JPH0843393A - Immunological measuring method and analyzing device - Google Patents

Abstract

PURPOSE:To make a wide-range, high precision measurement by measuring the turbidity change mount due to reaction of the component to be measured with a reaction component using a plurality of wavelengths differing from one another, preparing standard curve for each measuring wavelength, and using the measurements which have undergone a concentration conversion with the short wavelength side standard curve in the case of a low concentration specimen and with the long wavelength side standard curve in the case of a high concentration specimen. CONSTITUTION:The main part of a device is configured with a sample erection supply part 1, sampling mechanism 2, reaction cell disc 3, reagent supply part 4, and analyzing optical system 6. The optical system 6 may be of any commercially available sort, and beams of light having different wavelengths are fed from a light source device 7 and cast on a specimen placed in a reaction cell 28, and the transmitted light is reflected by a non-abberation concave diffraction grating 26 and received by a sensor 27, and therefrom a turbidity signal is forwarded to a plurality of standard curve preparing circuits 9. The circuits 9 prepare a standard curve for measuring low concentration using a turbidity signal with short wavelength and a high concentration standard curve using a turbidity signal due to long wavelength, and a concentration calculating circuit 10 makes conversion into the concentration of the specimen by the use of these standard curves and selects concentration signal and forwards it as output 11.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunological measuring method and analyzer.

[0002]

2. Description of the Related Art Recently, as a method capable of quantitatively measuring a target component with high sensitivity, a latex nephelometry (L
-TIA) is becoming popular.

L-TIA uses a latex agglutination measuring device or a biochemical automatic analyzer, and a predetermined amount of a sample in a sample cup placed in a sample rack is placed in a reaction optical cell and a predetermined amount of a buffer solution is added thereto at 37 ° C. Incubate for 5 minutes,
Add a certain amount of latex reagent, stir, and incubate for another 5 minutes at 37 ° C. Measure the absorbance immediately after adding latex reagent and the absorbance 5 minutes after adding latex reagent to obtain the amount of change in absorbance, and use the standard solution in advance. In general, the concentration of the component to be measured in the sample is determined from a standard curve obtained by the same operation.

The measuring range is determined according to the concentrations of the components to be measured, and when the measuring range is a low concentration of ng / ml to μg / ml, a highly sensitive latex reagent is required,
While latex particles having a relatively large particle size are used, low sensitivity latex reagents are required when the measurement range is high sensitivity of μg / ml to mg / ml, and latex particles having a relatively small particle size are used. It is generally used. As the measurement wavelength, a relatively long wavelength of 570 to 800 nm is selected for a latex reagent having a large particle size, and a relatively short wavelength of 340 to 500 nm is selected for a latex reagent having a small particle size.

In recent years, with the progress of medical treatment, it has been desired to improve the accuracy of measurement of in vitro diagnostic agents. For example, in the case of measuring C-reactive protein (CRP) in serum, 0 mg / dl to 50 mg.
Since the concentration range of / dl and the sample is extremely wide, there is a demand for a measurement reagent that has high sensitivity and does not cause a zone phenomenon in the high concentration range.

[0006]

However, when a latex reagent is used to increase the sensitivity so that CRP can be measured up to 0.1 mg / dl, latex particles having a large particle size are used. dl
When it becomes above, the reaction amount increases remarkably and the difference in absorbance exceeds the measurable range of the automatic analyzer.

Alternatively, when the CRP is 10 mg / dl or more, a zone phenomenon occurs, and the latex agglutination reaction becomes difficult to occur, and the difference in absorbance is reduced, and an accurate measured value cannot be obtained.

In order to extend the measuring range, latex reagents having a small particle size are used, and by shortening the measuring wavelength, a latex reagent which can be measured with high sensitivity and has a wide measuring range has been developed and is becoming popular. , IgG in serum, I
When measuring a component such as gA which is present at a higher concentration than CRP, a zone phenomenon occurs.

In order to avoid this zone phenomenon, a large amount of latex reagent having a small particle size is added to the reaction system and the measurement wavelength is lengthened to enable measurement of high concentration components.
By increasing the measurement wavelength, the sensitivity on the low concentration side is reduced. If the wavelength is shortened to increase the sensitivity in order to prevent a decrease in the sensitivity, the reagent absorbance becomes too high and approaches the absorbance measurement limit of the automatic analyzer, making it impossible to measure in the high concentration region.

[0010]

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned conventional problems, and comprises a component to be measured by an immunological reaction, a component that reacts with the component to be measured, and / Or the reaction with the carrier particles insolubilized components to react, by measuring a plurality of wavelengths different from each other, the turbidity change amount after reaction for a certain period of time is measured, and a standard curve is prepared for each measurement wavelength, and at least one of them is prepared. An immunological measurement method characterized by measuring a component to be measured in a sample from one standard curve, and a sample erection supply unit, a sampling mechanism, a reaction cell, a first reagent supply unit, and / Or, in an analyzer comprising a second reagent supply unit, a stirring mechanism, and an analysis optical system, a device for irradiating light beams having a plurality of different wavelengths, a light receiving unit, and each measurement wavelength in the light receiving unit A standard curve creation circuit that creates a standard curve from the turbidity signal of the obtained standard substance, and a standard curve created by the standard curve creation circuit from the turbidity signal of each measurement wavelength of the sample are to be used for measurement. According to another aspect of the present invention, there is provided an analyzer, comprising: a concentration calculation circuit for calculating the concentrations of the respective components and a selection output circuit for selecting and outputting concentration signals obtained from a plurality of wavelengths.

The immunological reaction applicable here includes an antigen-antibody reaction, a complement fixation reaction, a modified IgG-RF reaction and the like.

The component to be measured refers to any of the reaction components involved in the immunological reaction, and includes antigens, antibodies, complements, RF and the like.

The component reacting with the component to be measured refers to the other of the reaction components involved in the immunological reaction, and for example, when the component to be measured is an antigen, it becomes an antibody. When the reaction component is an antibody, either a polyclonal antibody or a monoclonal antibody can be used, and either a divalent antibody or a monovalent antibody may be used.

As the carrier particles, latex particles, metal colloid particles and the like are advantageous in terms of reactivity and dispersion stability. The production of finely divided carrier particles requires a high degree of skill, but those commercially available these days can be used. When the content of the component to be measured is high, the smaller the particle size is, the lower the reagent blank becomes, and it becomes possible to add a large amount to the reaction system. Can be

The carrier particles may have a particle size of 1.6 μm or less, but it is possible to combine a plurality of carrier particles having a particle size of 1.6 μm or less, for example, an average particle size of 0.10 μm.
It is advisable to combine carrier particles of m or more with carrier particles of 0.10 μm or less. Further, three or more kinds of carrier particles having different average particle diameters may be combined.

As a method for insolubilizing one component of the immunological reaction in the carrier particles, a general physical adsorption method or a chemical binding method using a water-soluble carbodiimide or a bifunctional reagent is used.

As the sample containing the component to be measured, there are used body fluids such as serum, plasma, blood, urine, ascites, pleural effusion, and cerebrospinal fluid, or culture fluids and extracts of cells and tissues.

As a means for detecting a reaction with a sample containing a component to be measured, a generally used optical detecting means is used. That is, the amount of change in turbidity after reaction for a certain period of time is measured as the amount of increase or decrease in absorbance and / or scattered light intensity at a wavelength of 0.3 to 2.4 μm. In order to increase the measurement sensitivity, it is advantageous to use a shorter measurement wavelength, but increasing the particle size of the latex increases the measurement sensitivity but also increases the reagent blank, so for example, the upper limit of the absorbance measurement of a general biochemical automatic analyzer Is 2.0 to 3.0, it is preferable to lengthen the wavelength so as not to exceed the upper limit of measurement by the optical detecting means. In addition, since the content of the component to be measured is high, it is preferable that the wavelength is long even when a wide measurement range is desired. A long wavelength of 1.3 times or more of a short wavelength is used.

A low-concentration measurement standard curve is created by handwriting or computer by the turbidity change amount measured by the short wavelength, and a high-concentration measurement standard curve is similarly created by the turbidity change amount measured by the long wavelength. Concentration conversion is performed by the turbidity change amount of the component to be measured, which is measured by the wavelength of.

When the standard curve on the low concentration side is prepared, the difference in the measured turbidity between the main wavelength and the sub wavelength may be used with the short wavelength as the main wavelength and the long wavelength as the sub wavelength.

Of the measured values of the component to be measured calculated from a plurality of standard curves, the higher value is selected and output.

The amount of change in turbidity after reaction for a certain period of time may be measured before or after addition of a component that reacts with the component to be measured and / or carrier particles insolubilized with the component that reacts. It may be measured later.

Also, the device of the present invention is

FIG. 6 and

As shown in FIG. 7, the sample erection supply unit 1 and
Irradiation with light beams having a plurality of different wavelengths in an analyzer including a sampling mechanism 2, a reaction cell disk 3, a first reagent and second reagent supply unit 4, a stirring mechanism 5, and an analysis optical system 6. Light source device 7 and light receiving unit 8
And a standard curve creating circuit 9 for creating a standard curve from the turbidity signal of the standard substance obtained at each measurement wavelength in the light receiving unit 8, and the standard curve creating circuit from the turbidity signal at each measurement wavelength of the sample. Concentration calculation circuit 10 for calculating the concentrations of the components to be measured using the standard curves created in 9 respectively.
And a selection output circuit 11 for selecting and outputting the density signals obtained from a plurality of wavelengths. In the figure, 12 is a reagent pipetting mechanism, 13 is a sample dispensing mechanism, 14 is a washing mechanism, 15 is a wash water pump, 16 is a reagent dispensing mechanism, 17 is a constant temperature bath, 18 is a sample cup, 19 is log conversion and A
A / D converter, 20 is a microcomputer, 21 is a printer, 22 is a CRT, 23 is a floppy disk device, 24 is an operation panel, and 25 is an interface.

[0024]

FIG. 7 shows a light source device 7 for irradiating light beams having a plurality of different wavelengths, and an aplanatic concave diffraction grating 26.
For the light receiving unit 8 including the detector 27, a device widely used at present is used as it is, and a known standard curve is created by creating a standard curve from the turbidity signal of the standard substance obtained for each measurement wavelength in the light receiving unit. A plurality of circuits 9 are provided.

The turbidity signal input to the standard curve forming circuit 9 measures the turbidity of the mixed solution of the sample and the first reagent in the reaction cell 28 at different wavelengths at regular intervals, and further measures the second reagent. The turbidity signal is used, in which the turbidity is measured at regular intervals after addition and stirring.

Although the standard curve for low concentration measurement uses the turbidity signal of a short wavelength, the difference between the turbidity signals of long and short wavelengths may be used with the short wavelength as the main wavelength and the long wavelength as the sub wavelength. Further, the standard curve for low concentration measurement may use the turbidity change amount after the addition of the second reagent.

Further, each is provided with a concentration calculation circuit 10 for calculating the concentration of the component to be measured from the turbidity signal for each measurement wavelength of the sample using the standard curve created by the standard curve creation circuit 9.

The density calculation circuit 10 can use a known straight line or non-linear standard curve calculation formula, and log logit or spline can be used as the non-linear type.

A selection output circuit 11 is provided which outputs the concentration signals from the concentration calculation circuit 10 as they are for each wavelength or selects the higher one and outputs it.

[0030]

[Function] A component to be measured by an immunological reaction,
When the reaction with the component to be measured and / or the reaction with the carrier particles in which the reactive component is insolubilized is measured by the analysis optical system 6 in the reaction cell 28 at a plurality of different wavelengths, a short wavelength The measured absorbance difference becomes large in the low concentration region of the component to be measured,
Higher detection sensitivity. Further, the difference in absorbance measured by the long wavelength becomes small in the low concentration region of the component to be measured, but becomes large in the high concentration region, and the measurement accuracy is good.

Therefore, when measuring the concentration of the component to be measured in the sample, high sensitivity measurement can be performed by using the standard curve by short wavelength measurement at low concentration, and long wavelength measurement at high concentration. By using the standard curve according to, it is possible to reliably measure high-value samples.

By making a standard curve using the difference in absorbance before and after the addition of the second reagent during the long-wavelength measurement, it is possible to measure a higher concentration. This is because the turbidity change amount is generally the largest immediately after the addition of the second reagent, and the turbidity change amount decreases with the passage of time. By measuring the turbidity change amount before and after the addition, a larger turbidity change amount can be obtained and the accuracy is improved.

When the amount of change in turbidity after the addition of the second reagent is to be measured, the stirring operation by the stirring mechanism 5 is performed in a short period of time, so the change in turbidity at the initial stage of the reaction is not used. The turbidity change remarkably occurs, and the turbidity change amount used for the calculation may not necessarily be large. Therefore, the density value calculated by the density calculation circuit 10 may be lower than the actual value.

However, in the case of measuring the turbidity change amount before and after the addition of the second reagent, all the turbidity change amount generated by the antigen-antibody reaction can be used for calculation, which is advantageous for the measurement of high-value samples.

Although the above description is for the case where the turbidity change amount increases in proportion to the reaction time, the same measurement can be performed when the turbidity change amount decreases in proportion to the reaction time. .

[0036]

【Example】

Example 1 A goat anti-CRP antibody is physically adsorbed on a commercially available polystyrene latex particle having a particle size of 0.05 μm by a conventional method for sensitization. 0.2M Glycine-N with 0.1% BSA
The sensitized latex reagent was obtained by floating with an aCl buffer (pH 7.2).

0.1 M glycine containing 0.1% BSA
C in 300 μl of NaCl buffer (used as reaction buffer)
The RP standard solution (5 μl) was added, and then the sensitized latex reagent was added, and the absorbance at 0.34 μm and 0.7 μm at 1 minute and 5 minutes after the start of the reaction was measured to determine the difference in absorbance.

The result is

As shown in FIG. 1, a highly sensitive standard curve of CRP 0 to 5 mg / dl is obtained at a measurement wavelength of 0.34 μm, and a wide range of standard curve of 0 to 20 mg / dl is obtained at a measurement wavelength of 0.7 μm. Was obtained.

Samples having a CRP concentration of 0 to 5 mg / dl were subjected to concentration conversion by a standard curve for low concentration by short wavelength measurement,
For samples up to 5 to 20 mg / dl, the concentration was converted by the standard curve for high concentration by long-wavelength measurement and the concentration was
L1 = 0.06 mg / dl, L2 = 0.22 mg / d
1, M1 = 3.22 mg / dl, M2 = 5.36 mg /
dl, H1 = 12.87 mg / dl, H2 = 18.49
It was mg / dl.

Example 2 Using the anti-CRP antibody-sensitized latex reagent prepared in Example 1, the absorbances at 0.34 and 0.70 μm were measured in the same manner as in Example 1, and a standard curve for low concentration was obtained. It was prepared from a turbidity signal obtained by subtracting the absorbance difference of the sub wavelength from the absorbance difference of the main wavelength with 0.34 μm as the main wavelength and 0.70 μm as the sub wavelength.

Reaction buffer 300 μl, CRP standard solution 5
μl and 50 μl of the sensitized latex reagent were reacted in the same manner as in Example 1 to determine the difference in absorbance.

The result is

2 is as shown in FIG.

Example 3 Using the anti-CRP antibody-sensitized latex reagent prepared in Example 1 above, the absorbance at 0.34 and 0.70 μm was measured at intervals of about 20 seconds after adding the first reagent to the sample. The measurement was performed for 5 minutes, and after the second reagent was added, the measurement was performed for 5 minutes at the same intervals.

The low-concentration standard curve was prepared by the difference in absorbance due to the difference between the main wavelength (0.34 μm) and the sub-wavelength (0.70 μm) after the addition of the second reagent, as in Example 2, and the high-concentration side was obtained. A standard curve was constructed using the long wavelength of 0.70 μm and the difference in absorbance before and 5 minutes after the addition of the second reagent.

The result is

[Fig. 3] As shown in Fig. 3, CR is measured by measurement on the short wavelength side.
A highly sensitive standard curve from P0 to 5.0 mg / dl was obtained, and a wide range standard curve from CRP0 to 20 mg / dl was obtained by measurement on the long wavelength side.

Example 4 Commercially available polystyrene latex particles having a particle size of 0.22 μm were sensitized with a goat anti-CRP antibody in the same manner as in Example 1 to obtain a sensitized latex reagent.

3 μl of CRP standard solution was added to 300 μl of reaction buffer, and then 50 μl of the sensitized latex reagent was added.
In addition, 0.57 μm and 0.80 for 1 minute and 5 minutes after starting the reaction
The absorbance at μm was measured to obtain the difference in absorbance.

The result is

[Fig. 4] As shown in Fig. 4, CR is obtained by measurement on the short wavelength side.
A highly sensitive standard curve from P0 to 10 mg / dl was obtained, and a wide range standard curve from CRP0 to 30 mg / dl was obtained by measurement on the long wavelength side.

Example 5 Commercially available polystyrene latex particles having a particle size of 0.1 μm were sensitized with goat anti-β2 microglobulin antibody in the same manner as in Example 1 to obtain a sensitized latex reagent.

To the reaction buffer (300 μl), β2 microglobulin standard solution (3 μl) was added, and then the sensitized latex reagent (50 μl) was added.
The absorbances at μm and 0.57 μm were measured, and the difference in absorbance was determined.

The result is

[Fig. 5] As shown in Fig. 5, β2 is measured by measurement on the short wavelength side.
A highly sensitive standard curve of 0 to 10 mg / l of microglobulin was obtained, and a wide range of standard curve of 0 to 50 mg / l of β2 microglobulin was obtained by measurement on the long wavelength side.

[0052]

As described above, according to the present invention, the following various excellent effects can be obtained.

1. According to the method of the present invention, a reaction between a component to be measured by an immunological reaction and a component or the like that reacts with the component to be measured is measured by measuring the turbidity change amount at each of a plurality of wavelengths, and measuring each measurement. Since a standard curve is created for each wavelength, low-concentration samples can be measured with a concentration converted from the short-wavelength side standard curve, allowing high-sensitivity measurement, and high-concentration samples can be converted into concentration with a long-wavelength side standard curve. Since the measured value can be adopted, high concentration can be measured with high accuracy.

2. By creating the short-wavelength side standard curve by the difference between the short-wavelength measured turbidity and the long-wavelength measured turbidity, the low-concentration sample can be measured more accurately.

3. By measuring the amount of change in turbidity after reaction for a certain period of time after adding a component that reacts with the component to be measured and / or carrier particles insolubilized with the component that reacts, the measurement range on the high concentration side is further increased. Can be expanded up to.

4. According to the analyzer of the present invention, an automatic analyzer which has been widely used conventionally is provided with a standard curve creating circuit and a concentration calculating circuit for each of a plurality of wavelengths, and selectively outputs a concentration signal obtained from a plurality of wavelengths. As a result, the low-concentration sample can be measured with high sensitivity, and the high-concentration sample can reduce the negative conversion due to the excess of the component to be measured.

5. According to the reagent and the apparatus of the present invention, the measurement range is remarkably expanded, so that the number of dilution re-measurements is reduced and the measurement time and the reagent cost are reduced.

[Brief description of drawings]

FIG. 1 is an explanatory diagram showing an effect of the present invention.

FIG. 2 is an explanatory diagram showing the effect of the present invention.

FIG. 3 is an explanatory diagram showing the effect of the present invention.

FIG. 4 is an explanatory diagram showing the effect of the present invention.

FIG. 5 is an explanatory diagram showing the effect of the present invention.

FIG. 6 is an explanatory diagram showing an example of an analyzer of the present invention.

FIG. 7 is an explanatory diagram of an analysis optical system in the analysis apparatus of the present invention.

[Explanation of symbols]

 1 Sample installation supply unit 2 Sampling mechanism 3 Cell disk for reaction 6 Optical system for analysis 7 Light source device 8 Light receiving unit 9 Standard curve creation circuit 10 Concentration calculation circuit 11 Selective output circuit

Claims (20)

[Claims] 1. A reaction between a component to be measured by an immunological reaction, a component that reacts with the component to be measured, and / or carrier particles in which the component that reacts is insolubilized by a plurality of wavelengths different from each other. , Measure the turbidity change amount after reaction for a fixed time, create a standard curve for each measurement wavelength,
An immunological measuring method, which comprises measuring a component to be measured in a sample from at least one standard curve. 2. The immunological measuring method according to claim 1, wherein the particle size of the carrier particles is 1.6 μm or less. 3. The immunological measurement method according to claim 1, wherein the measurement wavelength is selected from wavelengths of 2.4 μm or less. 4. The immunological measuring method according to claim 1, wherein at least one of visible light, near infrared light and laser light is used. 5. The immunological measuring method according to claim 1, wherein the plurality of different wavelengths are two or more kinds of wavelengths different from each other by at least 1.3 times or more. 6. The immunological measurement method according to claim 1, wherein the turbidity change amount is measured by absorbance and / or scattered light intensity. 7. The immunological measuring method according to claim 1, wherein the low-concentration region of the component to be measured is converted into concentration by a standard curve on the short wavelength side, and the high concentration region is converted into concentration by a standard curve on the long wavelength side. . 8. The immunological measuring method according to claim 1, wherein the standard curve on the short wavelength side is prepared by the difference between the short wavelength measured turbidity and the long wavelength measured turbidity. 9. The immunology according to claim 1, wherein the higher measured value among the measured values of the component to be measured calculated from a plurality of standard curves is used as the measured value of the component to be measured. Measurement method. 10. A carrier particle having a particle size of 0.1 μm or less, in which a component that reacts with the component to be measured is insolubilized according to the amount of the component to be measured by an immunological reaction, and the component to be measured. A plurality of wavelengths different from each other by combining a component that reacts with a component that reacts with at least one of carrier particles having a particle size of 0.1 μm or more insolubilized with the component that reacts with a sample containing the component to be measured. Immunoassay characterized by measuring the amount of change in turbidity after reaction for a certain period of time and measuring the component to be measured in the sample from at least one of the standard curves for each measurement wavelength. Method. 11. The immunological measurement method according to claim 9, wherein a plurality of types of carrier particles having a particle diameter of 0.1 μm or less are combined. 12. A combination of carrier particles having a particle diameter of 0.1 μm or less and carrier particles having a particle diameter of 0.05 μm or less.
1. The immunological measurement method described in 1. 13. The immunological measurement method according to claim 10, 11 or 12, wherein the amount of change in turbidity after reaction for a certain period of time is optically measured. 14. The immunological measurement method according to claim 12, wherein an increase in absorbance and / or scattered light intensity at a wavelength of 0.3 to 2.4 μm is measured. 15. The immunity according to claim 1, wherein the amount of change in turbidity after reaction for a certain period of time is measured before and after adding a component that reacts with a component to be measured and / or carrier particles insolubilized with the component that reacts. Measurement method. 16. The immunology according to claim 1, wherein the amount of change in turbidity after reaction for a certain period of time is measured after adding a component that reacts with a component to be measured and / or carrier particles insolubilized with the component that reacts. Measurement method. 17. The standard curve measured before and after the addition according to claim 15 is used as a high-concentration measurement standard curve, and the standard curve measured after the addition according to claim 16 is created as a standard curve for low-concentration measurement. The immunological measuring method according to claim 1. 18. An analyzer comprising a sample erection supply unit, a sampling mechanism, a reaction cell, a first reagent supply unit and / or a second reagent supply unit, a stirring mechanism, and an analysis optical system, A device for irradiating light beams having a plurality of different wavelengths, a light receiving unit, a standard curve creating circuit for creating a standard curve from the turbidity signal of the standard substance obtained for each measurement wavelength in the light receiving unit, and each of the samples From the turbidity signal for each measurement wavelength, the concentration calculation circuit that calculates the concentration of each component to be measured using the standard curve created by the standard curve creation circuit, and the concentration signal obtained from multiple wavelengths are selected. An analysis device comprising a selective output circuit for outputting. 19. The analysis optical system comprises visible light, near infrared light,
2. At least one of laser beams.
8. The analyzer according to item 8. 20. The analysis optical system is an integrating sphere.
8. The analyzer according to item 8.