JPH0511788B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a method for measuring antigen or antibody concentration. (Prior art and problems to be solved by the invention) Conventionally, antibodies or antigens are immobilized on insoluble carrier particles by physical adsorption or the formation of covalent bonds, and the antigen or antibody is immobilized on the antibodies or antigens immobilized on the carrier particles. The rate of the antigen-antibody reaction is measured from the increase in absorbance, that is, the decrease in transmittance, of the reaction mixture as the reaction progresses, or the absorbance or transmittance of the antibody in the reaction mixture at the end of the reaction,
Measure the difference in absorbance or transmittance of the antigen or antibody before the start of the reaction, and then quantify the concentration of the antigen or antibody in the sample from the speed or the difference in absorbance or transmittance before the start of the reaction and at the end of the reaction. There are known ways to do this. This method has the advantage of being able to quickly quantify the concentration of antigen or antibody with high accuracy. However, there are the following drawbacks. For example, when an antibody is immobilized on an insoluble carrier particle, in a region where the number of antigen molecules is small compared to the number of antibody molecules, the antigen-antibody reactant increases in proportion to the increase in the number of antigen molecules, and the number of antigen molecules increases. In a region where the number of antigen molecules exceeds the number of antibody molecules, the excess antigen neutralizes the antibody molecules that would normally contribute to aggregation, and the number of antigen-antibody reactants decreases as the number of antigen molecules increases. The former is generally called an antigen (antibody) deficient region, and the latter is generally called an antigen (antibody) abundant region. Due to this phenomenon, generally for one antigen-antibody reactant concentration,
Multiple antigen or antibody concentrations are supported. The same phenomenon is observed even if the antibody and antigen are replaced. In clinical tests, test fluids belonging to the above antigen-rich region generally appear less frequently, but if a test fluid belonging to the antigen-rich region is mistakenly evaluated as being from the antigen-poor region, it may be clinically serious. It would be a serious mistake.
Furthermore, measurement methods that cause such errors have little clinical significance. Traditionally, to prevent such errors from occurring,
A method of measuring two or more different dilution rates for the same test solution, or adding an additional antigen or antibody after the measurement is completed, measuring the concentration of the antigen-antibody reactant, and then adding the antigen or antibody. A method has been proposed for determining that a test liquid belongs to an antigen-excessive region or an antibody-excessive region when the antigen-antibody reactant concentration does not change. In either method, it is necessary to measure the same test liquid multiple times. However, with the recent appearance of automatic measuring devices that can measure a large number of test samples in a short period of time, it has become necessary to develop a measurement method that can determine whether the sample belongs to the antigen-excessive region or the antibody-excessive region within a single measurement operation. It has been desired. (Means for Solving the Problems) The present inventors have conducted extensive research with the aim of establishing a measurement method suitable for measuring a large number of test liquids in a short period of time using an automatic measuring device. As a result, the present inventors, for example, as shown in FIG. Add each of the two test solutions and stir for 2 seconds.
When the difference in absorbance was measured for a certain period of time after 60 seconds and after 60 seconds, the difference in absorbance exhibited by both test solutions was the same. In other words, this phenomenon indicates that multiple antigen concentrations correspond to one antigen-antibody reactant concentration. Therefore, it is not possible to determine whether the antigen or antibody concentration in a test solution with an unknown concentration is in an antigen (antibody) excess region or an antigen (antibody) deficiency region in a single measurement using a measurement method similar to that described above, making it impractical for practical use. Indicates that there is a problem. The present inventors investigated in detail the change in absorbance over time after adding the test solution, and found that
2 after 5 and 10 seconds and 10 and 15 seconds after adding the test solution
When the ratio of the difference in absorbance with respect to a specific time was determined, it was found that the values of the ratio of the difference between the two test liquids were significantly different. Furthermore, we conducted multiple measurements for each serum showing various antigen concentrations, and examined the reproducibility of the ratio of the difference in absorbance.We found that by using the ratio of the difference in absorbance, it is extremely easy to detect the antigen in the sample solution. Alternatively, the present invention was completed by confirming that the antibody concentration can be quantified. That is, in the present invention, an antibody or antigen is immobilized on an insoluble carrier particle, a known concentration of the antigen or antibody is reacted with the antibody or antigen immobilized on the carrier particle, and light is applied at a time that changes over time after the start of the reaction. , calculate the ratio of the difference in light absorbance or transmittance for two or more specific times in the above reaction, calculate the corresponding curve between the ratio and the antigen or antibody concentration, and then calculate the ratio of the difference in light absorbance or transmittance for two or more specific times in the above reaction, calculate the corresponding curve between the ratio and the antigen or antibody concentration, and then This is a method for measuring antigen or antibody concentration, which is characterized in that the ratio of the difference in light absorbance or transmittance for two or more specific times is determined, and the concentration corresponding to the ratio is determined based on the above-mentioned corresponding curve. The insoluble carrier particles used in the present invention may be organic polymers or inorganic compounds, as long as they are substantially insoluble under the conditions of use. Further, the shape thereof is not particularly limited, but examples of those generally suitably used are insoluble carrier particles having an average particle diameter of about 1.0 μm or less, preferably 0.05 to 0.4 μm. An antibody or antigen is immobilized on the insoluble carrier particles, and then the antigen or antibody in the test liquid is reacted, for example, from 400 to 1000 mm, preferably from 500 to 950 mm.
Using a light beam with a wavelength in the range of The concentration of can be measured. The antigen or antibody in the test solution in the above method is
Generally, one of these is included, but
Even a mixture of the antigen and antibody can be adequately handled. The light beam used for the above measurement has a relatively large absorbance or transmittance with respect to the progress of the reaction, has excellent sensitivity, and has a wavelength in the above wavelength range with relatively little interference from chyle, hemoglobin, bilirubin, etc. that usually coexist in the test liquid. suitable. Regarding the particle size of insoluble carrier particles, when the particle size is large, the amount of change in particle size due to aggregation is large, but the aggregation reaction rate tends to be slow, whereas when the particle size is small, Brownian motion is active and the aggregation reaction rate is slow. However, since the primary particle size is small, the amount of change in particle size due to the aggregation reaction tends to be small. For the above reasons, it is preferable to carry out the measurement by appropriately combining the above particle diameter and measurement wavelength. As described above, the insoluble carrier particles include polystyrene, styrene-butadiene copolymer, styrene-butadiene copolymer, and styrene-butadiene copolymer which are substantially insoluble in the liquid medium used for the measurement and have the above-mentioned average particle diameter.
Fine particles of organic polymer substances such as organic polymer latex obtained by emulsion polymerization such as methacrylic acid copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer, or silica, silica-alumina, alumina, etc. An inorganic oxide or an inorganic particle in which a functional group is introduced into an inorganic oxide through a silane coupling treatment or the like is used. In the present invention, antibodies or antigens are not particularly limited, and known antibodies or antigens can be used. Typical examples of those that are generally preferably used include denatured gamma globulin, antinuclear factor, human albumin, anti-human albumin antibody, immunoglobulin G (IgG), anti-human IgG antibody, and immunoglobulin A ( IgA), anti-human IgA antibody, immunoglobulin M
(IgM), anti-human IgM antibody, anti-human IgE antibody, streptolysin O, streptokinase, hyaluronidase, C-reactive protein (CRP), anti-human CRP
antibody, alpha fetoprotein (AFP), anti-human AFP antibody, carcinoembryonic antigen (CEA), anti-human
CEA antibody, human chorionic gonadotrobin (HCG),
Anti-HCG antibody, anti-estrogen antibody, anti-insulin antibody, hepatitis B surface antigen (HBs), anti-HBs antibody, Treponema pallidum antigen, rubella antigen, influenza antigen, complement C ₁ q, anti-C ₁ q antibody, anti-C ₃ antibody,
Anti- _C4 antibodies, anti-transferrin antibodies, etc. In the present invention, an antibody or antigen capable of reacting with an antigen or antibody in a test liquid to be measured is immobilized on such insoluble carrier particles. In this case, the immobilization method may be either physical adsorption or chemical covalent bond formation, but physical adsorption is suitable for immobilizing proteins with high physical adsorption capacity, such as antibodies and high molecular weight proteins. Formation of chemical covalent bonds is preferably used to immobilize hormones and haptens with low adsorption capacity. Many immobilization methods have already been proposed, and it is preferable to select an appropriate immobilization method according to the characteristics of the antibody or antigen to be immobilized. For example, the antibody or antigen may be mixed with insoluble carrier particles in a dispersion medium in the presence of a buffer or a crosslinking agent as required. The dispersion medium for the insoluble carrier particles on which the antibody or antigen is immobilized is not particularly limited, but from the viewpoint of the stability of the insoluble carrier particles during storage and the reproducibility of the reaction during the agglutination reaction, glycine-sodium hydroxide buffer Buffers such as tris-hydrochloric acid buffer, tris-hydrochloric acid buffer, and phosphate buffer are preferably used. The concentration of the insoluble carrier particles on which the antibody or antigen is immobilized is 0.005% by weight or more during the reaction, preferably
The insoluble carrier particles are suspended in the above dispersion medium at a concentration of 0.02 to 0.20% by weight to obtain a suspension of insoluble carrier particles on which antibodies or antigens are immobilized. A method for measuring the antigen or antibody concentration in a test liquid using the suspension can be carried out, for example, as follows. First, a certain antibody or antigen is immobilized on insoluble carrier particles having a certain average particle diameter to prepare the suspension. Next, an antigen or antibody that is the same or almost the same as the antigen or antibody contained in the test solution is diluted or concentrated using a medium that is the same or almost the same as that of the test solution, and then prepared as standards of various known concentrations. Prepare the test solution. Next, the suspension and the standard test solution are mixed under certain conditions, the absorbance or transmittance is measured at two or more specific times after the start of the reaction, and the amount of change, that is, the ratio of the difference, is calculated. do.
Next, when the value of the ratio of the difference is plotted, for example, on the vertical axis and the antigen or antibody concentration in the standard test solution is plotted on the horizontal axis, for example, the antigen or antibody concentration in the test solution as shown in FIG. A corresponding curve between the value of the ratio of the difference and the value of the ratio of the difference is obtained. Next, for a test solution of unknown concentration containing the same or almost the same antigen or antibody as the antigen or antibody in the standard test solution, the absorbance or transmittance was measured under the same conditions and in the same manner as those used to obtain the above corresponding curve. The amount of antigen or antibody contained in the test liquid can be detected by measuring the difference in the ratio, obtaining a value of the ratio, and comparing it with the corresponding curve. According to the method of the present invention, the concentration of antibodies or antigens immobilized on insoluble carrier particles for a test solution of the same concentration can be reduced to 1/10 or less compared to the conventional technique. On the other hand, for measurement items that require prior art dilution of the test solution to reduce the antigen or antibody concentration in the test solution, the method of the present invention allows the dilution factor to be reduced. The concentration can be reduced to 1/10 or less, and furthermore, in some cases, it becomes possible to measure the antigen or antibody concentration in the test liquid without performing a dilution operation. In the former case, the amount of antibody or antigen used for measurement and the amount of insoluble carrier particles used can be reduced;
In the latter case, measurement errors due to dilution operations can be reduced or eliminated. In the present invention, it is preferable to set the specific time in consideration of the following points. That is, after starting a reaction by adding a test liquid to a suspension of antibodies or antigens immobilized on insoluble carrier particles, preferably after stirring and mixing the suspension and test liquid under certain conditions. Furthermore, it is preferable to measure the amount of change in the absorbance or transmittance of the reactant at two or more specific times after 2 to 3 seconds have passed and the reaction system has become substantially stabilized. In this case, at least one of the specific times is defined as the time when the reaction system is substantially stabilized, that is, 2 to 3 seconds after stirring, and the absorbance or transmittance of the test liquid is determined. The value of the difference ratio tends to correspond sensitively to the antigen or antibody concentration in the test liquid. Therefore, it is preferable to set at least one of the specific times as early as possible in the reaction when photometry is possible. Regarding the setting of the specific time interval and other specific times, suitable conditions may be selected for each antigen or antibody to be measured, taking into consideration the amount of change in absorbance or transmittance. Typical embodiments that are generally suitably employed are explained below. For example, the absorbance or transmittance is measured after a certain period of time has elapsed (b seconds) from the reference time after the reaction has started (a seconds later), and the difference (△E ₁ ) is calculated. Set a reference time different from the above (c seconds later), measure the absorbance or transmittance after a certain period of time (d seconds later), and compare the difference with the reference time (c seconds later). (△E ₂ ) is calculated. The ratio between the two, ie, ΔE ₁ /ΔE ₂ is calculated, and a corresponding curve of the ratio at each known concentration is created. The above standard elapsed time (c seconds later) can also be set to b seconds after the previous absorbance or transmittance measurement; in this case, absorbance or transmittance measurements at at least 3 points can be set for the above two specified times. The ratio of the difference in absorbance or transmittance can be determined. The value of the ratio determined in this way is specific depending on the antibody or antigen concentration, for example,
A calibration curve as shown in the figure can be obtained. Next, with respect to the antibody or antigen concentration in the test solution of unknown concentration, the ratio of the difference in absorbance or transmittance for two or more specific times as described above is determined, and the antibody or antigen in the test solution is determined from the above calibration curve. The concentration of can be determined. (Effect of the invention) The method for measuring antigen or antibody concentration according to the present invention is as follows:
In the conventional technology, it was necessary to determine whether there is excess antigen or antibody, but it is possible to determine whether there is excess antigen or antibody for the range of antigen or antibody concentration in the test liquid without using complicated judgment operations. The antigen or antibody concentration in the test solution can be uniquely determined, and there is no need for retesting. In addition, the concentration of antibodies or antigens immobilized on insoluble carrier particles for the same test solution can be lowered compared to conventional techniques, and/or the concentration of the immobilized antigen or antibody for the same test solution can be measured by diluting the test solution. Errors can be reduced or eliminated. Furthermore, in the measuring method according to the present invention, absorbance or transmittance measurements can be performed at least three times. Therefore, the method for measuring antigen or antibody concentration according to the present invention is particularly useful in automatic measurements that process a large number of test liquids in a short period of time, has few restrictions on automatic measuring equipment, and can be used widely in general automatic measurements. Can be applied to measuring equipment. (Example) Hereinafter, the present invention will be explained in further detail with reference to Examples. Example 1 (1) Preparation of reagent for measuring C-reactive protein Polystyrene latex particles with an average diameter of 0.123 μm were diluted with Tris-HCl buffer (=7.5).
A suspension with a latex concentration of 1% by weight is prepared.
Next, C-reactive protein (hereinafter abbreviated as CRP)
The anti-CRP goat IgG fraction was fractionated by salting out from the anti-CRP serum obtained by immunizing goats with the following: diluted with Tris-HCl buffer (=7.5) to give a protein concentration of 2 mg/
Prepare a solution of ml. Add 1 volume of anti-CRP goat IgG fraction solution to 1 volume of the above latex suspension and heat at 37℃ for 2 hours.
Allow time to react. Further, bovine serum albumin was added to a final concentration of 0.05% by weight, followed by centrifugation, the supernatant was removed, and the precipitate was dissolved in Tris-HCl buffer (=
7.5) and redisperse the latex concentration to 0.08% by weight.
was prepared to obtain a CRP measurement reagent. (2) Measurement method Absorbance was measured using a device equipped with a temperature controller and a magnet type stirring device attached to the photometry section of a self-recording spectrometer U-3200 manufactured by Hitachi, Ltd. A cylindrical stirrer was placed in a glass optical cell with an optical path length of 10 mm, and then 2450 μ of the CRP measurement reagent obtained in (1) was added.
was dispensed, inserted into the photometer, and kept at 37°C. Next, while stirring the CRP measurement reagent using the stirring device, 50μ of the test liquid was added. Measurement of absorbance was started at the same time as the addition. Absorbance measurements were performed using a light beam with a wavelength of 580 mm. Note that stirring was stopped 3 seconds after the addition of the test liquid. (3) Measurement of known samples Serum with a CRP concentration of 22.4 mg/dl was diluted with Tris-HCl buffer (=7.5), and the CRP concentration was 0.41, 1.24,
Test solutions of 3.73, 5.60, 7.46, 11.2, and 16.8 mg/dl were obtained. On the other hand, the above serum was concentrated and the CRP concentration was 44.8,
A test solution of 89.6 mg/dl was obtained. Under the measurement conditions of (2), the above nine test solutions and
Absorbance was measured five times for serum with a CRP concentration of 22.4 mg/dl and for Tris-HCl buffer (=7.5). Among the absorbances obtained, 6 seconds after adding the test solution and
From the absorbance after 12 seconds, 18 seconds, and 60 seconds, calculate the difference in absorbance between 12 seconds and 6 seconds after adding the test solution as the ratio of the difference between the two specific times, and calculate the difference in absorbance between 18 seconds and 12 seconds after adding the test solution. A value was obtained by dividing by the difference in absorbance. On the other hand, the difference in absorbance over a certain period of time was obtained by subtracting the absorbance 6 seconds after addition of the test liquid from the absorbance 60 seconds after addition of the test liquid. The results are shown in Table 1.

【table】

[Table] The coefficient of variation was calculated.
(Note 2) Tris-HCl buffer that does not contain CRP.
Next, the vertical axis is the average value of the difference in absorbance over a certain period of time shown in Table 1, and the CRP in the added test solution is
A corresponding curve shown in FIG. 2 was obtained with concentration as the horizontal axis.
On the other hand, a corresponding curve according to the present invention shown in FIG. 3 was obtained, with the vertical axis representing the average value of the ratio of the difference in absorbance for two specific times and the horizontal axis representing the CRP concentration in the added test liquid. Here, let the value of the error ratio be x, and the CRP
If the concentration is y, the CRP concentration is 3.73 mg/dl to 89.6
The corresponding curve of the ratio of the difference in the mg/dl range was well approximated by y=exP(4.059× _O gx+2.697) (Equation 1). On the other hand, for the CRP concentration range below 5.60 mg/dl in the corresponding curve of the difference, if the value of the difference is x and the CRP concentration is g, then y=exP(0.9553× _O gx+2.585) (Formula 2) It was possible to approximate it well. ₀ in (Formula 1) and (Formula 2)
g is the natural logarithm. Generally, the CRP concentration in the serum of strongly CRP-positive patients is 40
mg/dl or less, and CRP in the test solution with known concentration
The highest concentration of 89.6 mg/dl is considered to be the upper limit of the range required for clinical measurement, and if the corresponding curve of the ratio of the difference according to the present invention is used, the CRP concentration can be reduced from 3.73 mg/dl to 89.6 mg/dl.
It is possible to unambiguously determine the antigen concentration within the concentration range of mg/dl, where it was impossible to unambiguously determine the antigen concentration and it was necessary to determine whether or not it belonged to the antigen-excess region. . Note that by applying the error correspondence curve of the conventional method to the CRP concentration range of less than 33.7 mg/dl, it is also possible to measure the CRP low concentration range. That is, when measuring a test liquid with unknown CRP concentration, measure according to the measurement method (2),
Obtain the difference value and the error ratio value for the test liquid with unknown CRP concentration. Next, compare the difference value of 0.2651 at a CRP concentration of 3.73 mg/dl with the difference value of the above test solution, and if the error value of the above test solution is 0.2651 or more, the ratio of the difference is Substitute the value into (Equation 1) to obtain the CRP concentration. On the other hand, if the value of the difference of the above test liquid is less than 0.2651, substitute the value of the difference into (Equation 2).
Obtain CRP concentration. (4) Measurement of unknown sample When serum with unknown CRP concentration was measured using the measurement method in (2), the difference was 0.5307 and the ratio of the difference was 1.196.
I got it. The value of the difference at a CRP concentration of 3.73mg/dl
Since the value of the above difference is larger than 0.2651, the value of the error ratio is applied to (Equation 1) and the CRP concentration is 30.7 mg/dl.
I got it. Next, the above serum was diluted with Tris-HCl buffer (PH=
When diluted 5 times and 10 times in 7.5) and measured using the measurement method in (2), the difference value for the 5 times diluted solution was
Obtain the ratio value of 0.4381 and the difference, 0.801, and use (Equation 1) to obtain
The CRP concentration was determined to be 6.03 mg/dl. For the 10-fold diluted solution, the difference value 0.2137 and the difference ratio value 0.711 were obtained, and the CRP concentration was determined to be 3.04 mg/dl using (Equation 2). Furthermore, the 5-fold diluted solution was added to LC Partigen, a CRP quantitative reagent manufactured by Hoechst using a one-way immunodiffusion method.
CRP concentration measured by CRP was 6.0mg/dl
In measuring CRP, whose concentration in serum is distributed over a wide range, using the method of the present invention, it is impossible to determine the CRP concentration unambiguously using the conventional method, and it is difficult to determine whether it belongs to the antigen-rich region. it was necessary
It has become possible to uniquely determine CRP concentration within the CRP concentration range. Furthermore, by implementing the conventional method in the CRP low concentration range, the CRP concentration was 0.41mg/dl.
In other words, the CRP concentration ranges from around the normal range to 89.6.
mg/dl, or abnormally high CRP concentration,
It is now possible to measure CRP concentration with a single measurement operation.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the amount of change in absorbance versus time after addition of a corresponding antigen to a suspension of insoluble particle carriers on which antibodies are immobilized. In the figure, the solid line shows the results for the test solution belonging to the antigen-poor region, and the dotted line shows the results for the test solution belonging to the antigen-excess region. FIG. 2 shows a corresponding curve in which the horizontal axis is the antigen concentration in the test solution and the vertical axis is plotted as the difference in absorbance over a certain period of time. FIG. 3 shows a corresponding curve according to the present invention in which the horizontal axis is the antigen concentration in the test liquid and the vertical axis is the ratio of the difference in absorbance for two specific times.

Claims (1)

[Claims] 1 Immobilizing antibodies or antigens on insoluble carrier particles,
The antibody or antigen immobilized on the carrier particles is reacted with a known concentration of the antigen or antibody, and light is irradiated at time points that change over time after the start of the reaction, and the light is applied at two or more specific times in the above reaction. Determine the ratio of the difference in absorbance or transmittance, determine the corresponding curve between the ratio and the antigen or antibody concentration, and then calculate the ratio of the difference in absorbance or transmittance of light for two or more specific times for a sample of unknown concentration. A method for measuring an antigen or antibody concentration, characterized in that the concentration corresponding to the ratio is determined using the above-mentioned corresponding curve.