JPS62291548A - Optical density measuring method for dry chemical analytical element - Google Patents

Abstract

PURPOSE:To prevent the lowering in measuring accuracy by effectively preventing the interference of bilirubin contained in a specimen liquid, by irradiating the specimen liquid with photometric light having only quantity necessary for measurement at the time of the measurement of optical density provided so as to have a predetermined time interval. CONSTITUTION:In a method wherein a specimen liquid containing enzyme is supplied onto a dry chemical analytical element to be held to constant temp. and the coloration chemical concn. of the substance having absorption at a specific wavelength such as p-nitrophenol coloring matter (PNP) formed by chemical reaction due to enzyme is measured by a reaction speed method, when optical density is measured at each time, light is allowed to irradiate the reagent layer 5a of a chemical analysis slide 5 only for a time necessary for the measurement of the reflection chemical concn. of said reagent layer 5a, and, after the photometry at this time, irradiation is blocked by a filter 73 even during the reaction time on the reagent layer 5a until the next measurement of concn. to prevent the irradiation of the reagent layer 5a with light. By this method, the irradiation quantity of light irradiating the reagent layer is reduced and inference due to the fading of bilirubin contained in the specimen liquid can be prevented effectively.

Description

Detailed Description of the Invention 3. Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a method for measuring enzyme activity using a dry chemical analysis element, and in particular, the present invention relates to a method for measuring enzyme activity using a dry chemical analysis element. This invention relates to a method for measuring optical density using a reaction rate method, which measures the color optical density caused by a product on an element and measures the change in density.

(Prior Art) Self-developing substrates that dissociate varanitrophenol dyes have been widely developed in recent years. In particular, various oligosaccharides (e.g. G5, G7) with varanitrophenol dyes (abbreviated as PNP) such as PNP-G5 and PNP-G
7 and a glucosidase enzyme, the amylase analysis method uses a conventional pigmented starch substrate (dyed starch with a reactive dye).
For example, starch is produced and quantified using the difference in molecular weight distribution using blue starch, amylopectin azure) [pigmented starch method (e.g. H, U, belbumi t-s
It is superior in terms of quantitativeness and accuracy compared to Methods-17 Enzymatic Analysis by J. Ergmeyer (see p. 894), and has the advantage of being able to continuously monitor and analyze enzyme reactions (late assay in a narrow sense). .

In addition, T-glutamyltransferase (abbreviated as GGT) can be used as a self-developing substrate to measure the activity of T-glutamyltransferase (abbreviated as GGT), which is important in the diagnosis of cancer, Myotong cancer, pancreatic cancer, etc.
p-Nitroanilide is known. This substrate liberates p-nitroaniline in the presence of glycylglycine.

For this reason, a dry chemical analysis slide (element) using a reagent layer containing a self-developing substrate as described above was developed, and enzyme activity is measured using such a dry chemical analysis slide. The method has been put into practical use. In this method, first, a predetermined amount of a sample solution containing the enzyme to be measured is dotted onto the reagent layer of a dry chemical analysis slide, and this is kept at a constant temperature (incubation) for a predetermined time in an incubator (constant temperature Ia). During this time, a method is used in which the color density due to the varanitrophenol dye or the like released from TI by the enzyme is optically read at appropriate time intervals. Incidentally, this optical reading is performed by irradiating the chemical analysis slide surface with measurement irradiation light containing a wavelength preselected according to the dye and reading the reflected optical density.

(Invention or problem to be solved) However, like PNP and paranitroaniline, 4
A dry chemical analysis element that optically detects the formation of a substance with a maximum absorption wavelength around 10 nm (400 nm in the solution method) uses hemoglobin (
Since the spectral absorption region of Yabilirubin (maximum absorption wavelength 414 nm) and the absorption maximum value of the substance overlap, they interfere with each other. In the reaction rate method commonly used to measure enzyme activity, the interference of bi-norphine has a negative effect on the results of enzyme activity measurement.
Generally produces a negative error. In particular, when the bilirubin concentration is high due to illness, the negative error due to bilirubin interference is greatly reduced. This error does not become a problem in colorimetric analysis using a solution method, but is a problem specific to dry analysis.

(Means for solving the problem) This defense explains that when measuring enzyme activity by the reaction rate method using dry chemical analysis slides (elements), interference of bilirubin is caused by irradiation of photometric light for optical density measurement. Taking note of the fact that bilirubin increases, the aim is to provide a method for measuring optical density that can effectively prevent bilirubin interference, and the method described below is considered as a means for this purpose.

In other words, in the measurement method of the present invention, a sample solution containing a predetermined enzyme is supplied onto a dry chemical analysis element, the sample solution is kept at a constant temperature, and the chemical reaction caused by the enzyme described above is performed to detect live-bitten P.
In a method of measuring the color optical density of a substance that absorbs at a specific wavelength, such as NP, using a reaction rate method, when measuring the optical density at predetermined time intervals,
The metering light is only irradiated with the brightness necessary for measurement.
Irradiation of light other than the irradiation during this measurement is blocked or attenuated. The predetermined time interval does not need to be a fixed interval, and may be appropriately selected depending on requirements such as measurement accuracy, and the interval may be changed.

(Function) According to the measurement method described above, when measuring by the reaction rate method, at least when optical density measurement is necessary, the light necessary for photometry is incident on the analytical element, or optical measurement is performed even during the reaction time. Since the light was not allowed to enter the analysis element or the amount of irradiation was reduced while the analysis was not carried out, even if bilirubin was contained in the sample solution, there was no negative effect on the measurement results by the reaction rate method due to discoloration of bilirubin. The error is reduced, and bi-norbin interference is effectively prevented.

(Example) Hereinafter, specific examples of the present invention will be described.

First, an example of a dry chemical analysis element used in the optical density measurement method according to the present invention will be described.This analysis element is manufactured as follows.

First, a water-soluble 8! After drying, apply to a weight of 10 Um (100 g,/m) and dry.

Gelatin 300 Cl surfactant 10G (Orlin Co., Ltd.) 5g Bolico (styrene-N methylmorpholinium methylstyrene-divinylbenzene) Polymerization ratio 55:43: 215% latex solution 280g water
2150Q (pH adjusted to 7.0 with dilute NaOH solution) Next, water was uniformly supplied to the gelatin layer at a rate of about 30C1/m over the entire surface to moisten it, and then a Tosocot knitted fabric (made of polyester) was placed on top of the gelatin layer. 40
Gauge) with light pressure to laminate and dry.

Next, an aqueous solution having the following composition was applied almost uniformly onto this cloth at a ratio of 200 cc/bottom, and dried to prepare an integrated multilayer analytical element for amylase measurement.

Varanitrophenyl-α-D-maltoheptaoside 60gα-glucosidase 2.15 million ILJ water
90 to 17,000 polyvinylpyrrolidone (average molecular weight 700,000) 140CI potassium phosphate 60C1(+)8
7.0) In the above chemical analysis element for amylase measurement, various concentrations (0, 5, 10.20%) of golirubin (BOVjne Gal 5tones S+) as shown in Table 1 were added.
control serum (amylase activity value 791;, /l-) supplemented with 10.gma). Dot uL and make this 37
The temperature was maintained at °C, and the reflection density at a wavelength of 400 nm was measured from 3 minutes to 6 minutes. Then, the change in reflex 'a' degree per minute was calculated from this measured value, and the amylase activity was calculated from a standard curve prepared in advance using control serum to which no bilirubin had been added. This measurement method includes a method according to the present invention in which the chemical analysis element is irradiated with measurement light for about 0.5 seconds every minute during the measurement period;
Table 1 shows the results obtained by the two methods of the present invention and the conventional method.

Table 1 As is clear from Table 1, the measurement method of the present invention is less affected by bilirubin than the conventional methods.

Next, the method of the present invention will be explained in detail by showing a chemical analysis apparatus used for carrying out the optical density measuring method according to the present invention. Figure 1 is a sectional view showing one example of this chemical analysis device, and Figure 2 is a sectional view showing this chemical analysis device along arrows ■-■ in Figure 1. explain.

A guide rail 2 having a slide conveyance groove 2a extending left and right in the figure is attached to the upper surface of the main body case 1. A chemical analysis slide 5 is placed on top a and is slidable in the left and right directions. An incubator 10 is provided at the upper left of the guide rail 2.
0 indicates a plurality of slide holders 118 to 1 that hold the chemical analysis slide 5 between the external cover 12 and the conveying groove 2a.
1d. Slide presser foot lla~ll
Is d the conveyance groove mentioned above? a, and are arranged so as to cover the groove 2a, and form a storage container extending in the transportation direction (lateral direction) between the transportation groove 2a, and chemical analysis slides are placed in this storage seedling. It is designed to hold 5.

The slide holders 118 to 11d each have the same width as the length of the chemical analysis slide 5 in the transport direction, and each of the slide holders lla to lld clamps each chemical analysis slide 5 as shown in the figure. There is. Each of the slide holders lla to lld has a built-in heater (not shown), and each chemical analysis slide 5 held in the storage IFF between it and the transport groove 2a is incubated (maintained at constant temperature).
Vru. Each chemical analysis slide 5 has a support, a reagent layer 58. A dry multi-purpose chemical analysis element is arranged, which is formed by depositing 53 in this order. The temperature is kept within the incubator 10 to cause a color reaction.

On the other hand, on the right side of this incubator 10, there is a spotter 2.
1 and a holding frame 22 is provided. The holding frame 22 is attached to the slide presser 11.
Each chemical analysis slide 5 is located on the right side of a and above the transport groove 2a, and is held in the storage chamber.
The chemical analysis slides 5 are held side by side in the transport direction (lateral direction) on the same plane.

Is this holding frame 22 an opening for dotting in the center? 2a, and a deployable holder 5s disposed at the center of the chemical analysis slide 5 is exposed upward through this spotting opening 22a. Therefore, the spotting tip 21a8 of the spotting device 21 is positioned above the spotting opening 22a, and a predetermined amount of sample liquid is manually or automatically applied from the lower end of the spotting tip 21a to the chemical analysis slide 5. It is now possible to supply points on WSs.

This score station? Is there a conveyance groove on the right side of O? A stocker 30 as a slide holding means capable of holding one or more chemical analysis slides 5 in a stacked manner is provided on top a, and roughly on the right side of this stocker 30, it is slidable left and right on the conveying groove 2a. An insertion lever 40 is provided which acts as a sliding insertion means. An inlet opening 31 into which the tip 41 of the insertion lever 40 is inserted is formed at the lower right end of the stocker 30, and one chemical analysis slide 5 of the lowest stage pushed by the tip 41 is placed at the lower left end. Exit frontage 3 for sending only to the left side
2 is formed.

An optical reading head 50 is disposed below the guide rail 2 and is supported by a head feeding mechanism 60 so as to be slidable in the left-right direction (lateral direction) in the figure.
The a structure 60 includes a support plate 62a fixed to the main body case 1,
62b, a feed rod 63 having a feed screw, a support rod 64 made of a round bar, and a feed motor 61 that rotates the feed rod 63.The feed rod 63 and the support rod 64 are parallel to each other between the support plates 62a and 62b. and are arranged to extend in the left and right direction.

The optical read head 50 reads the reagents of the chemical analysis slide 5 @
Irradiate the irradiation light onto 5a, and the reflected optical I! A head part 51 that detects the event, and a head support part 55 that supports the head part 51, is threadedly engaged with the feed rod 63, and is slidably fitted with the support rod 64 and supported by both rods 63 and 64. It consists of Therefore, when the feed rod 63 is rotated by each feed motor 61, the head support portion 55 screwed thereto is moved in the left-right direction together with the head portion 51. As shown in the figure, the guide rail 2 has a holding frame for the chemical analysis slide 5 at the bottom of the stocker 30 and the dotting station. Irradiation openings 38 to 3f are formed in a storage chamber opened in the incubator 10 and the chemical analysis slides 5 held by the slide holders 2 and 38 to 3f, which face the reagents of the chemical analysis slides 5 held by the respective slide holders 11a to 11d, The head portion 51 whose upper end faces the lower surface of the guide rail 2 is moved between positions facing each of the irradiation openings 38 to 3f.

One end of an optical fiber cable 76 is connected to the head section 51, and irradiation light is sent through this optical fiber cable 76. The irradiation light generating means 70 that sends irradiation light to the optical fiber cable 76 includes a light source 74 and a light source 74 that narrows down the light from the light source 74 to the optical fiber cable 76.
The filter 73 is attached to a filter wheel 72 and is driven by a pulse motor 71.
The type can be changed according to the rotation of the .

Next, FIG. 3 shows an enlarged view of the chemical analysis slide 5 held in the storage chamber by the slide holder 11a and the head portion 51 facing the reagent @ 5a of the chemical analysis slide 5 through the irradiation opening 3C. The measurement of reflective optical density by the head unit 51 will be explained.

The head section 51 includes a focusing lens 52 that focuses the irradiation light emitted from one end of the fiber cable 76 and irradiates it onto the reagent reservoir 5a of the chemical analysis slide 5, and this reagent reservoir 5a.
A photometric sensor 53 that detects the reflected light reflected from a
The detection information of the photometric sensor 53 is transmitted through the cable 53a.
The information is sent to ``Kakureso@'' (not shown) via .

Note that this chemical analysis apparatus is provided with a slide sensor 81 that detects the position of each chemical analysis slide 5 inserted into the incubator 10.

A method for measuring reflected optical density using the chemical analysis device configured as described above will be described.

First, one or more unused chemical analysis slides 5 are stacked in the stocker 30. Next, the operating portion 42 of the insertion lever 40 is pushed to the left so that its tip 41 protrudes from the inlet opening 31 into the stocker 30, and the lowermost chemical analysis slide 5 of the chemical analysis slides 5 stacked in the stocker 30 is inserted. Dotting station 2 for just one sheet
Send it within 0. Note that the insertion lever 40 is then returned to its original position by the urging force of a spring (not shown), etc.
Is the lowest chemical analysis slide 5 in the stocker 30 a transport groove? The whole unit is lowered one step so that it is located above a. When the chemical analysis slide 5 is sent to the spotting station 20, the holding frame 2? A predetermined amount of a sample solution containing an enzyme whose activity is to be measured is dripped onto the reagent layer. When this spotting is completed, the operating portion 42 of the insertion lever 40 is pushed again to send the lowest unused chemical analysis slide 5 in the stocker 30 into the spotting station 20. In this way, the end of the unused chemical analysis slide 5 fed by the insertion lever 40 is transferred to the spotted chemical analysis slide 5 on which the sample liquid has been spotted at the spotting station 20.
The spotted chemical analysis slide 5 is sent into the storage chamber between the slide holder 11a and the conveyance groove 2a of the incubator 10. In this storage chamber, the spotted chemical analysis slide 5 held by the weight of the slide holder 11a is incubated at a predetermined WIN by the heater in the slide holder lla. By repeating the above operations, the spotted chemical analysis slides 5 on which the sample liquid has been spotted and supplied at the spotting station 20 are sequentially pushed into the incubator 10, and the slide holders 11a to lld and the conveying groove 2a are successively pushed into the incubator 10. The cells are incubated in a storage chamber extending laterally between the cells. Note that in the device of this example, simultaneous 1. : 4 Ihigaku analysis slides 5
can be incubated.

While incubating in this way, the optical reading head 50 is moved left and right by the feed mechanism 60, and the reagents on the chemical analysis slides 5 in each storage chamber are transmitted through the irradiation openings 30 to 3f of the guide rail 2. By irradiating 5a with irradiation light, the color optical density of the substance that absorbs at a specific wavelength generated in reagent 1 by the chemical reaction of the reagent in the reagent layer by the enzyme in the sample solution is measured. . This measurement is performed by moving the optical reading head 50 as necessary based on the detection of the position of each chemical analysis slide 5 by the slide sensor 81. This measurement is based on a reaction rate method and is carried out at least twice, preferably three to ten times, within a period of one minute to several minutes after the sample liquid is spotted on each slide.

The time required for each optical density measurement varies slightly depending on the response characteristics of the optical density measuring means (optical reading head 50), but it is in the range of 0.001 seconds to 10 seconds, and the time required for each measurement is from 0.001 seconds to 10 seconds. It is sufficient to carry out the irradiation. Therefore, in the measurement method of the present invention, during each of the above optical density measurements, the irradiation light is irradiated onto the reagent layer 5a of the chemical analysis slide 5 only in the darkness of the above-mentioned time necessary for measuring the reflected optical density. However, after this photometry is completed, the reagent @ 5a is not irradiated with irradiation light even during the reaction time in the chemical analysis element (reagent layer) until the next measurement of reflected optical density ( (You can also simply reduce the illumination level.)

For this purpose, there is a method of turning off the open light source until the next photometry, but there is a possibility that the amount of light changes each time the photometry is performed. In order to interrupt (or attenuate) the irradiation with the light source turned on, a means for blocking the irradiation of the irradiation light onto the reagent layer 5a is required, but in this example, the filter wheel 72 is used.
A light-shielding filter 73 is disposed in the reagent layer, and the light-shielding filter 73 blocks irradiation of the irradiation light onto the reagent layer at times other than the above-mentioned measurement. Note that this light shielding means is not limited to using a light shielding filter as described above, but may also be other means. The chemical analysis slide 5 itself can be slid in a direction perpendicular to the plane of the drawing to position the reagent layer out of the optical path of the reading head's irradiation light. You can also do something like this.

When measurements are carried out in this way, the amount of light irradiated onto the reagent layer is reduced, so as is clear from the results shown in Table 1, interference due to discoloration of bilirubin contained in the sample solution may occur. can be kept to a minimum.

(Effects of the Invention) As explained above, according to the present invention, when measuring enzyme activity by a reaction rate method using a dry chemical analysis slide (element), irradiation of irradiation light for reflective optical density measurement is possible. or the time required for measurement.
Interference of bilirubin contained in the sample solution can be effectively prevented, and a decrease in the measurement accuracy of enzyme activity can be prevented.

[Brief explanation of the drawing]

FIG. 1 is a front cross-sectional view of a chemical analysis device used to carry out the optical 19-degree measuring method according to the present invention, and FIG. 2 is a side cross-sectional view taken along the arrow II-II in FIG. 1 without showing the chemical analysis device. , FIG. 3 is a partially sectional view showing an enlarged part of the incubator and the optical reading head in the above device. 1... Main body case 2... Guide rail 5.
...Chemical analysis slide 10...Incubator 11
a~lid...Slide foot 20...Dotting station? 1... Spot applicator 30.
... Stocker 40 ... Insertion lever 50 ...
・Optical reading head 60...Head feeding mechanism 61・
...Feed motor 70...Irradiation light generating means 72
... Filter wheel 73 ... Filter 76 ... Optical fiber cable Figure 2 Figure 3 Figure 1 Table of events a&$061 Special fW M No. 135
．． 841 @2, Name of the invention: Method for measuring optical density of dry chemical analysis elements 3, Relationship with Sekiji f[, which performs M correction Patent applicant Address: 210 Nakanuma, Minamiashigara City, Kanagawa Prefecture Name (
5201H Photo Film Co., Ltd. Substitute Minoru Onishi 4, Agent address 1lI37 5-2-1 Roppongi, Miyako-ku
1 Yorai Ya Pill 7R1; 1,” / 6, “Detailed Description of the Invention” column 7 of the specification to be amended, contents of the amendment 1) 1 after “irradiation light” on page 4, line 3 of the specification (white light is also acceptable)”. 2> On the same page, after "Irradiate", insert "J" near the above wavelength. 3) Page 6, line 11, 1-. ” followed by F The above effects of the present invention can also be used in the case of biochemical analysis using the so-called end-point method in which optical density measurement is performed only once at the final stage of an analytical chemical reaction, and the light is blocked or attenuated except when optical measurements are performed. This will prevent interference from Lilbin. ” is inserted.

Claims (1)

[Scope of Claims] After supplying a sample solution containing a predetermined enzyme onto a dry chemical analysis element for enzyme activity measurement having one or multiple reagent layers, the dry chemical analysis element is kept at a constant temperature, and the In the optical density measurement method of optically measuring the colored optical density of a substance having absorption at a specific wavelength generated by a chemical reaction of the reagent in the reagent layer at predetermined time intervals, the sample liquid is supplied. The optical density is measured by irradiating only the photometric light necessary for optical density measurement onto the dry chemical analysis element at the predetermined time intervals, and removing light other than the photometric light irradiation at the time of the measurement. A method for measuring optical density of a dry chemical analysis element, characterized in that the irradiation of the element is blocked or attenuated.