JPH0723871B2 - Multi-item biochemical analysis method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a multi-item biochemical analysis method. More specifically, it relates to a multi-item biochemical analysis method useful for measuring a sample in which suspended substances such as milky serum are mixed.

(B) Conventional technology Conventionally, a method using colorimetric analysis or turbidimetric analysis has been performed for multi-item biochemical analysis in biochemical samples such as serum, plasma and urine, and usually, for each item. A predetermined reaction reagent is mixed with the divided sample to make a measurement solution, and the absorbance at each predetermined wavelength of each measurement solution is measured by the endpoint method. Each item is quantified by multiplying by. And as the analysis items at this time, for example, TP, ALB, T-CHO,
PL, TG, T-BiL, D-BiL, GLV, Ca, iP and the like are mentioned, and the absorbance measurement wavelength is usually in the range of 340 to 750 nm.

(C) Problems to be Solved by the Invention However, so-called milky serum that is cloudy due to extremely high amounts of chylomicron and lipoprotein is among the samples. When measuring a certain item by the endpoint method,
In addition to the absorbance that changes depending on the reaction, in milky serum, turbidity causes light scattering to increase the apparent absorbance, which has a disadvantage of positively affecting the analysis result.

In order to correct this, for each item, prepare a sample blank solution in which the sample and the blank reaction reagent (each reaction reagent minus the reaction components) are mixed, and under the same measurement conditions as the above measurement solution. A method of measuring the absorbance and subtracting the absorbance of these sample blank solutions for each item from the absorbance of the measurement solution (sample blank method), or the actual sample blank solution,
A value obtained by converting the absorbance difference obtained by measuring at two determined wavelengths into the absorbance per unit turbidity obtained by measuring the turbidity standard liquid (polystyrene powder suspension) at the above two wavelengths in advance. Divide by, and multiply the value by the conversion constant to the other wavelength (absorption measurement wavelength of the reaction solution) that was obtained for the reference solution, and use it as the turbidity of the sample itself at that wavelength, from the absorbance of the reaction solution. Method of correction by subtraction (JP-A-54-6378)
No. 5) is known.

However, the former method has a problem that a large number of channels for blank solution measurement are required in the automatic analyzer. On the other hand, in the latter method, it is not easy to obtain a turbidity reference solution, and the spectrum will be different if the particle size of the turbidity component is different, so a single reference solution is used as a reference solution that summarizes the turbidity of specimens such as milky serum. It is impossible to say. That is, the particle size of chylomicron, which is the main turbidity component of milky serum, is about
0.5 μm, of the lipoproteins, pre-β-lipoprotein is about 0.08 μm, β-lipoprotein is about 0.035 μm, α-
Lipoproteins have different particle diameters of about 0.015 μm, and the shape of the spectrum varies depending on the origin of the turbidity of the sample. Accurate correction was difficult with the correction method used.

The present invention has been made to solve such a problem, and in particular, does not require a large number of blank channels for measuring one sample at different wavelengths, and further, the above turbidity reference solution, etc. It is intended to provide a multi-item biochemical analysis method capable of removing an error due to a turbid component as accurately as possible without using.

(D) Means for Solving the Problems Thus, according to the present invention, when performing multi-item biochemical analysis of a sample in which a suspended substance is mixed, a sample divided into each item is used for measuring each item. The reaction reagents were mixed to form a measurement solution, and the absorbance at each predetermined wavelength of each measurement solution was measured, and a plurality of items were quantified based on each of these absorbances. One sample blank solution is prepared, and the absorbance of this blank solution is measured by at least two kinds of wavelengths to obtain a and b of the following formula (1). From this formula (1), the measurement wavelengths for the above items are measured. A multi-item biochemical analysis method is provided, which comprises calculating a blank absorbance and correcting the absorbance of each of the above-mentioned measurement solutions by each blank absorbance.

A = a · λ ^b (i) The most characteristic feature of the present invention is that when performing multi-item analysis on a sample containing suspended substances such as milky serum, a single sample is used. The point is to use the sample blank solution of, and for such a single sample blank solution,
Absorbance at two or more kinds of wavelengths is measured to obtain a wavelength-absorbance regression function for a sample blank solution, A = a · λ ^b
Obtained, and based on this, calculate the estimated absorbance at the wavelength measured for each item, and subtract this from the absorbance of the reaction solution to correct chyle and to eliminate the need for a turbidity reference solution. .

As the reaction reagent used in the method of the present invention, various reaction reagents known in the art corresponding to the intended analysis item are used. These reaction reagents include, for example, an antiserum reagent for performing nephelometric analysis by reacting an antibody in a sample with an antigen-antibody.

The blank reaction reagent used in the present invention may correspond to any reaction reagent as long as it has no absorption of the reaction components from the reaction reagent,
For example, a buffer solution serving as a solvent for the reaction reagent, physiological saline, water or the like can be applied. One sample blank solution in which such a blank reaction reagent is mixed with a sample may be prepared for each sample.

The absorbance of the sample blank solution is measured with at least two kinds of wavelengths. In general, it is suitable to increase the measurement wavelength in order to obtain a more accurate regression function. Usually, since the measurement of each item is performed within the range of 340 to 750 nm, in order to obtain the wavelength-absorbance regression function in this range,
It is preferable to appropriately disperse and select 2 to 4 kinds of wavelengths within this range. However, if the range of the measured wavelength of each intended item is narrow, the regression function may be obtained based on at least two types of wavelengths near these ends. The method of obtaining the regression function will be described below.

First, the measurement wavelength λ (nm) is on the x-axis, and the absorbance A (Abs) is on the y-axis.
On the axis, the turbidity spectrum is as shown in FIG. 1, for example. Simultaneous measurement of a cloudy sample with n types of wavelengths λ ₁ , λ _2, ... λ
Assuming that the absorbances measured with n are A ₁ , A ₂ ... An, respectively, the relationship between the wavelength and the absorbance is A = aλ ^b (a and b are constants).
Can be approximated by Here, the constant a is determined by the degree of turbidity, and b is determined by the average particle size of the turbidity component. Therefore, the combination of wavelength and absorption (λ, A) = (λ ₁ , A ₁ ), (λ ₂ , A ₂ ) ... (λ
n, An) power regression (in this case the least squares method)
Since the values of the constants a and b can be determined, the absorbance of the blank solution at an arbitrary wavelength can be estimated based on the values.

In addition, as described above, the turbid components in milky serum are roughly classified into two types, chylomicron and lipoprotein. Therefore, in order to perform a more rigorous regression, the relationship between wavelength and absorbance should be A = a ₁ λ.
expressed as ^{_{b1 + a 2 λ b2, a}} 1, b 1 is from chylomicrons,
It is suitable that a ₂ and b ₂ are constants derived from lipoprotein, and a ₁ , a ₂ , b ₁ and b ₂ are obtained from at least four different wavelengths and used as a regression function of wavelength and absorbance. However, in practical analysis, it has been found that it is full intended to correct by one of the power regression of A = a, λ ^b from the average particle size, such as above.

In addition, when the mixing ratio of the sample in each measurement reaction solution and the sample blank solution is different, the volume correction may be performed, and the sample concentrations in these final reaction solutions may not necessarily be the same. Blank liquid volume V _B (ml), sample volume V _{B in it}
(Ml), measured liquid volume _VA (ml), sample volume _VA (m)
l), the absorbance calculated by the above regression function at the wavelength λ of the sample blank solution is A _B , and the absorbance at the wavelength λ of the measurement reaction solution is A _A , the absorbance Ac due to the substantial reaction at the wavelength λ of the measurement reaction solution is , Ac = A _A − (V _B · v _A / V _A · v _B ) · A _B.

The method of the present invention usually uses a multi-item automatic analyzer equipped with a plurality of analytical lines corresponding to multi-items, further, a dispensing means for adding a blank reaction reagent to a sample, and the absorbance of this sample blank solution. Is preferably performed by additionally providing a blank line equipped with a multi-wavelength photometer for measuring each of at least two wavelengths. Furthermore, a regression function of wavelength-absorbance is obtained for a sample blank solution of each sample in which suspended substances are mixed based on the absorbance of two or more types measured on the blank line, and the sample blank absorbance at an arbitrary measurement item / wavelength It is preferable to calculate and calculate the above-mentioned capacity as needed, and to automate the calculation by configuring a calculation unit for calculating the difference from the absorbance of the reaction solution by a program-controlled microprocessor. The structure of such a multi-item biochemical analyzer is shown in FIG. In FIG. 2, (1), (2), ..., Multiple analytical lines, (3), Single blank line, (4), (4), ..., Measuring solution, (4A), Sample blank solution, (5). (5) (5) ... is a sample dispenser,
(6) and (7) are reaction reagents, (8) is a blank reaction reagent, (9) and (10) are fixed wavelength optical measurement systems for measuring each item, and (11) is an optical measurement system capable of multi-wavelength photometry. At (11A)
Is a spectroscope, (12) is a photoelectric detector, and (13) is the above-mentioned correction calculator.

(E) Action According to the method of the present invention, the positive error in the absorbance due to the scattering of the suspended substances in the sample existing in the measurement liquid is corrected with higher accuracy.

(F) Example A blank solution was prepared by adding 2.5 ml of physiological saline as a blank reaction reagent to 50 μ of three real samples (milky serum), and measuring the absorbance at 340 nm and 700 nm. were calculated constants a and b in relation a = a? ^b of the wavelength and the absorbance by the least squares method.

Based on this regression function, 400,450,
The absorbance at 500, 550, 600 and 650 nm was calculated. On the other hand, the spectrum actually obtained by scanning between 340 and 700 nm was as shown in FIG.

Above, calculated absorbance of 400 ~ 650 nm based on the regression function,
Table 1 shows the results of comparison with the measured absorbance (FIG. 3) and the absorbance at 400 to 650 nm.

As described above, the value obtained by the regression function and the actual absorbance substantially match, and it can be understood that the absorbance of the blank solution at different wavelengths can be accurately estimated without actually measuring it. Therefore, it was revealed that the turbidity can be easily and accurately corrected for each item in the multi-item biochemical analysis by using the above regression function.

(G) Effect of the Invention According to the method of the present invention, a multi-item biochemical analysis by the endpoint method is performed without using a special turbidity reference solution as in the past and without requiring a large number of blank channels. Can be performed without causing an error. Therefore, it is a very useful method for a biochemical automatic analysis method and apparatus that handles a large amount of specimens.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an explanatory diagram for determining a wavelength-absorbance regression function in the method of the present invention, and FIG. 2 is a structural explanatory diagram illustrating an apparatus for carrying out the method of the present invention. FIG. 3 is a graph showing the relationship between wavelength and absorbance in the examples. (4) …… Measuring liquid, (4A) …… Sample blank liquid, (6) (7) …… Reaction reagent, (8) …… Blank reaction reagent, (11) …… Multiwavelength photometric optical measurement System, (13) …… Correction calculation part.

Claims (2)

[Claims] 1. When performing multi-item biochemical analysis of a sample in which suspended substances are mixed, a reaction liquid for measuring each item is mixed with a sample divided into each item to obtain a measurement liquid, and each of these measurements is performed. It consists of measuring the absorbance at a predetermined wavelength of the solution and quantifying multiple items based on each of these absorbances, and further preparing one sample blank solution in which the sample is mixed with a reagent for blank reaction. Absorbance is measured by at least two kinds of wavelengths to obtain a and b of the following formula (1), and the blank absorbance at the measurement wavelength for each of the above items is calculated from the formula (1). A multi-item biochemical analysis method, which comprises correcting the absorbance of a measurement solution. A = a · λ ^b (1) A ... Absorbance λ = wavelength a · b ... Constant 2. The analysis method according to claim 1, wherein the sample in which the suspended substances are mixed is milky serum.