JP4733595B2 - Liquid reagent for measuring albumin in liquid samples - Google Patents

Description

  The present invention relates to a liquid reagent for highly sensitive and specific measurement of albumin in a liquid sample.

  Measuring protein in a biological sample is important in pathological diagnosis. For example, when liver function is reduced, the amount of serum albumin is reduced. On the other hand, in the case of renal / urinary tract diseases such as nephritis, nephrotic syndrome, stones, and tumors, the amount of protein in the urine increases. In recent years, it has been pointed out that the amount of albumin in urine increases even in patients at risk such as myocardial infarction and cerebral infarction caused by lifestyle-related diseases such as diabetes, obesity, hypertension, or arteriosclerosis. ing. As a method for improving lifestyle-related diseases, urinary albumin decreases when various antihypertensive drugs are effective. Therefore, to measure albumin excreted in trace amounts in urine with high sensitivity, specifically, simply and inexpensively, for early diagnosis of the above-mentioned diseases, appropriate treatment policy, determination of therapeutic effect, It is also an important indicator for motivation to improve each individual's preventive awareness to prevent progression to serious lifestyle-related diseases.

  Various measuring methods have been developed for the purpose of measuring urinary albumin with high sensitivity. In particular, in recent years, the concept of metabolic syndrome (central obesity syndrome) with a high risk of developing myocardial infarction and cerebral infarction has been proposed, and metabolic syndrome has developed even below the diagnostic criteria for urinary albumin in diabetic nephropathy Has been reported, and there is a need for a more sensitive measurement method.

  The American Diabetes Society proposes that the diagnostic criteria for microalbuminuria, which is diabetic nephropathy, is 20 mg / L (2 mg / dL) or more and 200 mg / L or less. In order to accurately measure the vicinity of this diagnostic standard, a method capable of accurately measuring 20 mg / L (2 mg / dL) or less as a measurement method is desired. The minimum detection sensitivity of the method needs to be able to be measured up to 1 to 5 mg / L (0.1 to 0.5 mg / dL).

  The test paper method is widely used as a screening test for urinary protein containing albumin. In the test paper method, the protein error method is mainly used. In this method, for example, tetrabromophenol blue (TBPB) is used as a pH indicator, and the amino group of albumin shows basicity in the vicinity of the isoelectric point of albumin, and color develops TBPB which is a pH indicator. In order to use the test paper method, TBPB and a buffer agent are impregnated and dried on a filter paper and attached to a plastic film piece to produce a urine protein measurement test paper, which is widely used for screening. The minimum detection sensitivity of the test paper method by the protein error method using the pH indicator was 150 mg / L (15 mg / dL), and the sensitivity was insufficient.

  In recent years, a test paper method has been developed that improves the protein error method and can measure urinary albumin with high sensitivity. Bis (3 ′, 3 ″ -diiodo-4 ′, 4 ″ -dihydroxy-5 ′, 5 ″ -dinitrophenyl) -3,4,5,6-tetrabromosulfophthalein is used as a pH indicator. The detection sensitivity is 10 mg / L. Although the sensitivity is about 10 times higher than that of the TBPB method, there is still a problem of low sensitivity.

  Furthermore, a test paper method using a protein binding method (hydrophobic binding) capable of measuring urinary albumin with high sensitivity has been developed. Test paper using 4,5,6,7-tetrachloro-2 ′, 4 ′, 5 ′, 7′-tetraiodo-fluorescein disodium salt (common name: Acid Red 94, Rose Bengal) as albumin binding dye Method, the detection sensitivity is likewise 10 mg / L. (Patent Document 1)

  As a method for measuring trace albumin in urine with high sensitivity and specificity, an immunoturbidimetric method or an immunolatex agglutination method is generally used. These immunization methods are highly sensitive methods capable of measuring up to 1 mg / L (0.1 mg / dL), and are highly specific to albumin because they are immune reactions. However, even in urine samples that are determined to be normal when the albumin concentration is 20 mg / L or less, there are reports that there are cases in which many denatured albumins and globulins are detected by electrophoresis or liquid chromatography. The antibody used in the immunization method is prepared using one fraction of human serum albumin as an antigen. Albumin excreted in urine contains a large amount of denatured albumin (three-dimensional structure changed, drugs and bilirubin bound, glycated or sulfonated, low molecular weight albumin with side chains cleaved). Yes. It has been pointed out that various compounds bind to an antigen recognition site (epitope) of albumin or denatured albumin (immune non-responsive albumin) cannot be measured by this immunization method (Non-patent Document 1).

International Publication WO2004 / 015423 Clinical Chemistry 50: 2286-2291, 2004. Characterization of Immunochemically Nonreactive Urinary Albumin Tanya M. Osicka and Wayne D. Comper

  The minimum detection sensitivity of the test paper method using TBPB, which is frequently used as a screening test for urinary protein, as an indicator is 15 mg / dL, which is 20 mg / L (2 mg / dL), which is the diagnostic standard for microalbuminuria from the American Diabetes Association. There was a problem that albumin in the vicinity of a low concentration could not be measured. Naturally, the minimum detection sensitivity for satisfying the above diagnostic criteria is not detectable near 1 to 5 mg / L (0.1 to 0.5 mg / dL), or the concentration difference cannot be identified quantitatively.

  As a method for measuring various denatured albumins as described in Non-Patent Document 1, there is a method of separating and analyzing using liquid chromatography or electrophoresis, but an expensive special device is required. In addition, the operation is complicated and not practical. In order to improve the reliability, it is conceivable to prepare an antibody against each modified albumin and immunologically measure it with a mixed antibody, but the cost is further increased and it is extremely impractical.

  Therefore, the present invention specifically and easily modifies albumin in a liquid sample such as urine with a sensitivity comparable to that of an immunological assay, and denatured albumin (immune non-responsive albumin) that cannot be measured by an immunoassay. It is an object to provide a liquid reagent that can be measured with high sensitivity.

  The present invention provides an indicator having a chemical structure represented by the following chemical formula 1 or the following chemical formula 2, and at least one of a citrate buffer solution and an acetate buffer solution having a pH adjusted to 1.0 to 3.0. And a nonionic surfactant. A liquid reagent for specifically measuring trace albumin in a liquid sample.

  As a result of diligent research, the present inventors improved the albumin high-sensitivity measurement method described in Patent Document 1 described above, and found a prescription that is more sensitive and more specific to albumin, thereby completing the present invention. Thereby, it is possible to specifically measure a lower concentration of albumin while having sensitivity comparable to an immunological measurement method. In addition, the albumin sensitive measurement method described in Patent Document 1 is based on the principle of staining the protein itself and is not limited by the presence or nature of the antigen recognition site, and therefore cannot be measured by the immunological measurement method in the present invention. Immunologically unresponsive albumin can also be measured.

  In addition, when the liquid reagent of the present invention is used, a special device that is expensive and cumbersome using liquid chromatography or electrophoresis is not required to simultaneously measure immune non-responsive albumin. The liquid reagent of the present invention uses a reagent available at low cost as an indicator. Therefore, the liquid reagent of the present invention can measure albumin with high sensitivity at low cost.

  The liquid reagent of the present invention is Acid Red 92 (generic name: Phloxine B (generic name: Phloxine B) having a chemical structure represented by the following chemical formula 3 among halogenated xanthene dyes used in the dye binding method as an indicator for measuring microalbumin. )) Or Acid Red 94 (generic name: Rose Bengal) represented by the following chemical formula 4.

  The concentrations of Acid Red 92 (Chemical Formula 3) and Acid Red 94 (Chemical Formula 4) in the liquid reagent of the present invention can be changed according to the concentration of trace albumin to be measured, for example 0.005 to 0.2 mmol / L. Preferably, the concentration of the indicator contained in the liquid reagent is 0.01 to 0.1 mmol / L.

  The liquid reagent of the present invention further comprises at least one of a citric acid and an acetic acid buffer capable of adjusting the pH of the reaction system to a condition of 1.0 to 3.5, and a nonionic surfactant. And.

  As the buffer, when the indicator contained in the liquid reagent is Acid Red 92 (the above chemical formula 3), a buffer that can adjust the pH of the reaction system to 2.5 to 2.8 is used. preferable. In this case, it is preferable to use a citrate buffer as the buffer.

  On the other hand, when the indicator contained in the liquid reagent is Acid Red 94 (Chemical Formula 4 above), a buffer that can adjust the pH of the reaction system to 2.2 to 3.5 is used. Is preferred. In this case, it is preferable to use an acetate buffer as the buffer.

  The concentration of the buffer contained in the liquid reagent is, for example, 0.01 to 0.5 mol / L, preferably 0.05 mol / L.

  The nonionic surfactant contained in the liquid reagent is for improving the solubility of Acid Red 92 and Acid Red 94 which are indicators. As the nonionic surfactant, those which do not inhibit the reactivity between the indicator and albumin are preferably used, and polyoxyoctylphenyl ether series (ethoxyl group is 7 to 70) is preferably used. By using polyoxy (10) octylphenyl ether marketed as -100 (registered trademark of Union Carbide Chemicals and Plastic Co.), the specificity for albumin can be further increased. The concentration of the nonionic surfactant in the liquid reagent is, for example, 0.01 to 1.0%, and the optimum concentration is 0.1 to 0.5%.

  Basically, the liquid reagent according to the present invention can be preferably used in a one-component system or a two-component system. In the two-part form, the indicator (Acid Red 92 or Acid Red 94) and the nonionic surfactant are present independently as separate solutions (R1 and R2 reagents). R1 reagent is added to the liquid sample and mixed to measure absorbance (A), then R2 reagent is added and mixed to measure absorbance (B), and (B) − [(A) × volume coefficient It is possible to obtain a measurement value obtained by subtracting the specimen blind test from

  Here, the R1 reagent includes a nonionic surfactant and at least one of a citrate buffer solution and an acetate buffer solution.

  On the other hand, the R2 reagent contains an indicator (Acid Red 92 or Acid Red 94) and at least one of a citrate buffer and an acetate buffer.

  The concentrations of the nonionic surfactant and the buffer solution in the R1 reagent are set so that the final concentration when mixed with the R2 reagent falls within the concentration range described above. For example, the final concentration of the nonionic surfactant is 0.01-1.0%, the final concentration of the indicator is 0.005-0.2 mmol / L, and the final concentration of the buffer is 0.01-1.0%. 0.5 mol / L.

  The liquid reagent prepared as a two-component system can be used as a one-component system by previously mixing an R1 reagent containing a nonionic surfactant and a buffer solution and an R2 reagent containing an indicator and a buffer solution. In this case, the sample blind cannot be measured, but the reagent blind can be used instead.

  Table 1 shows an example of the measurement method when the liquid reagent prepared as a two-component system is used as a two-component system and when used as a one-component system. Here, in Table 1, for example, absorbance measurement A shows the case where 30 μL of urine sample is added to 200 μL of R1 reagent and the absorbance is measured, and absorbance measurement B shows the urine sample used for absorbance measurement A and the R1 reagent. The case where 100 μL of R2 reagent is added to the mixed solution and the absorbance is measured is shown. Regarding Table 1, the absorbance measurements C to I have the same view as the absorbance measurements A and B. Here, the absorbance measurement A in Table 1 corresponds to the case where the R1 reagent is used as a sample blind test. This measurement method is an example, and the sample amount and the amounts of the R1 reagent and R2 reagent can be appropriately changed depending on the type of automatic analyzer and the type of sample to be measured.

  The liquid reagent of the present invention can measure the albumin concentration in liquid samples such as urine, blood, spinal fluid, saliva, tears, gastric fluid, and albumin-containing liquids (eg, infusion solutions, tissue extracts, protein purification solutions, foods, etc.). It can be used to measure and is particularly useful when the liquid sample is a urine specimen.

  The invention will now be illustrated by the following examples. In addition, this invention is not restrict | limited to the following Example. In addition, the concentration of the indicator (Acid Red 92 or Acid Red 94) in the following examples was set to 0.05 mmol / L, which is a concentration at which 100 mg / dL of albumin can be measured. The reason is that the diagnostic criterion of 2 mg / dL for diabetic nephropathy is important for detecting premature nephropathy and arteriovascular disorder, and 100 mg / dL or more is severe due to overt nephropathy. Therefore, it is meaningless to measure a concentration higher than this.

  In this example, when Acid Red 92 and Acid Red 94 were used as indicators, the effect of the pH of the buffer in the liquid reagent on the reactivity with albumin was examined before the type of the buffer was optimized. .

  As a buffer solution contained in the liquid reagent, a tartrate buffer solution (0.1 mol / L) was used, and the pH was set to 2.3 to 3.5, and the reactivity with albumin was confirmed. The reactivity to albumin was confirmed by measuring the absorbance of each of three types of human serum albumin, α, β-globulin, and γ-globulin as liquid samples using a 20 mg / dL aqueous solution. As for the absorbance measurement results, the reactivity was evaluated as the relative value (%) of the absorbance of each globulin when the absorbance of albumin was 100%. Table 2 for Acid Red 92 and Table 3 for Acid Red 94 Respectively.

  As shown in Table 2, it was found that when Acid Red 92 was used as an indicator, the reactivity to albumin was high and the reactivity to globulin was low at pH 2.5 and pH 2.8.

  As shown in Table 3, when Acid Red 94 was used as an indicator, it was found that the reactivity to albumin was high and the reactivity to globulin was low in the range of pH 2.2 to pH 3.5.

  In this example, when Acid Red 92 and Acid Red 94 were used as indicators, the effect of the type of buffer on the reactivity with albumin was examined.

  When Acid Red 92 was used as the indicator, a buffer solution prepared by adjusting each buffer solution of citric acid, tartaric acid, phthalic acid, and glycine to 0.05 mol / L and pH 2.8 was used. The reactivity when using each buffer was measured for absorbance of human serum albumin, α, β-globulin, and γ-globulin using 20 mg / dL aqueous solutions in the same manner as in Example 1. Was confirmed. Regarding the measurement results of absorbance, reactivity was evaluated as relative values (%) of absorbance of each globulin when the absorbance of albumin was 100%, and the results are shown in Table 4.

  As can be seen from Table 4, citric acid (0.05 mol / L, pH 2.8) had the highest absorbance for albumin and the lowest reactivity with globulin, and was specific for albumin.

  On the other hand, when Acid Red 94 was used as the indicator, a buffer prepared by adjusting each buffer solution of citric acid, acetic acid, formic acid, and glycine to 0.05 mol / L and pH 2.8 was used. The reactivity when using each buffer was measured for absorbance of human serum albumin, α, β-globulin, and γ-globulin using 20 mg / dL aqueous solutions in the same manner as in Example 1. Was confirmed. Regarding the measurement results of absorbance, reactivity was evaluated as relative values (%) of the absorbance of each globulin when the absorbance of albumin was 100%, and the results are shown in Table 5.

  As can be seen from Table 4, when citrate buffer, acetate buffer (0.05 mol / L, pH 2.8) and glycine buffer were used as buffers, reactivity with globulin was low and specific for albumin. It was the target. Among these buffers, the acetate buffer has the highest absorbance for albumin. From this point, it can be seen that it is preferable to use acetic acid as the buffer when Acid Red 94 is used.

  In this example, when Acid Red 92 was used as an indicator, the effect of the type of nonionic surfactant on the reactivity with albumin was examined.

  In this example, Acid Red 92 (0.05 mmol / L), citrate buffer (pH 2.8, 0.05 mol / L), and nonionic surfactant (0.1%) were used as liquid reagents. Including one was used. As a nonionic surfactant, it is necessary to dissolve acid red 92 and to ensure the solubility of the bound substance of acid red 92 and albumin, which inhibits the reactivity of albumin and acid red 92. Non-ionic surfactants, general name Triton X-100 (registered trademark of Union Carbide Chemicals and Plastic Co.) and polyoxyethylene lauryl ether marketed under the general name Brij35 were used.

  The reactivity with albumin was confirmed by measuring the absorbance of human serum albumin, α, β-globulin, and γ-globulin in the same manner as in Example 1 using a 20 mg / dL aqueous solution. As for the measurement results of the absorbance, the reactivity was evaluated as the relative value (%) of the absorbance of each globulin when the absorbance of albumin was 100%, and the results are shown in Table 6.

  As shown in Table 6, when polyoxy (10) octylphenyl ether marketed under the general name Triton X-100 (registered trademark of Union Carbide Chemicals and Plastic Co.) is used as the nonionic surfactant As compared with the case where polyoxyethylene lauryl ether marketed under the general name Brij35 is used, it was found that the reactivity with albumin is the same, but the reactivity with globulin is low.

  In this example, the quantitative characteristics of the automatic analyzer in the liquid reagent formulation using Acid Red 92 or Acid Red 94 were evaluated as simultaneous reproducibility. In this evaluation, an automatic analyzer 7170 manufactured by Hitachi High-Technologies Corporation was used as an automatic analyzer. The simultaneous reproducibility is prepared so that the albumin concentration is 10 mg / dL, 30 mg / dL, 60 mg / dL and 100 mg / dL as an albumin solution, and each albumin solution is used as a sample from 20 simultaneous reproducibility, The coefficient of variation was evaluated.

(Measurement conditions for two-component systems)
In the case of a quantitative method using Acid Red 92 as an indicator, first, 30 μL of a sample (albumin solution) and 200 μL of a citrate buffer solution (0.05 mol / L, pH 2.8) containing 0.1% Triton X-100 are mixed. The mixture was incubated at 37 degrees for 5 minutes. Using the above automatic analyzer, the absorbance was measured at a main wavelength of 546 nm and a sub-wavelength of 660 nm using a reagent blind as a control to make a sample blind. Subsequently, the above mixed solution and 100 μL of citrate buffer solution (0.05 mol / L, pH 2.8) containing Acid Red 92 (0.05 mmol / L) are mixed and incubated at 37 degrees for 5 minutes. A sample color developing solution was prepared. The absorbance of this sample color developing solution was similarly measured using an automatic analyzer at a main wavelength of 546 nm and a sub-wavelength of 660 nm using a reagent blind as a control. A 100 mg / dL albumin standard solution separately tested was subjected to the same operation, and the absorbance of the sample color developing solution was divided by the absorbance of the standard solution, and multiplied by 100 mg / dL to determine the albumin concentration. The measurement results of the albumin concentration in each concentration of the sample solution are shown in Table 7. Table 7 shows the average value, standard deviation, and coefficient of variation of 20 measurements at the same time.

  As shown in Table 7, when a liquid sample having the above formulation is used, the coefficient of variation of 0.2 to 0.8% is very good in the sample solution having various concentrations, and the simultaneous reproducibility is excellent. I understood. Moreover, it can be seen from the results shown in Table 7 that the measured value when Acid Red 92 is used as an indicator shows linearity passing through the origin in the range of 10 to 100 mg / dL.

  The present inventors further show that although the data is not shown, for the minimum detection sensitivity of albumin with Acid Red 92, a 100 mg / dL albumin standard solution is serially diluted with purified water, and the reproducibility of purified water is +2 standard deviation. The reproducibility of the lowest albumin concentration was determined as the concentration at which −2 standard deviations did not cross. As a result, the minimum detection sensitivity of albumin was 0.5 mg / dL (5 mg / L). Therefore, it can be seen that the liquid indicator of the present invention using Acid Red 92 can measure albumin concentration in the range of 0.5 to 100 mg / dL, and can accurately measure albumin concentration in a wide range from low to high. It was.

  On the other hand, when Acid Red 94 was used as an indicator, an albumin standard solution was serially diluted with purified water in the same manner as Acid Red 92 except that an acetate buffer was used as a buffer, and the albumin concentration was measured. When the minimum detection sensitivity of albumin concentration under these conditions was determined in the same manner as Acid Red 92, the minimum detection sensitivity of albumin concentration was 0.125 mg / dL (1.25 mg / L). In addition, the liquid reagent of the present invention using Acid Red 94 had good linearity in the measurement result of albumin concentration as in Acid Red 92. Therefore, it can be seen that the liquid indicator of the present invention using Acid Red 94 can measure albumin concentration in the range of 0.125 to 100 mg / dL, and can accurately measure albumin concentration in a wide range from low concentration to high concentration. It was.

  As described above, the liquid reagent of the present invention has a coefficient of variation of 1% or less when measuring the albumin concentration from a low concentration to a high concentration, can obtain extremely good reproducibility, and can be used in clinical practice.

  The minimum detection sensitivity of the liquid reagent of the present invention is 0.125 to 0.5 mg / dL, which clears the minimum detection sensitivity of 0.1 to 0.5 mg / dL desired from the diagnostic criteria for diabetic nephropathy. It was confirmed that the sensitivity was equivalent to that of the immunization method.

The present invention is capable of easily measuring a small amount of albumin in a liquid sample such as a biological sample with high sensitivity, specifically for albumin, at a low cost and easily using the principle of the dye binding method, and comparable to an immunological measurement method. It is a liquid reagent having the ability to

Claims (13)

A liquid reagent for measuring trace albumin in a liquid sample,
An indicator having a chemical structure represented by chemical formula 1 or chemical formula 2 below:
at least one buffer of 1.0 to 3.5 two adjusted with citric acid buffer and acetic acid buffer pH,
A nonionic surfactant;
A liquid reagent for specifically measuring trace albumin in a liquid sample, comprising:

The liquid reagent according to claim 1, wherein the concentration of the indicator contained in the liquid reagent is 0.005 to 0.2 mmol / L. The liquid reagent according to claim 2, wherein the concentration of the indicator contained in the liquid reagent is 0.01 to 0.1 mmol / L. The indicator contained in the liquid reagent is the one described in Chemical Formula 1 above,
The liquid reagent according to claim 1, wherein the pH condition of the liquid reagent is 2.5 to 2.8. The liquid reagent according to claim 4, wherein the buffer contained in the liquid reagent is a citrate buffer. The indicator contained in the liquid reagent is the one described in Chemical Formula 2 above,
The liquid reagent according to claim 1, wherein the pH condition of the liquid reagent is 2.2 to 3.5. The liquid reagent according to claim 6, wherein the buffer contained in the liquid reagent is an acetate buffer. The liquid reagent according to claim 1, wherein the concentration of the buffer contained in the liquid reagent is 0.01 to 0.5 mol / L. The liquid reagent according to claim 1, wherein the nonionic surfactant contained in the liquid reagent is a polyoxyoctylphenyl ether series (ethoxyl group is 7 to 70). The liquid reagent according to claim 9, wherein the nonionic surfactant contained in the liquid reagent is polyoxy (10) octylphenyl ether (trade name: Triton X-100). The liquid reagent according to claim 1, wherein the concentration of the nonionic surfactant contained in the liquid reagent is 0.01 to 1.0%. The indicator according to claim 1, characterized in that the indicator and the nonionic surfactant are present as separate solutions (R1 reagent and R2 reagent) independently and can measure the sample blind. Liquid reagent. The liquid reagent according to claim 1, wherein the indicator and the nonionic surfactant can be measured as the same reagent.