JP2009291123A - Method for confirming addition of specimen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for visually confirming the addition of a specimen when adding a specimen to a diluting liquid or a dissolving liquid in an analysis necessitating the dilution or dissolution of a specimen derived from body fluid in a reaction vessel. <P>SOLUTION: The method for confirming the addition of a specimen to a diluting liquid or a dissolving liquid includes the detection of glucose in the specimen by a color reaction to use glucose as a substrate. The color reaction is carried out by using an enzyme selected from glucose oxidase, glucose dehydrogenase, and glucose-6-phosphate dehydrogenase. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for confirming whether or not a sample is added to a diluted solution or a dissolved solution.

  There are many opportunities to dilute or dissolve the sample as a pretreatment in general analysis and testing. When the sample is added to the diluted solution or dissolved solution in the reaction vessel, the presence or absence of the addition can be easily determined if the amount of the sample is sufficient. However, when the amount of the sample is very small, it is difficult to visually confirm the sample addition.

  Particularly in the field of clinical chemistry, there are many opportunities to use a very small amount of sample, and a method for confirming the presence or absence of sample addition is desired. For example, when diluting a sample in a well of a microplate, a diluent is first added, and then a small amount of sample is added. At this time, it is difficult to confirm whether the sample is added. In addition, the purpose is to confirm the presence or absence of sample addition, but the confirmation method needs to be a method that does not affect subsequent inspections and the like.

  Several patents have been issued as specimen dispensing confirmation methods. A representative method is a method using a pH indicator. As described above, in the method using the pH indicator, when the amount of the sample is very small, the color tone change of the color reaction may not be remarkable. Moreover, when using a pH indicator, since it must be made into specific pH, the target measurement reaction may be inhibited (patent document 1: Unexamined-Japanese-Patent No. 5-52853).

  In addition, there is a method of confirming the presence or absence of addition of a specimen by qualitatively and quantitatively determining the presence or absence of a stabilizer added to the specimen. This method requires a pretreatment of adding a stabilizer to the specimen, which is inconvenient when directly diluting in the microplate as described above. In addition, various stabilizers may not be used because some substances affect the measurement. (Patent Document 2: JP-A-5-264414).

  The blood glucose concentration is 70 to 109 mg / dL for healthy individuals on an empty stomach, and is one of the substances that are abundant as blood components. In addition, it is gradually decomposed after blood collection, and its concentration decreases. Therefore, the device which measures glucose correctly as an analysis object has been made.

In order to quickly and accurately measure the glucose concentration, the glucose measuring reagent has been improved by adding a glucose stabilizer such as sodium fluoride. In addition, it has been aimed at highly sensitive measurement of glucose concentration mainly for quantitative analysis (Patent Document 3: JP 2000-262299 A).
However, glucose is exclusively an object to be accurately measured, and glucose has not been considered as an object as an indicator of the presence or absence of a specimen sample.
JP-A-5-52853 JP-A-5-264414 JP 2000-262299 A

  The problem to be solved by the present invention is to provide a simple method for confirming whether or not a sample has been correctly added to a diluting solution or a lysing solution in the analysis of a specimen sample derived from a body fluid.

  In order to solve the above-mentioned problems, the present inventors decided to use glucose as an index for determining the addition of the specimen sample based on the fact that glucose is generally present in the biological specimen sample. As described above, since glucose is quickly decomposed without a stabilizer, it seems that it may be difficult to confirm the presence or absence of a sample over time. However, as a result of analysis by the present inventors, it was confirmed that the sample was present in the sample for several days even without a stabilizer in a general sample, and was sufficiently good as an indicator of the presence or absence of the sample.

  In the present invention, when a sample sample containing glucose is added to and mixed with a diluent or lysate in a reaction container, the presence of glucose is detected by detecting the color change of a color reaction using glucose as a substrate. A sample addition confirmation method characterized by confirming addition of a sample to a liquid or a solution.

  According to the above means, even if the amount of the sample is very small, the presence or absence of the sample can be easily confirmed visually by utilizing the color reaction of glucose.

  Furthermore, the confirmation method itself does not affect the inspection and analysis performed later.

  Hereinafter, the present invention will be described in detail.

  The method for confirming whether or not a sample is added according to the present invention is roughly configured by coloring a sample containing glucose by a reaction using glucose as a substrate. Here, “containing glucose” means that the sample contains glucose in addition to the object to be analyzed by the target analysis / measurement system. The concentration of glucose contained in the sample is not particularly limited. Since the color reaction of glucose in the present invention is not intended for quantitative or qualitative analysis, it is exhibited by increasing or decreasing the amount of the substance in the solution where the color reaction occurs according to the concentration of glucose present in the sample. This is because the purpose can be achieved if the composition is such that a color is produced.

  The target sample is a specimen sample derived from a body fluid, and examples of the body fluid include blood, plasma, serum, saliva, urine and the like. The sample may be a sample that originally contains glucose, or may be a sample that contains glucose by adding glucose or generating glucose by pretreatment. An example of a sample that originally contains glucose is a serum sample. The blood glucose concentration is 70 to 109 mg / dL in a healthy person on an empty stomach. A sample containing glucose produced by pretreatment is, for example, a sample containing a polysaccharide or oligosaccharide having glucose as a constituent unit, such as glucose produced by the action of a glycolytic enzyme.

  The method of the present invention includes a step of adding a color reagent for glucose detection to a diluting solution or a dissolving solution in order to perform a color reaction of glucose when diluting or dissolving a specimen sample in a reaction container. And In general, when dilution or dissolution is performed as a pretreatment for analysis or inspection, the diluent or dissolution solution is dispensed into a reaction vessel, and a sample is added (or the sample is added to the reaction vessel and then diluted. Alternatively, the solution may be dispensed). The glucose detection reagent may be added to the diluted solution or dissolved solution either before or after dispensing the diluted solution or dissolved solution into the reaction vessel.

  In the present invention, the “color reagent for detecting glucose” means a set of reagents containing substances necessary for a color reaction performed via an enzyme using glucose as a substrate. Examples of the color reaction using glucose as a substrate include reactions using glucose oxidase, glucose dehydrogenase, and glucose-6-phosphate dehydrogenase.

  The reaction using glucose oxidase is as follows.

  In the first reaction, glucose is oxidized by glucose oxidase in the presence of oxygen and water to produce gluconolactone and hydrogen peroxide. The second reaction is a color reaction in which hydrogen peroxide generated in the first reaction is reacted with a hydrogen donor and peroxidase to generate a colored body. Therefore, the color detection reagent for glucose detection in this case includes three kinds of substances, glucose oxidase, hydrogen donor, and peroxidase. Examples of the hydrogen donor combination include phenols and 4-aminoantipyrine. Typical phenols are p-chlorophenol, naphthalene derivatives such as 1,7-dihydronaphthalene instead of phenols, N-ethyl-N-2-sulfoethyl-m-toluidine, N, N-diethyl-m- Toluidine derivatives such as toluidine, dihydroindoles, tetrahydroquinolines, or N-ethyl-N-sulfopropyl-3-matequinaniline or N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxy Examples thereof include substituted aniline compounds such as aniline. As an alternative to 4-aminoantipyrine, a leuco dye can be used. Examples of leuco dyes include 2- (3,5-dimethoxy-4-hydroxyphenyl) -4- [4- (dimethylamino) phenyl] -5-phenethylimidazole and 2- (3,5-dimethoxy-4-hydroxyphenyl). -4- [4- (Dimethylamino) phenyl] -5-benzylimidazole and salts thereof, 2- (3,5-dimethoxy-4-hydroxyphenyl) -4- [4- (dimethylamino) phenyl] -5 -Phenethylimidazole acetate leuco dye and the like.

  The reaction using glucose dehydrogenase is as follows.

In the first reaction, glucose is oxidized by glucose dehydrogenase in the presence of oxidized nicotinamide adenine dinucleotide [NAD (P) ⁺ ] (glucose is oxidized and NAD (P) ⁺ is reduced), and reduced with gluconolactone. Type nicotinamide adenine dinucleotide [NAD (P) H]. In the second reaction, [NAD (P) H] produced in the first reaction is a reaction in which a color is generated by donating hydrogen to the reducing coloring dye by the action of diaphorase or an electron mediator to produce the dye. Therefore, the color detection reagent for glucose detection in this case contains four kinds of substances, oxidized nicotinamide adenine dinucleotide [NAD (P) ⁺ ], glucose dehydrogenase, diaphorase or electron mediator, and a reducing coloring pigment. Reduction type coloring dyes include 3- (4,5-dimethyl-2-thiazoyl) -2,5-diphenyl-2H-tetrazolium bromide (MTT) and 2- (4-iodophenyl) -3- (4-nitrophenyl) It is possible to use tetrazolium salts such as) -5- (2,4-disulfophenyl) -2H-tetrazolium, sodium salt (WST-1) (Dojindo Laboratories).

  The reaction using glucose-6-phosphate dehydrogenase is as follows.

In the first reaction, glucose is converted to glucose-6-phosphate by hexokinase in the presence of ATP. In the second reaction, glucose-6-phosphate produced in the first reaction is oxidized with glucose-6-phosphate dehydrogenase in the presence of [NAD (P) ⁺ ], and gluconolactone-6-phosphate and [ NAD (P) H] is generated. In the third reaction, [NAD (P) H] produced in the second reaction is a reaction in which hydrogen is supplied to the reducing color-developing dye by the action of diaphorase or electron mediator to produce the dye and color. Therefore, the color detection reagent for glucose detection in this case contains six kinds of substances: ATP, hexokinase, glucose-6-phosphate dehydrogenase, [NAD (P) ⁺ ], diaphorase or electron mediator, and reducing coloring pigment. . Reduction type coloring dyes include 3- (4,5-dimethyl-2-thiazoyl) -2,5-diphenyl-2H-tetrazolium bromide (MTT) and 2- (4-iodophenyl) -3- (4-nitrophenyl) It is possible to use tetrazolium salts such as) -5- (2,4-disulfophenyl) -2H-tetrazolium, sodium salt (WST-1) (Dojindo Laboratories).

  The above color reaction is basically the same as the reaction used for quantifying glucose concentration. However, in the present invention, these color reactions do not aim at accurate quantification of glucose, and Since it is important not to affect subsequent tests and analysis, it is desirable not to add a glycolysis inhibitor such as sodium fluoride to the sample. Further, in the case of quantification, the glucose concentration and the color density are related, but in the present invention, glucose is used only as an indicator of the presence or absence of the sample addition, so the presence or absence of the color density is a problem. Therefore, in the present invention, the coloration does not need to depend on the glucose concentration, and the color reaction is desirably saturated. Furthermore, in order to quickly confirm the coloration, it is desirable that the coloration reaches saturation within 10 minutes after addition of the specimen sample, or more preferably within 5 minutes.

  The present invention is a method for confirming the presence or absence of addition of a specimen sample to a dilute solution or a lysate, and can be used in all analyzes and tests that require a process for diluting or lysing a sample in a reaction vessel. -Any type of diluent may be used as long as it is an appropriate buffer according to the intended analysis or inspection. However, according to the present invention, since the addition of a small amount of sample can be visually confirmed by the color reaction of glucose, the effect is remarkable when used for analysis and inspection using a small amount of sample in the field of clinical chemistry. is there.

  Analyzes and tests suitable for use in the present invention, particularly those that handle a large number of trace samples for the purpose of detecting or measuring specific components in the sample, such as antigen-antibody reaction by diluting the sample in the well of a microplate And a suitable example includes ELISA, EIA, and RIA methods (including automatic analyzers).

  The method of the present invention uses a glucose color reaction for the purpose of confirming whether or not a sample is added to a reaction vessel, but this reaction affects the results of analysis / inspection performed in the same reaction vessel thereafter. It is important not to give. Further, it is desirable that the color reaction has a color tone different from that of the sample so that the coloration by glucose can be easily confirmed visually. Hereinafter, specific examples will be described.

  For example, in the examples described later, the case where the measurement object of ELISA is a minute amount of protein in a minute amount of serum sample is shown, but in general, the solution of a serum sample has yellow, orange, and red color. As a result, the color reaction of glucose is blue-purple, and it can be visually confirmed whether or not a serum sample is added to the well, and the color reaction of glucose itself is completely different from the antigen-antibody reaction of the subsequent ELISA. Since it is a different reaction and does not interfere with the color development reaction of ELISA, the result of the antigen-antibody reaction can also be obtained in a state where the sample addition is confirmed by glucose coloring.

As another example of ELISA, there is no problem in assays using luminescent systems, but in the case of chromogenic systems, depending on the enzyme used in ELISA, it may interfere with the combination of color reactions, so avoid that There is a need. For example, when glucose oxidase is used as a color reaction, peroxidase is used in this reaction. Therefore, if peroxidase is used in the color development of ELISA, the reaction is affected. In this case, the problem can be solved by using a color reaction using glucose dehydrogenase or glucose-6-phosphate dehydrogenase, or by changing the color of ELISA to alkaline phosphatase or the like.

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, it is not limited to this.

  In the present invention, it is not necessary to accurately measure the glucose concentration, but since the amount of glucose in the blood decreases with time, a sufficient amount remains that can be used as an indicator of the presence or absence of the sample sample. Need to be. In this example, it was confirmed that the serum from which blood cell components were separated contained relatively stably glucose even after a lapse of days (Table 1).

  In the glucose coloring reaction in the present invention, unlike the conventional quantitative determination, it is not desired that the color tone changes depending on the glucose concentration. Therefore, the color reaction must be saturated. Furthermore, because of the property of confirming the addition of the specimen, the color reaction must be performed promptly. It was confirmed that the color reaction was saturated quickly under the conditions of this example (Table 2).

The present invention is a method for confirming the presence / absence of addition of a specimen sample, and it is desirable that the specimen sample exhibiting a color reaction does not affect the target measurement system. The Example by the enzyme immunoassay (ELISA method) of the alpha-fetoprotein (AFP) used as a tumor marker of a clinical test is shown. In order to confirm the presence or absence of addition of the sample by coloration, 10 mM phosphoric acid, 0.15 M NaCl, 2.0 U / mL glucose dehydrogenase, 2.0 U / mL diaphorase, 1 mM NAD ⁺ , 0.1 M MTT are added to the buffer as a diluent. It was. 200 μL of this buffer solution was dispensed into a container, and 15 μL of serum sample was further added.

  A purple color started from 1 minute after the addition of the specimen, and a deep purple color was exhibited after 5 minutes, confirming the addition of the specimen.

Next, it was verified whether the glucose color reaction for confirming the presence or absence of the sample addition had an effect on the enzyme immunoassay. As shown in the graph of FIG. 1, when AFP readings were compared based on the presence or absence of a glucose color reaction, no significant difference was found between the two. Thus, it was found that the color reaction for confirming the addition of the specimen sample does not affect the enzyme immunoassay.

From the above results, by adding a glucose detection color reagent to the diluted solution, glucose present in the biological sample generally reacts, and the presence or absence of the sample can be easily confirmed. it can.

The correlation of the reading of AFP by the presence or absence of the color reaction by ELISA is shown.

Claims (6)

  In an analysis that requires a process of diluting or dissolving a specimen sample derived from a body fluid in a reaction container, a method for confirming whether or not the specimen sample has been added to the diluent or dissolved liquid in the reaction container, The method, wherein the presence or absence of addition of a specimen sample is confirmed using a color reaction as a substrate.   2. The method according to claim 1, wherein the sample sample is confirmed to be added by detecting glucose present in the sample sample using the color reaction.   The method according to claim 1 or 2, further comprising a step of adding a color reagent for glucose detection to a diluting solution or a dissolving solution.   The coloration reaction is a coloration reaction performed via any enzyme of glucose oxidase, glucose dehydrogenase, and glucose-6-phosphate dehydrogenase, according to any one of claims 1 to 3. the method of.   The method according to any one of claims 1 to 4, wherein the presence or absence of sample addition is visually confirmed by visually discriminating a result of the color reaction.   The method according to claim 1, wherein the body fluid is blood, plasma or serum.

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