JPS5823794A - Process for analyzing plurality of items - Google Patents

Abstract

PURPOSE:A metallic nozzle is used to add the reagent solution for the first analysis to the first sample to conduct the measurement for the first analysis, then the same nozzle is used to add the second sample and conduct the second analysis, thus compensating the analysis error caused by the nozzle. CONSTITUTION:A reagent solution for the first analysis which contains an enzyme with a lower isoelectric point than the pH of the reagent solution is sucked and held in a metallic nozzle and the solution in the nozzle is added to the first sample to conduct the first analysis. Then, the same nozzle is used to add the second sample that causes a specific enzymatic reaction with the above enzyme thereto and to conduct the second analysis. Thus, even when a metallic nozzle is used to add a plurality of reagent solutions for several analyses in order, the contamination of samples for other analyses, which is caused by adhesion of the enzyme to the nozzle, is prevented to give yhe analytical data with high reliability.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for analyzing a plurality of items, and in particular, it is an object of the present invention to provide an analysis method suitable for use when a plurality of analysis items are analyzed using the same enzyme reaction.

In conventional biochemical analyzers for multi-item analysis, reagent solutions for each analysis item are supplied into reaction vessels corresponding to the respective analysis items through independent pipetting devices and nozzles attached thereto. However, recently, analyzers have become smaller, and analyzers that can supply reagents for a wide variety of items using only a small number of dispensing devices have begun to appear.

In such an analyzer, a series of reaction vessels containing samples corresponding to each analysis item are transported, and at a predetermined position, the reagent liquid necessary for the analysis of each item is transferred.

A method of supplying with a 30-dispensing device is adopted.

In this case, the nozzle of the dispensing device sucks and holds a predetermined amount of reagent liquid from one reagent liquid tank near the nozzle tip, discharges it into the corresponding reaction container, and then performs suction and discharge operations for another reagent liquid tank. do. Therefore, a single nozzle supplies multiple reagent solutions.

However, if the reagent solution supply method described above is carried out using a nozzle made of a specific material, that is, a metal nozzle, large analysis errors often occur, and furthermore, because two types of analysis items that utilize the same enzyme reaction are used, The inventors have discovered that when two reagent solutions are supplied through the same metal nozzle, the error in the analysis value for one item becomes significantly higher.

The present invention was made based on the discovery of such a phenomenon. The reagent components adsorbed on the metal nozzle are hardly removed by ordinary washing with washing water.

An object of the present invention is to provide a solution that, even if each reagent solution for analysis of multiple items using the same enzyme reaction is supplied using a single metal nozzle, measurement results due to the metal nozzle for one of the analysis items may occur. The object of the present invention is to provide a multi-item analysis method that does not introduce errors.

A feature of the present invention is that an enzyme that causes an error in the measured value of the second analysis item is contained in the reagent solution for the first analysis item, and the enzyme has an isoelectric point that is lower than that of the reagent solution for the first analysis item. By satisfying the condition of having low pH and low pH, adsorption of the enzyme to the nozzle can be prevented and the first
The second analytical reagent solution and the second analytical reagent solution can be added using the same metal nozzle.

Prior to a detailed description of the present invention, knowledge regarding errors caused by nozzles obtained through experiments by the present inventors will be explained.

For example, glutamate pyruvate transaminase (
After adding the reagent for lactate dehydrogenase (LD)
H), when the reagent for LDH is added after the reagent for glutamate ogizaloacetate transaminase (GOT), when the reagent for free cholesterol is added after the reagent for total cholesterol, or When a free fatty acid reagent is added after a triglyceride reagent is added, a correct error often occurs in the measured value of the analysis item to which the reagent is added later. This error occurs when each reagent solution is added using the same metal nozzle, and occurs even when a subsequent reagent is added not immediately after the previous reagent but after a different batch of reagents is added. The reagent solutions added before each contain an enzyme that brings about a specific enzymatic reaction.

The metal nozzle sucks and holds a predetermined amount of reagent liquid from the first reagent liquid tank, then discharges the held reagent liquid into the first reaction vessel, and then performs the same process for the second and subsequent reagent liquids. When such a metal nozzle is immersed in a reagent solution, the nozzle is charged to a negative potential based on the ionization tendency of the metal. A stainless steel nozzle is charged to several tens to hundreds of mV, although this varies depending on the reagent solution that is the electrolyte. Therefore, it is thought that the enzyme in the reagent solution is electrostatically adsorbed to the nozzle.

The enzyme generally called LDH has 5 different physical properties.
The following types of isomers are known: M4゜MsH, M, H,
, MH, and H6.

A notable difference in the physical properties of these isomers is their isoelectric points. The LDH derived from rabbit 6-zo and pig 6-zo is H4 type and has an isoelectric point of about 4.7. LDH derived from pig muscle
is M4 type and has an isoelectric point of about 9.5.

The adsorption phenomenon is caused by the relationship between I)H of the reagent solution and the isoelectric point of the enzyme. That is, if the isoelectric point is higher than the pH of the reagent solution, the enzyme will be adsorbed to the negatively charged metal nozzle. The GPT reagent solution is adjusted to pH 7.4, and the isoelectric point of M in this reagent solution is 9.5.

More than 99.9% of type LDH is +NH3-4-C
It exists in the OOHO form and is thought to have a positive charge. Furthermore, about 99.5% of H type LDH, which has an isoelectric point of 4.7, exists in the form of NH, -R-COO- in a solution of pH 7.4, and is considered to have a negative charge. Therefore, the M4 type is adsorbed, but the H4 type is not. Therefore, in the present invention, when using LDH, a type with an isoelectric point lower than that of the reagent solution I) is adopted. The isoelectric point is the pH of the reagent solution
If it is lower than 2 or more, it is particularly difficult to adsorb.

Example I An example will be shown in which LDH measurement errors caused by GPT measurement reagents can be prevented. Table 1 shows examples of compositions of reagents for measuring GPT, and Table 2 shows examples of compositions of reagents for measuring LDH.

Table 1 Table 2 In Tables 1 and 2, I indicates the first solution and ■ indicates the second solution. The LDH of formulation A in Table 1 is derived from rabbit 6zo, and the LDH of formulation B is derived from pig 6zo. The isoelectric point of each LDH is approximately 4.7, and the pH of the reagent solution
2.7 lower than 7.4.

In an automatic analyzer, a series of transparent reaction vessels are transferred to a reaction line shaped like a turntable and maintained at a predetermined temperature, for example 37C. The reaction container is transported through a sample discharge position, a reagent addition position, and a photometry position,
Each reaction container corresponds to each analysis item to be measured. On the other hand, the metal nozzle for reagent dispensing is moved between a reagent suction position, a reagent addition position, and a nozzle cleaning position. At the reagent suction position, corresponding reagent vessels are sequentially positioned in association with the analysis items of the reaction vessels positioned at the reagent addition position. The reagent dispensing nozzle is made of stainless steel pipe and is connected to the syringe that performs suction and discharge operations.
It can move up and down in each of the above positions.

When the reaction container into which 20 μt of the serum sample to be measured for GPT has been added comes to the reagent addition position, the first liquid tank in Table 1 has been transferred to the reagent suction position, and the reagent is added to the first liquid tank. A dispensing nozzle is inserted and 400 μt is sucked and held. Subsequently, the nozzle is moved to the reagent addition position, and the first liquid for GPT held in the reaction container is discharged. Thereafter, the reaction vessel is rotated, while the nozzle is washed with approximately somz pure water at the nozzle washing position in preparation for the next reagent solution addition. After 5 minutes of pre-incubation, when the same reaction vessel is brought to the reagent addition position, 100μ is added using the same nozzle as in the 2nd solution for GPT in Table 1. Thereafter, the reaction vessel is positioned at a photometric position at predetermined time intervals, and
The change in absorbance of the reaction solution with respect to light with a wavelength of 0.011 m is measured, and the GPT activity value is determined.

During this time, in the reaction vessel, pyruvic acid and glutamic acid are produced by the action of GPT in serum using DL-alanine and α-ketoglutaric acid as substrates. The generated pyruvate reacts with nicotinamide adenine nucleotide reduced form (NADH) in the presence of LDH. Because NADH changes to oxidized form (NAD") through the reaction,
Ultraviolet absorption at 340 nm is reduced.

After that, when the reaction container into which 20 μt of the serum sample to be measured for LDH was added came to the reagent addition position, the first solution in Table 2 was transferred to the reagent suction position, and the same nozzle as above was used to collect 400 μt of serum sample. One liquid of the first liquid for LDH is added to the reaction vessel. After incubation (5 minutes), add the second solution from Table 2 using the same nozzle. The reaction vessel was repeatedly positioned at the photometric position, and the photometer measured 34% of the reaction solution.
The absorbance change at 0 nm is measured and quantified by kinetic method. In the reaction vessel, pyruvic acid and NADH
is reacted by LDH in the serum sample, and lactate and NAD
” is changed to

Table 3 shows the analysis results obtained in this example,
These are the results using control serum (Monitrol IIX) as a sample. Prescriptions A and B in Table 3 correspond to A and B in Table 1. In prescription C in Table 3, the concentration of the reagent solution is the same as in prescription B in Table 1, but LDH derived from the free muscle with an isoelectric point of 9.5 is used.

Table 3 () After measuring the PT activity value, the LDH activity value was measured in three consecutive samples, and only in the case of prescription C, approximately 359 mu
A positive error of /ml was produced. This is probably because LDH containing 1200 mu/fTl or more in the GPT reagent solution was adsorbed by the metal nozzle, and this was mixed in when adding the LDH measurement reagent solution, causing an error.

The normal value range for analysis item LDH is 150 to 35 Qmu/
mt, a positive error of 35 Q mu/mt is fatal. In the case of prescriptions A and H, in which the isoelectric point of DH in the GPT trial solution is lower than the pH of the solution, no abnormal correct error occurs.

Example 2 This example is an example in which LDH measurement errors due to GOT measurement reagents can be prevented. Table 4 shows an example of the composition of the GOT measurement reagent.

In this example, the same analyzer as in Example 1 is used, and GOT
After measuring the activity value, the LDH activity value was measured.

The reagent solution for LDH measurement is the same as that in Table 2.

During GOT measurement, oxaloacetate produced using L-aspartic acid and α-ketoglutarate as substrates is
and malate dehydrogenase (MDI).
The GOT activity value is determined from the rate of decrease in NADH.

LDH with an isoelectric point of 4.7 as shown in Table 4 (derived from Buta 6 Kura)
In the LDH activity value measurement results after using the GOT trial liquid chemical solution containing
A positive error of about 35 Q mu/mt occurred in the LDH activity value after using the GOT trial solution containing LDH (derived from pig muscle).

Example 3 Next, an example will be shown in which a total cholesterol reagent solution is added to a serum sample, and then a free cholesterol reagent solution is added using the same metal nozzle.

First, total cholesterol in a serum sample is analyzed using the analyzer shown in Example 1. The composition of the reagent solution for total cholesterol is shown in Table 5. As the cholesterol esterase, one produced by Sue Hirotake and having an isoelectric point of 4.1 was used. When the reagent solution shown in Table 5 is added to the reaction container containing the serum sample using a metal nozzle, 1) the action of cholesterol esterase in the H7,9 buffer converts the tennis-like cholesterol to free cholesterol; Cholesterol is oxidized by cholesterol oxidase to produce hydrogen peroxide and Δ1 cholestenone. The produced hydrogen peroxide is condensed with phenol and 4-aminoanthibiry/ in the presence of peroxidase to produce a red quinone.

The concentration of total cholesterol was determined by measuring absorbance based on this red quinone.

The free cholesterol reagent solution is then added to the corresponding reaction vessel using the same gold layer nozzle.

The reagent solution for free cholesterol consists of a composition not containing cholesterol esterase among the reagent solution compositions shown in Table 5. The reaction for producing red quinone is the same. As a result of the measurement,
The concentration of free cholesterol in the serum sample is 85 mg/dl
Met. Since the isoelectric point of the cholesterol esterase in Table 5 is lower than the pH of the buffer (7,9), this enzyme is easily adsorbed to the nozzle and therefore does not introduce an abnormal positive error in the free cholesterol assay. Analysis error is 1
mgAdt or less.

For comparison, the isoelectric points were 8 and 4 for the same serum sample.
After adding a reagent solution for total cholesterol containing cholesterol esterase (produced by microorganisms), the same measurement as above was performed using the same metal nozzle. As a result, free cholesterol measurements had a positive error of 50-100 mg/dz. This is because cholesterol esterase, which has an isoelectric point higher than the pH (7,9) of the buffer solution, is adsorbed to the nozzle, and the ester-form cholesterol mixed in during free cholesterol analysis is converted to free cholesterol.

Example 4 This is a triglyceride test for serum samples.
This is an example in which after adding a sample reagent solution, a reagent solution for free fatty acid measurement is added using the same metal nozzle.

When preparing a reagent for triglyceride measurement, if lipoproteinase (LPL) with an isoelectric point (6,9) having an isoelectric point (6,9) that is similar to the pH of the reagent solution is used,
The free fatty acid adsorbed to the nozzle causes a positive error in the constant value, but the free fatty acid has a low isoelectric point (derived from bacteria of the genus Pseudomonas).
4, 8) When LPL is used, it is possible to prevent the appearance of abnormal positive errors.

Claims (1)

[Claims] 1. A reagent solution for the first analysis item containing an enzyme with an isoelectric point lower than the pH of the reagent solution is added to the first analysis item using a metal nozzle.
After the first step, a reagent solution for a second analysis item is added to the sample using the metal nozzle, and a second sample that can cause a specific enzymatic reaction with the enzyme is added. and a step of measuring the first analysis item after the first process and measuring the second analysis item after the second process. How to analyze.