JP2018004323A - Immunological measurement method which makes dextran coexist - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunological measurement method using an antigen-antibody reaction, the method using molecules which improve an antigen-antibody reaction to increase the measurement performance, and the molecules which improve an antigen-antibody reaction.SOLUTION: The immunological measurement method is for immunologically measuring a measurement target substance in a sample, and more specifically makes a measurement by making dextran with an average molecular weight of 60,000 daltons or larger and preferably of 200,000 daltons or larger coexist with a measurement system in a concentration in a range of preferably 15 g/L to 25 g/L, both inclusive, and more preferably 15 g/L to 20 g/L, both inclusive.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an immunoassay method in which dextran coexists. More specifically, it relates to the addition of a water-soluble polymer that promotes an antigen-antibody reaction to an antigen-antibody reaction solution.

  Measurement of biomolecules using antigen-antibody reaction is widely used not only in the research field but also in some medical devices for testing purposes and simple diagnostic kits. There are a wide variety of uses in medical equipment such as ELISA, EIA, latex agglutination, and immunochromatography. In these methods, the antigen-antibody reaction occurs mainly in an aqueous solution containing the antigen and the antibody, and in the solution in which the respective molecules are dispersed, the antigen-antibody reaction binds only with a combination having a certain binding constant. Specificity of the measurement is obtained by causing a reaction. That is, in order to improve the performance of the medical device, it is important to increase the reaction efficiency of the antigen-antibody reaction. To date, many attempts have been made to increase antibody binding by creating antibodies with higher binding ability, modifying antibody amino acid sequences, and the like, contributing to higher performance of many medical devices. As an attempt to increase the efficiency of the antigen-antibody reaction, it is also important to increase the accuracy of the relatively specific antigen-antibody reaction by suppressing non-specific reactions. The removal of a portion that contributes to non-specific binding of a representative antibody, chemical modification or substitution of an amino acid residue, and the like are performed. At the same time, regarding the aqueous solution that causes antigen-antibody reaction, it is considered important to have an appropriate chemical composition, and the buffer type, pH, salt concentration, etc. are adjusted to suit each antibody-antigen reaction. The composition is formulated. For such optimization of the composition, a composition imitating the environment in the living body is usually used, but there is no generalized rule, and adjustment is made according to the target antibody and antigen combination.

  Until now, various molecules have been used for the reaction solution where antigen-antibody reaction occurs. In particular, since molecules derived from living bodies including antibodies have a hydrophilic surface, hydrophilic molecules such as saccharides are often used.

  Patent Document 1 discloses an immunoassay containing pullulan in order to provide an immunoassay reagent and an immunoassay method that suppress non-specific binding and aggregation between carriers in a latex agglutination method and have a high aggregation promoting effect upon antigen addition. Reagents are presented. However, it is described that dextran, which is a water-soluble polysaccharide similar to pullulan, did not show the effect of pullulan. Patent Document 2 describes a homogeneous immunoassay method such as a turbidimetric method, and non-saccharide water-soluble polymers such as PEG are sometimes used. It also describes that there is a harmful effect due to an increase in viscosity. Patent Document 3 describes that in an immunochemical reaction, dextran is allowed to coexist in a reaction solution to promote an antigen-antibody reaction. However, for dextran, various molecular weights in a certain range may be fractionated and used. It is said that the effect does not change even if the molecular weight is mixed.

  In other words, in the case of a measurement reagent that requires measurement accuracy such as an in vitro diagnostic drug, the composition of the reaction solution of the immune reaction needs to be determined in consideration of the influence on the measurement accuracy. For homogeneous immunoassay methods such as latex agglutination and turbidimetric methods, the effect of adding hydrophilic polymers to the reaction solution has been demonstrated, while heterogeneous systems using solid phase carriers such as the ELISA method have been shown. The effect of promoting antigen-antibody reaction by adding hydrophilic polymer in the immunoassay method is unknown.

Japanese Patent No. 3396231 Japanese Patent No. 4512492 JP-A-61-79164

  An object of the present invention is to provide a measurement method using a molecule that improves an antigen-antibody reaction in order to improve measurement performance in an immunological measurement method using an antigen-antibody reaction.

As a result of intensive studies to solve the above-mentioned problems, the present inventor has found that dextran, which is a water-soluble polymer, promotes an antigen-antibody reaction almost without promoting nonspecific reaction, and further has a measurement accuracy. As a result, the present invention has been completed.
That is, the present invention is as follows.
(1) In the immunological measurement method for measuring a substance to be measured in a sample by an immunological method, the dextran having an average molecular weight of 60,000 daltons or more is allowed to coexist in a measurement system.
(2) The method according to (1), wherein the average molecular weight of dextran is 200,000 daltons or more.
(3) The method according to (1) or (2), wherein the concentration of dextran to be coexisted is 15 g / L or more and 25 g / L or less.
(4) The method according to any one of (1) to (3), wherein the concentration of dextran to coexist is 15 g / L or more and 20 g / L or less.

  The present invention is described in further detail below.

  In the present invention, the sample is not particularly limited, and examples thereof include human body fluids, and specific examples include blood, serum, plasma, urine, and ascites. There are no particular limitations on the substance to be measured, and examples include antigens, haptens, antibodies, and various diagnostic markers (for example, human ribonuclease 1). Moreover, the immunological measurement method measured by an immunological method is a measurement method using an antigen-antibody reaction. Although not particularly limited, ELISA method, EIA method, latex agglutination method, immunochromatography method and the like are mentioned, and it refers to a measurement method involving at least one antigen-antibody reaction. Moreover, a sandwich method, a competitive method, etc. are mentioned. In the present invention, for example, the reaction between human ribonuclease 1 and anti-human ribonuclease 1 antibody can be mentioned, and the anti-human ribonuclease 1 antibody is preferably MrhRN0614.

  In the present invention, dextran coexists in such a measurement system. In the measurement system, the antigen-antibody reaction is often carried out in a solution, so dextran may be allowed to coexist in the reaction solution. The dextran used in the present invention has an average molecular weight of 60,000 daltons or more. The average molecular weight is preferably 200,000 daltons or more. The upper limit of the average molecular weight of dextran is not particularly limited, but is preferably about 1,000,000. In the present invention, the concentration of dextran coexisting in the measurement system is not particularly limited, but is preferably 15 to 25 g / L, and more preferably 15 to 20 g / L.

  According to the present invention, an antigen-antibody reaction is promoted, and a highly sensitive measurement is possible in a measurement based on an immune reaction. This effect enables measurement of a smaller amount of sample and measurement in a shorter time.

It is a figure which shows the average and standard deviation of the measured value in Example 3.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by an Example.

  In the following examples, a fully automatic enzyme immunoassay device (AIA-CL2400, manufactured by Tosoh Corporation) is used as an immunoassay device, and an anti-RNase1 antibody described in JP-A-2006-42083 is used as an immunoassay reagent. The total RNase 1 amount measuring reagent prepared was used by the method shown below. Each measurement was performed by a two-step method.

  The RrhRN203 antibody-immobilized magnetic fine particles are obtained by activating carboxyl groups exposed on the surface of magnetic fine particles (MX200 / Carboxyl) manufactured by JSR with EDC, and 4 ng with respect to 1 μg of dry weight of the magnetic fine particles via the activated amino groups. Of RrhRN203 antibody was prepared by covalent binding. The magnetic fine particles were subjected to blocking treatment after the antibody was immobilized. A buffer solution (20 mM TrisHCl (pH 7.4), 150 mM NaCl, 1% BSA, 5%) in which magnetic fine particles of 50 μg are suspended in a first reaction tank of a magnetically permeable container having two reaction tanks in a dry weight equivalent. FBS, 5% mannitol, 5% sucrose) was added, and the other second reaction tank contained a buffer containing 20 ng of alkaline phosphatase labeled MrhRN0614 antibody (20 mM TrisHCl (pH 7.4), 150 mM NaCl, 1% BSA, 5% FBS, 5% sucrose) was added and lyophilized. In the buffer solution containing the antibody added to the second reaction tank, a water-soluble polymer was added in the range of 0.5% to 2.5% as necessary in the following examples.

  Using this reagent and a commercially available fully automatic enzyme immunoassay apparatus (AIA-CL2400, manufactured by Tosoh Corporation), the amount of human ribonuclease contained in the sample was measured. The measurement principle is as follows. That is, 50 μL of a measurement sample was added to the first reaction vessel, and the magnetic fine particles were reacted at 37 ° C. for 5 minutes while stirring. After washing the magnetic fine particles with the washing solution, 50 μL of the buffer solution containing the alkaline phosphatase-labeled antibody solubilized with the lysis solution is transferred from the second reaction vessel to the first reaction vessel, and the magnetic fine particles are stirred at 37 ° C. for 3 Reacted for 1 minute. After the reaction, a B / F separation operation was performed to separate and remove the free labeled antibody, and detection was performed using a chemiluminescent substrate that measures alkaline phosphatase activity.

Example 1 Acceleration of antigen-antibody reaction depending on molecular weight of water-soluble polymer A reagent for measurement was prepared by adding the water-soluble polymer shown in Table 1 at a concentration of 25 g / L to a buffer containing an alkaline phosphatase-labeled antibody. . In addition, a reagent without adding a water-soluble polymer was prepared as a reference reagent.

各試薬を用いて、５０ｎｇ／ｍＬのヒトリボヌクレアーゼ１を含む標準品を測定して得られた結果を表１に示す。結果は、３回測定を実施した測定値の平均を、試薬１で５０ｎｇ／ｍＬのヒトリボヌクレアーゼ１を含む標準品を測った測定値を１００％とした時の相対値として示す。

Table 1 shows the results obtained by measuring a standard product containing 50 ng / mL human ribonuclease 1 using each reagent. A result shows the average of the measured value which measured 3 times as a relative value when the measured value which measured the standard goods which contain 50 ng / mL human ribonuclease 1 with the reagent 1 is set to 100%.

  From the above results, it can be seen that the measured value increases as the average molecular weight of dextran increases. It can also be seen that the addition of pullulan shows the same effect as dextran 200K. In particular, the molecules that showed an increase in the measured value of 40% or more were dextran having an average molecular weight of 60,000 Da, 150,000 Da, 200,000 Da, and pullulan. In addition, using the same measurement reagent, the measurement value when measuring a buffer solution not containing human ribonuclease 1 is 100%, and the measurement value obtained by measuring a standard product containing 50 ng / mL human ribonuclease 1 with reagent 1 is 100%. The relative value is shown. It was found that the addition of the water-soluble polymer did not cause a significant non-specific reaction in all the reagents, and the S / N ratio indicating the measurement performance was inferior to that of the reference reagent to which no water-soluble polymer was added. There is no high price.

Example 2 Evaluation of antigen-antibody reaction promoting effect of water-soluble polymer For reagent 1 and reagent 6 in Table 1, only the magnetic fine particle suspension in the first reaction tank was freeze-dried to label the alkaline phosphatase in the second reaction tank. Table 2 shows the results obtained by simultaneously preparing a reagent that was not lyophilized for the buffer containing the antibody and comparing the buffer containing the alkaline phosphatase labeled antibody in the second reaction tank with lyophilized. The measurement conditions are the same as in Example 1.

凍結乾燥の有無で、デキストラン添加の効果は変わらず、分子量が大きいものがより測定値が高いことが明らかとなった。このことは、水溶性高分子が凍結乾燥の際に抗体の安定性を保つことに寄与しているのではなく、水溶性高分子の添加によって抗原抗体反応が促進されていることを示す。

The effect of dextran addition did not change with and without lyophilization, and it was revealed that the measured value was higher when the molecular weight was higher. This indicates that the water-soluble polymer does not contribute to maintaining the stability of the antibody during lyophilization, but that the antigen-antibody reaction is promoted by the addition of the water-soluble polymer.

Example 3 Evaluation of Antigen-antibody Reaction Promotion Effect by Dextran Addition Concentration The effects of dextran addition concentration were evaluated on reagents 4, 5, and 6 that showed a high antigen-antibody reaction promotion effect in Example 1. Results of adjusting the reagent with addition concentrations of 5 g / L, 15 g / L, and 25 g / L by the method described in Example 1 and comparing with the reference reagent 1 (25 g / L) prepared at the same time (relative value) ) Is shown in Table 3. The measurement conditions are the same as in Example 1.

表３の結果は、水溶性高分子の添加による抗原抗体反応の促進効果は、添加する水溶性高分子の濃度に依存して高くなることを示す。表３の結果からは、４０％以上の測定値の増加を示す条件は、各分子量の水溶性高分子ともに２５ｇ／Ｌであり、試薬６に使用した平均分子量が２００，０００Ｄａのデキストランでは、１５ｇ／Ｌでも４０％の測定値の増加を示した。

The results in Table 3 show that the effect of promoting the antigen-antibody reaction by the addition of the water-soluble polymer is increased depending on the concentration of the water-soluble polymer to be added. From the results of Table 3, the conditions showing an increase in the measured value of 40% or more were 25 g / L for each water-soluble polymer of each molecular weight, and 15 g for dextran having an average molecular weight of 200,000 Da used for Reagent 6. / L also showed a 40% increase in measured value.

  Next, for the reagent 6 to which dextran having an average molecular weight of 200,000 Da was added, the measurement accuracy of the immunoassay reagent according to the concentration of dextran added was examined. As in Example 1, a standard product containing 50 ng / mL human ribonuclease 1 was measured three times per condition, and the average and standard deviation of the measured values at each concentration are shown in FIG.

  The measured value increases depending on the concentration of dextran added, so it can be seen that there is an effect of promoting antigen-antibody reaction, but the standard deviation indicating the variation in measured value also increases depending on the concentration of dextran added. Became clear. An immunoassay reagent that requires high measurement accuracy needs to have a variation in measurement below a certain standard. When the standard is 15%, it is desirable that the concentration of dextran added is 25 g / L or less, which is even stricter. When it was set to 10%, it became clear that the dextran addition concentration is desirably 20 g / L or less.

  Based on the above results, it was shown that the concentration of dextran that promotes the antigen-antibody reaction is within the range of 15 g / L to 25 g / L within the range where the immunoassay reagent is kept at high measurement accuracy. The concentration was found to be the addition of dextran having an average molecular weight of 200,000 Da in the range of 15 g / L to 20 g / L.

Claims (4)

An immunological measurement method for measuring a substance to be measured in a sample by an immunological method, wherein the dextran having an average molecular weight of 60,000 daltons or more coexists in a measurement system. The method according to claim 1, wherein the average molecular weight of dextran is 200,000 daltons or more. The method of Claim 1 or 2 whose density | concentration of the dextran to coexist is 15 g / L or more and 25 g / L or less. The method according to any one of claims 1 to 3, wherein the concentration of dextran to be coexisted is 15 g / L or more and 20 g / L or less.

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