to be detected visually or optically. The average particle size is preferably 0.02 to 1.6 μm, particularly preferably 0.03 to 0.5 μm.

  The surface of the latex particles is sensitized with a protein, preferably an antibody or an antigen, against the substance to be measured. The sensitizing method is not particularly limited, and a known method can be used. For example, a method of physically adsorbing an antibody or an antigen on the latex particle surface and a method of sensitizing the latex particle surface having a functional group by covalent bond or immunological bond can be mentioned.

The sensitizing antibody is not particularly limited, and examples thereof include a monoclonal antibody and a polyclonal antibody. The monoclonal antibody can be produced and obtained using a monoclonal antibody-producing fusion cell line prepared in the field of cell fusion technology. The shape of the antibody is not particularly limited, and antibody digests such as Fab and F (ab ′) ₂ can be preferably used in addition to antibodies such as IgG itself. You may use an antibody individually or in combination of multiple types.
Examples of the antigen to be sensitized include a native antigen present in nature, a recombinant antigen obtained by genetic manipulation, an antigen bound to a polymer such as bovine serum albumin (BSA), and the like.

  The concentration of the protein-fixed latex particles in the suspension is not particularly limited, but is preferably 0.1 to 10 mg / mL.

  The method for preventing spontaneous aggregation of protein-immobilized latex particles of the present invention can be suitably used for the reagent for latex aggregation reaction of the present invention. That is, the reagent for latex agglutination reaction of the present invention is obtained by suspending the above protein-immobilized latex particles in a specific buffer solution at a concentration of 3 to 18 mM.

  Specific buffers include phosphoric acid, 2-amino-2-hydroxymethyl-1,3-propanediol (Tris), 2- (N-morpholino) ethanesulfonic acid (MES), 3- (N-morpholino) 2-hydroxypropanesulfonic acid (MOPSO), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), 3- (N-morpholino) propanesulfonic acid (MOPS), bis (2 -Hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), N- (2-acetamido) iminodiacetic acid (ADA), N- (2-acetamido) -2-aminoethanesulfonic acid (ACES) or N- 2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) can be used. These buffers may be used alone or in combination of two or more. The concentration of the buffer is 3 to 18 mM, but 3 to 10 mM is particularly preferable.

  In addition, a stabilizer such as BSA and sucrose and a preservative such as sodium azide may be appropriately added to the latex agglutination reagent of the present invention. The reagent for latex agglutination reaction of the present invention may be further combined with a diluent to form a latex agglutination reaction kit. The above-mentioned stabilizer and preservative can be added to the diluted solution as necessary.

  The substance to be measured in the sample is prepared by mixing latex particles sensitized with an antibody (or antigen) against the substance to be measured and the sample in a solution, and forming an aggregate of latex particles by an antigen-antibody reaction. It can be measured by measuring. Specifically, the sample can be dispensed into a reaction vessel, incubated, and then added and mixed with the reagent for latex agglutination reaction of the present invention. The temperature of the antigen-antibody reaction is preferably 25 to 40 ° C, particularly preferably 30 to 38 ° C.

Examples of the target substance that can be measured using the reagent for latex agglutination reaction of the present invention include physiologically active substances contained in biological samples such as serum and plasma. Examples of such target substances include cancer markers such as AFP (human α-fetoprotein), CEA (carcinoembryonic antigen), CA19-9 (sugar chain antigen 19-9); PLG (human plasminogen), Blood coagulation / fibrinolytic enzymes such as α ₂ -PI (human α ₂ -plasmin inhibitor) and ATIII (human antithrombin III); HBs antibody, HBs antigen; others, ferritin, β ₂ M (β ₂ microglobulin), Lp (a) (lipoprotein (a)), CRP (C-reactive protein), FDP (fibrinogen / fibrin degradation product), D-dimer (fibrin degradation product), MMP-3 (matrix metalloproteinase-3), PSA (prostate) Specific antigen) and the like.

  The method for measuring the aggregate of latex particles obtained using the reagent for latex aggregation reaction of the present invention is not particularly limited. For example, when measuring the aggregate qualitatively or semi-quantitatively, the measurement target Can be visually determined from a comparison with turbidity when a known sample is measured. In the case of quantitative measurement, it is preferable to measure optically from the viewpoint of simplicity and accuracy.

  As an optical measurement method, a known method can be used, but an absorbance method is preferable. Absorbance is measured using a general-purpose spectrophotometer, biochemical automatic analyzer based on spectrophotometric measurement (Hitachi 7150, 7070, 7170, etc. (manufactured by Hitachi); Toshiba TBA-120FR, etc. (Toshiba) )), Devices with near infrared measurement wavelength (LPIA, etc. (Mitsubishi Kasei Co., Ltd.)), devices with integration sphere turbidity as measurement principle (EL system, etc. (Kyowa Hakko)), scattered light intensity It can be performed using an optical measuring instrument such as a measuring device (BNA system or the like (manufactured by Boehringer)).

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these.

Example 1
(1) Preparation of beta ₂ microglobulin (beta ₂ M) First reagent for measurement
A 0.05M glycine buffer (pH 9.0) containing 4% NaCl and 1% bovine serum albumin (BSA) was prepared and used as the first reagent.
(2) Preparation of second reagent for β ₂ M measurement Average in 5 mL of a mixture of goat anti-human β ₂ M antibody (Incstar) mixed with 0.25 M glycine buffer (pH 9.5) at a concentration of 1.4 mg / mL 5 mL of a 2% suspension of polystyrene latex (manufactured by Sekisui Chemical Co., Ltd.) having a particle size of 0.1 μm was added and stirred at 4 ° C. for 2 hours to sensitize the antibody to the latex. After removing the supernatant by centrifugation, 0.05 M glycine buffer (pH 9.5) containing 2% BSA was added to the precipitate, and the mixture was stirred overnight at 4 ° C. The precipitate was collected by centrifugation, suspended in 160 mL of 5 mM phosphate buffer (pH 7.5) containing 0.02% BSA as the final suspension, and suspended with the second reagent (anti-β ₂ M antibody-immobilized particle suspension). Suspension).
(3) Absorbance measurement Using a Hitachi 7150 automatic analyzer, 200 μL of the first reagent was added to 6 μL of physiological saline (saline), and about 5 minutes later, 200 μL of the second reagent was added and stirred. About 5 minutes after adding the second reagent, the absorbance at a wavelength of 600 nm was measured. The results are shown in Table 1 together with the following Example 2 and Comparative Examples 1 to 6.

Example 2
A second reagent for β ₂ M measurement was prepared in the same manner as in Example 1 except that 5 mM Tris buffer (pH 7.5) was used instead of 5 mM phosphate buffer (pH 7.5), and the absorbance was measured.

Comparative Examples 1-3
A second reagent for β ₂ M measurement was prepared in the same manner as in Example 1 except that 20, 50, and 100 mM phosphate buffer (pH 7.5) were used instead of 5 mM phosphate buffer (pH 7.5). The absorbance was measured.

Comparative Examples 4-6
A second reagent for β ₂ M measurement was prepared in the same manner as in Example 2 except that 20, 50, and 100 mM Tris buffer (pH 7.5) were used instead of 5 mM Tris buffer (pH 7.5). Was measured.

A second reagent for β ₂ M measurement was prepared in the same manner as in Example 1 except that purified water was used instead of 5 mM phosphate buffer (pH 7.5), and the absorbance was measured in the same manner (control example). .

As apparent from Table 1, the reagent for latex aggregation reaction of the present invention did not show an increase in absorbance due to spontaneous aggregation of antibody-sensitized latex particles (anti-β ₂ M antibody-immobilized particles). In ˜6, an increase in absorbance was observed.

Example 3
(1) Preparation of first reagent for measuring lipoprotein (a) (Lp (a)) 0.05M glycine buffer (pH 9.0) containing 1% NaCl and 1% BSA was prepared and used as the first reagent. .
(2) Preparation of second reagent for Lp (a) measurement Monoclonal antibody which is obtained from a mouse by a conventional method using purified human apolipoprotein (a) (apo (a)) as an immunogen, and causes immunoaggregation when used alone [independent An antibody produced by Hybridoma 28205 (FERM BP-3755) deposited at the Patent Organism Depositary (IPOD) of the National Institute of Advanced Industrial Science and Technology (AIST) at a concentration of 1.4 mg / mL, 0.25 M glycine buffer (pH 9.5) 5 mL of a mixture of 2% suspension of polystyrene latex (manufactured by Sekisui Chemical Co., Ltd.) with an average particle size of 0.1 μm was added to 5 mL of the solution mixed with the above) and stirred at 4 ° C. for 2 hours to sensitize the antibody to the latex . After removing the supernatant by centrifugation, 0.05 M glycine buffer (pH 9.5) containing 2% BSA was added to the precipitate, and the mixture was stirred overnight at 4 ° C. The precipitate is collected by centrifugation, suspended in 4 mM phosphate buffer (pH 7.0) containing 0.02% BSA as a final suspension, so that the absorbance at a wavelength of 600 nm is 1.6 OD. (Anti-Lp (a) antibody-immobilized particle suspension).
(3) Measurement of absorbance Using Hitachi 7150 type automatic analyzer, 300 μL of the first reagent was added to 5 μL of the raw food, and about 5 minutes later, 100 μL of the second reagent was added and stirred. About 5 minutes after adding the second reagent, the absorbance at a wavelength of 600 nm was measured (initial value). Further, the same measurement was performed using a second reagent stored at 37 ° C. for 2 weeks. The results are shown in Table 2 together with the following Examples 4 to 8 and Comparative Examples 7 to 9.

Examples 4-8
Example 3 was used except that 9, 18 mM phosphate buffer (pH 7.0), 4, 9, 18 mM Tris buffer (pH 7.0) was used instead of 4 mM phosphate buffer (pH 7.0). A second reagent was prepared and the absorbance was measured.

Comparative Examples 7-9
The second reagent was used in the same manner as in Example 3 except that 36 mM phosphate buffer (pH 7.0), 45, 90 mM Tris buffer (pH 7.0) was used instead of 4 mM phosphate buffer (pH 7.0). And the absorbance was measured.

Example 9
(1) Preparation of first reagent for C-reactive protein (CRP) measurement A 0.02 M Tris buffer (pH 8.5) containing 2% NaCl and 1% BSA was prepared and used as the first reagent.
(2) Preparation of Second Reagent for CRP Measurement Monoclonal antibody [hybridoma 08204 deposited with IPOD (FERM P- 16765) is mixed with 0.25 M glycine buffer (pH 9.5) at a concentration of 1.4 mg / mL in 5 mL of a polystyrene latex (manufactured by Sekisui Chemical Co., Ltd.) 2 % Suspension was added and stirred at 4 ° C. for 2 hours to sensitize the antibody to latex. After removing the supernatant by centrifugation, 0.05 M glycine buffer (pH 9.5) containing 2% BSA was added to the precipitate, and the mixture was stirred overnight at 4 ° C. The precipitate was collected by centrifugation, suspended in 4 mM phosphate buffer (pH 7.0) containing 0.02% BSA as a final suspension so that the absorbance at a wavelength of 600 nm was 2 OD. Anti-CRP antibody-immobilized particle suspension).
(2) Absorbance measurement Using a Hitachi 7150 automatic analyzer, 210 μL of the first reagent was added to 3 μL of the raw food, and about 70 minutes later, 70 μL of the second reagent was added and stirred. About 5 minutes after the addition of the second reagent, the absorbance at a wavelength of 600 nm was measured (initial value). Moreover, the light absorbency in wavelength 600nm was similarly measured using the 2nd reagent preserve | saved at 37 degreeC for 4 weeks. The results are shown in Table 3 together with the following Examples 10 to 34 and Comparative Examples 10 to 22.

Examples 10-34, Comparative Examples 10-22
A second reagent was prepared in the same manner as in Example 9 except that the buffering agent shown in Table 3 was used at the concentration shown in the table instead of the 4 mM phosphate buffer (pH 7.0), and the absorbance was measured.

  From Tables 2 and 3, it was found that the latex agglutination reagent of the present invention can maintain a stable absorbance even when stored for a long period of time.

Example 35, Comparative Example 23 [Latex agglutination reaction]
Instead of 4 mM phosphate buffer (pH 7.0), the second reagent (anti-CRP antibody-fixed latex particle suspension) was used in the same manner as in Example 9 except that the buffer of Table 4 was used at the concentration shown in the table. Was prepared. Absorbance was measured for each sample of raw food and CRP concentration (1, 5, 10, 20, 30 mg / dL) in the same manner as in Example 9 (initial value). In addition, using the second reagent after storage at 4 ° C. for 7 months, the absorbance in the case of using a sample with saline and a known CRP concentration was measured in the same manner.
The results are shown in Table 4. In addition, the measured value of the sample with known CRP concentration in Table 4 is a value obtained by subtracting the absorbance when using raw food.

  As is apparent from Table 4, the latex agglutination reagent of the present invention has good dispersibility even when stored for a long period of time, and therefore no increase in the absorbance of saline is observed, and the CRP antigen in the sample is highly sensitive. It turned out that it can measure. On the other hand, in Comparative Example 23, not only an increase in the absorbance of raw food was observed due to natural aggregation, but also a decrease in measurement sensitivity was observed.

Claims (4)

  Protein-fixed latex particles consist of 3-18 mM phosphoric acid, 2-amino-2-hydroxymethyl-1,3-propanediol, 2- (N-morpholino) ethanesulfonic acid, 3- (N-morpholino) -2- Hydroxypropanesulfonic acid, N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid, 3- (N-morpholino) propanesulfonic acid, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane, N Suspended in a buffer selected from-(2-acetamido) iminodiacetic acid, N- (2-acetamido) -2-aminoethanesulfonic acid and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. A reagent for latex agglutination reaction.   The reagent for latex agglutination reaction according to claim 1, wherein the concentration of the buffer solution is 3 to 10 mM.   The reagent for latex agglutination reaction according to claim 1 or 2, wherein the latex particles are polystyrene latex particles. Protein-immobilized latex particles were added to 3-18 mM phosphoric acid, 2-amino-2-hydroxymethyl-1,3-propanediol, 2- (N-morpholino) ethanesulfonic acid, 3- (N-morpholino) -2-hydroxy. Propanesulfonic acid, N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid, 3- (N-morpholino) propanesulfonic acid, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane, N- Suspend in a buffer selected from (2-acetamido) iminodiacetic acid, N- (2-acetamido) -2-aminoethanesulfonic acid and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. A method for preventing spontaneous aggregation of the latex particles during storage, characterized by