JPS60100764A - Method for measuring concentration of antigen or antibody - Google Patents

Abstract

PURPOSE:To enable automation of a nephelometry or a turbidimetry utilizing an antibody antigen reaction by making the calibration curve with which the concn. of the antigen or antibody, scattering intensity and absorbancy attain a linear relation only with the calibrator for a concn. rate and the known concn. at an optional one point. CONSTITUTION:A sample vessel 44 is supplied to a sampling position and a specified amt. of serum is held in a probe 41 by a pipetter 40, is transferred to a discharge position 45 and is discharged into a reaction vessel 22. The vessel 22 passes through a luminous flux 26 during rotation of a disc 21. The output from spectroscope 27 is taken into a central processing unit 50 via an A/D converter 54. The unit 50 detects the concn. of an antigen or antibody by utilizing the calibration curve which is formed only from the calibrator of a concn. rate and the known concn. at optional one point and at which the concn. of the antigen or antibody, scattering intensity and absorbancy attain a linear relation.

Description

[Detailed Description of the Invention] [Field of Application of the Invention] The present invention relates to serum immunoprecipitation carried out by measuring the concentration of an antigen or antibody in a sample through an antigen-antibody reaction, and in particular to a method for use in an automatic analyzer. The present invention relates to a method for measuring antigen or antibody concentration suitable for.

[Background of the invention]

In general, when the reaction between an antigen and an antibody in a solution is expressed by the relationship between the antigen concentration and the concentration of the immunoconjugate produced, that is, the scattering intensity by the nephelometric method or the absorbance by the nephelometric method, when the antibody concentration is kept constant, It becomes a sigmoid curve as shown in Figure 1. According to FIG. 1, when the antigen concentration is extremely low as in region A shown in FIG. 1, the rate at which large immune complex molecules are produced, that is, the rate at which scattering intensity or absorbance increases is low. This is because the antibody concentration is excessive compared to the antigen preparation, resulting in an antibody excess region where it is difficult to form macromolecules. When the antigen concentration increases to a certain extent as shown in region B in Figure 1, a macromolecular immune complex is generated in proportion to the antigen concentration, and the scattering intensity or absorbance increases linearly. Therefore, this region is the optimal region in which the antigen concentration in the sample can be accurately measured from the measured values of scattering intensity or absorbance. Furthermore, when the antigen concentration approaches the antibody concentration, the rate of production of macromolecules decreases again, and the rate of increase in scattering intensity or absorbance slows down as shown in region C in FIG. Further, in the so-called antigen excess region where the antigen concentration is higher than the antibody concentration, as shown in the region shown in FIG. 1, as the antigen concentration increases, the amount of macromolecules produced decreases, and the scattering intensity or absorbance decreases. This is a well-known phenomenon called the prozone phenomenon.

When measuring antibodies in a so-called sample in which the antigen concentration is kept constant and the antibody concentration is varied with l-, results similar to those for the antigen shown in FIG. 1 can be expected.

In other words, when measuring an antigen (or antibody), if the concentration of the antibody (or antigen) in the reagent is made sufficiently large, it is possible to move the upper limit of the measurement target range to the above-mentioned optimal region shown in Figure 1. It is. However, as the antibody concentration (or antigen concentration) increases, the area in which the calibration curve at low concentrations of the measuring object draws a curve, as shown in FIG. 1, expands.

Therefore, only the optimum region (region C shown in FIG. 1) moves. Therefore, in conventional serum immunoassays using the nephelometric method or nephelometric method, which utilize antigen-substance reactions, the calibration curve is not a straight line in most cases.

FIG. 2 shows an example of a calibration curve for measuring transferrin with a laser nephrometer.

That is, the relationship between the scattering intensity and the concentration of transferrin (antigen) to be measured exhibits a typical sigmoid curve in the measurement range of transferrin from 0 to 1000 mg/dt.

Therefore, with a laser nephelometer, 5 to 6 calibrators with known concentrations are measured, and each calibrator is
A calibration curve is determined by connecting the curves with a polygonal line or by performing curve regression using LOG-LOGIT or a cubic polynomial.

Therefore, in such conventional methods for measuring antigen-antibody reactions, a large number of calibrators with known concentrations are required in order to establish an accurate calibration curve, and the measurement required to establish the calibration curve requires a large amount of effort. This method has the drawback of not being able to automate the nephelometric method or nephelometric method, which utilizes antigen-antibody reactions, because it takes time and the processing of measurement data is complicated.

[Purpose of the invention]

An object of the present invention is to provide a method for measuring the concentration of an antigen or antibody that can automate the nephelometric method or nephelometry that utilizes antigen-antibody reactions.

[Summary of the invention]

The present invention is a comparative method that utilizes the antigen-antibody reaction by creating a calibration curve in which the antigen or antibody concentration has a linear relationship with the scattering intensity and absorbance using only the concentration O (zero) and a calibrator whose concentration is known at one arbitrary point. The idea is to automate the law and turbidimetry.

[Embodiments of the invention]

Examples of the present invention will be described below.

Almost all reagents for nephelometric or turbidimetric methods currently on the market have a sigmoidal curve as shown in FIG. 3 in the measurement target range. That is, the curvature shown in Figure 3 A, which is near the upper limit of the measurement target range, can be resolved by increasing the concentration of antibody (or antigen) in the reagent. Increasing the concentration of antibody (or antigen) in the reagent shown in FIG.
As shown by bw+, it is possible to correct the curvature shown in A of FIG. 3 near the upper limit of the measurement target range. In the meantime,
As is clear from the comparison between Figures 3 and 4, the curve near the lower limit of the measurement range shown in Figure 3B also applies to the antibody in the reagent (
(or antigen) is enlarged as shown in FIG. 4B. On the other hand, since the antigen-antibody reaction depends on the ratio of the two concentrations, the width of the optimal region where the line becomes a straight line expands as the antibody (or antigen) in the reagent increases. The enlargement is the lower part of the measurement target range shown in Figure 3B.
ift (much larger than the expansion of the bend near τJ.

Therefore, the linearity of the calibration curve can be ensured by eliminating the curvature near the lower limit τ1 of the measurement range shown in FIG. 3B.

Therefore, a two-liquid system is used instead of the conventional one-liquid system. That is, the first reagent is a slow (iti substance (e.g., phosphate buffer, etc.) that differs depending on the type 1 of the analyte) and a one-time analyte (antigen or antibody) shown by Cm in Figure 4. The main components shall be pure products.The second reagent should contain a sufficiently larger amount (e.g., twice or more) of the antibody or antigen and a buffer substance (compared to the buffer substance of the first reagent) than the upper limit concentration CM of the measurement range. same).

First, since the identity of the sample (for example, antigen) to be measured is known in advance, a first reagent is prepared using a pure version of this sample under the above conditions, and then a second reagent is prepared. Next, by using deionized water (concentration 0) and multiple samples with known concentrations for each concentration, and measuring the scattering intensity or absorbance for each sample, a straight line is obtained over the entire measurement target range as shown in Figure 5. Become. Therefore, a calibration curve can be easily established using deionized water (concentration 0) and one sample of known concentration.

Once this calibration curve is established, measuring the concentration of antigen or antibody based on this calibration curve involves first mixing the collected sample with an unknown concentration with the first reagent, then mixing it with the second reagent and initiating the reaction. This can be easily done by starting.

Furthermore, the antigen concentration of the antibody in the second reagent is sufficient to avoid bending the calibration curve at the upper limit of the range, in most cases 2 to 3 times the upper limit concentration; When detecting the lower limit, the curve of the line also increases, causing an increase in the concentration of the pure substance in the first reagent, resulting in unnecessary consumption of the first and second reagents.

FIG. 6 shows a schematic configuration diagram of an automatic analyzer to which the present invention is applied.

In the figure, a sample disk 10 is provided on which several samples θv of each object to be measured can be placed. These multiple samples are sequentially placed on the sample disk for each measurement target.
It is configured so that it can be arranged on top of 0. Further, the reaction disk 21 has a plurality of reaction containers 22 that also serve as measurement cells on its circumference, and is configured to be rotatable.Moreover, the sample is transferred by a sampling probe 41, and the reagent The dispensing is performed by dispensers 38, 39. The spectrometer 27 is of a multi-wavelength simultaneous photometry type having a plurality of detectors, and faces the light source lamp 25, with the reaction disk 21 in a rotating state.

The array of reaction vessels 22 is configured such that a light beam 26 from a light source lamp 25 passes through the row of reaction vessels 22 when the reaction vessel 22 is turned on. The light beam 26 is emitted from, for example, the 31st reaction container 46 counting clockwise from the discharge position 45 when the reaction disk 21 is in a stopped state.
It is arranged so that it passes through the center. A draining device and a cleaning device 24 are arranged between the position of the light beam 26 and the discharge position 45.

The overall configuration of the control device includes a multiplexer, a logarithmic conversion amplifier 53, an A/D converter 54, and a read-only memory (
Random access memory (hereinafter referred to as ROM), random access memory (hereinafter referred to as ROM),
(hereinafter referred to as RAM), printer 55, operation panel 5
2. It consists of a mechanism drive circuit 35, and an A/D converter 54
Furthermore, the central processing unit 5 via the interface 50
Connected to 1. A microcomputer is used as the central processing unit 51, which controls the entire apparatus including the mechanical system and performs general data processing such as preparing a calibration curve by multiple regression and calculating concentration.

Next, the operation will be explained.

First, since the type of antigen or antibody to be measured is known in advance, deionized water (concentration 0) and a sample of known concentration 1
Measure the scattering intensity or absorbance of each sample using two samples, and store a calibration curve of the object to be measured based on the two measurements (11'1).

Next, when the E sample container 44 containing the sample is supplied to the zangling position, the tip of the glove 41 of the pipetter 40 is immersed into the sample container, and a certain amount of serum is aspirated.
It is held within the probe 41.

Thereafter, the glove 41 moves to the discharge position 45 of the reaction disk 21 and discharges the serum held 17 by the probe 41 into the reaction container 22 which has been transferred to the discharge position 45. When the above sampling operation is completed, the reaction disk 21
begins an intermittent rotational movement in the counterclockwise direction and stops at rotation l- until one more reaction vessels 22 than the total number of reaction vessels 22 have passed through the discharge position.

As the reaction disk 21 rotates, the reaction container 2 containing the sample sampled in the sampling operation is rotated.
No. 2 comes to a position which has advanced one pitch of the reactor 6 in the counterclockwise direction from the discharge position 45 and has stopped. During the rotation of the reaction disk 21, all the reaction vessels 22 on the reaction disk 21 pass through the light beam 26. Therefore, when each reaction vessel 22 passes through the light beam 26, the spectrometer 27
Optical absorption measurement is performed by the spectrometer 27, and a multi-blephr selects a signal of the currently required measurement wavelength from the output of the spectrometer 27, which is taken into the central processing unit 50 by the A/D converter 54 and stored in the RAM.

The time during which the reaction disk 21 is rotated and stopped is set to a reaction time (5 to 15 minutes, although it varies depending on the type of object to be measured) so that the reaction is constant when measuring after adding and stirring the second liquid. ). The above operation is repeated with this time as one cycle. As the cycle progresses, the position of the sampled sample to be measured moves counterclockwise by one pitch of the reaction container when the reaction disk 21 is stopped. Dispenser 36 and dispenser 37
The reagent is discharged from the reaction disk 21 while the reaction container 22 containing the sample advances counterclockwise by one pitch of the reaction container and stops at a discharge position of 1 it 46.47 on the reaction disk 21. When looking at a particular sample, a certain amount of the first reagent is added at the ejection position 47, and a certain amount of the tn 2 nt drug is added at the ejection position 46, thereby starting an antigen-antibody reaction. When we look at a specific sample of the reaction disk 21 in one cycle with the above operation, we can see that the sample's reaction density is #
-, L Measured 31 times every 1 minute 30 seconds to 5 minutes 30 seconds,
Measurement data from 48 to 165 minutes are recorded in R and AM.1. ((
be done.

The central processing unit operates according to the program in the ROM, and
Extract 31 pieces of measurement data in AM and perform calculation processing.

The calibration curve drawn in this way is a straight line passing through the origin with respect to the R degree of the measured object l'( In particular, the present invention provides a reagent composition and a measurement method for anti-1.1 formula (also known as an antibody) in a sample and an antibody (also known as an antibody) in a reagent.
It is effective in the nephelometric method and nephelometric method, in which a large immune complex is formed by reaction with a large immune complex (or an antigen), and the resulting increase in scattering intensity or absorbance is detected.

Next, the results of measuring IgG (immunoglobulin G) in serum by turbidimetry using the present invention will be explained.

The remaining 12 measurements performed by the conventional method and the method according to the present invention using automatic analyzers are shown in Table 1.

a￥ 1 In Table 1, there is a difference between the one-liquid method and the two-liquid method, but test fF included, final dilution rate, and measurement? The length and other details are the same. In the 2nd table I cut out (1L and 21''; j: +?f of the trial result of Ming's method)
1 configuration is shown.

Under these conditions, the IgG standard blood concentration (3000 m
A conventional example determined by a dilution series of g/dtWJ(0),
The concentration versus absorbance relationship of the present invention is shown in FIG.
The middle part of FIG. 7 shows the conventional example, and the part B shows the measurement results according to the present invention. According to FIG. 7, it can be seen that the conventional method requires the preparation of a calibration curve using gear calibrators with known concentrations at 5 to 6 points, but in Kenpo, it is possible to create a calibration curve using only a calibrator of one arbitrary concentration. In a similar manner, I gA, I gM, CR, P, RF
.

We succeeded in linearizing the calibration curves for turbidimetry for more than 10 serum immunoassays such as haptogrons, transferrin, α-lid-antitrypsin, ceruloplasmin, C3, and C4. Further, since the reactant (antigen or antibody) and the added pure product (antigen or antibody) are separated into the first liquid and the second liquid, the stability of each reagent is not a problem at all.

Therefore, according to this example, it became possible to automate many serum immunological tests using antigen-antibody reactions. Furthermore, a similar effect can be obtained in the nephelometric method.

As explained above, according to the present invention, it is possible to automate the nephelometric method and nephelometric method that utilize antigen-antibody reactions.

[Brief explanation of the drawing]

Figure 1 shows the conventional scattering intensity (or absorbance) /l"v characteristic diagram when the antigen concentration is varied with the antibody at 0 degrees constant. Figure 2 shows the conventional method when measuring transferrin with a laser nephrometer. Figure 3 is a sigmoid curve i diagram showing the measurement target range, Figure 4 is a measurement diagram when the antibody in the reagent shown in Figure 3 is increased by 5 degrees, and Figure 5 is a diagram according to the present invention. A calibration curve diagram, Fig. 6 shows an I'i/Y diagram of an automatic analyzer to which the present invention is applied, and Fig. 7 shows a conventional example of measuring IgG in serum using a nephelometric method and measurement results according to the present invention. It is a figure. 22... Reaction container, 26... Spectrometer, 36.37.
...dispenser, 40... pipettor, 44... sample container. Agent Patent attorney Tatsuyuki Unuma 1st Meka original liquid level −□ $ 2 Figure concentration (Morning p/d Nono 3rd area Bt/!!/rC/″ ′ Also thorough concentration CM Vmu area

Claims (1)

[Scope of Claims] 1. In a method for measuring the concentration of an antigen or antibody by measuring the increase in scattering intensity or absorbance due to a macromolecular immune complex generated by an antigen-antibody reaction, the first reagent contains an object to be measured. a first predetermined concentration of the same antigen or antibody, a second predetermined concentration of the antibody or antigen, and after mixing the measurement object and the first reagent, the second reagent is mixed with the first reagent. A method for measuring the concentration of an antigen or antibody, characterized in that the scattering intensity or absorbance is measured by mixing the two. 2. The method for measuring the concentration of an antigen or antibody according to the invention as set forth in claim 1, wherein the first predetermined concentration is a lower limit concentration of an optimal region. 3. The invention according to claim 1 or 2, wherein the second predetermined concentration is twice or more the upper limit concentration of the antigen or antibody in the measurement range. Method for measuring antibody concentration.