JPH06217794A - Method of measuring chemoluminescence - Google Patents

Abstract

PURPOSE:To measure a whole blood specimen as it is without pretreatment of the specimen and to completely remove influence of Hb. CONSTITUTION:Quenching effect (negative interference) of Hb existing in a specimen on chemoluminescence is suppressed or eliminated by a spectral element or solution filter effect and a chemical component concentration and Hb concentration in blood are obtained in combination with measured values before and after using the spectral element.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring specific components in blood, serum and plasma samples by chemiluminescence.

[0002]

2. Description of the Related Art The chemiluminescence method is known as a method for analyzing glucose, lactate, urea, cholesterol, phospholipids, etc. in blood, serum and plasma samples.

This is because after adding a specific oxidase to a blood, serum or plasma sample or contacting it with the same enzyme immobilized on resin beads to generate an exciter such as hydrogen peroxide, luminol or the like is added thereto. Is added to a catalyst such as an exciting substance or red blood salt to generate chemiluminescence such as luminol, and the spectrum is measured.

For example, when lactic acid (LA) in blood is measured by chemiluminescence, the following reaction is caused.

[0005]

[Formula 1]

[0006]

However, in the conventional measuring method, no particular consideration is given to the influence of hemoglobin (Hb) mixed in blood, serum and plasma samples, and therefore one of the spectra of chemiluminescence is not considered. The department had received interference from Hb.

In order to eliminate this effect, it is conceivable to remove red blood cells (containing Hb) by centrifugation before measuring the sample, but if some red blood cells were broken at the stage of blood collection, centrifugation was performed. Since Hb is mixed in the supernatant, the effect of Hb cannot be completely removed even by this method, and a pretreatment step is required before the measurement, which requires a large amount of time until the measurement is completed. Was there.

[0008] Therefore, it is an object of the present invention to provide a new method capable of measuring a whole blood sample as it is without pretreatment of the sample and completely eliminating the influence of Hb.

[0009]

[Means for Solving the Problems] In order to solve the above-mentioned problems, the first invention is that the oxidase is added to or brought into contact with blood, serum and plasma samples to form an exciter, In the method of measuring a specific component in a sample by adding a substance to be excited and a catalyst to it and causing chemiluminescence, a wavelength range of 450 nm or less showing absorption of hemoglobin in the sample between the chemiluminescence measurement liquid storage part and the detector. Is arranged to measure chemiluminescence by disposing a spectroscopic element which does not transmit the light.

Here, the oxidase is determined by the component to be measured in the sample. For example, when measuring lactic acid, lactate oxidase, glucose is glucose oxidase, uric acid is uricase, pyruvate is pyruvate oxidase, and cholesterol is cholesterol. Examples of oxidases and phospholipids include, but are not limited to, hollilipase, free fatty acids,
L, D-amino acids, creatinine, etc. can be measured by selecting an enzyme suitable for them.

The generated exciter is, for example, hydrogen peroxide, and the substance to be excited is luminol, a luminol derivative,
Examples include, but are not limited to, lucigenin, N-methylacridinium, and the like. Further, examples of the catalyst include transition metal compounds such as red blood salts and amine complexes of metal ions such as copper.

The spectroscopic element may be, for example, a filter, but is not limited to this and may be any element as long as it does not transmit light in the wavelength region of 450 nm or less which substantially absorbs most hemoglobin.

The spectroscopic element may be arranged between the chemiluminescence measurement liquid storage part and the detector by mounting the spectroscopic element on a chopper or the like so that the spectroscopic element can be put in and taken out, or fixedly arranged in front of the detector. good.

Incidentally, the chemiluminescence measurement liquid storage part is a place where an exciter, a substance to be excited and a catalyst are mixed, and all things such as a reaction coil, a beaker, an optical cell, etc. correspond, but when performing a flow injection analysis. Reaction coils are preferred. The detector may be, for example, a photomultiplier tube or a photon counter.

Next, in the second aspect of the present invention, an oxidase is added to or brought into contact with blood, serum and plasma samples to generate an exciter, and then a substance to be excited and a catalyst are added thereto to cause chemiluminescence. In the method for measuring a specific component in a sample, the chemiluminescence is measured by increasing the concentration of the catalyst more than the concentration at which the highest sensitivity of chemiluminescence is obtained to suppress the interference of hemoglobin in the sample. .

Here, the catalyst is the same as that described above, and to raise the concentration higher than the concentration at which the maximum chemiluminescence sensitivity is obtained is to act as a so-called "solution filter". For example, when lactic acid in blood is measured by chemiluminescence of red blood salt-catalyzed luminol, the maximum chemiluminescence sensitivity is obtained at a red blood salt concentration of 1 g / dl. At least about 6.6 g / dl is required to suppress the interference of.

A third aspect of the present invention is to calculate the chemical components and hemoglobin concentration in blood by utilizing the first aspect of the present invention. That is, assuming that the measured value when the spectroscopic element is arranged (spectral light amount measurement) is C _SP, and the measured value when the spectroscopic element is not arranged (non-spectral light amount measurement) is C _{sp '} ,
C _SP represents the chemical component concentration in blood, and C _{sp '} represents the chemical component concentration in blood + Hb concentration.

The Hb concentration (C _Hb ) is calculated by the following formula (where F _Hb is a conversion coefficient for _obtaining the Hb concentration). C _Hb = [(C _SP −C _{sp ′} ) / C _SP ] × F _{Hb As} a device for obtaining the measurement value, a spectroscopic element is attached to a chopper or the like, and a chemiluminescence measurement liquid storage unit and a detector are provided. One that sequentially obtains both measured values by putting in and out the space, or by arranging the detection parts in two directions from the chemiluminescence measurement liquid storage part,
It is conceivable that a spectroscopic element is arranged in front of the detection unit for measuring the amount of spectroscopic light and the non-spectral light and the amount of spectroscopic light are detected separately, but the present invention is not limited thereto.

[0019]

The present invention suppresses or eliminates the quenching effect (negative interference) on the chemiluminescence due to Hb coexisting in the sample due to the spectroscopic element and the solution filter effect, and combines the measured values before and after using the spectroscopic element with blood. The chemical component concentration and the Hb concentration in the inside can be obtained.

[0020]

【Example】

<Example 1: Removal of influence of Hb by spectroscopic element> The effect of removing the influence of Hb by the spectroscopic element was measured using the apparatus of FIG. In the figure, 1 is a sample cup, 2 is a sample sampling pump, 3 is a buffer solution delivery pump, 4 is a buffer solution reservoir, 5 is an immobilized enzyme column, 6 is a luminol solution reservoir, 7 is a luminol solution delivery pump, and 8 is A red blood salt solution reservoir, 9 is a red blood salt solution sending pump, 10 is a reaction coil, 11 is a spectroscopic element, and 12 is a detector.

In such a device, a sample is weighed by the sample sampling pump 2 and sent to the immobilized enzyme column 5 as a buffer solution. In the immobilized enzyme column 5, a specific component in the sample reacts with oxygen to produce an organic substance and hydrogen peroxide. Luminol and red blood salt are added to this hydrogen peroxide to generate chemiluminescence in the reaction coil 10, which is detected by the detector 12 via the spectroscopic element 11.

The measurement conditions are as follows. Measurement sample; 20-fold diluted blood 8 × 10 ⁻³ ml Immobilized enzyme column; Lactate oxidase buffer solution; Na ₂ HPO ₄ (190 mg / l) / KH ₂ PO ₄
(160mg / l) (pH = 7.5) Luminol solution; Luminol (26mg / l) / KOH (11g / l)
/ Boric acid (12.4 g / l) / NaCl (117 g / l) red blood salt; 0.13 to 13 g / dl detector; photomultiplier tube spectroscopic element; filter V-Y45, V-Y47 (manufactured by Hoya Glass)

The measurement results are shown in FIG. FIG. 2 is an emission spectrum of luminol. In FIG. 2, the solid line shows the original spectrum, the one-dot chain line shows the spectrum when the filter V-Y45 was attached, and the two-dot chain line shows the spectrum when the filter V-Y47 was attached. The maximum spectral wavelength of chemiluminescence of luminol is 425 to 430 nm, but the absorption maximum of the red blood salt used as a catalyst is 500 nm or less, so the apparent maximum wavelength is 470 nm).

FIG. 3 is a diagram showing the filter used for measuring the emission spectrum of luminol and the transmittance curve of Hb. From these figures, it is understood that the influence of Hb can be eliminated by the spectroscopic element of the present invention.

<Example 2: Hb due to solution filter effect
Removal of the effect of Hb> by using the same apparatus and conditions as in Example 1 without using a spectroscopic element and gradually changing the concentration of red blood salt.
The effect of was removed.

FIG. 4 shows a transmittance curve of red blood salt and Hb in the reagent for measuring lactic acid (LA). From this figure, it can be seen that by changing the concentration of red blood salt, a transmittance curve equivalent to that of the filter shown in FIG. 3 can be provided, and most of the absorption of Hb can be removed.

Next, in order to determine the optimum red blood salt concentration, the relationship between the potassium ferricyanide concentration in the red blood salt solution and the Hb concentration in the sample is shown in FIG. From this figure, it is understood that at least a red blood salt concentration of about 6.6 g / dl is necessary to avoid Hb interference.

The relationship between red blood salt concentration and LA measurement sensitivity is shown in FIG. From this figure, it is understood that the LA measurement sensitivity is highest when the red blood salt concentration is around 1 g / dl. Therefore, from the viewpoint of measurement sensitivity, the red blood salt concentration is optimal at around 1 g / dl, but considering Hb interference, about 6.6 g / dl is required.

<Embodiment 3; Calculation of chemical components in blood and hemoglobin concentration> FIG. 7 shows a schematic view of an apparatus for achieving this embodiment. 7, the same parts as those in FIG. 1 are designated by the same reference numerals. Reference numeral 11 'in FIG. 7 is a spectroscopic element (filter), which is greatly different from that in FIG. The chopper is driven by the motor 13. Reference numeral 14 is a light amount counter, 15 is a C _SP / C _{sp ′} calculation unit, 16 is a C _Hb calculation unit, and 17 is an output unit (where C _SP is a measured value when a spectroscopic element is arranged (spectral light amount measurement), C _{sp '} is the measured value when no spectroscopic element is arranged (non-spectral light amount measurement), C
_Hb is the Hb concentration).

The light quantity counter 14 separately counts the spectral light quantity and the non-spectral light quantity in synchronization with the rotation of the chopper motor 13, and based on these light quantities, C _SP and C _{sp '.}
Is calculated.

Under the above apparatus, the glucose concentration and Hb concentration in blood were measured under the following measurement conditions. Measurement sample; Blood 20 times diluted blood 8 × 10 ^-3
ml immobilized enzyme column; glucose oxidase buffer solution; Na ₂ HPO ₄ (190 mg / l) / KH ₂ PO ₄
(160mg / l) (pH = 7.5) Luminol solution; Luminol (26mg / l) / KOH (11g / l)
/ Boric acid (12.4 g / l) / NaCl (117 g / l) red blood salt; 0.66 g / dl detector; photomultiplier tube spectroscopic element; filter V-Y47 (Hoya Glass)

FIG. 8 shows the interference of hemoglobin in the glucose analysis. From this figure, it can be seen that when the chemiluminescent light quantity is measured as it is without being spectrally separated, the quantitative value of glucose shows a low value depending on the Hb concentration. On the other hand, when glucose is quantified from the amount of spectroscopic light, it becomes a correct value, so that the Hb concentration can be obtained from the difference between the two.
The glucose (GLU) concentration and the Hb concentration in blood are quantified by the following equations.

(1) Calibration (GLU and H
b concentration conversion coefficient -K _G and K _Hb - calculation of)

[Table 1] From the relationship of FIG. 8, the Hb concentration conversion coefficient (K _Hb ) is C _Hb = K _Hb
(1-C _NG / C _DG ) holds, and K _Hb = C _Hb / (1-C _NG
/ C _DG ) See below for C _NG and C _DG (2) Quantification of GLU and Hb in blood-non-spectrophotometric measurement value: _INS , spectrophotometric measurement value: I _DS [GLU concentration measurement (C _DG )] = K _DG (I _DS −I _DO ) ... (a) Hb is quantified from C _DG and apparent GLU concentration (C _NG ).

[Apparent GLU concentration (C _NG )] = K _NG (I _NS −I _NO ) Hb (C _Hb ) is obtained from the following equation.

C _Hb = K _Hb / (1−C _NG / C _DG ) ... (B) The values of the equations (B) and (B) are output as the GLU value and the Hb value, respectively.

However, when the interference of Hb cannot be completely avoided due to the performance of the spectroscopic element, I _D > I _{D ′} at the time of calibration, so the GLU value is corrected by the following equation.

[True GLU value] = _CDG + b ( _{CDG- CNG} ) = _CDG + _ID / _{ID '} ( _{CDG- CNG} ) b = _ID / _ID' is calculated at the time of calibration. Keep it.

Although the above description is for the case where the apparatus of FIG. 7 is used, the same measurement can be performed with the detection system shown in FIG. 9, for example. FIG. 9 shows the reaction coil 10.
The detectors 12 and 12 ′ are arranged in two directions from the above, and the spectroscopic element 11 is arranged in front of the detection unit for measuring the amount of spectral light. 14 'is a light quantity counter, 15' is a C _SP calculator, 1
5 ″ is a C _{sp ′} calculation unit, and other than that is the same as in FIG. 7.

[0039]

EFFECTS OF THE INVENTION According to the present invention, it is possible to avoid the influence of Hb mixed by hemolysis in the pretreatment process of serum, plasma sample and the like. In addition, whole blood samples can be analyzed, pretreatment for obtaining serum and plasma is not required, and rapid analysis can be performed. Furthermore, a specific component and Hb concentration in blood can be simultaneously measured using a whole blood sample.

[Brief description of drawings]

1 is an apparatus diagram for carrying out the method of the present invention.

Figure 2: Luminol emission spectrum

FIG. 3 is a diagram showing a filter used for measuring an emission spectrum of luminol and a transmittance curve of Hb.

FIG. 4 Permeability curve of red blood salt and Hb in a reagent for measuring lactate

FIG. 5 is a diagram showing the relationship between the concentration of potassium ferricyanide in red blood salt and the concentration of Hb in a sample.

FIG. 6 is a diagram showing red blood salt concentration and lactate measurement sensitivity.

FIG. 7 is a device diagram for calculating chemical components and Hb concentration in blood.

FIG. 8 is a diagram showing Hb interference in glucose analysis.

FIG. 9 is a diagram of another embodiment of the apparatus for calculating the chemical composition and Hb concentration in blood.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... Sample cup 5 ... Immobilized enzyme column 6 ... Luminol liquid reservoir 8 ... Red blood salt liquid reservoir 10 ... Reaction coil 11 ... Spectroscopic element 12 ... Detector

Claims (3)

[Claims] 1. A specific component in a sample is measured by adding or contacting an oxidase to a blood, serum or plasma sample to generate an exciter, and then adding a substance to be excited and a catalyst to chemiluminescence. In the method, a chemiluminescence is measured by disposing a spectroscopic element that does not transmit light in a wavelength range of 450 nm or less showing absorption of hemoglobin in the sample between the chemiluminescence measurement liquid storage part and the detector. Chemiluminescence measurement method. 2. A specific component in a sample is measured by adding or contacting an oxidase to a blood, serum, or plasma sample to generate an exciter, and then adding a substance to be excited or a catalyst thereto and causing chemiluminescence. In the method, a chemiluminescence measuring method is characterized in that the chemiluminescence is measured by further increasing the concentration of the catalyst beyond the concentration at which the highest sensitivity of chemiluminescence is obtained to suppress the interference of hemoglobin in the sample. 3. The method according to claim 1, wherein a measured value when the spectroscopic element is arranged and a measured value when the spectroscopic element is not arranged are obtained, and the chemical components in blood and hemoglobin are obtained based on these both measured values. A method for measuring chemiluminescence, which comprises calculating a concentration.