JP3005400B2 - How to measure antigen or antibody concentration - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring the concentration of an antigen or antibody in a test solution. More specifically, the present invention relates to a method for measuring the concentration of an antigen or an antibody capable of accurately and accurately measuring the concentration of an antigen or an antibody in a test solution exceeding a measurement limit of a reagent.

[0002]

2. Description of the Related Art Conventionally, insoluble carrier particles (hereinafter abbreviated as immobilized carrier particles) in which an antibody or an antigen is immobilized by physical adsorption or formation of a covalent bond on the insoluble carrier particles are used in a test solution such as serum or urine. BACKGROUND ART An immunological measurement method for measuring a corresponding antigen in a test solution by observing an agglutination reaction or an agglutination inhibition reaction based on an antigen-antibody reaction with a corresponding antigen is known.

[0003] As a method for quantifying the state of aggregation of the insoluble carrier in a reaction solution obtained by reacting a reagent comprising a dispersion of the above-mentioned immobilized carrier particles with a plurality of test solutions, light is applied to the reaction solution. Irradiation and a method of determining the concentration of the antigen or antibody in each test solution from the intensity of the scattered light or transmitted light are common.

[0004] Such a method is widely used by using an automatic analyzer capable of automatically quantifying the concentration of an antigen or an antibody in a test solution because measurement can be automated.

A description will be given of a case where a conventional general measuring method is used for clinical diagnosis. In the case where the concentration of antigen or antibody is expected to differ significantly between individuals or at the time of collection, first, Measure the concentration of the antigen or antibody under predetermined measurement conditions, and for the test solution in which the concentration of the antigen or antibody is out of the measurement range of the reagent, of the measurement conditions, the conditions relating to the reagent do not substantially change, In general, a method of increasing and decreasing the concentration of a test solution to be subjected to measurement and performing the same measurement again has been employed.

For example, when the concentration of antigen or antibody in a sample is high and there is a sample which exceeds the upper limit of measurement at the first measurement under predetermined measurement conditions, (A) the test solution is diluted to a predetermined multiple and the test solution is diluted. And measuring again by adjusting the concentration of the sample, and multiplying the obtained measured value by a predetermined dilution factor, and (B) a method of measuring the amount of the test liquid by subtracting it from the initial measurement condition.

When the concentration of the antigen or antibody in the sample is low and there is a sample which is less than the lower limit of measurement at the time of the first measurement under the predetermined measurement conditions, (C) the test solution is concentrated to a predetermined multiple and the measurement is performed again. And (D) a method in which the amount of the test solution is measured by increasing the amount compared to the initial measurement conditions.

[0008]

However, the above-mentioned conventional method has the following disadvantages.

In other words, the method (A) has some strengths depending on the measurement items, but the measured values may be interfered by the properties of the diluent, and it may be difficult to select the diluent. In addition, it is necessary to pay attention to the method of performing the dilution operation accurately and accurately, and there is a drawback including an error factor when the dilution is performed manually. In addition, when the dilution function is added to the automatic analyzer, better precision and accuracy than manual dilution can be expected,
Usually, this method has a drawback that the number of measurements per unit time, that is, the processing capacity is halved, and the use of the diluent is restricted and may not be used. In addition, in the dilution by the automatic analyzer, if there is no test liquid having a volume equal to or more than a predetermined liquid amount in the fractionation, mechanical dilution becomes impossible, and there is a problem that a test liquid that is more valuable than necessary is used. .

In the method (B), there is a lower limit of the amount of the test liquid that can be dispensed with good reproducibility by the automatic analyzer, and this method cannot be adopted if the initial measurement conditions are close to the lower limit of the automatic analyzer. In addition, even if the sample dispensing volume for the initial measurement and the re-measurement with the test sample volume reduced can be set within the upper and lower limits of the sample dispensing volume of the automatic analyzer, the dispensing accuracy of the automatic analyzer is In general, the remeasured value is relatively inaccurate because it is inversely proportional to the sample amount.

Furthermore, the method (C) has the disadvantage that the test solution is subject to considerable denaturation during the concentration / reduction operation, and the concentration operation itself is complicated and inferior in speed. Not suitable for rapid measurement.

Further, according to the method (D), the automatic analyzer is designed so that even a small amount of the test liquid can be dispensed with good reproducibility, but the upper limit of the amount of test liquid that can be dispensed with good reproducibility. Exists. Therefore, this method cannot be adopted if the initial measurement conditions are close to the upper limit of the dispensed amount of the test liquid. In addition, even if each sample dispensing volume for initial measurement and remeasurement with increased sample volume can be set within the upper and lower limits of the sample dispensing volume of the automatic analyzer,
Since the concentration of the analyte in the reaction solution increases in proportion to the increase in the test solution, not only the measurement target in the test solution but also the concentration of the substance that interferes with the reaction in the reaction solution increases in proportion. Therefore, this method has a disadvantage that a nonspecific reaction other than the originally expected reaction occurs, and the concentration of the antigen or antibody cannot be accurately measured.

[0013]

Means for Solving the Problems The present inventors have found that even when an automatic analyzer is used, a large number of samples that may contain a test solution whose antigen or antibody concentration is out of the measurement range of the reagent can be obtained. Researches have been made earnestly for the purpose of establishing a suitable measuring method for accurately measuring a test solution in a short time.

As a result, for the test liquid whose antigen or antibody concentration is out of the measurement range of the reagent, the ratio of the liquid not involved in the reaction can be reduced without substantially changing the ratio of the test liquid in the reaction liquid. This problem can be solved by increasing or decreasing the ratio of the insoluble carrier particles in the reagent to the concentration of the insoluble carrier particles in the reaction solution after mixing with the reagent, and measuring the antigen or antibody concentration again. And completed the present invention.

That is, the present invention provides a method comprising reacting a reagent comprising a dispersion of insoluble carrier particles on which an antibody or an antigen is immobilized with a large number of test liquids, and reacting the reagent with the aggregation state of the insoluble carrier particles in the reaction solution. In the method of measuring the concentration of antigen or antibody in a test solution, most of the test solution is measured with a reagent in which the specific concentration of the insoluble carrier particles has been reduced in advance, and most of the measured values are less than a predetermined reference value. And then re-measuring the remaining test liquid having a predetermined reference value or more with a reagent having a specific concentration, and then measuring the antigen or antibody concentration.

The insoluble carrier particles used in the present invention may be any known organic polymer or inorganic polymer without particular limitation, as long as they are substantially insoluble in the liquid that comes into contact during storage and measurement. Can be used. For example, organic polymers such as organic polymer latex particles obtained by an emulsion polymerization method such as polystyrene, styrene butadiene copolymer, styrene-methacrylic acid copolymer, polyglycidyl methacrylate, and acrolein-ethylene glycol dimethacrylate copolymer. Examples include fine particles of a substance, or particles made of a material such as an inorganic oxide such as silica, silica-alumina, and alumina, or an inorganic particle having a functional group introduced into the inorganic oxide by an operation such as silane coupling treatment.

The shape of the insoluble single particles is not limited. When generally used ones are exemplified, the average particle diameter is about 1.0 μm or less, preferably 0.1 μm or less.
Insoluble carrier particles having a particle size of 0.5 to 0.4 μm.

In the present invention, the antibody or antigen immobilized on the insoluble carrier particles is not particularly limited, and any known antibody or antigen can be used. Representative examples generally used preferably include modified gamma globulin, antinuclear factor, human albumin, anti-human albumin antibody, immunoglobulin G
(IgG), anti-human IgG antibody, immunoglobulin A
(IgA), anti-human IgA antibody immunoglobulin M (I
gM), anti-human IgM antibody, anti-human IgE antibody, streptolysin O, streptokinase, hyaluronidase, C-reactive protein (CRP), anti-human CRP antibody,
Anti-human alpha-fetoprotein (AFP) antibody, anti-human carcinoembryonic antigen (CEA) antibody, anti-human chorionic gonadotropin (hCG) antibody, anti-estrogen antibody, anti-insulin antibody, hepatitis B surface antigen (HBs), anti-HBs
Antibodies, Treponema pallidum (TP) antigen, rubella antigen, influenza antigen, complement C1q, anti-C1q antibody, anti-C3 antibody, anti-C4 antibody, anti-transferrin antibody and the like.

In the present invention, an antibody or antigen capable of reacting with an antigen or antibody in a test solution to be measured is selected and immobilized on the insoluble carrier particles. Such an immobilization method may be either physical adsorption or formation of a chemical covalent bond, but physical adsorption is suitable for immobilizing a protein having a high physical adsorption capacity, such as an antibody or a high molecular weight protein. Formation of a chemical covalent bond is preferably used for immobilization of hormones and haptens having low adsorption ability. Many methods have been proposed for these immobilization methods, and an appropriate immobilization method may be selected from known methods according to the characteristics of the antibody to be immobilized. For example, the antibody or the antigen may be mixed with the insoluble carrier particles in a dispersion medium in the presence of a buffer or a crosslinking agent as required.

The dispersion medium for immobilizing the antibody on the insoluble carrier particles and the dispersion medium for the immobilized carrier particles are not particularly limited, and known ones may be used. It may be appropriately selected from the viewpoint of the reproducibility of the reaction at the time of the agglutination reaction. When the above-mentioned crosslinking agent is used, it is necessary to use a dispersion medium in which the components in the dispersion medium do not react with the crosslinking agent. Suitable dispersion media include buffers such as phosphate buffer, glycine-sodium hydroxide buffer, Tris-HCl buffer, ammonium chloride-ammonia buffer.

In the present invention, the reagent can be obtained by suspending insoluble carrier particles having immobilized antibodies or antigens in a liquid that does not substantially participate in the reaction with the test solution. The liquid that does not substantially participate in the reaction with the test liquid is not particularly limited as long as it has such a function. Generally, it is selected from the above-mentioned dispersion media, but water, physiological saline and the like can also be used effectively.

Although the concentration of the insoluble carrier particles in the reagent is not particularly limited, it is generally determined at the time of the reaction in a range of 0.005% by weight or more, preferably 0.01 to 0.5% by weight. Is preferred.

Further, the reagent is not only a one-pack type reagent consisting of only a suspension of the immobilized carrier particles, but also a liquid containing the suspension containing the immobilized carrier particles and another reagent component or the suspension. A two-part reagent consisting of two components consisting of a diluent of a suspension,
It can be used as a three-part reagent containing other reagent components.

In the present invention, the reagent and the test solution are mixed at a fixed ratio, and the concentration of the antigen or antibody in the test solution is measured based on the state of aggregation of the insoluble carrier particles in the reaction solution.

In general, such a method for measuring the concentration has a relatively large difference in the intensity of scattered light or transmitted light with respect to the progress of the reaction, is excellent in sensitivity, and which is usually coexistent in a test solution.
It reacts with light having a wavelength range in which the interference of hemoglobin and bilirubin is relatively small and the linearity of the optical system mounted on the automatic analyzer is relatively good, for example, 400 to 1000 nm, preferably 500 to 950 nm. Irradiate the solution, measure the difference in the intensity of scattered light or transmitted light of the reaction solution at two or more specific times after the start of the reaction, and determine the concentration of antigen or antibody in each test solution from a calibration curve prepared in advance. It is performed by asking.

Regarding the particle size of the insoluble carrier particles, when the particle size is large, the amount of change in the particle size due to aggregation is large, but the agglutination reaction rate tends to be slow. Although the agglutination reaction speed is high, the amount of change in the particle diameter due to the agglutination reaction tends to be small because the primary particle diameter is small.

Therefore, in consideration of these points, it is preferable that the above-described particle diameter and the measurement wavelength are appropriately combined so as to increase the measurement sensitivity.

In the present invention, an important requirement is that the re-measurement of the antigen or antibody concentration of the test solution whose antigen or antibody concentration is out of the measurement range of the reagent is performed in the reaction solution at the time of the first measurement. Ratio between the carrier concentration and the particle concentration at the time of the re-measurement (hereinafter, abbreviated as particle concentration ratio).
It is important to change the ratio by increasing or decreasing the ratio of the liquid that does not substantially participate in the reaction between the reagent and the test liquid to the insoluble carrier particles.

The method of changing the particle concentration ratio is not particularly limited as long as the ratio of the test solution in the reaction solution is not substantially changed.

For example, as a method of lowering the particle concentration ratio, a method of diluting a reagent containing insoluble carrier particles with a liquid that does not substantially participate in the reaction between the reagent and the test solution, In addition to the above-described dilution method, the reagent component containing the insoluble carrier is reduced, and the liquid corresponding to the reduced amount is supplied to the reaction system separately with a liquid that does not substantially participate in the dilution or reaction of other reagent components. Method.

As a method for increasing the particle concentration ratio,
There is a method of increasing the reagent concentration by reducing the amount of the dispersion medium mixed with the insoluble carrier particles during the preparation of the reagent.

In general, the particle concentration ratio is changed so that the particle concentration ratio is 2/3 or less when diluting, and the particle concentration ratio is 1.5 or more when concentrating. A good effect can be obtained by setting such that.

The method of measuring the concentration of an antigen or antibody in a test solution according to the method of the present invention can be carried out, for example, as follows.

First, a reagent is set in an automatic analyzer to be used in order to set initial measurement conditions. Common automatic analyzers include sample dispensing, reagent dispensing, stirring of a reaction solution consisting of a sample and a reagent, keeping the reaction solution warm, measuring transmitted light or scattered light intensity of the reaction solution, calculating the concentration, and measuring results. It has a function to automatically output the data, etc., and is equipped with an optical cell that performs a reaction and a spectrophotometer that can measure multiple wavelengths. In addition, there is an automatic analyzer to which various functions such as preservation of reagents, cleaning of an optical cell, and dilution of a test solution are added.

When a two-component reagent is used, in an automatic analyzer, a buffer solution as a first reagent component is referred to as a reaction solution 1 (hereinafter abbreviated as R1) in the order of addition of the reagents, and a second solution is referred to as R1. The suspension containing the immobilized carrier particles, which is a reagent component of, is referred to as a reaction liquid 2 (hereinafter, abbreviated as R2).

Next, a test solution containing an antibody or an antigen to be measured is set.

Prior to the measurement, the measurement wavelength is determined. Since a suitable measurement wavelength range exists depending on the particle size of the immobilized carrier particles, the linearity and sensitivity of the spectrometer of the automatic analyzer are within this wavelength range. And the concentration of the immobilized carrier particles at the time of the reaction.

In setting the initial measurement conditions, provisional amounts of the test solution, R1 and R2 are set and measured, and the reaction solution at two or more times after the start of the reaction with the plurality of test solutions is measured. The intensity of scattered light or transmitted light (hereinafter referred to as optical density) is measured, and the optical density is plotted against the elapsed time after the start of the reaction to obtain a so-called time course. Two specific time points are selected between the two time points when the time course of each test liquid monotonically increases, and the difference in optical density between the two time points is large for each test liquid. Next, the difference in optical density between the two time points is plotted against the concentration of the test solution to obtain a calibration curve. The calibration curve is approximated by a calibration curve equation selected from an arbitrary function regardless of a straight line or a curve to determine the coefficient of the approximate equation. Some automatic analyzers also automatically create a calibration curve.

Next, various test liquids are measured using this calibration curve, and various test liquids are measured using the above calibration curve under provisional measurement conditions. For example, a test solution diluted at regular intervals is measured to evaluate dilution linearity, and the same test solution is repeatedly measured a plurality of times to evaluate reproducibility, thereby determining a measurement range in which specificity and reproducibility are good. If this range does not include clinically significant boundary values or mode values of the concentration distribution, or if the range is biased against the upper or lower limit of the normally existing concentration distribution width, provisionally determined measurement conditions should be used. Review and repeat the examination of measurement conditions.

When the initial measurement conditions have already been determined by another automatic analyzer, the conditions may be converted into the conditions of the automatic analyzer and set.

If the initial measurement conditions are determined, a test solution having a general concentration can be measured. When such initial measurement is performed on a plurality of test liquids and there is a test liquid that is out of the measurement range, according to one aspect of the method, in the present invention, the fixation in R2 at the time of the first measurement is performed. The particle concentration of R2 for re-measurement different from the concentration of the activated carrier particles is set.

That is, when the concentration of the measurement object in the test solution exceeds the upper limit of the measurement in the initial measurement conditions, for example, the particle concentration ratio is set to 2 and the particle concentration at the time of re-measurement is twice the initial concentration. Set to. Conversely, when the concentration of the measurement target in the test solution exceeds the lower limit of the first measurement condition, for example, the particle concentration ratio is set to 1/2, and the particle concentration at the time of remeasurement is set to 1/2 of the first measurement. I do.

In the above method, the measurement conditions other than the concentration of the insoluble carrier particles in the reagent are basically not changed, but the measurement wavelength and the like may be appropriately set in consideration of the optical density under the re-measurement conditions.

The concentration of the insoluble carrier particles in the reagent for re-measurement may be adjusted appropriately by the above-described method. in this case,
If there is room for the reagents to be set in the automatic analyzer, the re-prepared reagents for re-measurement can be set together with the reagents for the first measurement, and there is sufficient space for the reagents to be set in the automatic analyzer. If there is no, the present invention can be carried out by a simple operation of simply replacing the already set initial measurement reagent and re-measurement reagent.

In the above method, a calibration curve is generally prepared separately under each measurement condition.

The test liquid exceeding the measurement range of the initial measurement conditions is measured under the re-measurement conditions set as described above. Even in such a case, if the measurement is out of the measurement range, the particle concentration may be set again.

Depending on the type of the automatic analyzer, it is possible to automatically determine whether the measurement is within or outside the measurement range in the first measurement and perform the re-measurement, and output the final measurement result. Also, when this work is performed manually, the first measurement value is compared with its measurement range to determine whether re-measurement is necessary, and if necessary, only the instruction to start re-measurement is obtained to obtain the final result. be able to.

In the measuring method of the present invention, when the intensity of the scattered light or transmitted light of the reaction solution exceeds the usable range of the spectroscopic system due to an increase in the concentration of the insoluble carrier particles, the measurement wavelength is set longer than the initial measurement condition. Just change to the side. Conversely, because the concentration of the insoluble carrier particles is reduced, if the intensity of the scattered light or transmitted light of the reaction solution decreases and the reproducibility of the optical system becomes problematic, the measurement wavelength can be changed to a shorter wavelength than the initial measurement conditions. good.

[0049]

As will be understood from the above description, according to the present invention, the concentration of the antigen or antibody in a plurality of test liquids can be reduced by using the dispersion of the insoluble carrier particles on which the antibody or antigen is immobilized. And a plurality of test liquids are reacted at a fixed ratio, and when measuring the concentration of the antigen or antibody in each test liquid based on the aggregation state of the insoluble carrier particles in the reaction liquid, the measurement range of the reagents Measuring the antigen or antibody concentration by increasing or decreasing the ratio of the liquid that does not substantially participate in the above reaction to the insoluble carrier particles, and changing the concentration of the insoluble carrier particles in the reaction solution. Thereby, it is possible to measure the concentration of the antigen or antibody in a large number of test liquids which may include the test liquid in which the concentration of the antigen or antibody is out of the measurement range of the reagent in a short time and with high accuracy. .

Therefore, it is possible to effectively prevent the following disadvantages of the conventional measuring method in which the concentration of the reagent is kept constant and the test liquid is operated to measure the test liquid outside the measurement range.

When it is necessary to dilute the test solution or to reduce the amount of the test solution, the test solution is wasted. If the test solution is small, remeasurement is impossible. It is difficult to select a diluent. There are problems such as errors caused by the measurement and errors in collecting a small amount of sample. When the concentration of the test solution or the amount of the test solution needs to be increased, denaturation during the concentration operation, the concentration operation itself is complicated and inferior in speed, the concentration of the interfering substance also increases in proportion to the concentration of the test solution in the reaction solution. Non-specific reactions other than those originally expected occur and increase abnormal values.

According to the method of the present invention, such a measurement can be carried out in a simple manner in which a reagent is prepared by changing the concentration of the immobilized carrier particles in the reaction solution in advance and a calibration curve is prepared. In particular, measurement suitable for mass rapid processing using an automatic analyzer becomes possible.

In other words, in the measurement method using an automatic analyzer, the initial measurement can be carried out simply by replacing a reagent bottle filled with reagents of different concentrations, for which a calibration curve has been independently prepared in advance. Thus, the concentration of the antigen or antibody can be measured without impairing the measurement accuracy and accuracy of the test solution outside the measurement range of the reagent.

[0054]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 (1) Preparation of C-Reactive Protein Measurement Reagent Polystyrene latex particles having an average particle size of 0.123 μm were treated with a Tris-HCl buffer (pH = 7.5, hereinafter abbreviated as Tris buffer). Dilute to prepare a suspension with a latex concentration of 1% by weight. Next, C-reactive protein (hereinafter referred to as “C
RP) is diluted with Tris buffer from an anti-CRP goat IgG fraction obtained by immunizing a goat with anti-CRP antiserum by salting out to prepare a solution having a protein concentration of 2 mg / ml. Anti-CRP goat Ig is added to one volume of the above latex suspension.
One volume of the G fraction is added and reacted at 37 ° C. for 2 hours. Further, bovine serum albumin was added at a final concentration of 0.05% by weight, followed by centrifugation. After removing the supernatant, the precipitate was redispersed in Tris buffer to adjust the latex concentration to 0.25%. ) Got.

On the other hand, 0.05 mol of sodium chloride was added to the Tris buffer.
/ L, a buffer solution (R1) containing bovine serum albumin at 0.5% by weight and choline chloride at 5% by weight was prepared.
A CRP measurement reagent consisting of R1 and R2 was obtained.

(2) Setting of initial measurement conditions CRP shows an abnormally high concentration exceeding 50 mg / dl depending on the individual and disease state, while 0.1 mg / dl is shown in a healthy person.
It is a protein antigen that is also distributed below. A TBA-80FR type automatic analyzer manufactured by Toshiba Corp. was used as an automatic analyzer, and the R1 of the CRP measurement reagent obtained above was analyzed in the apparatus by 37%.
300 μl was dispensed into an optical cell kept at a temperature of 30 ° C., 3 μl of a human serum was directly added as a test solution to the optical cell as a test solution, and the mixture was stirred and kept at 37 ° C. for about 5 minutes. After that, CR
50 μl of P measurement reagent R2 was added and stirred to start an agglutination reaction.

The above operation is automatically performed, and the wavelength 572 for about 90 seconds between about 30 seconds and 120 seconds after the start of the reaction.
The change in absorbance in nm was measured.

(3) Preparation of Calibration Curve and Measurement of Samples with Known CRP Concentrations Serum having a CRP concentration of 3.40 mg / dl and Tris buffer were each used as test liquids, and the measurement was performed five times under the above initial measurement conditions. As a result, the amounts of change in the absorbances of the serum having a CRP concentration of 3.40 mg / dl and the Tris buffer were 0.194 and 0.001, respectively, on average. From this value, the calibration curve was y = (x−0.001) /0.0568.

Next, the serum having a CRP concentration of 29.8 mg / dl was diluted with Tris buffer to give a CRP concentration of 0.14.
0.41, 1.24, 3.73, 5.60, 7.46,
Test liquids of 11.2, 14.9, 22.4, and 29.8 mg / dl were obtained and similarly measured under the first measurement conditions described above.

The average value, standard deviation,
And the standard deviation was divided by the mean to determine the coefficient of variation expressed as a percentage.

The average value obtained by substituting the average value into the above calibration curve was divided by the known concentration of each test solution to obtain an average recovery rate. Table 1 summarizes the obtained results.

From Table 1, it can be seen that the average recovery rate, which is excellent in accuracy, is 0.1%.
The concentration range of 95 to 1.05 is 0.41 to 16.8 mg /
dl, 0.14 and 22.4, 29.8 mg
/ Dl was out of the measurement range.

The range of the coefficient of variation with excellent accuracy within 5% is 0.41 mg / dl or more, and 0.14 mg / dl or more.
dl was out of the measurement range.

(4) Re-measurement conditions Latex test solution (R2) of CRP reagent obtained in (1) above
Was diluted 2.5-fold with Tris buffer to prepare R2 having a latex concentration of 0.10%. The measurement was performed in the same manner as in (3) except that the latex concentration during the reaction was changed.
A calibration curve Y = (X-0.002) /0.0253 was obtained.

As a result of changing the reagent to the one using R2, the recovery rate was 0.98 and the reproducibility was 4% for the sample having the CRP concentration of 0.14 mg / dl.
Up to 14 mg / dl.

[0067]

[Table 1]

Example 2 and Comparative Example 1 In an optical measurement method using immobilized carrier particles, rheumatoid factor (hereinafter abbreviated as RF) has reactivity with the immobilized antibody and is erroneously detected. It is known that high values may be shown.

Regarding five test solutions containing serum with an abnormally high RF value of an RF concentration of 1000 international units / ml or more, first, although the measurement principle is different from the method of the present invention, among the various CRP measurement methods, it is relatively low. Hoechst's "trade name:" uses the simple immunodiffusion method, which is hardly affected by interfering substances, as the measurement principle.
LC PARTIGEN CRP ", the CRP concentration was measured to be 0.27, 4.3, 1.15, 7.9, 10.3 respectively.
8 mg / dl was shown.

Table 2 shows the results of the measurement of the test liquid according to the method performed in Example 1.

On the other hand, in the above, with respect to the sample liquid out of the measurement range, measurement was carried out by the same operation as that using the same reagent as in the above-mentioned first measurement except that the amount of the test liquid was increased by 2.5 times. Also shown in Table 2 as Comparative Example 1.

In Example 1, the recovery rate of LC-partitigen was close to 1.00 for the strongly strongly positive test liquid of the test liquid out of the measurement range, indicating good accuracy. The value had a large error, and was considered to be affected by the interfering substance in the test solution.

[0073]

[Table 2]

Example 3 As a condition for re-measurement of a sample solution in which the CRP concentration in the test solution exceeded the CRP concentration of 14.9 mg / dl, which is the upper limit of the measurement in the first measurement condition, obtained in (1) of Example 1 above. The latex concentration of the CRP measurement reagent in the latex reagent solution (R2) was 2
It was adjusted twice to obtain R2 having a latex concentration of 0.50%.

Using the above-mentioned reagent using R1, the same procedure as in (3) of Example 1 was carried out except that the latex concentration and the measurement wavelength during the reaction were changed to 700 nm.
= (X-0.004) /0.0306.

As a result, the CRP concentrations were 22.4 and 2
The recovery rate for the test liquid of 9.8 mg / dl was 0.99 and 0.97, the reproducibility was 1.0 and 0.9%, and the upper limit of the measurement range was up to 29.8 mg / dl.

As a more specific embodiment of the above method, an experiment was performed using 200 test liquids for which outpatients of a hospital were requested to measure CRP.

First, when the measurement was carried out under the initial measurement conditions of Example 1, 9 test solutions exceeded the upper limit of measurement of 14.9 mg / dl. In the first embodiment, a sample cup containing a test solution exceeding the upper limit of measurement is collected, and a reagent bottle with a changed concentration is set in an automatic analyzer, and the measurement can be easily performed again by simply performing the measurement again. Was.

On the other hand, when the conventional method for diluting a test liquid was attempted, a large amount of the test liquid was required to be applied to a diluting apparatus, and the remaining sample contained 90 μl and 110 μl.
There were test liquids as small as possible, and these two test liquids had little residual liquid of the original test liquid, so that other additional measurements could not be performed.

Example 4, Comparative Example 2 (1) Preparation of reagent for measuring syphilis TP antibody 1 × 10 ⁹ / ml dispersion of syphilis antigen, Treponema pallidum (hereinafter abbreviated as TP), and 1% n- An equal amount of octyl-β-D-glucoside aqueous solution was mixed and incubated at room temperature for 1 hour.
After centrifugation for 0 minutes, a supernatant containing the antigen was obtained. This supernatant was diluted 20-fold with a 0.02 M phosphate buffer (pH = 7.2) to obtain a sensitizing antigen.

A polystyrene latex particle having an average particle diameter of 0.154 μm was added to a 0.02 M phosphate buffer (pH = 7.
2, hereinafter abbreviated as phosphate buffer. ) To prepare a suspension having a latex concentration of 1% by weight. Next, 1 volume of the sensitizing antigen solution is added to 1 volume of the above latex suspension and reacted at 37 ° C. for 2 hours. Further, bovine serum albumin was added at a final concentration of 0.05% by weight, followed by centrifugation. After removing the supernatant, the precipitate was redispersed in a phosphate buffer, and centrifuged again to remove the supernatant. The dispersion was redispersed in a phosphate buffer to adjust the latex concentration to 0.20% to obtain a reagent latex reagent solution (R2).

Separately, a phosphate buffer solution containing 0.05% salt was added.
mol / l, bovine serum albumin was added at 0.5% by weight and choline chloride at 5% by weight.
1) was prepared, and a syphilis TP antibody measurement reagent consisting of R1 and R2 was obtained.

(2) Initial Measurement of Individual Test Solution As an automatic analyzer, a fully automatic immunochemical analyzer 501X manufactured by Analytical Instruments Co., Ltd. was used. , R2 were set.

The initial measurement conditions were as follows: the test solution was 15 μl, R1 was 300 μl, and R2 was 50 μl. In order to create a calibration curve, a syphilis TP antibody standard solution (500 units / ml) obtained by valuing with a WHO standard was set at a predetermined position, and a two-fold dilution series was automatically created, and a wavelength of 660 nm was obtained. The scattered light intensity was measured. The test solution was diluted with R1, kept at 37 ° C. for about 4 minutes, and then added with R2 and stirred.
The increase in the optical density at two specific times (150 seconds and 375 seconds) was determined from the data measured at seconds, 195 seconds, and 420 seconds. Using the increase in optical density between 45 and 420 seconds after stirring as the first calibration curve to measure the low concentration range, and using the increase in optical density between 45 and 195 seconds after stirring as the second calibration curve Each was approximated by a third-order polynomial to obtain two calibration curves.

First calibration curve Y = 1.097 × 10 ⁻⁹ X ³ −5.694
^{× 10- 6 X 2 + 4.283 ×} 10 -2 X-2.813 second calibration curve ^{Y = 2.683 × 10 -9 X 3} -4.313 × 10- 6 X 2 +0.
1595X-21.96 The lower limit of measurement was 10 units / unit based on the results of the reproducibility test.
ml. Here, two calibration curves are used in combination, but from 0 to 100 units / ml, the first calibration curve is used.
The threshold value was set so that the calibration was performed using the second calibration curve from 100 to 500 units / ml.

[0086] The test solution of a patient clinically diagnosed as syphilis negative is a test method which is different in principle from the method of the present invention, and is generally used as a TPHA reagent manufactured by Fujirebio "Trade name: 5 determined to be suspended (positive to antibody titer × 40, ± 80 for × 80)
The test solution was measured using the syphilis TP antibody measurement reagent obtained in (1). Table 3 shows the above measurement results.

Next, among the initial measurement conditions, the R2
A calibration curve was prepared in the same manner as the initial measurement conditions except that the concentration of the immobilized carrier particles in the medium was reduced by half, and the measurement was performed again.

On the other hand, as a comparative example,
Remeasurement was carried out in the same manner as the initial measurement conditions except that the amount of the test solution was doubled among the initial measurement conditions.

Table 3 shows the results.

The concentration of the immobilized carrier particles in the reaction solution under the initial measurement conditions was 0.027%, and the concentration of the immobilized carrier particles in the comparative example was 0.026%. It can be handled as the same concentration.

From the results shown in Table 3, the results of the first measurement and the comparative example in the test liquids 1, 2, and 4 were significantly different from those of the negative examples in which the determination was suspended by the cellodia TP. I found it to be.

Although the cause is unknown, it is considered that the interference component in the test solution has acted critically to cause a non-specific reaction.

On the other hand, in the example, a measured value well corresponding to the first measurement can be obtained, and it can be determined that the test liquid having the first measurement lower limit or less can be measured.

[0094]

[Table 3]

Example 5 A latex test solution (R) of the syphilis reagent obtained in Example 4 (1)
2) 4-fold dilution with water to make latex concentration 0.05%
In the same manner as in Example 3, R1 and the dilution R2 were set in a fully automatic immunochemical analyzer 501X. A calibration curve was prepared in the same manner as in Example 4 except that the test solution was 15 µl, R1 was 300 µl, and R2 was 50 µl. However, Example 4
Since the tendency for the rise of the reaction to be delayed was recognized as compared with, the time course was observed in detail, and a specific time interval at which the optical density monotonously increased was determined. That is, unlike Example 4, R2 was added and stirred, and 105 seconds after stirring, 255
Seconds, two types of specific times (1
Two calibration curves were obtained to determine the increase in optical density between (50 seconds and 375 seconds).

First calibration curve Y = 2.463 × 10 ⁻⁹ X ³ −7.695
× 10 ⁻⁶ X ² + 0.1565X−0.048 Second calibration curve Y = 8.094 × 10 ⁻⁹ X ³ −3.311 × 10 ⁻⁶ X ² +0.
2591X-45.14 The measurement range was 3 to 150 units / ml.

The above measurement conditions were measured as the initial measurement conditions for 100 cases of sera collected from inpatients and outpatients such as pregnant women and stomach examinations. As a result, 96 cases were less than 3 units / ml and 4 cases were remaining. Was greater than 4, 7, 58 and 150 units / ml.

From among these, 10 of the positive test liquids which were separately determined to be clinically positive under the initial measurement conditions of Example 4
Two cases which were higher than the lowest value of 12 units / ml and considered positive for 0 cases were measured using the initial measurement conditions of Example 4 (latex concentration 0.20%) as the re-measurement conditions.

As a result, 60 units / ml, 47
It became 3 units / ml. These two test liquids were also positive for cellodia TP, and subsequent investigations revealed that they had been infected with syphilis in the past but had already been cured, but they had retained the anti-TP antibody.

Claims (2)

(57) [Claims] 1. A reagent comprising a dispersion of insoluble carrier particles having an antibody or antigen immobilized thereon is reacted with a large number of test solutions, and the antigen in the test solution is determined by the state of aggregation of the insoluble carrier particles in the reaction solution. Alternatively , in a method for measuring the concentration of an antibody, a reagent in which the specific concentration of insoluble carrier particles has been reduced in advance.
Measure all of the test solution and measure the value
After confirming that most of the test solution is below the value,
Reagents with specific concentrations for remaining test liquids that are above the reference value
A method for measuring the concentration of an antigen or an antibody, wherein the measurement is carried out again . 2. A reagent having a specific concentration reduced to 2/3 or less.
The method according to claim 1, which is used .