JPS6331733B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for optical measurement of liquid samples. In particular, the present invention relates to a method of accurately measuring the optical properties of a very small amount of sample. It is well known to conduct a reaction in an optical measurement cell and optically track changes in the optical properties of the sample as the reaction progresses, such as changes in absorbance, to determine the progress of the reaction.This method can be used in various ways. applied to analytical methods. For example, one example is the measurement of antigens or antibodies by antigen-antibody reactions using sensitized latex, which has recently been in the spotlight. In a typical example of an antigen-antibody reaction, a sensitized latex in which an antigen or antibody is supported on an insoluble carrier with an average particle size of 1.6 microns or less is placed in an optical measurement cell, and a sample containing the antibody and/or antigen is placed in an optical measurement cell. is added to generate an antigen-antibody reaction under stirring. The cell has a wavelength
0.6-2.4μ, more than 1.1 times the average particle size of latex,
In particular, we irradiate the latex with 1.5 times more light and measure the optical properties of the latex, usually changes in the amount of transmitted light. Measurements are usually made as measurements of either the rate of change of absorbance or transmittance, the absorbance after a certain period of time, or the time to reach a certain absorbance (see USP No. 4,118,192). In measuring such an antigen-antibody reaction, it is necessary to stir the liquid within the cell with appropriate strength in order to make the inside of the cell uniform and to promote the reaction. A commonly used stirrer occupies only a portion of the volume of the cell and is located outside the optical path of the irradiated light. When the stirrer is installed in the optical path, the stirrer is taken out of the optical path during measurement to prevent light from being absorbed or scattered by the stirrer and resulting in inaccurate measurements. However, such a method is generally inappropriate as a measurement method using a trace amount of sample. As a method for accurately measuring the optical properties of a minute amount of sample, the present inventor first put the sample into a cell equipped with a large prismatic, translucent stirrer inside, and the optical path of the measuring light beam. proposed a method of measuring while rotating a stirrer in the
74253). Although this method is excellent as a method for measuring trace amounts of samples, it has been found that the rotation of the stirrer causes air bubbles to be entrained, which sometimes interferes with the measurement. This phenomenon becomes noticeable when the amount of solution in the cell is significantly reduced or when the stirrer is rotated at high speed. The present invention provides a measurement method that is less susceptible to interference due to bubble entrainment. According to the present invention, an antigen-antibody reaction is performed using a sensitized latex in an optical measurement cell, and the progress of the reaction is measured by tracking the optical properties of the reaction mixture as the reaction progresses. In this method, a liquid sample is placed in an optical measurement cell equipped with a stirring bar whose lower part is made of a translucent material in the shape of a thick polygonal column and whose upper part is formed into a cylindrical shape. By positioning the columnar part in the optical path of the measuring light beam and the columnar part on the liquid surface of the sample, and measuring the optical properties of the sample while rotating the stirrer, a small amount of the sample can be used. It is possible to measure its optical properties with high precision. To explain the present invention in detail, the measuring device used in the present invention includes an optical measurement cell containing a sample solution, a stirrer inserted into the cell, a light source unit that irradiates the cell with a measurement light beam, and a cell. and a light receiving section that measures the measurement light beam that has passed through the sensor. As the light source section and the light receiving section, those used in similar general optical measuring devices can be used as they are. The cell also usually has a rectangular or square cross-sectional shape, which is used in general optical measurement devices. On the other hand, a stirring bar is different from the one normally used in that at least a part of the part immersed in the sample solution is made of transparent material such as glass or synthetic resin such as polyethylene, polystyrene, or polymethyl methacrylate. It is made of a polygonal material and has a polygonal columnar cross section. A stirrer with a polygonal cross section can achieve a higher stirring effect than a stirrer with a circular cross section. The length of the portion having a polygonal cross section is preferably such that it usually occupies 30% or more of the depth of the solution when immersed in the solution. The cross-sectional shape of this polygonal portion is preferably a rectangle or a regular hexagon in which opposing sides are parallel to each other. Such a shape is effective in reducing light scattering by the stirrer. In terms of stirring effect and light scattering, a stirring bar with a square cross section is considered to be the most preferable. The part of the stirrer located at the liquid level has a circular cross section. When a stirrer whose part is located near the liquid surface is in the shape of a polygonal column, is rotated at high speed, air bubbles may be trapped at the liquid surface, causing errors in measurements. However, a stirrer having a cylindrical shape near the liquid surface significantly reduces entrainment of air bubbles. Usually at least 1mm above the liquid level
If the lower part, preferably 1 to 5 mm from the liquid level, is made cylindrical, problems caused by air bubbles can be avoided. It is preferable that the stirrer be as large as possible within the limit of rotation within the cell. Usually, the cross-sectional area of the stirrer is set to be 25% or more, preferably 30% or more, of the cross-sectional area of the cell. If the stirrer has a large cross-sectional area, a small amount of sample solution can fill the space in the cell. Additionally, the distance that the measurement light beam travels through the solution is reduced, which reduces the scattering of light by the solution.
Absorption is reduced. Therefore, even sample solutions with large light scattering and absorption can be measured with high accuracy.
Normally, if the minimum gap between the stirrer and the inner wall of the cell is 0.5 to 2 mm in the rotating state, it is possible to achieve both the free rotation of the stirrer and the above-mentioned advantages of using a large stirrer. To measure the optical properties of a solution in a cell using the method of the present invention, a stirrer as described above is inserted into the cell,
Position the polygonal column-shaped part within the optical path of the measurement light beam,
Irradiate the cell with light while rotating the stirrer. The distance that the light beam travels through the solution, ie the optical path length, and the proportion of light scattered by the stirrer vary periodically depending on the rotation of the stirrer. The optical properties measured at each moment therefore change periodically, even if no changes occur in the sample. However, when the rotational speed is large, such as several hundred to several thousand revolutions per minute, a normal measuring device automatically measures the average value of periodic changes, so there is no problem at all with measurement. If the rotational speed is small and periodic changes due to rotation affect the measurement, the optical characteristics may be measured by integrating them over a certain period of time. Note that, as a matter of course, the rotation speed is set to be sufficiently higher than the rate of change in the optical properties of the sample, so that the change in the optical properties of the sample does not affect the removal of the influence of rotation. According to the method of the present invention, the presence of the stirrer does not interfere with measurement, so measurements can be performed immediately after stirring with the stirrer, and the influence of stirring on measurements can be minimized. can. Furthermore, since the effective optical path length is shortened, the absorbance and scattering degree of the sample per unit optical path length can be increased. Therefore, in the case of the antigen-antibody reaction of the present invention using a sensitized latex, the sensitivity of the measurement can be improved by increasing the concentration of the latex or using a latex with a large particle size. Furthermore, the most commonly used propeller-type (turbine-type) stirring blades apply shearing force to the inside of the liquid as they rotate, but the shearing force is not so strong in the polygonal columnar stirrer used in the method of the present invention. . Therefore, in the case of an antigen-antibody reaction using a sensitized latex, when the rotation speed is increased, the aggregates formed by the reaction may be destroyed with propeller-type stirring blades, but this can be avoided with the method of the present invention. It almost never happens. EXAMPLES Next, the present invention will be explained in more detail with reference to examples, but the present invention is not limited to the following examples unless it exceeds the gist thereof. In the following examples, a transparent plastic cell with a 7 mm square cross section was used as the optical measurement cell. As a stirrer,
A transparent plastic prism with a square cross-section of 4 mm square and a flat bottom (= Stirrer A) and Stirrer A
A stirrer (=stirrer B) was used in which the bottom surface was round, the height of the prismatic part was 6.5 mm, and the upper part was a cylinder with a diameter of 4 mm. When inserting the stirrer into the cell, the center of the stirrer was aligned with the center of the cell, and the bottom of the stirrer was placed at a height of 2 mm from the bottom of the cell. The light source is an infrared light emitting diode (manufactured by Monsanto)
ME7124, center wavelength 0.94μ, wavelength half width 0.05μ) was used. The luminous flux had a diameter of 3 m/m, and its center was positioned at a height of 6 mm from the bottom of the cell. A silicon photodetector (509-10 manufactured by Bell Howell) was used to detect the transmitted light. The anti-AFP antibody sensitized latex prepared by the following method was used. Preparation of anti-AFP antibody sensitized latex (AFP = α-fetoprotein) Glycine buffer solution of anti-AFP antibody (concentration: 2
mg/ml) to 10ml of polystyrene latex with an average particle size of 0.234μ (manufactured by Dow Chemical Company, solid content concentration:
1 ml of 10% by weight) was added, stirred at room temperature for 30 minutes, then heated to 40°C and further stirred for 30 minutes, followed by centrifugation (12000 rpm) for 50 minutes while cooling at 2-4°C. Decant the precipitate and separate the
The AFP antibody-sensitized latex was suspended in a bovine serum albumin solution (concentration: 0.2% by weight) to obtain an anti-AFP antibody-sensitized latex reagent with a sensitized latex particle concentration of 1.0% by weight. Example 1 0.1 ml of physiological saline containing 0.2% bovine serum albumin, 0.05 ml of anti-AFP antibody sensitized latex, and 0.25 ml of glycine buffer solution were added to the cell. (in this case, the cylinder is located at the liquid level). Changes in transmittance over time were measured while stirring at 1000 rpm with a stirrer. When stirring bar B was used, almost no change in transmittance over time was observed. However, it was found that when stirrer A was used, the transmittance changed irregularly due to the inclusion of air bubbles, which could seriously impede measurement. Examples of transmittance charts when using stirrers A and B are shown in FIGS. 3 and 4, respectively. In addition, in the above example, when the rotation speed of the stirrer is 800 rpm, and when the rotation speed of the stirrer is 0.2
When 0.2 ml of physiological saline containing % bovine serum albumin was used, almost no change in transmittance over time was observed regardless of which stirrer was used. Example 2 Measurement of agglutination reaction of latex (1) Measurement of standard transmittance Put 0.2 ml of physiological saline containing 0.2% bovine serum albumin and 0.05 ml of anti-AFP antibody-sensitized latex into a cell, and add 0.25 ml of glycine buffer. solution was added.
The transmittance (T ₀ ) was measured while stirring with stirring bar B at 1000 rpm. (2) Measurement of transmittance Add 0.2ml of standard AFP solution and 0.05ml of anti-
AFP antibody sensitized latex and 0.25 ml of glycine buffer solution were added. While stirring at 1000 fpm with stirring bar B, measure the average transmittance (T ₁ ) from 20 seconds to 24 seconds after the start of stirring and the average transmittance (T ₂ ) from 44 seconds to 48 seconds. did. The average rate of change in absorbance V was calculated as V=60/24log(T ₁ /T ₂ ). The results are shown in Table 1. 【table】

[Brief explanation of the drawing]

FIG. 1 is a front view and a plan view of a stirrer A used in Examples. FIG. 2 is a front view and a plan view of the stirrer B used in the example. The curvature of the bottom is
2.5R, and the length of the prism part (L in the diagram) is 6.5mm.
It is. FIG. 3 is an example of a chart of transmittance when the stirrer shown in FIG. 1 is used. FIG. 4 is an example of a chart of transmittance when the stirrer shown in FIG. 2 is used. 1...Rotating shaft, 2...Cylindrical part, 3...Prismatic part,
4...Bottom surface.

Claims (1)

[Claims] 1. Performing an antigen-antibody reaction using a sensitized latex in an optical measurement cell, and measuring the progress of the reaction by tracking the optical properties of the reaction mixture as the reaction progresses. In the method of Optics of a liquid sample, characterized in that a polygonal columnar part is located in the optical path of a measuring light beam and the columnar part is located on the sample liquid surface, and the optical properties of the sample are measured while rotating a stirrer. measurement method. 2 The cylindrical part of the stirrer is at least 1 mm below the liquid level.
The optical measuring method according to claim 1, characterized in that the optical measuring method continues to a depth of . 3. The optical measurement method according to claim 1, wherein the cylindrical portion of the stirrer extends 1 to 5 mm below the liquid surface. 4. The optical measurement according to any one of claims 1 to 3, wherein the length of the polygonal column-shaped portion of the stirring bar accounts for at least 30% of the liquid depth in the cell. Law. 5. The optical measurement method according to any one of claims 1 to 4, wherein the shortest distance between the stirrer and the inner wall of the cell during rotation of the stirrer is 0.5 to 2.0 mm. 6. The optical system according to any one of claims 1 to 5, wherein the cross-sectional area of the polygonal columnar portion of the stirring bar is 25% or more of the cross-sectional area of the cell in the portion where the stirring bar is located. measurement method. 7. The optical measurement method according to any one of claims 1 to 6, wherein the polygonal columnar portion of the stirrer has a rectangular cross-sectional shape. 8. The optical measuring method according to any one of claims 1 to 6, wherein the polygonal columnar portion of the stirrer has a square cross-sectional shape. 9. The optical measurement method according to any one of claims 1 to 8, wherein the cell has a rectangular cross section. 10. The optical measurement method according to any one of claims 1 to 8, wherein the cell has a square cross section. 11. The optical measurement method according to any one of claims 1 to 10, which measures the transmittance of light in a sample solution.