JP2008046053A - Liquid reagent for measuring total protein in liquid sample - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a liquid reagent reacting with various proteins containing albumin when the total protein in a liquid sample such as a biosample or the like is measured on the basis of the theory of a coloring matter bonding method corresponding to screening inspection to measure the total protein with extremely high sensitivity. <P>SOLUTION: The liquid reagent for measuring the total protein in the liquid sample contains a protein measuring indicator having a chemical structure represented by the chemical formula 1, a formic acid buffer solution the pH of which is adjusted to 1.0-3.5 and a nonionic surfactant. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a liquid reagent for measuring a protein-containing sample solution, particularly total protein present in urine.

  In clinical examinations, various methods are used for quantifying total protein. The total protein quantification method is refractometer method, 280nm specific absorption based method, turbidimetric method, Kjeldahl method, bicinchoninic acid method, peptide bond is complexed with copper under strong alkaline condition, red purple There is a biuret method that develops a color (around 550 nm). In particular, the biuret method is used in many fields because it has a constant color development sensitivity and can be easily colorimetrically determined regardless of the types of proteins present.

  However, this biuret method has the biggest problem of low sensitivity. In addition, since a strong alkali is used, waste liquid treatment has also been a problem. Although the biuret method has these drawbacks, it is rather convenient when quantifying the total protein in the serum because the protein exists in the order of several grams in the serum. However, since proteins in urine and cerebrospinal fluid exist on the order of milligrams, there are problems that cannot be measured by the biuret method, and the development of a reagent capable of measuring total protein with high sensitivity has been demanded.

  For example, urinary proteins include not only albumin but also many low molecular weight proteins and denatured proteins such as α1-microglobulin, β2-microglobulin, Bence Jones protein, and retinol-binding protein. Each of these proteins has clinical significance, but is mainly measured by electrophoresis.

  There is an immunological method for measuring total protein with high sensitivity. There are several tens of proteins in urine in addition to the representative proteins as described above, and antibodies for each of these proteins are used. The total protein can be measured by combining all the proteins by immunoturbidimetry, but it is not practical because the reagent cost of the antibody becomes very expensive.

  A ternary complex method using pyrogallol-Mo has been developed as a high-sensitivity measurement method for total protein, and the resulting ternary complex conjugate has the advantage of less deposition on optical measurement cells. It came to be used in the field (patent document 1). However, since the minimum detection sensitivity is around 2 to 5 mg / dL, a trace amount of protein cannot be measured. In addition, it is difficult to say that the total protein can be measured because of low reactivity with proteins other than albumin (mainly Bence-Jones protein and globulin).

  A similar ternary complex method, Bromopyrogallol Red-In method, has improved the reactivity with proteins other than albumin (mainly Bence-Jones protein and globulin), so it is now possible to measure total protein. However, the minimum detection sensitivity can be measured only up to about 1 to 5 mg / dL. (Patent Document 2)

JP 61-155757 A International Publication WO2004 / 015423

  There has been a demand for a simple and inexpensive method capable of comprehensively measuring total protein in urine in a screening and comprehensive manner. If an abnormality is found in the screening test, each component can be quantified immunologically, or separated and analyzed by electrophoresis or liquid chromatography to identify the disease state.

  Therefore, the present invention measures the total protein with extremely high sensitivity by reacting with a wide range of proteins including albumin when measuring the total protein in a liquid sample such as a biological sample based on the principle of a dye binding method suitable for a screening test. It is an object to provide a liquid reagent that can be used.

  The present invention is a liquid reagent for measuring the total protein in a liquid sample, which is an indicator for protein measurement having a chemical structure represented by the following chemical formula 1 or 2 and a pH of 1.0 to 3.5. A liquid reagent for measuring total protein in a liquid sample, comprising a formic acid buffer adjusted to 1 and a nonionic surfactant.

  As a result of intensive research, the present inventors have improved the dye binding method, which is a high-sensitivity measurement method for total protein that is different from the ternary complex method, and can measure total protein with higher sensitivity, and is more specific to total protein. The formulation was found and the present invention was completed. Thereby, it becomes possible to measure the total protein from a lower concentration to a higher concentration over a wide range.

  The liquid reagent of the present invention is Acid Red 92 (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 protein measurement. ) Or Acid Red 94 (generic name: Rose Bengal) represented by Chemical Formula 4 below.

  The concentration of Acid Red 92 (Chemical Formula 3) and Acid Red 94 (Chemical Formula 4) in the liquid reagent of the present invention is, 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 contains a formic acid buffer that can adjust the pH of the reaction system to a condition of 1.0 to 3.5, and a nonionic surfactant.

  As the formate buffer, when Acid Red 92 (the above chemical formula 3) is used as an indicator, it is preferable to use a solution capable of setting the pH of the reaction system to 2.3 to 3.5. In the case of using Red 94 (the above chemical formula 4), it is preferable to use one that can make the pH of the reaction system 2.3 to 2.8.

  The concentration of the buffer solution 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 that do not inhibit the reactivity between the indicator and the total protein are preferably used, sorbitan surfactants are preferably used, and polyoxyethylene lauryl marketed under the general name Brij35 By using ether, the specificity for the total protein can be increased. The concentration of the nonionic surfactant in the liquid reagent is, for example, 0.1 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). For example, the merit of the two-component system is that the absorbance (A) is measured after adding and mixing the R1 reagent to the liquid sample. Furthermore, the R2 reagent is added and mixed, and the absorbance (B) is measured, and a measurement value obtained by subtracting the sample blind from (B)-[(A) × volume coefficient] can be obtained. By this, the coloring and turbidity of the sample can be eliminated.

  When used in a one-component system, the R1 reagent and the R2 reagent can be mixed in advance and used as a coloring reagent. In this case, the sample blind cannot be measured, but the reagent blind can be used instead. If there is no coloration or turbidity of the specimen, it is sufficient to blind the reagent.

  In the case of a two-component system, the R1 reagent is a nonionic surfactant and contains a formate buffer. On the other hand, the R2 reagent contains an indicator (Acid Red 92 or Acid Red 94) and a formic acid buffer.

  When the R1 reagent and the R2 reagent are mixed, the concentration of the nonionic surfactant contained in the R1 reagent and the indicator (acid red 92 or acid red 94) contained in the R2 reagent is relatively diluted. The final density when mixed is set to the density range described above. For example, the final concentration of the nonionic surfactant is 0.01 to 1.0%, and the final concentration of the indicator (Acid Red 92 or Acid Red 94) is 0.005 to 0.2 mmol / L. The final concentration of formate buffer is 0.01 to 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 A indicates 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 is a mixed solution of the urine sample and R1 reagent used for absorbance measurement A. In the figure, 100 μL of R2 reagent is added to the sample and the absorbance is measured. Table 1 assumes that the absorbance measurements C to I are viewed in the same manner 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. Note that 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 is obtained by analyzing the total protein in a liquid sample such as urine, blood, spinal fluid, saliva, tear fluid, gastric fluid, and albumin-containing fluid (eg, infusion solution, tissue extract, protein purified solution, food). 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 capable of measuring 100 mg / dL of total protein.

  In this example, when Acid Red 92 and Acid Red 94 are used as indicators, the effect of the pH of the buffer in the liquid reagent on the reactivity with the total protein is examined before the type of the buffer is optimized. did.

  As the buffer solution contained in the liquid reagent, tartrate buffer solution (0.1 mol / L) is used for Acid Red 92, and formate buffer solution (0.1 mol / L) is used for Acid Red 94, and the pH is 2.3 to 2.3. The reactivity with respect to total protein was confirmed as 3.5. For Acid Red 94, TritonX100 (0.5%) was included in the liquid reagent.

  The reactivity with respect to the total protein 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, when Acid Red 92 was used as the indicator, it was found that the specificity to the total protein was good when the pH ranged from 2.3 to 3.5. On the other hand, at pH 2.3, the absorbance at the time of measuring 20 mg / dL of albumin decreased and sensitivity decreased. At pH 3.5, the reagent was colored pink and the reagent blank rose. Therefore, the pH of the buffer solution is preferably pH 2.8, which has high sensitivity and a low reagent blank.

  As shown in Table 3, when Acid Red 94 was used as an indicator, specificity to total protein was observed in the range of pH 2.3 to 2.8, and specificity to total protein was observed at pH 2.8. It was found to be the best.

  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 total protein was examined.

  When Acid Red 92 was used as an indicator, a buffer solution prepared by adjusting each buffer solution of citric acid, tartaric acid, phthalic acid, glycine and formic acid 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. 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 4 for Acid Red 92 and Table 5 for Acid Red 94 Respectively.

  As shown in Table 4, when Acid Red 92 was used as the indicator, when formic acid buffer was used as the buffer, the absorbance to albumin was high, and the color development of globulin when the color development of albumin was 100% The value was as high as 75.3%. In addition, when the formate buffer was used, the reactivity with albumin was about 26.6% higher than when the tartrate buffer was used as the buffer. Therefore, when measuring the total protein in a liquid sample using Acid Red 92, it is preferable to use a formic acid buffer as the buffer.

  As shown in Table 5, when Acid Red 94 was used as an indicator, when formic acid buffer was used as the buffer, color development (absorbance) of globulin when albumin color development (absorbance) was 100%, A high value of 94% was shown. Therefore, when measuring the total protein in a liquid sample using Acid Red 94, it is preferable to use a formic acid buffer as the buffer.

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

  In this example, a liquid reagent containing Acid Red 92 (0.05 mmol / L), formic acid buffer (pH 2.8, 0.05 mol / L), and a surfactant (0.1%) is used. used. Surfactants are commercially available under the generic name Triton X-100 (registered trademark of Union Carbide Chemicals and Plastic Co.) and the generic name Brij35, which are nonionic surfactants that have similar reactivity between albumin and globulin. The polyoxyethylene lauryl ether used was used.

  The reactivity with the total protein was confirmed by measuring the absorbance of human serum albumin, α, β-globulin, and γ-globulin using 20 mg / dL aqueous solution in the same manner as in Example 1. . 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 of using polyoxyethylene lauryl ether marketed under the general name Brij35, it was found that Brij35 has improved reactivity with globulins other than albumin.

  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. For simultaneous reproducibility, various concentrations of albumin solutions, which are representative of proteins, were prepared and used as protein solutions. As the concentration of the solution, 10 mg / dL, 30 mg / dL, 60 mg / dL, and 100 mg / dL were prepared, and 20 simultaneous measurements were performed for each concentration solution, and the coefficient of variation was evaluated.

(Measurement conditions for a two-component system using Acid Red 92)
In the case of a quantitative method using Acid Red 92 as an indicator, first, 30 μL of a sample solution (total protein solution) and 200 μL of formic acid buffer solution (0.05 mol / L, pH 2.8) containing 0.1% Brij35 are mixed, This mixture was incubated at 37 degrees for 5 minutes. Using the previous 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 previous mixture was mixed with 100 μL of formic acid buffer (0.05 mol / L, pH 2.8) containing Acid Red 92 (0.05 mmol / L), 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. The absorbance of the sample color developing solution was divided by the absorbance of the 100 mg / dL albumin standard solution separately tested, and multiplied by 100 mg / dL to obtain the total protein concentration. Table 7 shows the measurement results of the protein concentration in each concentration of the sample solution. Table 7 shows simultaneously the average value, standard deviation, and coefficient of variation in 20 measurements.

  As shown in Table 7, when the liquid reagent having the above formulation is used, the coefficient of variation is very good at 0.2 to 0.8% in each concentration of protein solution, 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.

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

  On the other hand, when Acid Red 94 was used as an indicator, a measurement sample was prepared in the same manner as Acid Red 92, except that Acid Red 94 was used instead of Acid Red 92, and the total protein concentration was measured. . When the minimum detection sensitivity of the total protein under these conditions was determined in the same manner as in Acid Red 92, the minimum detection sensitivity of the total protein was 1.0 mg / dL (10.0 mg / L). In addition, the liquid reagent of the present invention using Acid Red 94 had good linearity in the measurement result of the total protein concentration as in Acid Red 92. Therefore, it can be seen that the liquid reagent of the present invention using Acid Red 94 can measure total protein in the range of 1.0 to 100 mg / dL, and can accurately measure total protein in a wide range from low concentration to high concentration. It was.

The present invention uses the principle of the dye binding method for the total protein in a liquid sample such as a biological sample, shows high reactivity not only with albumin (100%) but also with globulin, and is simple and extremely low cost. A liquid reagent can be provided.

Claims (12)

A liquid reagent for measuring total protein in a liquid sample,
An indicator for protein measurement having a chemical structure represented by the following chemical formula 1 or 2;
a formate buffer having a pH adjusted to 1.0 to 3.5;
A nonionic surfactant;
A liquid reagent for measuring total protein 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 2.3 to 3.5. 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 2.3 to 2.8.   The liquid reagent according to claim 1, wherein the concentration of the formic acid buffer 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 selected from sorbitan surfactants.   The liquid reagent according to claim 7, wherein the nonionic surfactant contained in the liquid reagent is polyoxyethylene lauryl ether (generic name: Brij35).   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. 2. The liquid sample according to claim 1, wherein the liquid sample is urine, blood, spinal fluid, saliva, tear fluid, gastric fluid, and albumin-containing fluid (eg, infusion solution, tissue extract, protein purified solution, food, etc.). Liquid reagent.