JPS63149564A - Method for judging antigen-antibody reaction - Google Patents

Abstract

PURPOSE:To accurately judge whether the first antigen-antibody reaction is performed in either one of an antigen excessive region and an antibody excessive region, by performing the second reaction by further adding an antibody or antigen to the system after the first antigen-antibody reaction. CONSTITUTION:An antigen or antibody and an antibody or antigen generating the antigen-antibody reaction with said antigen or antibody are reacted in a liquid medium to form the first reaction system having an antigen-antibody bonded substance formed therein and said reaction system is irradiated with the first and second wavelength lights having predetermined wavelengths to measure the intensities of transmitted or scattering lights. Next, the second reaction system wherein a new antigen or anti body is added to the system after the first reaction to perform reaction is irradiated with the first and second wavelength lights having the same predetermined wavelengths to respectively measure the intensities of transmitted or scattering lights. Next, the ratio of the measured values due to the first and second wavelength lights of the first and second reaction systems is calculated. A judge index is calculated from the ratio of said measured values and compared with a judge index due to a preliminar ily known amount of the antigen and the antibody to make it possible to accurately judge whether the first reaction is performed in either one of an antigen excessive region and an antibody excessive region.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for determining whether an antigen-antibody reaction is occurring in an antigen-excessive region or an antibody-excessive region.

(Prior art) As a method for measuring II acid components in body fluids, a target substance to be measured (antigen or antibody) is reacted with a substance (antibody or antigen) capable of antigen-antibody reaction with the substance, and the resulting antigen-
Methods have been employed to measure the degree of agglutination caused by antibody conjugates. For that.

For example, an immunoturbidimetric method in which a specimen containing the substance to be measured is directly reacted with the substance capable of reacting with the antigen-antibody, or a method in which a reagent in which the substance capable of reacting with the antigen-antibody is supported on an insoluble carrier is applied to the specimen (e.g. .

latex agglutination reaction method). In both cases, the reaction is carried out in a liquid medium and the resulting agglutination reaction is measured with a measuring device. Measurement methods include a method in which light is made incident on the reaction system and the intensity of the transmitted light is measured; a method is made in which light is made incident on the reaction system and the intensity of the scattered light is measured.

For example, CRP (c-reactive protein) contained in serum can be obtained by mixing the serum with a solution containing anti-CRP antibodies.

The amount of antigen-antibody conjugate produced is determined by measuring absorbance. The amount of antigen-antibody complex produced is cRp
The absorbance increases as the a degree increases and the particle size of the bond increases. However, CRP
(antigen) present in large excess relative to the anti-CRP antibody, the antigen-antibody bridging effect by the antigen disappears,
The particle size of the antigen-antibody conjugate decreases, resulting in a decrease in absorbance. For example, CRP as shown in Figure 2? a
The relationship between power and absorbance (measured at 750 nm) is obtained.

According to FIG. 2, 1, for example, C corresponds to an absorbance of 0.3.
Two RP concentrations (5μg/- and 150μg/-)
Therefore, the cRp concentration for a certain absorbance cannot be uniquely determined. Therefore, the CRP value of a sample containing CRP with an abnormally high value may be determined to be low.

A method has been proposed for accurately measuring a substance to be measured by determining whether the antigen-antibody reaction is occurring in an antigen-excessive region or an antibody-excessive region. For example, JP-A-60-7
No. 9269 discloses a method of reacting with an antigen or antibody in a sample by changing the amount of the corresponding antibody or antigen (for example, setting the amount of antibody to 2) and measuring the resulting antigen-antibody bond. There is. By performing the reaction multiple times at different concentrations in this manner, it can be determined whether the first reaction occurred in the antigen-excessive region or the antibody-excessive region, and the concentration of the analyte can be uniquely determined. However, it is necessary to use a plurality of reaction vessels, and the measurement process is complicated.

In addition, 1. For example, by measuring absorbance, we can track the antigen-antibody reaction over time and determine whether the antigen-antibody bond is increasing over time based on the rate of change in absorbance over 1 hour.
Alternatively, a method has also been adopted in which it is determined whether the 9 reaction is occurring in an antigen-excessive region or an antibody-excessive region. For example, Tokko Sho 61-1
The immunoturbidimetric analysis method disclosed in Publication No. 0775 corresponds to this method. However, even with this method, measurement is difficult when the antigen or antibody is present in extremely large excess relative to the corresponding antibody or antigen.

(Problem to be solved by the invention) The present invention solves the above-mentioned conventional drawbacks.

The purpose is to provide a method that can accurately determine by simple operations whether the antigen-antibody reaction is occurring in an antigen-excessive region or an antibody-excessive region.

(Means for Solving the Problems) The method for determining an antigen-antibody reaction of the present invention includes 1(a) reacting an antigen or antibody with an antibody or antigen capable of antigen-antibody reaction with the antigen or antibody in a liquid medium; (b) irradiating the first reaction system with a first wavelength light and a second wavelength light each having a predetermined wavelength individually or simultaneously, and producing the transmitted light; Step 1 (C) of measuring the intensity or scattered light intensity, respectively. Second reaction step 1 (d) Adding the new antigen or antibody to the system after the first reaction and causing the reaction. Step 1 (e) of irradiating one-wavelength light and second-wavelength light individually or simultaneously and measuring the transmitted light intensity or scattered light intensity, respectively. Measured values using first-wavelength light in the first and second reaction systems. The ratios A1 (i.e., a+/ax) and A, (i.e., a, l/, 21) of a, and alo to the measured values a2 and a2' by the second wavelength light
(J)
A step of comparing B with a determination index B0 set in advance based on a known amount of the antigen and the antibody, and determining whether the first reaction is occurring in an antigen-excess region or an antibody-excess region. , thereby achieving the above objective.

Furthermore, the method for determining an antigen-antibody reaction of the present invention includes 1(a) reacting an antigen or an antibody with an antibody or antigen capable of performing an antigen-antibody reaction with the antigen or antibody in a liquid medium to form an antigen-antibody combination. First reaction step for generation 1(b) Adding a new antigen or antibody to the first reaction system at any time after the starting point (t0) at the time when the first reaction is substantially completed. Second reaction step 1 (C) After the start of the second reaction, from the base point (L0) to a predetermined time point (1,
) in.

Step 1 (d) of irradiating the second reaction system with first wavelength light and second wavelength light having a predetermined wavelength, individually or simultaneously, and measuring the transmitted light intensity or scattered light intensity, respectively. After the measurement by the process, the base point (t
Step 1 (e) performing the same operation as step (c) from step 0) to predetermined time (t2); step 1 (c1) and step (d
) The ratio A, (i.e. b1/b2) and A2゛ (i.e. b1'/bZ ') Step 1 of calculating each (') corresponding A
2 and A tl, and (g) comparing the Bt with the determination index 80'' set by performing first and second reactions with known amounts of the antigen and the antibody in advance. However, the method includes a step of determining whether the first reaction is occurring in an antigen-excessive region or an antibody-excessive region, thereby achieving the above object.

The term "antigen or antibody" as used in the present invention refers to any substance capable of causing an antigen-antibody reaction, including, for example, IgG, IgA, and IgM detected in clinical tests.

Fibrinogen, FDP-D, FDP-E,
Rheumatoid factor (RF), C-reactive protein (cRP)
, anti-streptolysin-〇 (ASO), α-fetoprotein (AFP).

Includes HCG, CEA, etc.

For example, the antibody (antigen) corresponding to the above antigen (antibody) is
It can be obtained by known methods such as general immunization and purification.

For example, an anti-human AFP goat antibody can be obtained by immunizing a goat with human FP. These substances may be supported on insoluble carrier particles that are substantially insoluble in the liquid medium used. As the insoluble carrier particles, both inorganic fine particles and organic polymeric substance fine particles can be used. Examples of inorganic substance particles include inorganic oxide particles such as silica powder.

Metal oxide fine particles such as alumina powder, inorganic/metal oxide fine particles such as kaolin and bentonite, various mineral fine particles, etc. are used. As the organic polymer substance fine particles, biological cells (for example, chicken red blood cells) and synthetic resin fine particles (for example, styrene resin) are used. In particular, latex in which polystyrene or styrene copolymer particles are uniformly suspended is preferably used. Latex reagents in which latex particles support the above-mentioned known antibodies or antigens are commercially available and can also be used.

In the method of the present invention, the wavelength of the light used to measure the antigen-antibody reaction is 300 rv or more,9 and usually 300 to 10 rv.
00 nm. The difference in wavelength between the first wavelength light and the second wavelength light is set to 50 nm or more. If the difference in wavelength between the second wavelength light and the first wavelength light is small, the measured value with the first wavelength light and the second wavelength light may differ.
Since the difference from the measured value with the wavelength light is small, it becomes difficult to accurately measure the substance to be measured.

Next, the method of the present invention will be explained using the reaction between CRP and anti-CRP antibody as an example.

First, a predetermined amount of anti-CRP antibody is added to a solution containing CRP and reacted in a liquid medium (first reaction).

This first reaction solution is irradiated with light (first wavelength light) of a predetermined wavelength (for example, 550 nm), and its absorbance a is measured.

Next, a wavelength different from the first wavelength light (for example, 750 nm) is applied.
m) is irradiated with light (second wavelength light), its absorbance a2 is measured, and the ratio A1 between +al and a2 is calculated.

Next, for example, a known amount of anti-CRP antibody is added to perform a second reaction. The reaction solution after the second reaction is irradiated again with the first wavelength light and the second wavelength light, respectively. The absorbance ratios A and l are calculated from the absorbances a, ゛ and a2°. A above
A determination index B is calculated from t, A, and I.

For example, FIG. 1 is a graph showing the results of first and second antigen-antibody reactions using CRP at known concentrations. In the first reaction shown by the solid line.

The region where the CRP 'tQ degree is O to 40 μg/ml is the antibody excess region. In this region, as the CRP concentration increases, the particle size (average particle size) of the antigen-antibody conjugate increases, so the absorbance increases (Fig. 2), and the absorbance ratio (550 nm/750 nm) conversely decreases. do(
Figure 1). This means that the particle size of suspended solids in a suspension (
The inventors' knowledge that there is a correlation between the average particle diameter) and the ratio of the measured values when the transmitted light or scattered light (e.g. absorbance) of the suspension is measured at two wavelengths (reference example described below) ). Since the concentration range of 40 μg/ml or more is an antigen-excessive region, cRpyH
Conversely, as the temperature increases, the absorbance ratio increases.

In this first reaction, 9 For example, the absorbance ratio ^1 is 2.0
When, C RP at the time of the first reaction? degree is about 5 μg/d (antibody excess area) or about 200 μg/d
111 (antigen-rich region). After the second reaction (indicated by the dashed line), if the CRP concentration is 5 μg/-, the absorbance ratio A, 1, will be higher than A, and if the CRPtm degree is 200 μg/m
On the other hand, if it is l, it shows a low value.

Therefore, for example, let a+//l, t be the determination index B1.

Judgment index B that was tested in advance and set to an appropriate value
By comparing it with 0, it is determined whether the first reaction is occurring in the antigen-excessive region or the antibody-excessive region.

Rather than calculating the measured value (for example, absorbance ratio) of transmitted light intensity or scattered light intensity as in the above method.

It is better to adopt a method that compares the measured value (absorbance) itself measured at a certain wavelength (for example, 750 nm).

It is considered to be simpler. However, especially when using a reagent in which an antibody or antigen is supported on an insoluble carrier (such as a latex reagent), the degree of absorbance or scattered light intensity due to the carrier itself is large, so the absorbance or scattered light intensity due to the antigen-antibody reaction is It is difficult to accurately measure changes in

In this way, in addition to the method of determining based on the amount of antigen-antibody bond produced, a method of determining based on the rate of production of the bond may also be adopted.

In this method, first, a predetermined amount of anti-CRP antibody is added to a solution containing CRP, and a reaction (first
react). The time point at which this first reaction is substantially completed is defined as the base point (t6; for example, 0 minutes). At any time thereafter, a new anti-CRP antibody (or CRP) is added to the first reaction system to perform a second reaction. After the start of the second reaction, at a predetermined time point (tl) from the base point (t0)
; For example, after 3 minutes), this second reaction system is irradiated with light (first wavelength light) having a predetermined wavelength (for example, 550 nm), and its absorbance is measured. Next, light (second wavelength light) having a wavelength different from the first wavelength light (for example, 750 nm) is irradiated, the absorbance b2 thereof is measured, and the ratio At between +bl and b2 is calculated. Furthermore, after these measurements, at a predetermined time (tt; for example, 3 minutes and 30 seconds after) from the base point (t,), measurements using the first wavelength light and the second wavelength light are performed in the same manner as above, and each Measured values b1″ and b
2", calculate the ratio A, l. Then.

The determination index B2 is calculated from the above A2 and A2". For example, the rate of change over time of the ratio of the measured values [(A2"-
A2) / (tz - tt)) as the determination index B''
Judgment index B, which was set and tested in advance,
By comparing with 1, it is determined whether the first reaction is occurring in the antigen-excessive region or the antibody-excessive region. For example, if the first reaction is performed in an antigen-excessive region, adding an antibody in the second reaction increases the particle size of the antigen-antibody conjugate. By that (82”-
Since the value of A in 82) is negative, the rate of change B'' is a negative value.

If the first reaction is carried out in the antibody-excessive region, the particle size of the antigen-antibody conjugate may not change by adding antibody in the second reaction, or the particle size may become smaller due to loss of the cross-linking effect of the antigen. There is. If the antigen is not contained in the sample to be measured in the first reaction, A2''-Az=0, so the rate of change B' is O, and if it is in the antibody excess region (A21-A2
) is a positive value, so the rate of change B' is a positive value.

In the method of measuring the rate of change described above, measurements are performed three or more times after the second reaction, and each ratio value (At, Az''
+A2-...) to determine the rate of change may also be advantageously adopted. In each of the above methods, the first wavelength light and the second wavelength light
Wavelength light refers to continuous wavelength light that includes these components, and by separating and extracting the components with a diffraction grating, it is also possible to simultaneously measure the first wavelength light and the second wavelength light by irradiating it. .

Furthermore, in both of the methods for measuring the amount of antigen-antibody conjugate produced and the method for measuring the production rate, it is also possible to perform measurement at a wavelength different from the first and second wavelength light.

For example, measurement is performed using third wavelength light (900 nm).

An index is calculated by adding the obtained absorbance ratio A3+A:l. When multiple wavelength light of 3 or more is used in this way.

More accurate measurements can be made.

As a device for measuring the antigen-antibody reaction product in the reaction system, a conventional spectrophotometric needle or a device for measuring the scattering intensity of light is used. Instruments with built-in spectrophotometers, such as automatic biochemical analyzers, specialized devices used for immunoturbidimetry, and specialized devices for measuring latex agglutination reactions, are also advantageously used. In addition, if a method is adopted in which an antigen-antibody reaction is performed in the wells of a microtiter plate and the absorbance is measured using a plate reader, a large amount of determination can be made in a short time using a small device.

(Function) According to the present invention, in order to perform the second reaction by further adding an antibody or antigen to the system after the first antigen-antibody reaction, the above-mentioned reaction can be easily carried out without preparing a plurality of second reaction vessels. It is determined whether one reaction is occurring in an antigen-excess region or an antibody-excess region. Even if the antigen or antibody is in extreme excess.

Judgments are made accurately. For measurement, the wavelength is 5onII+
Since two or more different wavelengths are used, compared to the conventional measurement using one wavelength, it is especially difficult to use a reagent (in which an antibody or antigen is supported on an insoluble carrier).
It is possible to accurately measure the state of production of antigen-antibody bonds due to antigen-antibody reactions in latex reagents, etc.).

(Example) Examples of the present invention will be described below.

A suspension (0.25%) of polystyrene latex with a particle size of 0.1 μm in distilled water was prepared and placed in a cell with an optical path length of 2. This was irradiated with 550 nm light (first wavelength light), and its absorbance a was measured. Next, 750 nm light (
2nd wavelength light) and measure its absorbance a2. + a
The ratio between l and a2 (8+=a+/ax) was calculated.

Next, the particle size of Nilatex is 0.2 μm and the iJm degree is 0.
1%), the absorbance due to the first wavelength light and the absorbance b2 due to the second wavelength light were measured in the same manner, and the ratio between b and b2 (A2 = tll /b2) was calculated. The particle size of latex is 0.45 μm (concentration 0.036%) + 1
．． 0 μm (concentration 0.017%), 1.97. czm
(concentration 0.008%) and A3 (cI/
C2), 84. (dt/at) and As (e+
/e2) was calculated. The ratio of latex particle size to absorbance (
8,~^,) is shown in Figure 3.

From Figure 3, the ratio of the particle size of latex to the absorbance (A1~^
) has a certain correlation with the above two wavelengths (550 nm and 750 nm), and the larger the latex particle size
It can be seen that the ratio of absorbance measured at ) becomes smaller. This fact means that the above ratio decreases as the antigen-antibody bond is produced by the antigen-antibody reaction and the particle size (average particle size) included in the reaction system becomes larger.

Anti-CRP on polystyrene latex with a microfoam particle size of 0.2μm
Anti-CRP with 0.5% latex solid content by adsorbing antibodies
An antibody sensitization latex solution (water suspension) was prepared. Separately,
A CRP aqueous solution containing CRP at a predetermined concentration was prepared. 40 μl of the above latex solution and 20 μl of CRP aqueous solution
Place the μl into a glass transparent cell with an optical path length of 51 cm and hold at 37°C.
The mixture was allowed to react for 15 minutes. This includes 1% bovine serum albumin.
Phosphate saline buffer (pH 7,0) containing 13001! and absorbance a1 at 550 nm and 750
Absorbance a2 in nm was measured.

Next, 40 μl of a new anti-CRP antibody-sensitized latex solution was added to the above reaction solution, and after reacting at 37° C. for 15 seconds, absorbances aI' and 82° at 550 nm and 750 nm were measured in the same manner as above.

The ratio of a and a2 above (a l/ a z ” A
r) + and the ratio of + and a2゛ (a, l/
a, 1 = a+') were calculated. Above CR
P? The M degree was set to 6 types from O to 200 μg/- as shown in FIG. 1, and reactions were performed for each. CRP>
The relationship between CRP concentration and A1 (first reaction) is shown in FIG. 1 as a solid line, and the relationship between CRP concentration and A and I (second reaction) is shown in FIG. 1 as a broken line.

In the solid line in Figure 1, CRP >74 degrees is 0~40 μg/
ml range, the absorbance ratio A1 decreases as the cRp concentration increases. This is (: RP
This shows that the average particle size in the reaction system increases as the t7M degree increases, that is, the region is in the antibody excess region. On the contrary, CRP:A degree of 40 μg/- or more indicates an antigen-excessive region.

For example, when the absorbance ratio is 2.0, CRI'
1ffi degree 5μg/- antibody excess area or 200μ
g/ml of antigen excess. By the second reaction, C
When the RP concentration was 5 μg/-, the absorbance ratio increased.

Conversely, it decreases at 200 μg/ytrl. Therefore.

For example, decide on the judgment index A t / A+ ',
If you set this to an appropriate value in advance, CR
When performing an antigen-antibody reaction on a sample with unknown P concentration.

It is easily determined whether this occurs in the antigen-excessive region or the antibody-excessive region.

(Effects of the Invention) According to the method of the present invention, whether the antigen-antibody reaction is occurring in the antigen-excess region or the antibody-excess region can be easily determined by a simple operation. The method of the present invention can be advantageously used, for example, to discover samples with abnormal values when measuring a large amount of samples using an automatic biochemical analyzer.

Figure 1 shows the relationship between the CRP concentration in each reaction solution and the absorbance ratio of two different wavelengths when the first and second reactions were performed according to the method of the present invention. The graph shown in Figure 2 is
C in the reaction solution when measuring CRP using the conventional method
A graph showing the relationship between RP concentration and absorbance at a predetermined wavelength; and FIG. 3 is a graph showing the relationship between the latex particle size in the latex suspension and the absorbance ratio at two different wavelengths.

that's all

Claims (1)

[Claims] 1. (a) A first reaction in which an antigen or antibody and an antibody or antigen capable of performing an antigen-antibody reaction with the antigen or antibody are reacted in a liquid medium to produce an antigen-antibody bond. (b) irradiating the first reaction system with a first wavelength light and a second wavelength light having a predetermined wavelength, respectively or simultaneously, and measuring the transmitted light intensity or scattered light intensity, respectively, (c ) a second reaction step of adding and reacting the new antigen or the antibody to the system after the first reaction; (d) applying the first wavelength light and the second wavelength light to the second reaction system individually or (e) Measured values a_1 and a_1' of the first wavelength light in the first and second reaction systems and measured values of the second wavelength light in the first and second reaction systems; The ratio A_1 with a_2 and a_2' (i.e. a_
1/a_2) and A_1' (i.e. a_1'/a_
2'), (i) calculating the determination index B_1 from the A_1 and the A_1', and (j) calculating the determination index B_0 set in advance based on known amounts of the antigen and the antibody A method for determining an antigen-antibody reaction, comprising the step of comparing the first reaction with B_1 and determining whether the first reaction is occurring in an antigen-excessive region or an antibody-excessive region. 2. The judgment indicators B_1 and B_0 are A_1 and A_
1'. 3. The wavelength of the first wavelength light and the second wavelength light is 300n.
The determination method according to claim 1, wherein the difference in wavelength between the first wavelength light and the second wavelength light is 50 nm or more. 4. The determination method according to claim 1, wherein the measured value is absorbance or transmittance calculated from transmitted light intensity. 5. The determination method according to claim 1, wherein the antibody or antigen is supported on a carrier made of organic polymeric substance fine particles or inorganic substance fine particles that are substantially insoluble in the liquid medium. 6. The determination method according to claim 5, wherein the organic polymer substance fine particles are synthetic resin fine particles or biological cells. 7. The determination method according to claim 6, wherein the synthetic resin is a styrene resin. 8. The determination method according to claim 5, wherein the inorganic substance is a metal oxide and/or an inorganic oxide. 9. Claim 1, wherein the transmitted light intensity is measured by an absorbance meter; or an automatic biochemical analyzer incorporating an absorbance meter, a measuring device exclusively for immunoturbidimetry, or a measuring device exclusively for latex agglutination reaction. Judgment method described in. 10. The determination method according to claim 1, wherein the first and second antigen-antibody reactions are performed on a microtiter plate, and the transmitted light intensity is measured with a plate reader. 11. (a) A first reaction step in which an antigen or antibody and an antibody or antigen capable of performing an antigen-antibody reaction with the antigen or antibody are reacted in a liquid medium to produce an antigen-antibody conjugate; (b) The starting point is the time when the first reaction has substantially completed (t_
0) and adding a new antigen or antibody to the first reaction system at any time thereafter; (c) a predetermined step from the base point (t_0) after the start of the second reaction; At the time point (t_1), a step of individually irradiating the second reaction system with a first wavelength light and a second wavelength light having a predetermined wavelength, and measuring the transmitted light or scattered light, respectively; (c) After the measurement in the process, the base point (t_
0) to a predetermined time point (t_2), performing the same operation as the step (c); (e) the measured values b_1 and 1 of the first wavelength light obtained in the steps (c) and (d); The ratios A_2 (i.e. b_1/b_2) and A_2' (i.e. b_1') of b_1' and the measured values b_2 and b_2' by the second wavelength light
/b_2'), (f) said A_2
and (g) calculating a determination index B_2 from A_2', and (g) using a previously known amount of the antigen and the antibody.
and a step of comparing the determination index B_0' set by performing a second reaction with the B_2 and determining whether the first reaction is performed in an antigen-excessive region or an antibody-excessive region. How to judge the reaction. 12. Claim 11, wherein the determination indices B_2 and B_0' are represented by the rate of change over time of the ratio of the measured values after the second reaction [(A_2'-A_2)/(t_2-t_1)] Judgment method described in. 13. The wavelength of the first wavelength light and the second wavelength light is 300.
12. The determination method according to claim 11, wherein the difference in wavelength between the first wavelength light and the second wavelength light is 50 nm or more. 14. The determination method according to claim 11, wherein the measured value is absorbance or transmittance calculated from transmitted light intensity. 15. The determination method according to claim 11, wherein the antibody or antigen is supported on a carrier made of organic polymer particles or inorganic particles that are substantially insoluble in the liquid medium. 16. The determination method according to claim 15, wherein the organic polymer substance fine particles are synthetic resin fine particles or biological cells. 17. The determination method according to claim 16, wherein the synthetic resin is a styrene resin. 18. The determination method according to claim 15, wherein the inorganic substance is a metal oxide and/or an inorganic oxide. 19. The transmitted light intensity is measured using an absorbance meter; or an automatic biochemical analyzer incorporating an absorbance meter. The determination method according to claim 11, which is carried out using a measuring device exclusively for immunoturbidimetry or a measuring device exclusively for latex agglutination reaction. 20. The determination method according to claim 11, wherein the first and second antigen-antibody reactions are performed on a microtiter plate, and the transmitted light intensity is measured with a plate reader.