JPH0599925A - Method for measuring concentration of antigen or antibody - Google Patents

Abstract

PURPOSE:To make it possible to measure the concentration of antigen (or antibody) highly sensitively by casting the lights having the different wavelengths on the composite of antibody and antigen, using the calibration curve of an absorbance ratio in a low concentration region, and using the calibration curve of absorbance difference in a high-concentration region. CONSTITUTION:In antigen-antibody reaction, latex agglutination is generated by using latex on which the antibody (or antigen) is carried and dispersed, suitable buffer solution and a sample such as, e.g. blood. The lights having the different wavelengths lambda1 and lambda2 are cast into the obtained reaction latex solution. Two calibration curves, which indicate the relationship between the concentration when the diluted series of the sample containing the antigen (or antibody) whose concentration is known is made to react for a specified time and the absorbance ratio Alambda1/Alambda2 and the relationship between the concentration and the absorbance difference (Alambda1-Alambda1 blank)-(Alambda2-Alambda2 blank), are obtained. These calibration curves are used, and the absorbances of the two different concentrations of the antigen (or antibody) in the sample, whose concentration is unknown, are measured. In response to the predetermined concentration region for every measuring item, the calibration curve of the absorbance ratio is used in the low-concentration region, and the calibration curves of the absorbance difference is used in the high concentration region. Thus, the concentration of the antigen (antibody) is determined.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for measuring the concentration of an antigen or antibody in a sample. More specifically, it relates to a method for quantifying an antigen (or antibody) by measuring the absorbance by spectroscopic analysis.

[0002]

2. Description of the Related Art In recent years, in the medical field, it has become an important subject to quantitatively detect the concentration of an antigen or an antibody for diagnosing a disease. Components that only exist, for example β ₂ M (β ₂ M
The development of a highly sensitive quantitative method that can quantitatively measure (eg, microglobulin) has become an issue.

Conventionally, (1) a latex carrying an antibody (or an antigen) is dispersed in a solvent, and this is reacted with the antigen (or antibody) to increase the turbidity (absorbance) associated with the agglutination reaction of the latex at a wavelength. A method for quantifying an antigen (or antibody) by measuring at 600 to 2400 nm (JP-A-58-58).
11575), (2) In the above method, the change in the absorbance due to the latex aggregation caused by the antigen-antibody reaction is smaller than the absorbance of the latex solution. And a two-point method for measuring the change in absorbance between two points after a lapse of a certain period of time, and (3) light of _two different wavelengths λ ₁ and λ _{2 for} the test solution containing the antigen-antibody complex. Method of determining the concentration of the antigen (or antibody) in the test liquid (sample) by irradiating, measuring the absorbances Aλ ₁ and Aλ ₂ of each wavelength, and changing the ratio of the absorbances Aλ ₁ / Aλ ₂ (end point method) ) (JP-A-63-138266).

(4) Recently, a solution containing agglomerated latex particles is divided into one agglomerate in a sheet flow,
A method for quantifying an antigen (or antibody) by analyzing the degree of aggregation by a light scattering detection method using a laser light source has also been developed.

[0005]

However, the above method has the following problems. In the method (1), the change in absorbance due to latex aggregation is smaller than that in the latex solution itself, and even if an attempt is made to increase the absorbance change by selecting the measurement wavelength, the absorbance of the latex solution itself becomes large. There is a problem that it does not improve / N.

The method (2) is a two-point method in which the change in the absorbance is measured at a fixed time after the start of the antigen-antibody reaction in the same reaction solution and at two points after the fixed time has elapsed. Therefore, it is difficult to use the so-called end point method (1 point method), which subtracts the absorbance of only the Latex solution, which is the reagent, from the absorbance after a sufficient reaction time. An automatic analyzer controlled by the above is required. It is not impossible to measure with a spectrophotometer, but after inhaling the sample, it will take a long time to get the measurement result after the spectrophotometer, because the measured value is not stable unless it reacts in the flow cell for a long time. There is a problem that it is inefficient when treating a large number of samples in a hospital or the like because the ability is reduced.

The method (3) can be easily measured in a relatively short time by using a general-purpose spectrophotometer, and the change rate is large and the sensitivity is high in the low concentration range, but in the high concentration range. Since the rate of change is small, there is a problem that the measured value changes greatly even with a slight change in absorbance, and the accuracy decreases.

In the method (4), the agglutination of latex and the measurement result correspond to each other in a one-to-one manner, and the endpoint method can be adopted. The longer the reaction time, the higher the agglutination and the higher the sensitivity. Although the former drawbacks such as the fact that the sensitivity does not change even if the concentration is reduced are improved, a laser light source is required because it is necessary to have a seed flow structure and the scattered light from one particle is detected. There was a problem that it was necessary and it had to be a dedicated device.

The present invention has been made in view of the above circumstances, and the concentration of an antigen (or antibody) can be measured with high sensitivity by an endpoint method using a general-purpose spectrophotometer without using a dedicated device. The purpose is to provide a method.

[0010]

[Means for Solving the Problems]
Therefore, the method for measuring the concentration of an antigen or antibody of the present invention is
Inert carrier of particles carrying antibody (or antigen)
Dispersed in a solvent and reacted with the antigen (or antibody)
The resulting antibody-antigen complex has different wavelengths λ. ₁, Λ
₂The light of each wavelength, the absorbance of each wavelength Aλ₁, Aλ₂Measure
And the ratio of the absorbances Aλ₁/ Aλ₂And absorbance difference (A
λ₁-Aλ₁Blank)-(Aλ₂-Aλ ₂blank)
In the low concentration range, use the calibration curve of the absorbance ratio to
In the concentration range, the antibody (or
(Antigen) concentration is measured.

The latex in which an antibody (or an antigen) is carried on a fine particle size insoluble carrier and dispersed in a solvent is not particularly limited, but usually has a particle size of 0.1 to 0.
It is about 8 μm, and a commercial product carrying various antibodies (or antigens) can be easily obtained. Antigen-
The antibody reaction is carried out by an antibody-antigen reaction using a latex in which such an antibody (or an antigen) is supported and dispersed, and usually an appropriate buffer solution and a test solution (sample) such as blood or serum. -Agglutination of the latex results in the antigen complex.

In the present invention, the reaction latex solution thus obtained is irradiated with light having different wavelengths λ ₁ and λ ₂ . Depending on the type of spectrophotometer, in a normal spectrophotometer wavelength range may be selected two wavelengths as appropriate over the range from the wavelength of for example 325～1100Nm, the wavelength lambda _2, especially long wavelength side In order to avoid absorption by water, it is preferable to select a wavelength of 1000 nm or less. Also, since the absorbance range is 4 ABS or less, it is desirable to adjust the concentration of the latex reagent so that the absorbance at 500 nm is usually 4 ABS or less depending on the diameter of the latex and the like.

In the present invention, first, a dilution series of a sample containing an antigen (or antibody) of known concentration is reacted for a certain period of time in the same manner as above, and then the concentration and the absorbance ratio Aλ ₁ / Aλ ₂
And the relationship between concentration and absorbance difference (Aλ ₁ -Aλ ₁ blank)-(Aλ ₂ -Aλ ₂ blank) are obtained in advance, and these calibration curves are used to similarly determine the unknown concentration. The absorbance of two different wavelengths of the antigen (or antibody) in the sample is measured, and the calibration curve is based on the absorbance ratio in the low concentration range and the absorbance in the high concentration range according to the concentration range determined in advance for each measurement item. A calibration curve based on the difference is used to determine the concentration of the antigen (or antibody). As to what concentration is to be defined as the low concentration region and the high concentration region, it depends on the type of latex used, particle size, antigen, type of antibody, etc., so depending on the calibration curve created by the absorbance ratio and the calibration curve created by the absorbance difference, From low concentration to high concentration, the sensitivity of the calibration curve by absorbance ratio decreases,
The point at which the calibration curve based on the absorbance ratio is switched to the calibration curve based on the absorbance difference and is read at a concentration at which the sensitivity of the calibration curve based on the absorbance difference is improved may be set in advance.

The difference in absorbance (Aλ ₁ -Aλ ₁ blank)-(Aλ ₂ -Aλ ₂ blank), Aλ ₁ blank,
The A λ ₂ blank indicates the absorbance at wavelengths λ ₁ and λ ₂ when the antibody (or antigen) concentration is 0. As a sample for measuring the absorbance of this blank, the same latex solution and buffer as the above-mentioned antibody (or antigen) are usually used in the same amount, but the antigen (or antibody) to be tested such as serum is used. It can be adjusted by using, for example, the same amount of physiological saline instead of the sample containing).

[0015]

The present invention is different from an antibody-antigen complex formed by dispersing an antibody (or an antigen) supported on an inactive carrier of fine particles in a solvent and reacting this with the antigen (or the antibody). The light having wavelengths λ ₁ and λ ₂ is irradiated, the absorbances Aλ ₁ and Aλ ₂ of the respective wavelengths are measured, the absorbance ratio and the absorbance difference are determined, and the calibration curve of the absorbance ratio has a large change rate in the low concentration range, There is little variation in measured values, so sensitivity is good,
Higher accuracy, the calibration curve of the absorbance difference has a larger rate of change than the calibration curve of the absorbance ratio in the high concentration range, and thus the sensitivity is better and the accuracy is higher. Using the calibration curve and the absorbance difference calibration curve in the high concentration range, the concentration of the antigen (or antibody) is measured by a general-purpose spectrophotometer in a relatively short time by the endpoint method, which is the same easy operation as before. It is possible to provide a method for measuring with high sensitivity.

The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto.

[0017]

EXAMPLES Table 1 shows a reagent comprising 180 μl of latex in which a β ₂ M (β ₂ -microglobulin) antibody is supported on a latex having a particle size of about 0.2 μm and 380 μl of Tris-HCl buffer, and serum having a known concentration of β ₂ M. After 15 minutes of reaction with 20 μl at room temperature, each wavelength λ ₁ = 700 nm, λ ₂ =
Absorbance at 950 nm A ₇₀₀ and A ₉₅₀ and absorbance ratio A
₇₀₀ / A ₉₅₀ , absorbance difference (A ₇₀₀ -A ₇₀₀ blank)-
Shows the (A ₉₅₀ -A ₉₅₀ blank). A blank sample having a concentration of β ₂ M of 0 ng / ml was prepared by using 20 μl of physiological saline in place of 20 μl of serum in the above.

[0018]

[Table 1]

FIG. 1 shows a calibration curve of the absorbance ratio and the absorbance difference created based on the data in Table 1. In FIG. 1, the solid line X shows the calibration curve of the absorbance difference, and the solid line Y shows the calibration curve of the absorbance ratio. The ranges indicated by the dotted lines X ₁ and X ₂ and the ranges indicated by the dotted lines Y ₁ and Y ₂ respectively represent the difference in absorbance and the absorbance ratio at each concentration when serum with a known concentration of β ₂ M was similarly measured. Indicates the variation of the value.

As is clear from FIG. 1, the absorbance ratio has a large change rate in the low concentration range and a high sensitivity, and the variation is small, but in the high concentration range, the change rate of the calibration curve is high. Is small and measurement errors are likely to occur.
On the other hand, the absorbance difference has a large rate of change up to the high concentration range, indicating that the sensitivity is good. Therefore, there is a drawback that an error is likely to occur. At a high concentration, the difference in absorbance with the reagent blank is large, and therefore the variation in the measured values does not work very well.

Therefore, the concentration of the antibody or the antigen can be accurately measured by using the calibration curve of the absorbance ratio method up to a concentration having a certain degree of inclination and using the calibration curve of the absorbance difference method above the concentration. You can do it.

In Table 2, the concentration is known in the low concentration range (item β ₂
M, concentration of 250 ng / ml) for the comparison, using the calibration curve of the absorbance ratio method and the reproducibility data of the measurement results when using the calibration curve of the absorbance difference method, respectively, Table 3 shows known concentrations in the high concentration range (item β
₂ M, concentration of 1200 ng / ml), the reproducibility data of the measurement results using the calibration curve of the absorbance ratio method and the calibration curve of the absorbance difference method are shown for comparison. ..

In Tables 2 and 3, n represents the n number of each measurement (measurement experiment number), X is the arithmetic mean of each measurement value, SD is the standard deviation, and CV is the coefficient of variation (standard deviation / arithmetic mean) × 10
It is indicated by 0.

[0024]

[Table 2]

[0025]

[Table 3]

As is clear from Tables 2 and 3, it is understood that in the low concentration region, the absorbance ratio method has an arithmetic mean value closer to the actual concentration and a smaller coefficient of variation. On the other hand, in the high concentration range, it is recognized that the absorbance difference method has an arithmetic mean value closer to the actual concentration and a smaller coefficient of variation. This data also shows that it is preferable to use the calibration curve by the absorbance ratio method in the low concentration range, and it is preferable to use the calibration curve by the absorbance difference method in the high concentration range.

[0027]

According to the present invention, a general-purpose spectrophotometer can be used easily in a short time without using a dedicated automatic absorbance change measuring device or a special device using a sheath flow and laser light scattering method. In addition, the concentration of the antigen (or antibody) in the antigen-antibody reaction utilizing latex aggregation can be accurately measured with high sensitivity.

[Brief description of drawings]

FIG. 1 is a graph showing a calibration curve of an absorbance ratio and an absorbance difference and variations in measured values at respective concentrations.

[Explanation of symbols]

 Calibration curve for X absorbance difference Calibration curve for Y absorbance ratio

Claims (1)

[Claims] 1. An antibody-antigen complex produced by dispersing fine particles of an inert carrier carrying an antibody (or an antigen) in a solvent and reacting this with the antigen (or an antibody) and different wavelengths λ. The light absorbency Aλ ₁ and Aλ ₂ at the respective wavelengths are measured by irradiating with light of ₁ and λ ₂ and the ratio of the light absorbency Aλ ₁ / Aλ
₂ and absorbance difference (Aλ ₁ -Aλ ₁ blank)-(Aλ
_2- Aλ ₂ blank) is determined, and the concentration of the antibody (or antigen) is measured using the calibration curve of the absorbance ratio in the low concentration range and the calibration curve of the absorbance difference in the high concentration range. Method for measuring the concentration of an antigen or antibody.