JP3443891B2 - Protein adsorption inhibitor - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a protein adsorption inhibitor used in biochemical analysis methods and the like, more specifically in a two-site method (sandwich measurement method) widely used in the field of clinical reagents and the like. , An antibody-binding solid phase, an antigen-binding solid phase, and a protein adsorption inhibitor that prevents protein adsorption to the solid phase itself and the like.

[0002]

2. Description of the Related Art Immunometric assays widely used in the fields of clinical diagnostics are generally two-si
The solid phase method based on the te method (sandwich measurement method) is used. This measurement method is based on the substance to be measured (test substance, A
g) two kinds of antibodies having different epitopes (Ab ₁ , A)
b ₂ ) is used. First, Ab ₁ is immobilized on the surface of a solid phase (SP) made of a synthetic polymer or the like, and Ag is added to this to bind. Then, a labeled antibody (Ab ₂ *) is reacted and washed to remove free Ab ₂ *, and the standard activity of Ab ₂ * (bound type, B) bound to the solid phase is measured. In this case, B increases in accordance with the amount of Ag, and a standard curve is obtained between the two. The amount of antigen in the sample is measured from this standard curve. Further, there is also used a method in which an antigen and an antibody are reversed, that is, a labeled antigen is used to measure the amount of antibody in a sample (measured using Ag ₁ , Ag ₂ * and Ab). Enzymes, fluorescent substances, luminescent substances, etc. are usually used for these labels.

One of the main factors that influence the sensitivity of these sandwich methods is the adsorption of the labeled antibody to the antibody-bound solid phase or the adsorption of the labeled antigen to the antigen-bound solid phase. It can be fully predicted that these adsorptions depend in part on the properties of the enzyme, fluorescent substance, luminescent substance, etc. used for labeling. The adsorption of the enzyme-labeled antibody was 1 / 30,000 or less than the amount of any of the alkaline phosphatase-labeled antibody, the glucose oxidase-labeled antibody, and the peroxidase-labeled antibody, and was nonspecific for the β-D-galactosidase-labeled antibody. Adsorption is 1/2000 (enzyme immunoassay,
Medical School). These adsorptions are responsible for the reduced sensitivity and lack of reproducibility in the sandwich method.

[0004] Conventionally, in order to prevent these adsorptions, a method of performing an immunoassay with a weakly acidic (pH 5 to 6) buffer solution, or after adsorbing Ab ₁ , an extra protein-binding site on the solid phase is treated with egg white. A method of blocking with albumin, bovine serum albumin, fetal bovine serum, normal serum, etc. is known.

However, the weakly acidic operation and blocking with various proteins do not have sufficient protein adsorption preventing ability, and there is a demand for the development of a more excellent protein adsorption inhibitor in the field of clinical diagnosis and the like.

Further, commercially available ovalbumin, bovine serum albumin, fetal bovine serum, etc. are often contaminated with immunoglobulins, enzymes, hormones, etc., which affects the reaction and causes an error in the analytical value, which is a problem. ing.

[0007]

The object of the present invention is not limited to a labeling substance such as an enzyme, a fluorescent substance or a chemiluminescent substance, and a substance to be measured, without affecting the measuring system. To provide a protein adsorption inhibitor that suppresses protein adsorption such as adsorption to an antibody-bound solid phase, adsorption to an antigen-bound solid phase, or adsorption to a solid phase, and makes it possible to analyze a target substance with high accuracy. It is in.

[0008]

According to the present invention, 2-methacryloyloxyethylphosphorylcholine (hereinafter referred to as MP
C), n-butyl (meth) acrylate, and (meth) acryl
Comprises a copolymer of acrylic acid methyl or styrene, and the
Provided is a protein adsorption inhibitor for use in biochemical analysis, characterized in that the copolymer is dissolved in a buffer solution and physiological saline. Further, according to the present invention, there is provided the above protein adsorption inhibitor, which is used for a blocking agent used in a liquid state in a biochemical analysis method.

The present invention will be described in more detail below.

The protein adsorption inhibitor of the present invention includes, for example, proteins, polypeptides, steroids, lipids, hormones and the like, more specifically, generally enzyme reactions of various antigens, antibodies, receptors, enzymes and the like or antigen-antibody reactions of immunoglobulins. It is a reagent that can be used in a method for analyzing a biologically-related substance, which is measured by using. Specifically, known radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), latex nephelometry, etc. are particularly preferable, and enzyme immunoassay (EIA) and fluorescence immunoassay are particularly preferable. Measurement method (F
IA), latex nephelometry, etc., and in these known biochemical analysis methods, after binding an antibody or an antigen to the surface of the solid phase, an extra protein binding site on the solid phase is identified. MPC copolymer (hereinafter referred to as polymer A
Block ) as the active ingredient.

[0011] Heavy that Ru used for protein adsorption inhibitor of the present invention
Coalescence A is a copolymer of MPC and other vinyl monomers. The MPC content in this polymer A component is
Against the, preferably at least 5 mol%. Further , the polymer A preferably has a number average molecular weight of from 1000 to 1,000, though it varies depending on the polymerization temperature, the amount of the polymerization initiator used, the use of the polymerization degree adjuster and the like.
1,000,000, particularly preferably 2000-70000
Is the range.

[0012] The in preparing the polymer A is, for example, the presence of a specific polymerization initiator, MPC and other copolymerizable JP
It can be obtained by a method of polymerizing with a specific vinyl monomer-containing component.

[0013] As the MPC can be copolymerized with other specific vinyl <br/> Rumonoma, for example (meth) acrylic acid n- butyl, (meth) acrylic acid methylation or styrene.

The polymerization initiator is not particularly limited as long as it is an ordinary radical polymerization initiator, but for example, 2,2'-azobisisobutyronitrile, benzoyl peroxide, diisopropyl peroxydicarbonate,
t-butylperoxy-2-ethylhexanoate, t
-Butyl peroxypivalate, t-butyl peroxydiisobutyrate, persulfate, persulfate-hydrogen sulfite and the like can be mentioned. The amount of the polymerization initiator used is preferably 0.01 to 10 parts by weight, particularly preferably 0.1 to 5 parts by weight, based on 100 parts by weight of all the raw material monomers.

The polymerization conditions for preparing the polymer A are preferably 30 to 80 ° C., particularly preferably 40 to 7
It is desirable to polymerize at 0 ° C. for 2 to 72 hours. At this time, a solvent may be used in order to carry out the polymerization reaction more smoothly, and the solvent may be water, methanol, ethanol, propanol, t-butanol, benzene, toluene, dimethylformamide, tetrahydrofuran, chloroform or a mixture thereof. Etc. can be mentioned.

The protein adsorption inhibitor of the present invention is not particularly limited as long as the polymer A is used as an active ingredient, and a solvent for dissolving the polymer A may be added and used. As the solvent for dissolving the polymer A,
Preferable examples include phosphate buffer, acetate buffer, carbonate buffer, citrate buffer, various physiological saline, etc., and these solutions include surfactants such as trade name "Tween 20" (manufactured by ICI) or An organic solvent such as dimethyl sulfoxide, tetrahydrofuran, or N, N-dimethylformamide can be added preferably in an amount of 0.01 to 20% by weight, and it is particularly preferable to use a phosphate buffer solution, various physiological saline solutions and the like.

The protein adsorption inhibitor of the present invention can be used by, for example, a method of binding the antibody or antigen to the solid phase surface to be used for analysis of various biological substances and then washing with the protein adsorption inhibitor. The treatment may be performed, and the treatment may be performed at any time before adding the serum or the labeled antibody or the labeled antigen. Further, even in the case of the analysis target substance in which the adsorption of the analysis target substance in the serum to the solid phase is not a problem, it may be before the addition of the labeled antibody or the labeled antigen, and the solution of the present invention may be added to all solutions to be analyzed. It is also possible to add and use a protein adsorption inhibitor. At this time, the amount of the protein adsorbent used is calculated in terms of the polymer A component,
It is preferably 0.0001 to 5% by weight.

The material of the solid phase for preventing the adsorption of the protein is not particularly limited, but examples thereof include polystyrene, polyvinyl chloride, polypropylene, acryl, polymethylmethacrylate, glass, metal, ceramics and silicon rubber. be able to. The shape of these materials is not particularly limited, but examples thereof include a test tube, a titer plate, latex, and magnetic fine particles.

[0019]

EFFECTS OF THE INVENTION Since the protein adsorption inhibitor of the present invention has a strong protein adsorption inhibiting ability at a low concentration, it can be used for the analysis of various bio-related substances and the like to obtain a reproducible and highly accurate analysis value. be able to.

[0020]

The present invention will be described in more detail with reference to Synthesis Examples, Examples and Comparative Examples, but the present invention is not limited to these.

[0021]

[0022]

[Synthesis Example 1] MPC-n-butyl methacrylate (hereinafter referred to as B
(Referred to as MA) copolymer synthesis MPC / BMA monomer charge molar ratio MPC / BMA = 40/60, total monomer concentration 1.0 mol / liter, and polymerization initiator to be 1 mol% based on the monomer. And MPC1.4
35 g (4.9 mmol), BMA 2.153 g (1
(5.1 mmol) was weighed into a glass reaction tube for polymerization, and AIBN 0.0328 g (0.2 m) was added as a polymerization initiator.
mol) and 20 ml of methanol as a polymerization solvent were added. After thoroughly purging the inside of the reaction tube with argon, seal it 2
The polymerization reaction was carried out by heating to 60 ° C. for 4 hours. The obtained reaction mixture was ice-cooled and then added dropwise to 400 ml of diethyl ether to precipitate a polymer. This was filtered off, washed thoroughly with diethyl ether, and dried under reduced pressure to obtain 2.019 g of a white powdery polymer. The polymerization rate was 56.3%. Further, the tetrahydrofuran solution of the obtained polymer was analyzed by GPC and the molecular weight was measured. As a result, it was 320 in terms of polystyrene.
It was 00. Furthermore, the molar composition ratio is from the result of elemental analysis,
MPC / BMA = 38.5 / 61.5.

[0023]

[Reference Example 1] Preparation of fluorescein isothiocyanate-labeled antibody anti-human carcinoembryonic antigen mouse antibody (hereinafter referred to as anti-human CEA
2.0 mg of fluorescein isothiocyanate (hereinafter referred to as FITC) was added to 2.0 mg of 20 mg / ml (referred to as mouse IgG) (0.2 M sodium carbonate buffer, pH 9.0). Then, after reacting at 4 ° C. overnight, the antibody bound to FITC was purified using a Sephadex G-25 column equilibrated with sodium phosphate buffered saline (hereinafter referred to as PBS). Next, the activated DEAE-cellulose was equilibrated with PBS, and FIT was eluted with the Sephadex G-25 column.
Add antibody bound to C and elute with PBS, then FITC
A labeled antibody was prepared.

[0024]

Example 1 4.0 ml of a 20 μg / ml rabbit antibody PBS solution was introduced into a polystyrene test tube having an inner diameter of 10 mmφ × 75 mm, incubated at 4 ° C. overnight to physically adsorb, and then 4.0 ml was added. Washing was performed 3 times with PBS. Then, the molar composition and the molecular weight synthesized according to Synthesis Example 2 were MPC / BMA = 30.4 / 69.6, M
4.0 ml of a PBS solution containing 0.005% by weight of a copolymer of n = 26000 was added thereto, the mixture was incubated at 4 ° C. overnight, and then washed with 4.0 ml of PBS three times.
Then, 4.0 ml of 20 μg / ml FITC-labeled anti-human CEA mouse IgG prepared in Reference Example 1 was added, and 4
After incubating at 0 ° C. overnight, the plate was washed 3 times with 4.0 ml of PBS. By further adding 4.0 ml of PBS containing 2% by weight of sodium dodecyl sulfate, the adsorbed FITC-labeled anti-human CEA mouse IgG was added.
Was removed from the test tube. The detached FITC-labeled anti-human CEA mouse IgG was measured at an excitation wavelength of 495 nm and a measurement wavelength of 520 nm. The amount of adsorption was expressed as a percentage of the added FITC-labeled anti-human CEA mouse IgG, and the measurement results are shown in Table 1.

[0025]

Example 2 Instead of the copolymer of MPC and BMA,
Copolymer of MPC and methacrylic acid methyl ester (hereinafter referred to as MMA) (having a molar composition and molecular weight of MPC / M
The same procedure as in Example 1 was performed except that MA = 34.4 / 65.6, Mn = 32000) was used. The measurement results are shown in Table 1.

[0026]

[0027]

Example 3 Instead of the copolymer of MPC and BMA,
Copolymer of MPC and styrene (hereinafter referred to as ST) (having a molar composition and molecular weight of MPC / ST = 38.5 / 61.
5 , Mn = 26000) was used, and the same procedure as in Example 1 was performed. The measurement results are shown in Table 1.

[0028]

[0029]

Comparative Example 1 MPC / BMA = 30.4 / 69.6, M
Example 1 was repeated except that a PBS solution containing 1% by weight of bovine serum albumin was used instead of the PBS solution containing 0.005% by weight of the copolymer of n = 26000. The measurement results are shown in Table 1.

[0030]

[Table 1]

As compared with Comparative Example 1 in which bovine serum albumin was added, it was found that the protein adsorption inhibitor of the present invention prevented adsorption of FITC-labeled anti-human CEA mouse IgG at a low concentration.

[0032]

Example 4 The molar composition and molecular weight synthesized according to Synthesis Example 2 are MPC / BMA = 30.4 / 69.6 (Mn
= 26000) 0.005 wt% polymer and 0.6 wt% bovine serum albumin in PBS solution 4.0
After adding ml to a polystyrene test tube having an inner diameter of 10 mmφ × 75 mm and incubating at 4 ° C. overnight, 4.0 m
It was washed 3 times with 1 l of PBS. Next, after adding 4.0 ml of a PBS solution containing 1% by weight sodium dodecyl sulfate, the amount of bovine serum albumin in this solution was adjusted to PIER.
CE brand name "Micro BCA Protein Assay Reagen
t ". The results are shown in Table 2.

[0033]

Example 5 Instead of the copolymer of MPC and BMA,
Copolymer of MPC and MMA (Molecular composition and molecular weight: MPC / MMA = 34.4 / 65.6, Mn = 3200
The same procedure as in Example 4 was carried out except that 0) was used. The measurement results are shown in Table 2.

[0034]

[0035]

Example 6 Instead of the copolymer of MPC and BMA,
Copolymer of MPC and ST (Molecular composition and molecular weight M
PC / ST = 38.5 / 61.5 , Mn = 26000)
The same procedure as in Example 4 was carried out except that was used. The measurement results are shown in Table 2.

[0036]

[0037]

Comparative Example 2 MPC / BMA = 30.4 / 69.6, M
Example 4 was repeated except that a PBS solution was used instead of the PBS solution containing 0.005% by weight of the polymer of n = 26000. The measurement results are shown in Table 2.

[0038]

[Table 2]

As compared with Comparative Example 2 using a PBS solution, it is clear that the protein adsorption inhibitor of the present invention prevents protein adsorption.

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kazuhiko Ishihara 6-5-9-201, Kamimizumotocho, Kodaira-shi, Tokyo (56) Reference JP-A-3-39309 (JP, A) JP-A-4-283653 (JP, A) JP-A-4-122858 (JP, A) JP-A-1-217266 (JP, A) JP-A-62-231169 (JP, A) (58) Fields investigated (Int. Cl. ⁷⁾ , DB name) G01N 33/53-33/579

Claims (2)

(57) [Claims] 1. 2-methacryloyloxyethylphosphorylcholine , n-butyl (meth) acrylate, and (meth) acrole
Comprises a copolymer of acrylic acid methyl or styrene, and the
A protein adsorption inhibitor used in a biochemical analysis method, wherein the copolymer is dissolved in a buffer solution and physiological saline. 2. The protein adsorption inhibitor according to claim 1, which is for a blocking agent used in a liquid state in a biochemical analysis method.