JP5062410B2 - Non-specific adsorption inhibitor, probe-binding particles and method for producing them - Google Patents

Description

  The present invention relates to a non-specific adsorption inhibitor applicable to, for example, the surface of immunodiagnostic particles, probe-binding particles, and a method for producing the same.

  In recent years, for the purpose of early detection of diseases, etc., it has been required to increase the sensitivity of tests, and improving the sensitivity of diagnostic agents has become a major issue. In diagnostic agents using a solid phase such as magnetic particles, for the purpose of improving sensitivity, a method using enzyme color development as a detection method is being switched to a method using fluorescence or chemiluminescence that can obtain higher sensitivity. With the development of these detection techniques, it is theoretically said that it has reached a level at which even the presence of a single molecule of a test substance can be detected, but in reality, sufficient sensitivity has not been obtained. The reason for this is that in the diagnosis of detecting specific substances in the presence of biomolecules such as serum, coexisting biomolecules, secondary antibodies, luminescent substrates, etc. adsorb non-specifically to the solid phase, resulting in noise. Increasing the sensitivity is hindering. Therefore, in immunodiagnostic measurement, albumin, casein, etc. are usually used in order to reduce the decrease in sensitivity due to adsorption of non-specifically bound substances to the solid phase surface used for immune reactions (non-specific adsorption). By using a biological substance such as gelatin as a blocking agent (or also referred to as “non-specific adsorption inhibitor”), non-specific adsorption is suppressed and noise is reduced.

  However, even if such a blocking operation is performed, non-specific adsorption still remains, and when such a biologically derived blocking agent is used, there is a possibility of biological contamination represented by BSE, etc. Development of a high-performance blocking agent by chemical synthesis is desired.

As a blocking agent by chemical synthesis, a vinyl monomer copolymer having polyoxyethylene in the side chain has been proposed (Patent Document 1), but the production of such a copolymer has poor reproducibility of molecular weight. As a result, the reproducibility of the blocking effect is poor. In addition, polyoxyethylene having two amino groups at one end has been proposed (Patent Document 2), but such a blocking agent has insufficient interaction with the solid phase, and the added blocking agent Many remain in the water phase and are not economical. Furthermore, a polyoxyethylene / pentaethylenehexamine block copolymer has been proposed as a blocking agent (Patent Document 3). In the synthesis of such a copolymer, both ends of polyoxyethylene are modified with pentaethylenehexamine. The produced compounds are easy to produce and require difficulty in production and purification. In addition, as a blocking agent similar to a polyoxyethylene / pentaethylenehexamine block copolymer, a production method using an acetal-terminated polyoxyethylene as an intermediate is disclosed (Patent Document 4). In this production method, Many dimers of polyoxyethylene are produced, which causes problems in production and purification.
JP 11-287802 A Japanese Patent No. 34079797 JP 2006-226882 A Republished Patent No. WO2005 / 010529

  An object of the present invention is to provide a chemically synthesized non-specific adsorption inhibitor and probe-binding particles which are easy to produce and have a sufficient noise reduction effect, and methods for producing them.

  In order to solve this problem, the present inventors have found a synthesis method that is easy to produce as a non-specific adsorption inhibitor, and further, using this non-specific adsorption inhibitor, treats immunodiagnostic particles. Thus, it was found that the particles exhibited a signal enhancing effect, and the present invention was completed.

A method for producing a non-specific adsorption inhibitor according to one embodiment of the present invention includes:
(A) tosylated product of polyoxyethylene monomethyl ether;
(B) A polyamine having 3 to 12 amino groups and imino groups (—NH—) or any one of them in total is reacted.

In the method for producing the non-specific adsorption inhibitor,
The tosylate of (A) polyoxyethylene monomethyl ether is:
Polyoxyethylene monomethyl ether,
p-toluenesulfonic acid chloride,
It can be a reaction product reacted in the presence of an amine compound.

In the method for producing the non-specific adsorption inhibitor,
The amine compounds include trimethylamine hydrochloride, trimethylamine hydrobromide, trimethylamine hydrofluorate, trimethylamine sulfate, trimethylamine nitrate, trimethylamine phosphate, triethylamine, tripropylamine, and N, N, N ′, N It can be one or more selected from the group consisting of '-tetramethylethylenediamine.

In the method for producing the non-specific adsorption inhibitor,
The amine compound may be a mixture of trimethylamine hydrochloride and an amine compound other than trimethylamine.

  The nonspecific adsorption inhibitor according to one embodiment of the present invention includes a compound represented by the following general formula (1).

・・・・・（１）
［式中、ｎは４〜２０００で表される数であり、Ｒ^１およびＲ^２は、それぞれ独立に、水素原子、または、アミノ基およびイミノ基あるいはいずれか一方を１〜１１個有する基を示し、かつ、Ｒ^１およびＲ^２に含まれるアミノ基およびイミノ基の合計は２〜１１個である。］
上記非特異吸着防止剤において、上記一般式（１）において、Ｒ^１およびＲ^２がそれぞれ下記一般式（２ａ）および（２ｂ）で表される基であることができる。

(1)
[In the formula, n is a number represented by 4 to 2000, and R ¹ and R ² each independently represent a hydrogen atom, an amino group, an imino group, or a group having 1 to 11 of either one. And the total number of amino groups and imino groups contained in R ¹ and R ² is 2 to 11. ]
In the non-specific adsorption inhibitor, in the general formula (1), R ¹ and R ² can be groups represented by the following general formulas (2a) and (2b), respectively.

・・・・・（２ａ）

(2a)

・・・・・（２ｂ）
［式中、ｘ１およびｘ２はそれぞれ１〜４で表される数であり、ｙ１およびｙ２はそれぞれ０〜１１で表される数であり、ｙ１＋ｙ２は２〜１１である。］
本発明の一態様に係るプローブ結合粒子の製造方法は、
粒子の表面にプローブを結合させる工程と、
上記非特異吸着防止剤を用いて、前記プローブが結合された前記粒子を処理する工程と、を含む。

(2b)
[Wherein, x1 and x2 are each a number represented by 1 to 4, y1 and y2 are each a number represented by 0 to 11, and y1 + y2 is 2 to 11. ]
A method for producing probe-bound particles according to an aspect of the present invention includes:
Binding a probe to the surface of the particle;
Treating the particles to which the probe is bound using the non-specific adsorption inhibitor.

  In the method for producing a probe-bonded particle, in the step of bonding the probe, the particle may have at least one group selected from a carboxyl group, an active ester group, a tosyl group, and an epoxy group.

  In the method for producing probe-bonded particles, the particles may be magnetic particles.

  The probe-coupled particle according to one embodiment of the present invention is obtained by the above-described method for producing a probe-coupled particle.

  The probe-binding particle according to one embodiment of the present invention has the non-specific adsorption inhibitor on the surface.

  The non-specific adsorption inhibitor is easy to manufacture and has no possibility of biological contamination because it is a chemically synthesized product. In addition, the non-specific adsorption inhibitor has a noise reduction effect compared to conventionally used non-specific adsorption inhibitors. high.

  Moreover, the said nonspecific adsorption | suction inhibitor can express a signal enhancement effect, when it uses for the particle | grains (for example, magnetic particle) used for an immunodiagnosis.

  Hereinafter, the non-specific adsorption inhibitor, the probe-binding particle and the production method thereof according to an embodiment of the present invention will be described.

1. Non-specific adsorption inhibitor and method for producing the same Non-specific adsorption inhibitor according to an embodiment of the present invention includes (A) tosylation product of polyoxyethylene monomethyl ether and (B) amino group and imino group (-NH-) or It is a reaction product with a polyamine having 3 to 12 of either one in total. The non-specific adsorption inhibitor according to this embodiment is suitable as a signal enhancer for particles, for example. In addition, the non-specific adsorption inhibitor according to the present embodiment may be the reaction product itself, or may contain a solvent as necessary.

  Hereinafter, the production and configuration of the nonspecific adsorption inhibitor according to the present embodiment will be described.

1.1. (A) Tosylate (A) Tosylate of polyoxyethylene monomethyl ether is α-methyl-ω-tosyl polyoxyethylene obtained by tosylating polyoxyethylene monomethyl ether. In the present invention, “tosylation” refers to conversion of a hydroxyl group (—OH) to a p-toluenesulfonyloxy group (—OTs group).

  In this embodiment, polyoxyethylene monomethyl ether can use a well-known thing, for example, various types as Nippon Oil & Fats Uniox M series, Nippon Emulsifier MPG series, Lion Corporation Leosolv PEM series, etc. Those of molecular weight are commercially available.

  The molecular weight of polyoxyethylene monomethyl ether is preferably 200 to 100,000, more preferably 1,000 to 10,000. When the molecular weight is less than 200 or more than 100,000, the noise reduction effect and the signal enhancement effect may be insufficient.

  As a method for tosylating polyoxyethylene monomethyl ether, a known method can be applied. For example, polyoxyethylene monomethyl ether can be obtained by reacting polyoxyethylene monomethyl ether with p-toluenesulfonate. The hydrogen atom of the terminal hydroxyl group is converted to a tosyl group. Although it does not specifically limit as p-toluenesulfonic acid salt, p-toluenesulfonic acid chloride etc. can be mentioned. In this step, typically, polyoxyethylene monomethyl ether is dissolved in an organic solvent such as pyridine, dichloromethane, acetonitrile or the like, and if necessary, an amine catalyst composed of an amine compound is used in combination with 1 mol of polyoxyethylene monomethyl ether. This is carried out by adding 1 to 5 mol of p-toluenesulfonic acid chloride and reacting at room temperature for 10 minutes to 24 hours. As described above, α-methyl-ω-tosylpolyoxyethylene is obtained.

  Examples of the amine catalyst include trimethylamine hydrochloride, trimethylamine hydrobromide, trimethylamine hydrofluorate, trimethylamine sulfate, trimethylamine nitrate, trimethylamine phosphate, triethylamine, tripropylamine, N, N, N ′, N′-tetramethylethylenediamine or the like is preferably used alone or in combination. In addition, it is particularly preferable to use trimethylamine hydrochloride and other amine compounds in combination for shortening the reaction time and improving the tosylation rate. The preferred use amount of the amine catalyst is 1.5 to 10 mol per mol of polyoxyethylene monomethyl ether when the amine catalyst is used alone, and when the trimethylamine hydrochloride and other amine are used in combination. , Trimethylamine hydrochloride 0.1-3 mol, other amine 1.5-10 mol. The α-methyl-ω-tosylpolyoxyethylene after the reaction may be purified by precipitation with diethyl ether, hexane or the like. By such precipitation purification, the remaining p-toluenesulfonate can be removed, and side reactions with the following polyamines can be prevented.

1.2. (B) Polyamine The polyamine that can be used for the production of the nonspecific adsorption inhibitor according to this embodiment is (B) a polyamine having 3 to 12 amino groups and / or imino groups in total.

  When an amine compound having an amino group and / or imino group in total of 2 or less, that is, monoamine or diamine is used, the interaction between the non-specific adsorption inhibitor and the solid phase is insufficient and added. Since most of the nonspecific adsorption inhibitor remains in the aqueous phase, it is not economical, and is easily detached by washing after the treatment with the nonspecific adsorption inhibitor, so that the noise reduction effect is insufficient. In addition, the desorbed non-specific adsorption inhibitor may inhibit the protein added to the test system and reduce the signal.

  On the other hand, when a polyamine having at least 13 amino groups and / or imino groups in total is used, the solid phase surface after treatment with the nonspecific adsorption inhibitor becomes cationic based on the amino groups, and nonspecific adsorption is prevented. It may increase and worsen the noise.

  (B) As a polyamine having 3 to 12 amino groups and imino groups in total, specifically, dimethylenetrimine, trimethylenetetramine, tetramethylenepentamine, pentamethylenehexamine, hexamethyleneheptamine, Polymethyleneamines such as heptamethyleneoctamine, octamethylenenonamine, nonamethylenedecamine, decamethyleneundecamine, undecamethylenedodecamine; diethylenetrimine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, hexaethyleneheptamine, Polyethyleneamines such as heptaethyleneoctamine, octaethylenenonamine, nonaethylenedecamine, decaethyleneundecamine, undecaethylenedodecamine; spermine, spermi Propylene, such as emissions - butylene-based amines can be mentioned, which may be used alone or in combination of two or more kinds. In view of water solubility and noise reduction effect as a non-specific adsorption inhibitor, (B) the polyamine is preferably polyethyleneamine, and may be triethylenetetramine, tetraethylenepentamine, or pentaethylenehexamine. More preferably, it is more preferably pentaethylenehexamine.

1.3. Production of non-specific adsorption inhibitor (A) The reaction of polyoxyethylene monomethyl ether tosylate and (B) a polyamine having 3 to 12 amino groups and / or imino groups in total is typically (A) A solution obtained by dissolving 2 to 100 times (B) polyamine with respect to the number of moles of tosylate is reacted at room temperature to 60 ° C. while dropping the solution of (A) over 1 to 24 hours. Can be done. Solvents that can be suitably used in this step are aprotic polar solvents such as acetonitrile, N, N-dimethylformamide, dimethyl sulfoxide, dimethylacetamide and the like. The nonspecific adsorption inhibitor according to the present embodiment is generated by the above steps.

  The produced nonspecific adsorption inhibitor is preferably purified by precipitation with an organic solvent such as diethyl ether or isopropyl alcohol. As the organic solvent at this time, a mixed solvent of a nonpolar solvent and a polar solvent, for example, a mixed solvent of hexane and ethyl acetate can be suitably used. As another purification method, the produced non-specific adsorption inhibitor solution may be dissolved in water, and the low molecular weight compound may be removed by a dialysis tube, a dialyzer, an acylator, or the like. In this case, the organic solvent may be removed by an evaporator before dissolving in water. By such purification, residual polyamine, tosyl compound, catalyst and the like can be removed, and the noise reduction effect can be maintained high.

1.4. Structure of non-specific adsorption inhibitor A typical structure of the non-specific adsorption inhibitor according to this embodiment is α-methyl-ω-polyaminated polyoxyethylene.

  In the non-specific adsorption inhibitor according to the present embodiment, the terminal primary amino group of the polyamine and the terminal of polyoxyethylene may be bonded, or the non-terminal secondary amino group of the polyamine and the terminal of polyoxyethylene May be combined.

  Two or more molecules of polyoxyethylene may be bonded to one molecule of polyamine. That is, both the terminal primary amino group and the non-terminal secondary amino group of the polyamine may be bonded to the terminal of the polyoxyethylene.

  From the viewpoint of noise reduction effect and signal enhancement effect, the structure of the preferred non-specific adsorption inhibitor according to this embodiment is a structure in which one molecule of polyoxyethylene is bonded to one molecule of polyamine. A nonspecific adsorption inhibitor in which one molecule of polyoxyethylene is bonded to one molecule of polyamine may be isolated by column purification. Note that how many molecules of polyoxyethylene are bound to one molecule of polyamine can be determined by measuring the molecular weight by liquid chromatography.

  In the nonspecific adsorption inhibitor according to the present embodiment, when the terminal primary amino group or secondary amino group of the polyamine is bonded to the terminal of polyoxyethylene, the nonspecific adsorption inhibitor according to the present embodiment is, for example, The compound which is a structure represented by following General formula (1) can be included.

・・・・・（１）
［式中、ｎは４〜２，０００で表される数であり、Ｒ^１およびＲ^２は、それぞれ独立に、水素原子、または、アミノ基およびイミノ基あるいはいずれか一方を１〜１１個有する基を示し、かつ、Ｒ^１およびＲ^２に含まれるアミノ基およびイミノ基の合計は２〜１１個である。］
上記一般式（１）において、ｎは２０〜２００であるのが好ましい。

(1)
[Wherein, n is a number represented by 4 to 2,000, and R ¹ and R ² each independently have 1 to 11 hydrogen atoms, amino groups, imino groups, or any one thereof. And the total of amino groups and imino groups contained in R ¹ and R ² is 2 to 11. ]
In the said General formula (1), it is preferable that n is 20-200.

In this case, in the general formula (1), R ¹ and R ² may be groups represented by the following general formulas (2a) and (2b), respectively.

・・・・・（２ａ）

(2a)

・・・・・（２ｂ）
［式中、ｘ１およびｘ２はそれぞれ１〜４で表される数であり、ｙ１およびｙ２はそれぞれ０〜１１で表される数であり、ｙ１＋ｙ２は２〜１１である。］
上記一般式（２）において、ｘ１およびｘ２は２であるのが好ましく、ｙ１およびｙ２はそれぞれ１〜６（ただし、ｙ１＋ｙ２＝２〜１１を満たす。）であることがより好ましい。

(2b)
[Wherein, x1 and x2 are each a number represented by 1 to 4, y1 and y2 are each a number represented by 0 to 11, and y1 + y2 is 2 to 11. ]
In the said General formula (2), it is preferable that x1 and x2 are 2, and it is more preferable that y1 and y2 are 1-6 (however, y1 + y2 = 2-11 is satisfy | filled), respectively.

1.5. Use of non-specific adsorption inhibitor and production of probe-bound particles The non-specific adsorption inhibitor according to the present embodiment further replaces non-specific adsorption by replacing albumin, casein, gelatin and the like used in conventional immunodiagnostic measurements. Can be suppressed and noise can be reduced.

  For example, in the plate method, after binding a probe such as an antibody to the plate, the non-specific adsorption inhibitor according to this embodiment can be added to treat the plate surface.

  Moreover, the nonspecific adsorption inhibitor according to the present embodiment can be suitably used, for example, for producing probe-bound particles. The method for producing a probe-bound particle according to the present embodiment includes a step of binding a probe to the surface of the particle, and a step of processing the particle to which the probe is bound using the non-specific adsorption inhibitor according to the present embodiment. Including.

  Here, the step of processing the particle to which the probe is bound using the non-specific adsorption inhibitor according to the present embodiment is, for example, bringing the non-specific adsorption inhibitor according to the present embodiment into contact with the surface of the particle for a predetermined time. Can be done. Thereby, nonspecific adsorption | suction on the particle | grain surface can be suppressed and noise can be reduced. Moreover, this process can be performed in the state which disperse | distributed the particle | grains in the solution of the nonspecific adsorption inhibitor which concerns on this embodiment, for example.

  In this case, the particle to which the probe is bound preferably has at least one group selected from a carboxyl group, an active ester group, a tosyl group, and an epoxy group on the surface (the reason will be described later). In this case, the particles are preferably magnetic particles.

  More specifically, for example, in immunochromatography, after binding a probe such as an antibody to colored particles, the nonspecific adsorption inhibitor according to this embodiment is added, and the surface of the colored particles is treated, Probe-bound particles can be obtained. Further, for example, in an assay method such as EIA, CLIA, CLEIA, after binding a probe such as an antibody to the surface of the magnetic particle, the non-specific adsorption inhibitor according to the present embodiment is added to the surface of the magnetic particle. By processing the above, probe-bound particles can be obtained.

  As explained above, according to the non-specific adsorption inhibitor according to the present embodiment, non-specific adsorption can be suppressed and noise can be reduced.

  When the particles having at least the active groups such as carboxyl group, active ester group, tosyl group and epoxy group (for example, magnetic particles) are treated using the non-specific adsorption inhibitor according to this embodiment, non-specific adsorption Since the amino group in the inhibitor and the active group on the particle surface form a covalent bond and improve the orientation of the immunodiagnostic antibody, the effect of enhancing the signal appears. Further, since the desorption of the nonspecific adsorption inhibitor can be suppressed, the test reagent is extremely stable with respect to buffers containing a surfactant or the like.

  The carrier on which the nonspecific adsorption inhibitor according to the present embodiment exhibits a particularly good effect is particles having a carboxyl group (for example, magnetic particles). As a preferable treatment method, there is a method in which the carboxyl group-containing particles are converted into active esters with water-soluble carbodiimide or the like, and after the immunodiagnostic probe is bound, the particles are treated with the nonspecific adsorption inhibitor (signal enhancer) according to this embodiment. Can be mentioned.

  The probe binding particles according to this embodiment can be obtained by the above-described method for manufacturing probe binding particles. For example, the probe-binding particles according to the present embodiment can have the above-mentioned non-specific adsorption inhibitor on the surface.

2. Examples Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

2.1. Example 1
10 g of polyoxyethylene monomethyl ether having an average molecular weight of 4,000 (manufactured by NOF Corporation, trade name “Uniox M-4000”) was dissolved in 100 g of pyridine, and placed in a separable flask equipped with a stirrer. In a separate container, a solution obtained by dissolving 2 g of p-toluenesulfonic acid chloride in 20 g of pyridine was dropped into the separable flask over 1 hour, and further reacted at room temperature for 6 hours. The obtained reaction product was dropped into 1 L of diethyl ether and purified by precipitation to remove the remaining p-toluenesulfonate, thereby obtaining 8 g of a polyoxyethylene monomethyl ether tosylate (A-1).

  Further, while stirring 50 g of an aqueous solution in which 2 g of pentaethylenehexamine (B-1) was stirred, the reaction was performed while dropping 80 g of an aqueous solution in which 8 g of tosylate (A-1) was dissolved over 2 hours at room temperature. I let you. The obtained reaction product was dropped into 1 L of diethyl ether, and the remaining pentaethylenehexamine (B-1) was removed by precipitation purification, followed by vacuum drying, whereby 6 g of nonspecific adsorption inhibitor (C-1). Got. The molecular weight distribution of this nonspecific adsorption inhibitor (C-1) by liquid chromatography was a broad peak with a molecular weight of 4,200 as the main peak, and a slight shoulder was seen on the high molecular weight side. The base number by titration was 1.1 mmol / g.

  Next, an aqueous solution of 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride (manufactured by Dojindo) is added to an aqueous dispersion having a solid content concentration of 1% in which 1 mg of magnetic particles having a carboxyl group (MAG1101 manufactured by JSR) is dispersed. The carboxyl group was activated by adding and rotating and stirring at room temperature for 2 hours. This was magnetically separated and the supernatant was discarded, and then 10 μg of an antibody against human α-fetoprotein (hereinafter referred to as “AFP”), which is a tumor marker (hereinafter referred to as “anti-AFP antibody”, manufactured by Cosmo Bio). For 3 hours at room temperature. After the reaction, 125 μL of the aqueous dispersion of the particles was added to a 0.4% aqueous solution of the nonspecific adsorption inhibitor (C-1), and the mixture was further reacted at room temperature for 15 hours. This was magnetically separated, washed repeatedly with a cleaning solution (containing 25 mmol / L Tris-HCl, pH 7.4, 0.01% Tween 20), diluted with a cleaning solution to a particle concentration of 0.5%, and used as a primary probe. Probe-bound particles (immunoassay particles) bound with an anti-AFP antibody were obtained. Take 10 μl of the probe-bound particle dispersion (corresponding to 50 μg of particles) in a test tube and mix with 50 μl of a standard specimen of AFP antigen (manufactured by Nippon Biotest) diluted to 100 ng / mL with fetal calf serum (FCS). And allowed to react at 37 ° C. for 10 minutes. After separating the particles by magnetic separation and removing the supernatant, an anti-AFP antibody labeled with alkaline phosphatase (hereinafter referred to as “ALP”) as a secondary antibody (manufactured by Fujirebio Inc., attached to Lumipulse AFP-N) Using reagent) 40 μl was added and allowed to react at 37 ° C. for 10 minutes. Next, after magnetic separation and removal of the supernatant, the particles obtained by repeating washing three times with PBS were dispersed in 50 μl of 0.01% Tween 20, and transferred to a new tube. After adding 100 μl of ALP substrate solution (Lumipulse substrate solution: manufactured by Fujirebio Inc.) and reacting at 37 ° C. for 10 minutes, the amount of chemiluminescence was measured.

  For the measurement of chemiluminescence, a chemiluminescence measuring device (trade name: Lumat LB9507) manufactured by Bertol Japan KK was used. As a result, the signal intensity was 123563 RIU. The noise intensity was measured in the same manner as above except that 50 μl of FCS not containing AFP antigen was used instead of 50 μl of the standard specimen of AFP antigen diluted to 100 ng / mL with fetal calf serum (FCS). Met.

2.2. Comparative Example 1
In Example 1, the nonspecific adsorption inhibitor of Comparative Example 1 was obtained in the same manner as in Example 1 except that ethylenediamine was used instead of pentaethylenehexamine (B-1). Comparative Example 1
The molecular weight distribution of the non-specific adsorption inhibitor by liquid chromatography was a broad peak with a molecular weight of 4000 as the main peak. The base number by titration was 0.2 mmol / g. The signal intensity was 109104 RIU, and the noise intensity was 105 RIU.

2.3. Comparative Example 2
Comparative Example 2 was carried out in the same manner as in Example 1 except that polyethyleneimine having a molecular weight of about 1200 (average number of 28 amino groups in one molecule) was used instead of pentaethylenehexamine (B-1) in Example 1. A non-specific adsorption inhibitor was obtained. The molecular weight distribution of the nonspecific adsorption inhibitor of Comparative Example 2 by liquid chromatography was a broad peak with a molecular weight of 11,000. The base number by titration was 2.5 mmol / g. The signal intensity was 65503 RIU and the noise intensity was 180 RIU.

2.4. Comparative Example 3
In Example 1, signals and noise were measured in the same manner as in Example 1 except that bovine serum albumin was used instead of the nonspecific adsorption inhibitor (C-1). The signal intensity was 92762 RIU and the noise intensity was 83 RIU.

2.5. Comparative Example 4
In Example 1, signals and noise were measured in the same manner as in Example 1 except that the nonspecific adsorption inhibitor (C-1) was not used. The signal intensity was 94673 RIU and the noise intensity was 123 RIU.

  From the above results, the non-specific adsorption inhibitor (C-1) of Example 1 was a total of 3 (A) tosylated product of polyoxyethylene monomethyl ether and (B) amino group and imino group. Compared with the case of using bovine serum albumin as a non-specific adsorption inhibitor (Comparative Example 3) and the case of not using a non-specific adsorption inhibitor (Comparative Example 3) because it is a reaction product with ˜12 polyamines Thus, it has been confirmed that it has a sufficient noise reduction effect.

  On the other hand, the nonspecific adsorption inhibitor of Comparative Example 1 is a reaction product of (A) polyoxyethylene monomethyl ether tosylate and a polyamine having two or less amino groups and imino groups in total. Therefore, the noise reduction effect was insufficient. In addition, the non-specific adsorption inhibitor of Comparative Example 2 is a reaction product of (A) polyoxyethylene monomethyl ether tosylate and polyamine having at least 13 amino groups and / or imino groups in total. The noise increased.

2.5. Example 2
100 g of polyoxyethylene monomethyl ether having an average molecular weight of 5,000 (manufactured by Fluka), 5 g of trimethylamine hydrochloride, 8 g of tripropylamine, and 8 g of p-toluenesulfonic acid chloride are dissolved in 300 g of acetonitrile, and placed in a separable flask equipped with a stirrer. By reacting at 30 ° C. for 2 hours with stirring, a solution of polyoxyethylene monomethyl ether tosylate (A-2) was obtained.

  Also, 47 g of pentaethylenehexamine (B-2) is dissolved in 230 g of acetonitrile, put into another separable flask with a stirrer, and kept at 40 ° C. while stirring, and the tosylated product (A-2) is dropped over 1 hour. The mixture was further stirred for 9 hours to be reacted. After the reaction, the mixture was allowed to stand at room temperature for 16 hours, and the precipitated by-product was removed by decantation. The supernatant obtained by decantation is concentrated with an evaporator, dissolved in 500 g of water, filtered, and further purified using a dialyzer to obtain a 2% aqueous solution of the nonspecific adsorption inhibitor (C-2). Got. The molecular weight distribution of the nonspecific adsorption inhibitor (C-2) by liquid chromatography was a peak having a molecular weight of 5,200 as a main peak, and a slight shoulder was observed on the high molecular weight side. From the ratio of the peak area of CH adjacent to the imino group by proton NMR and the peak area of CH of the ether bond, it was confirmed that polyoxyethylene monomethyl ether and pentaethylenehexamine were bonded at a ratio of 1: 1.

  Next, an aqueous solution of 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride (manufactured by Dojindo) is added to an aqueous dispersion having a solid content concentration of 1% in which 1 mg of magnetic particles having a carboxyl group (MAG2303 manufactured by JSR) are dispersed. The carboxyl group was activated by adding and rotating and stirring at room temperature for 2 hours. This was magnetically separated and the supernatant was discarded, and then 10 μg of an antibody against human α-fetoprotein (hereinafter referred to as “AFP”), which is a tumor marker (hereinafter referred to as “anti-AFP antibody”, manufactured by Cosmo Bio). For 3 hours at room temperature. After the reaction, 125 μL of the aqueous dispersion of the particles was added to a 0.4% aqueous solution of the nonspecific adsorption inhibitor (C-1), and the mixture was further reacted at room temperature for 15 hours. This was magnetically separated, washed repeatedly with a cleaning solution (containing 25 mmol / L Tris-HCl, pH 7.4, 0.01% Tween 20), diluted with a cleaning solution to a particle concentration of 0.5%, and used as a primary probe. Probe-bound particles (immunoassay particles) bound with an anti-AFP antibody were obtained. Take 10 μl of the probe-bound particle dispersion (corresponding to 50 μg of particles) in a test tube and mix with 50 μl of a standard specimen of AFP antigen (manufactured by Nippon Biotest) diluted to 100 ng / mL with fetal calf serum (FCS). And allowed to react at 37 ° C. for 10 minutes. After separating the particles by magnetic separation and removing the supernatant, an anti-AFP antibody labeled with alkaline phosphatase (hereinafter referred to as “ALP”) as a secondary antibody (manufactured by Fujirebio Inc., attached to Lumipulse AFP-N) Using reagent) 40 μl was added and allowed to react at 37 ° C. for 10 minutes. Next, after magnetic separation and removal of the supernatant, the particles obtained by repeating washing three times with PBS were dispersed in 50 μl of 0.01% Tween 20, and transferred to a new tube. After adding 100 μl of ALP substrate solution (Lumipulse substrate solution: manufactured by Fujirebio Inc.) and reacting at 37 ° C. for 10 minutes, the amount of chemiluminescence was measured.

  For the measurement of chemiluminescence, a chemiluminescence measuring device (trade name: Lumat LB9507) manufactured by Bertol Japan KK was used. As a result, the signal intensity was 182983 RIU. The noise intensity was measured in the same manner as above except that 50 μl of FCS not containing AFP antigen was used instead of 50 μl of the standard specimen of AFP antigen diluted to 100 ng / mL with fetal calf serum (FCS). Met.

2.6. Comparative Example 5
In Example 2, the nonspecific adsorption inhibitor of Comparative Example 5 was obtained in the same manner as in Example 2 except that ethylenediamine was used instead of pentaethylenehexamine (B-2). The molecular weight distribution of the nonspecific adsorption inhibitor of Comparative Example 5 by liquid chromatography was a peak having a molecular weight of 5000 as the main peak. As for the base number by titration, the signal intensity was 1358582 RIU and the noise intensity was 107 RIU.

2.7. Comparative Example 6
In Example 2, Comparative Example 6 was performed in the same manner as in Example 2 except that polyethyleneimine (average number of 28 amino acids in one molecule) having a molecular weight of about 1200 was used instead of pentaethylenehexamine (B-2). A non-specific adsorption inhibitor was obtained. The molecular weight distribution by liquid chromatography of the nonspecific adsorption inhibitor of Comparative Example 6 was a broad peak with a molecular weight of 12,000. The signal intensity was 72377 RIU, and the noise intensity was 193 RIU.

2.8. Comparative Example 7
In Example 2, signal and noise were measured in the same manner as in Example 2 except that bovine serum albumin was used instead of the nonspecific adsorption inhibitor (C-2). The signal intensity was 129246 RIU and the noise intensity was 90 RIU.

2.9. Comparative Example 8
In Example 2, signals and noise were measured in the same manner as in Example 2 except that the nonspecific adsorption inhibitor (C-2) was not used. The signal intensity was 127195RIU and the noise intensity was 131RIU.

  From the above results, the nonspecific adsorption inhibitor (C-2) of Example 2 is a total of 3 (A) tosylated product of polyoxyethylene monomethyl ether and (B) an amino group and / or an imino group. Compared with the case of using bovine serum albumin as a non-specific adsorption inhibitor (Comparative Example 7) and the case of not using a non-specific adsorption inhibitor (Comparative Example 7) because it is a reaction product with ˜12 polyamines Thus, it has been confirmed that it has a sufficient noise reduction effect.

  On the other hand, the non-specific adsorption inhibitor of Comparative Example 5 is a reaction product of (A) polyoxyethylene monomethyl ether tosylate and a polyamine having two or less amino groups and imino groups in total. Therefore, the noise reduction effect was insufficient. In addition, the non-specific adsorption inhibitor of Comparative Example 6 is a reaction product of (A) polyoxyethylene monomethyl ether tosylate and a polyamine having at least 13 amino groups and / or imino groups in total. The noise increased.

Claims (4)

(A) tosylated product of polyoxyethylene monomethyl ether;
(B) A method for producing a non-specific adsorption inhibitor, comprising reacting an amino group and an imino group (—NH—) or a polyamine having 3 to 12 of either one in total. The tosylate of (A) polyoxyethylene monomethyl ether is:
Polyoxyethylene monomethyl ether,
p-toluenesulfonic acid chloride,
The method for producing a nonspecific adsorption inhibitor according to claim 1, which is a reaction product reacted in the presence of an amine compound.   The amine compounds include trimethylamine hydrochloride, trimethylamine hydrobromide, trimethylamine hydrofluorate, trimethylamine sulfate, trimethylamine nitrate, trimethylamine phosphate, triethylamine, tripropylamine, and N, N, N ′, N The manufacturing method of the nonspecific adsorption inhibitor of Claim 2 which is 1 or more types chosen from the group which consists of '-tetramethylethylenediamine.   The method for producing a nonspecific adsorption inhibitor according to claim 2 or 3, wherein the amine compound is a mixture of trimethylamine hydrochloride and an amine compound other than trimethylamine.