JPH07113635B2 - Method for determining prozone in immune reaction - Google Patents

Description

TECHNICAL FIELD The present invention relates to a prozone determination method in immune reaction. More specifically, it relates to prozone detection of a test liquid containing an antigen-antibody complex in an immunoturbidimetric method.

(B) Conventional technology As a method for quantifying the antigen in serum, there is an immunoturbidimetric method.
This is a method in which the turbidity of a test liquid due to an antigen-antibody complex formed as a result of an antibody contained in a reagent specifically binding to an antigen as a measurement target substance is measured as an absorbance.

(C) Problems to be Solved by the Invention However, in the above method, the turbidity should generally increase with the increase of the antigen concentration, but actually, the turbidity decreases on the high concentration side. Can be seen (see FIG. 5). That is, the concentration of antigen contained in serum is high, whereas the concentration of antigen in the test liquid is apparently low, which is called prozone. This prozone means a state in which there are more antigens than antibodies, that is, an antigen excess state.In this state, even though the antigen concentration is high, there is insufficient antibody and an antigen-antibody complex of an appropriate size cannot be produced, resulting in turbidity. The degree will be reduced. When the prozone occurs, a problem arises in which two antigen concentrations are measured for one measured absorbance (apparent absorbance). Therefore, in the immunoturbidimetric method, it is necessary to check whether the test liquid is in the prozone range. For this reason, in the past, the antigen concentration was diluted in several steps, and it was confirmed whether there was a place where higher absorbance was obtained at a low concentration, or when the antigen was again dispensed into the reaction solution to react and the absorbance decreased. Methods such as determining the region to be in the prozone region and determining that the region to be in the prozone region if the rate of change in absorbance immediately after antibody dispensing is a certain value or more have been used. However, since the former one prepares a dilution series and reacts each, it takes time and cost,
Further, the latter two have problems that they are difficult to check without an automatic analyzer and that the measurement of the turbidity immediately after the antibody is dispensed is difficult due to the influence of the fluctuation of the liquid.

The present invention has been made in view of such circumstances, and particularly, it aims to provide a method for easily determining whether or not a test liquid is in the prozone region.

(D) Means for Solving the Problems Thus, according to the present invention, the test liquid containing the antigen-antibody complex is irradiated with light and the apparent absorbance thereof is measured at two different wavelengths in the visible region. A method for determining a prozone in an immune reaction, characterized in that the prozone of a test liquid is determined based on the obtained ratio of two kinds of absorbance.

The most characteristic point of the method of the present invention is that the ratio of the apparent absorbances at two different wavelengths is used as a parameter of the particle size of the antigen-antibody complex particles suspended in the test solution. Whether or not it is a pro zone is determined based on the magnitude relationship between the absorbance ratio and a preset determination level value.

In the prozone area, there is an excess of antigen, and when a reagent with a low antibody titer is used, a large number of antigen-antibody complexes with a small particle size are produced, resulting in an excess of antigen (Fig. 6 (a)). (See FIG. 6B), and when a reagent with a high antibody titer is used, a small number of large antigen-antibody complexes are produced (see FIG. 6 (b)). Therefore, the particle size of the antigen-antibody complex particles generated in the prozone region is extremely small or large and there is a large variation in the particle size, in other words, the generated antigen-antibody complex particles Particle size is almost uniform (usually 0.5-1.0μ
m)) is in the optimum state (see FIG. 6 (c))
It means that the immune reaction is progressing in the area other than the prozone area. From this, it was found that the change in particle size of the antigen-antibody complex particles generated in the test solution corresponds to the change in the apparent absorbance ratio, and that the absorbance ratio is used as a determination factor for the prozone. Is a feature of the method of the present invention.

It will be explained below that the ratio of the absorbances based on two different wavelengths is a function of the particle size of the particles suspended in the test liquid. That is, the intensity of transmitted light (absorbance is the logarithm of the reciprocal of the transmitted light intensity) obtained when a liquid medium containing suspended particles is irradiated with light of a certain wavelength, The absorbance of the suspension, which is decreased by the scattering effect and calculated by setting the absorbance of the liquid medium itself to 0, apparently increases. This apparent absorbance differs depending on the scattering action of the suspended particles, and in particular the angular distribution of the scattered light intensity tends to differ depending on the wavelength of the irradiation light, so the measured absorbance depends not only on the particle size of the suspended particles but also on the irradiation wavelength. Change. Under such a relationship, when a suspended particle having a particle size of 2 μm or less is targeted, a ratio of apparent absorbances of two different wavelengths becomes a function of the particle size.

The two types of wavelength light used in the method of the present invention may be those that undergo different scattering effects on the generated antigen-antibody complex particles, and are generally used in the visible light region (about 340 to 7).
It is convenient to select two different wavelengths (00nm).
As the two kinds of wavelengths, it is possible to use those having different wavelengths close to each other by adapting the accuracy on the side of the absorbance detector and the measurement particle size range, but usually 340 to 450 nm and 600 nm, respectively.
It is suitable to select two wavelengths of ~ 750 nm.

As the determination level used in the determination criteria of whether it is a prozone in the method of the present invention, a calibration curve is prepared by measuring the absorbance at a wavelength on the high sensitivity side of the test liquid in advance according to each measurement item, At the concentration at the boundary of the prozone region of this calibration curve, the ratio obtained by measuring the absorbance for the two different wavelengths is used.

In the method of the present invention, the determination as to whether the zone is prozone or not is made by comparing the magnitude of the determination level with the apparent absorbance ratio. When a reagent with a high antibody titer is used, the area where the value of the above ratio is smaller than the determination level is judged to be prozone, and when a reagent with a low antibody titer is used, the value of the above ratio is Areas larger than the judgment level are judged to be pro zones.

The method of the present invention can be carried out using an ordinary absorptiometer, photoelectric colorimeter, etc., and it is not necessary to use a special device such as a light scattering photometer. Therefore, as the measurement cell, a normal square cell can be used, and a normal cell thickness can be used, but it is preferable to use a thin cell thickness to prevent multiple scattering, usually 5 to 10 mm. It is suitable to use a grade. However, since the transmitted light is targeted, the influence of multiple scattering is small, and the conventional strictness of the cell thickness is not required.

However, it is suitable to carry out the method of the present invention by using an apparatus that has a two-wavelength absorbance measuring means and can output the absorbance ratios thereof. It is to be noted that this measuring means is preferably configured so as to measure the apparent absorbance for at least two wavelengths by using a continuous wavelength light source to disperse the irradiation light or the transmitted light.

(E) Action According to the present invention, a large change in particle size of the antigen-antibody complex particles produced near the boundary of the prozone becomes a judgment factor of the prozone, and therefore, it is based on two wavelengths that are parameters of the particle size. By measuring the apparent absorbance ratio and comparing the magnitude with the preset apparent absorbance ratio in the vicinity of the prozone boundary, whether or not the test liquid is the prozone is determined.

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

(F) Example FIG. 1 is a structural explanatory view of an example of a measuring apparatus used for carrying out the method of the present invention. In the figure, this measuring device comprises a prismatic measuring cell (3; 10 mm thick), a light source (1), a collimator lens (2), a condenser lens (4), an entrance slit (5),
Diffraction grating (6), two photoelectric conversion elements (7) (7 '),
It is composed of an optical measurement system including two preamplifiers (8) and (8 ') and two absorbance calculating circuits (9) and (9'), and a comparator including a comparative measurement circuit (10). The elements (7) and (7 ') are arranged so as to correspond to predetermined wavelengths λ ₁ and λ ₂ .

In such a device, the white light from the light source (1) is converted into a parallel light flux by the lens (2) and is applied to the prismatic measurement cell (3), and the light flux transmitted through the cell (3) is condensed by a condensing lens (4).
Is focused on the entrance slit (5) by the slit (5)
The light passing through is split by the diffraction grating (6). The light of wavelengths λ ₁ and λ ₂ of the split light is applied to the corresponding elements (7) and (7 ′), and the outputs of the preamplifiers (8) and (8 ′) are sent to the calculation circuits (9) and (9 ′). It is set to enter.

First, put pure water in the cell (3), and then the preamplifier (8)
The outputs I ₀ (λ ₁ ) and I ₀ (λ ₂ ) of (8 ′) are stored in the calculation circuits (9) and (9 ′). Next, the target sample is put into the cell (3), and its preamplifier output I (λ ₁ ), I
Using (λ ₂ ), the respective absorbances in the calculation circuits (9) and (9 ′): A (λ ₁ ) = − log [I (λ ₁ ) / I ₀ (λ ₁ )] A (λ ₂ ) = −log [I (λ ₂ ) / I ₀ (λ ₂ )] is calculated.

The outputs A (λ ₁ ) and A (λ ₂ ) are the ratio measuring circuit (1
0) and the ratio A (λ ₁ ) / A (λ ₂ ) is output.

IgA and IgG were selected as items, and standard serum was used as a sample. Further, as a reagent using immunoturbidimetric IgA, IgG and IgG for nephelometry, 9300 mg / dl for IgA for immunoturbidimetric, 43000 mg / dl for IgG, 900 mg / dl for IgG for nephelometry. A dilution series was prepared using dl samples, and A for λ ₁ = 340 nm and λ ₂ = 700 nm
(Λ ₁ ), A (λ ₂ ), and A (λ ₁ ) / A (λ ₂ ) were measured. The measurement results are shown in Figs.

In FIG. 2, the prozone phenomenon has occurred at the absorbance at 340 nm (A ₃₄₀ ), but the prozone phenomenon has not yet occurred at the absorbance at 700 nm (A ₇₀₀ ). Here, the value of the ratio of absorbance (A ₃₄₀ / A ₇₀₀ ) was calculated and plotted (×
If the mark) is below the judgment level [dotted line in the figure (calculated based on the calibration curve of A ₃₄₀ )], it is judged that the prozone phenomenon is occurring and this is indicated by A _340. It agrees well with the zone area.

In Fig. 3, the prozone phenomenon occurs at both the absorbance at 340 nm and 700 nm, and in this case as well, the ratio of the absorbance (A ₃₄₀
/ A ₇₀₀ ) value is calculated and plotted (X mark)
If it is below the judgment level [dotted line in the figure (calculated based on the calibration curve of A ₃₄₀ )], it is judged that the prozone phenomenon is occurring, which is often the prozone area pointed out by A _340. Match.

In Fig. 4, the prozone phenomenon occurs at both the absorbance at 340 nm and 770 nm (the curve connecting the circles in the upper part of the figure shows the absorbance at 340 nm). In this case, the ratio of the absorbances (A ₃₄₀ / A ₇₀₀ ) Is calculated and plotted (×
If the mark) is above the judgment level [dotted line in the figure (calculated based on the calibration curve of A ₃₄₀ )], it is judged that the prozone phenomenon is occurring and this is indicated by A _340. It agrees well with the zone area.

The plot value may exceed the judgment level even in the small dilution series, but it is natural that the antigen excess state does not occur in the extremely small dilution series, so data in this range should be considered. Do not put in the box (the absorbance ratio data is displayed together with the dilution ratio so that it can be easily identified).

(G) Effect of the Invention According to the present invention, the ratio of the apparent absorbance measured at two different wavelengths at a certain concentration is compared with a predetermined determination level to determine whether the test liquid is prozone phenomenon easily. Whether or not it is occurring can be determined, and complicated labor such as a check with a diluted sample or a check with antigen re-addition is unnecessary. Further, since the absorbance at the end point of the reaction is measured, stable measurement can be always performed.

[Brief description of drawings]

FIG. 1 is an explanatory view of the configuration of an example of a measuring apparatus used for carrying out the method of the present invention, and FIGS. 2 and 3 show the case where each standard serum is measured with each immunoturbidimetric reagent by the apparatus of FIG. FIG. 4 is a graph showing the absorbance ratio and the judgment level obtained based on the results and the measured values, FIG. 4 is a view corresponding to FIG. 2 when measuring standard serum with a reagent for nephelometry, and FIG. 5 explains the prozone phenomenon. FIG. 6 is a schematic diagram for explaining the state of particles of the antigen-antibody complex produced in the test liquid. (1) ... light source, (2) ... collimator lens, (3) ... rectangular measuring cell, (4) ... condensing lens, (5) ... incident slit, (6) ... diffraction grating, (7) (7 ') ... photoelectric conversion element, (8) (8') ... preamplifier, (9) (9 ') ... absorbance calculation circuit, (10) ... comparative measurement circuit.

Claims (1)

[Claims] 1. A test solution containing an antigen-antibody complex is irradiated with light to measure its apparent absorbance with respect to two kinds of wavelengths in different visible regions, and based on the ratio of the two kinds of absorbance thus obtained. A method for determining a prozone in an immune reaction, which comprises determining the prozone of a test liquid.