JP2009300280A - Highly sensitive enzyme immunoassay - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sandwich ELISA method capable of accurately measuring as low a level of antigen as possible. <P>SOLUTION: In the enzyme immunoassay comprising: reacting a solid phase antibody being a primary antibody with a low level of allergen of not more than 10 ng/ml which is a measuring object; reacting the reacted allergen with a secondary antibody labeled with an enzyme; and thereafter detecting an enzyme reaction product obtained by adding a chromogenic or luminescent substrate reacting with the enzyme, reaction conditions of the allergen with the primary antibody and those of the secondary antibody with the allergen are adjusted to ≤23°C and ≥6 hours. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a high-sensitivity enzyme immunoassay, and more particularly to a high-sensitivity enzyme immunoassay by a sandwich ELISA method in which an antigen to be measured is an allergen.

  Among immunoassays, enzyme immunoassay (EIA method) characterized by enzyme labeling is cheaper and less dangerous than radioimmunoassay (RIA method) using radioisotopes. It is known that the measurement sensitivity and accuracy are comparable.

  In the EIA method, when a solid phase is used, it is referred to as an ELISA method (Enzyme-Linked Immunosorbent Assay). Among them, there are improved measurement methods such as a competitive method and a sandwich method.

Enzyme immunoassay using labeled enzyme to improve measurement accuracy and sensitivity for the quantification of microorganisms such as bacteria, shorten reaction time, perform rapid measurement, and perform more stable measurement It is known that a reaction temperature of 11 to 30 ° C. is adopted as a reaction temperature between a solid phase antibody to be measured and a measurement target, and a temperature condition for reaction by adding a labeling enzyme is 11 to 22 ° C. (Patent Document 1, Patent Document 2).
For such microorganisms such as fungi, it is considered that detection of about 1.0 × 10 ⁴ cells / ml or more is sufficient, and it is considered that quantitative measurement at a lower concentration is not required.

  In order to increase the sensitivity of the immunoassay, it is better to set the reaction conditions between the solid phase antibody and the measurement target substance for several hours and the reaction conditions between the measurement target substance and the enzyme-labeled antibody for a long time. (Non-Patent Document 1).

According to the guidelines of the Ministry of Health, Labor and Welfare and the school hygiene standards of the Ministry of Education, Culture, Sports, Science and Technology, the measurement accuracy required when actually measuring allergens is a hygienic standard value that is less than 100 mites / m ² or less Since the allergen amount corresponding to 100 ticks is 10 μg, accurate quantification is required with relatively high sensitivity.

Thus, in the immunoassay method for measuring allergens, there is a need for accuracy capable of accurately quantifying allergens with a tick number of 100 or less in the dust in the measurement target area of 1 m ^2. There are few problems with this measurement method.

JP-A-5-307030 JP-A-5-307036

Eiji Ishikawa, “Ultra-sensitive immunoassay”, published in 1994, published by the Academic Publishing Center (page 129)

However, when conducting research and development of allergen removal agents, allergen removal methods, or allergen reduction agents, when conducting efficacy evaluations or comparisons of efficacy, etc., a precise examination on a scale considerably smaller than the above-mentioned hygienic standard values In addition, there is a problem that a method and means for measuring an extremely small amount of allergen have not been established even in a target area where 1 m ² cannot be secured.

  Also, under conditions where the concentration of antigen and antibody is low, it takes a long time for the reaction to occur until immunoassay is possible, but it is common knowledge that the temperature condition is as high as possible to cause the reaction as soon as possible. Then, the accuracy of quantification is impaired.

  Therefore, the object of the present invention is to solve the above-mentioned problems, and when using the sandwich ELISA method as an antigen to be measured as an allergen as a highly sensitive enzyme immunoassay, even for an antigen having a concentration as low as possible. It is a highly sensitive enzyme immunoassay that can be measured and can be accurately quantified.

  In order to solve the above-mentioned problems, in the present invention, a solid phase antibody is reacted with an allergen, the antigen-antibody complex produced is reacted with an enzyme-labeled antibody, and a substrate that reacts with the enzyme is added. In the enzyme immunoassay for detecting the resulting enzyme reaction product, each reaction temperature in the reaction conditions of the allergen to the solid phase antibody and the reaction conditions of the enzyme-labeled antibody to the antigen-antibody complex is 23 ° C. or less, and This is a highly sensitive enzyme immunoassay characterized by a reaction time of 6 hours or longer.

  The high-sensitivity enzyme immunoassay method of the present invention configured as described above is based on the reaction conditions of the allergen to the solid phase antibody and the reaction of the enzyme-labeled antibody to the antigen-antibody complex, as will be apparent from the test results described later. By adjusting each reaction temperature under the conditions to a low temperature of 23 ° C. or less and setting each reaction time to a long time of 6 hours or more, it is as short as 1 to 3 hours at a relatively high temperature exceeding 23 ° C. It becomes a highly sensitive enzyme immunoassay that can detect even a low concentration of allergens such as 10 ng / ml or less, which is difficult to detect by the reaction of time, and can be accurately quantified.

  The type of allergen to be measured is not particularly limited, and the concentration of the measurement target is, for example, a low concentration of 10 ng / ml or less, and further a low concentration allergen of 0.1 to 5 ng / ml can be measured. It can be measured without problems even at concentrations higher than that range.

  In addition, if the enzyme reaction product is a chromogenic or luminescent enzyme reaction product, the amount of antigen can be quantified by measuring absorbance or luminescence intensity.

  In addition, the reaction conditions of the allergen to the solid phase antibody and the reaction conditions of the enzyme-labeled antibody to the antigen-antibody complex are 2 to 10 ° C. and 12 hours or more. And the recovery rate calculated from the theoretical value is in the range of 80 to 110%, and is preferable for obtaining an accurate quantitative result.

  In this invention, the reaction temperature in the reaction conditions of the allergen to the solid phase antibody in the enzyme immunoassay and the reaction conditions of the enzyme-labeled antibody to the antigen-antibody complex is 23 ° C. or less, and each reaction time is 6 hours or more. As a result, it is possible to accurately quantify even the lowest possible antigen concentration of 10 ng / ml or less, especially with a coefficient of variation of 10 or less and a recovery rate calculated from a theoretical value of 80 to There is an advantage that it is an enzyme immunoassay method capable of accurate quantification in the range of 110%.

  The highly sensitive enzyme immunoassay method of the present invention is an improved method generally called a sandwich ELISA method, and is an antibody that reacts specifically with a protein allergen that is a kind of antigen, preferably a monoclonal antibody Is immobilized on a carrier to form a solid phase, and an antigen-antibody complex is formed by reacting such a solid phase antibody with an antigen that specifically reacts therewith. The reaction between the solid phase antibody and the antigen allergen is hereinafter referred to as a primary reaction.

  Furthermore, an antibody that specifically reacts with the antigen and has an enzyme bound to the antigen-antibody complex, preferably a monoclonal antibody that reacts specifically with the antigen and has the enzyme bound thereto (hereinafter referred to as an enzyme-labeled antibody). To form an antigen-antibody complex in which a solid phase antibody and an enzyme-labeled antibody are bound to the antigen. The reaction between the antigen-antibody complex and the enzyme-labeled antibody is hereinafter referred to as a secondary reaction.

  Then, the enzyme reaction product produced by adding a substrate that reacts with the enzyme is detected, for example, a color is developed by reacting the chromogenic substrate of the enzyme, and the absorbance of this color is measured to accurately determine the amount of the antigen. It is.

  The highly sensitive enzyme immunoassay method of the present invention can be carried out in the same manner as a normal enzyme immunoassay. For example, a microwell plate or the like can be used as a carrier, and a substrate on which an antibody that specifically binds to an analyte is fixed can be used. Usually, there is no need to shake for the reaction.

  The allergen in the present invention refers to a substance (antigen) itself that reacts with an antibody to cause an allergic symptom, but may be a substance containing the antigen, and the type of allergen to be measured is not particularly limited. Specific examples of such allergens include mites, plant pollen, animals (such as dust and insects), foods, microorganisms (such as molds and other fungi), and other known allergens. Generally, the amount of allergen in the indoor environment is determined based on the amount of mite allergen.

  Both reaction conditions in the reaction between the solid phase antibody and the antigen in the substance to be measured and the reaction between the antigen in the substance to be measured and the enzyme-labeled antibody are adjusted to a temperature condition of 23 ° C. or less, preferably 20 ° C. or less, and 6 hours or more. The reaction time is preferably set to 12 hours or longer.

  Depending on the reaction conditions, even with an antigen solution having an antigen concentration of several ng / ml or less, for example, 10 ng / ml or less, the coefficient of variation calculated from the measured value is 10 or less, and the recovery calculated from the theoretical value and the measured value is used. Accurate measurement results with a rate of 80% to 110% can be obtained.

  In order to perform more accurate quantification in the highly sensitive enzyme immunoassay method of the present invention, the temperature of each reaction in the reaction between the solid phase antibody and the allergen and the reaction between the antigen-antibody complex and the enzyme-labeled antibody is set to 2 to 2. It is preferable that the temperature is 10 ° C. and the reaction time is 12 hours or longer.

[Examples 1 to 24, Comparative Examples 1 to 48]
A purified mite antigen Derf2 (manufactured by Asahi Breweries) was prepared with a phosphate buffer at a known concentration. With respect to the antigen solution of this known concentration, the amount of antigen was measured by sandwich ELISA.

  The phosphate buffer is pH 7.3 and contains 0.1% by weight bovine serum albumin and 0.05% by weight polyoxyethylene (20) sorbitan monolaurate. Hereinafter, a phosphate buffer solution having the same composition is referred to as a phosphate buffer solution (pH 7.3).

  First, 100 μl per well of anti-Derf2 monoclonal antibody (manufactured by Asahi Breweries) diluted to 2 μg / ml with phosphate buffer (pH 7.3) was added to a plate (F16MAXISORP NUNC-IMMNO MODULE plate: NUNC). The plate was allowed to stand at 4 ° C. for 1 day or longer to be immobilized on the plate.

  Discard the solution in the well after solid-phase formation and add 200 μl of 1 wt% bovine serum albumin and phosphate buffer (pH 7.3), which are blocking reagents, and react at 37 ° C. for 60 minutes. It was. After this reaction, the plate was washed with a phosphate buffer (pH 7.3).

  After washing, 100 μl of the known antigen solution was dropped per well, and the reaction was carried out for a predetermined time under the reaction conditions (primary reaction) shown in Table 1. After this reaction, the plate was washed with a phosphate buffer (pH 7.3).

  After washing, a peroxidase-labeled anti-Derf2 monoclonal antibody (manufactured by Asahi Breweries) dissolved in a phosphate buffer solution (pH 7.3) to 200 μg / ml is further dissolved in a phosphate buffer solution (pH 7.3). The solution diluted 1200-fold was added by 100 μl per well, and the reaction was carried out under the reaction conditions (secondary reaction) shown in Table 1. After this reaction, the plate was washed with a phosphate buffer (pH 7.3).

After washing, ortho-phenyldiamine dihydrochloride (26 mg Table, manufactured by SIGMA CHEMICAL CO.) And 13 μl of hydrogen peroxide solution were added to 13 ml of 0.1 mol / L phosphate buffer (pH 6.2), and 100 μl per well was added. The solution was added in portions and reacted at 37 ° C. for about 5 minutes. Thereafter, 2 mol / L H ₂ SO ₄ was immediately added at 50 μl per well to stop the reaction.

[Absorbance measurement test according to temperature and reaction time]
About the enzyme immunoassay method in each reaction temperature condition and reaction time condition of Examples 1-24 and Comparative Examples 1-48, after said reaction stop, using a microplate reader (Bio-Rad Laboratories Inc.), 490 nm Absorbance was measured, and the relationship between absorbance for each known antigen concentration and reaction time is shown in FIGS.

When the temperature condition is as high as 25 ° C. or more as in reaction conditions 1 to 4, the absorbance is constant in a relatively short time of 2 hours or more, whereas the temperature is constant as in reaction conditions 5 to 12 When the condition is 20 ° C. or lower, the absorbance is constant for 6 hours or less, but the absorbance is constant from 6 hours. For this reason, a reaction time of 2 hours or more is required at high temperature conditions such as reaction conditions 1 to 4, and a reaction time of 6 hours or more is required at 20 ° C. or less as in reaction conditions 5 to 12.
Thus, under low temperature reaction conditions of 2 to 20 ° C. such as reaction conditions 5 to 12 in the examples, the reaction time is long, but the absorbance at an antigen concentration of 0.1 to 6.25 ng / ml exceeds 6 hours. It can be seen that the primary reaction and the secondary reaction are particularly stable up to 24 hours at 2 to 10 ° C., and it is possible to measure a low concentration of antigen.
On the other hand, when the temperature condition is 25 ° C. or more as in the reaction conditions 1 to 4 which are Comparative Examples 1 to 24, when the reaction time exceeds 3 hours, the low concentration absorbance is higher than that of the high concentration measurement object. Absorbance was particularly unstable for samples with an antigen concentration of 6.25 ng / ml or less.

[Examples 25-29, Comparative Examples 49-51]
An antigen solution having a known concentration was prepared from purified mite antigen Derf2 (manufactured by Asahi Breweries) with a phosphate buffer (pH 7.3). The antigen amount was measured by sandwich ELISA using this antigen solution of known concentration as Derf2.

  First, 100 μl per well of anti-Derf2 monoclonal antibody (manufactured by Asahi Breweries) diluted to 2 μg / ml with a phosphate buffer (pH 7.3) was added to a plate (F16MAXISORP NUNC-IMMNO MODULE plate: NUNC). The plate was allowed to stand at 4 ° C. for 1 day or longer to be immobilized on the plate.

  After forming the solid phase, the liquid in the well of the plate was discarded, and 200 μl of a mixture of 1% by weight bovine serum albumin and phosphate buffer (pH 7.3) was added as a blocking reagent at 37 ° C., 60 ° C. Reacted for 1 minute. After the reaction, the plate was washed with a phosphate buffer (pH 7.3).

  After washing, 100 μl of the known antigen solution was dropped per well, and the reaction was performed under the reaction conditions (primary reaction) shown in Table 2. After the reaction, the plate was washed with a phosphate buffer (pH 7.3).

  After washing, a peroxidase-labeled anti-Derf2 monoclonal antibody (manufactured by Asahi Breweries) dissolved in a phosphate buffer solution (pH 7.3) to 200 μg / ml is further dissolved in a phosphate buffer solution (pH 7.3). A solution diluted 1200-fold was added at a rate of 100 μl per well, and the reaction was carried out under the reaction conditions (secondary reaction) shown in Table 2. After the reaction, the plate was washed with a phosphate buffer (pH 7.3).

  After washing, 100 ml per well containing 13 ml of ortho-phenyldiamine dihydrochloride (26 mg Table, manufactured by SIGMA CHEMICAL CO.) And 13 ml of hydrogen peroxide solution in 13 ml of 0.1 mol / L phosphate buffer (pH 6.2) The solution was added in portions and reacted at 37 ° C. for about 5 minutes. Immediately thereafter, 2 mol / L H2SO4 was added at 50 μl per well to stop the reaction.

[Accuracy confirmation test of antigen solution of known concentration]
About the enzyme immunoassay method in each reaction temperature condition and reaction time condition of Examples 25-29 and Comparative Examples 49-51, after the above-mentioned reaction stop, using a microplate reader (Bio-Rad Laboratories Inc.), 490 nm Absorbance was measured. The measurement results (antigen amount, standard deviation, coefficient of variation, recovery rate) are shown in Tables 3-10.

Under reaction conditions 13, 14, and 15 at a temperature of 25 ° C. or higher, if the antigen concentration is less than 0.5 ng / ml, the coefficient of variation is 10 or more, and the recovery rate is not in the range of 80 to 110%. It turns out that quantification is not completed.
In addition, the coefficient of variation is 10 or less even under a temperature condition of less than 25 ° C. as in reaction conditions 16 to 20 (preferably less than 0.5 ng / ml and more than 0.1 ng / ml). The recovery rate was 80% or more and 110% or less, and it was confirmed that accurate quantification was possible.

Chart showing the relationship between the reaction time and absorbance of the primary reaction at a temperature of 37 ° C. Chart showing the relationship between the reaction time and the absorbance of the secondary reaction at a temperature of 37 ° C. A chart showing the relationship between the reaction time and absorbance of the primary reaction at a temperature of 25 ° C. Chart showing the relationship between the reaction time and absorbance of the secondary reaction at a temperature of 25 ° C. A chart showing the relationship between the reaction time and absorbance of the primary reaction at a temperature of 20 ° C. Chart showing the relationship between reaction time and absorbance of secondary reaction under temperature condition of 20 ° C Chart showing the relationship between the reaction time and absorbance of the primary reaction at a temperature of 15 ° C. Chart showing the relationship between reaction time and absorbance of secondary reaction under temperature condition of 15 ° C Chart showing the relationship between the reaction time and absorbance of the primary reaction at a temperature of 10 ° C. Chart showing the relationship between reaction time and absorbance of secondary reaction under temperature condition of 10 ° C Chart showing the relationship between the reaction time and absorbance of the primary reaction under temperature conditions of 2 ° C Chart showing the relationship between reaction time and absorbance of secondary reaction at 2 ° C in temperature condition

Claims (5)

In an enzyme immunoassay method in which an allergen is reacted with a solid phase antibody, an antigen-labeled antibody complex is reacted with an antibody labeled with an enzyme, and a substrate that reacts with the enzyme is added to detect an enzyme reaction product. ,
A highly sensitive enzyme characterized in that each reaction temperature in the reaction between the solid phase antibody and the allergen and the reaction between the antigen-antibody complex and the enzyme-labeled antibody is 23 ° C. or less and each reaction time is 6 hours or more. Immunoassay.   2. The highly sensitive enzyme immunoassay method according to claim 1, wherein the allergen to be measured is an allergen having a low concentration of 10 ng / ml or less.   The sensitive enzyme immunoassay method according to claim 1, wherein the allergen to be measured is an allergen having a low concentration of 0.1 to 5 ng / ml.   The reaction temperature in the reaction between the solid phase antibody and the allergen and the reaction between the antigen-antibody complex and the enzyme-labeled antibody is 2 to 10 ° C, and each reaction time is 12 hours or more. A highly sensitive enzyme immunoassay described in 1.   The coefficient of variation of the measured value of the enzyme reaction product to be detected is 10 or less, and the recovery rate of the enzyme reaction product calculated from the theoretical value is 80 to 110%. Sensitive enzyme immunoassay.

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