JP2005091111A - Reagent for quantitative determination of total hemoglobin in urine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an immunoassay reagent capable of determining quantitatively, easily and precisely total hemoglobin in an urine sample mixed with erythrocytes, without requiring specific and special pretreatment. <P>SOLUTION: In this quantitative analytical reagent of the present invention, a surfactant for hemolyzing the erythrocytes mixed in an urine, and a polymer for promoting a particle aggregating reaction are added preliminarily in the reagent, in quantitative immunoassay for the total hemoglobin in the urine by a gold colloid particle flocculation method. Since the reagent is applicable for a versatile automatic analyzer, a treatment capacity is enhanced when treating a large amount of specimens as in a group examination, and labor saving and efficiency are expected to be enhanced. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a quantitative reagent for urinary total hemoglobin using a colloidal gold particle aggregation method. More specifically, the present invention relates to a method for rapidly and completely lysing erythrocytes in urine and accurately quantifying a trace amount of total hemoglobin in urine.

  In hematuria caused by diseases of the renal urinary tract, red blood cells and hemoglobin are often mixed in the urine. Conventionally, as a method for inspecting a very small amount of hematuria that cannot be visually determined, a occult blood reaction using a test paper (test paper method) and a sediment-formed test using a microscope are generally used. However, the test paper method can qualitatively determine hemoglobin in urine easily, but there is a problem that mixed red blood cells have a spot-like coloration and are determined to be low in device determination. The test paper method is based on the redox reaction by the peroxidase-like activity of hemoglobin, so it is affected by reducing substances present in urine such as ascorbic acid and other substances having peroxidase-like activity such as myoglobin. There was also a problem that it was easy and low in specificity.

Then, in order to raise specificity, the urinary hemoglobin measuring method using an antigen antibody reaction is tried (nonpatent literature 1). That is, as a result of analyzing a urine sample by a particle agglutination method using polystyrene latex sensitized with an anti-hemoglobin antibody, it is described that hemoglobin in urine can be specifically qualitatively determined without being affected by ascorbic acid or myoglobin. ing. However, in the case of a urine sample in which erythrocytes are mixed in urine, this cannot be detected and there is a problem that a negative result is obtained.

On the other hand, it is disclosed that the total amount of hemoglobin in urine can be accurately quantified by analyzing urine sample mixed with red blood cells at -80 ° C and re-thawing urine sample by polystyrene latex method sensitized with anti-hemoglobin antibody. (Non-Patent Document 2). However, there is a problem that it is necessary to add a hemoglobin stabilizing reagent to the urine sample at the time of freezing, and further, it takes a lot of time, labor and equipment for the freezing treatment and re-thawing operation at -80 ° C. .

In Patent Document 1 and Patent Document 2, a surfactant is used as a reagent for flow cytometry or the like in order to hemolyze red blood cells present in a biological sample. However, if there is a surfactant in the reaction system in the quantitative immunoassay using the particle agglutination method, depending on the type of surfactant, the antigen in the analysis target and the antibody in the reagent may be denatured and no reaction will occur. Cause the phenomenon. Furthermore, even if the surfactant has a mild protein denaturing action, there is a problem in that since the particle aggregation is suppressed, the reactivity is remarkably lowered and the antigen present in a trace amount in the sample cannot be accurately measured.

Japanese Patent Laid-Open No. 06-109725 JP 09-329596 A Clinical examination vol. 39 969-973 1992 Clinical pathology vol. 51 pp. 403-408 2003

  The present invention solves the above-mentioned problems in quantifying total hemoglobin in urine samples containing red blood cells, and provides a reagent for easily and accurately quantifying a small amount of total hemoglobin in urine without requiring pretreatment. It is the purpose.

  The present invention relates to a surfactant that hemolyzes erythrocytes mixed in urine in a quantitative immunoassay of urinary total hemoglobin by a colloidal gold particle agglutination method, and a high molecular polymer that enhances the reactivity by promoting particle agglutination. Is previously contained in the reagent.

That is, in a reaction system using two types of reagents widely used in general-purpose automatic analyzers used in clinical tests, the quantitative analysis reagent of the present invention contains a surfactant in the first reagent, The above-described problem is solved by containing the polymer at a predetermined concentration in either the reagent or the second reagent, or in both the first reagent and the second reagent.

According to the quantitative analysis reagent of the present invention, even a very small amount of total hemoglobin in a urine sample in which red blood cells are mixed can be specifically and easily quantified with no special pretreatment.

The reagent used in the present invention is composed of a first reagent containing a surfactant having an action of lysing red blood cells and a second reagent containing a colloidal gold particle carrier to which an antibody against hemoglobin is bound. The polymer polymer that promotes the particle agglutination reaction, which is the other essential component, is contained at a predetermined concentration in either the first reagent or the second reagent, or in both the first reagent and the second reagent.

As the surfactant used in the present invention, a polyoxyethylene nonionic surfactant having an action of hemolyzing red blood cells can be used. For example, polyoxyethylene octyl phenyl ether (addition mole number 5-20) or polyoxyethylene nonylphenyl ether (addition mole number 5-20) etc. are mentioned. In particular, use of polyoxyethylene octyl phenyl ether (addition mole number 10) or polyoxyethylene nonylphenyl ether (addition mole number 10) is preferable. Also, the above can be combined. The content of these surfactants is 0.01 to 0.5% by weight.

Moreover, as the high molecular polymer used in the present invention, polyethylene glycol having an average molecular weight of 4,000 to 25,000 having an action of promoting the reaction can be used. The content of the high molecular polymer is 1 to 10% by weight.
Furthermore, it is advantageous to use a phosphate buffer solution, an ammonium chloride buffer solution, a Good buffer solution or the like in order to maintain a constant pH.

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

  (Preparation of reagent) 0.05 M containing 0.2 wt% polyoxyethylene octyl phenyl ether (added mole number 10), 6 wt% polyethylene glycol 6000 (average molecular weight 6,000), 0.1 wt% sodium azide A phosphate buffer (pH 6.8) was used as the first reagent.

According to the method described by Flens et al. (Nature Phys. Sci., Vol. 241, p20-22 (1973)), chloroauric acid was reduced with sodium citrate to prepare a colloidal gold solution. After adding 6 μg of anti-human hemoglobin / mouse monoclonal antibody described in JP-A-2-141665 per 1 ml of colloidal gold solution, 0.5 mL of 1% bovine serum albumin solution was added. Subsequently, the antibody-bound gold colloid as a dark red precipitate was collected by centrifugation at 10,000 rpm for 10 minutes, and the resulting solution was collected in 0.05M phosphate buffer (pH 6.5) containing 1% bovine serum albumin at a wavelength of 540 nm. The suspension was suspended so that the absorbance was 2.4, and used as the second reagent.

(Measurement) Human blood collected from heparin was diluted 100 times with physiological saline to prepare a human erythrocyte suspension. Those further diluted 1,000, 2,000, and 4,000 times with physiological saline were measured as non-hemolyzed samples. A general-purpose automatic analyzer (7070 type automatic analyzer manufactured by Hitachi, Ltd.) was used for the measurement. The reaction system was sample: 5 μl, first reagent: 130 μl, second reagent: 140 μl, and the difference in absorbance immediately after the start of the reaction and after 5 minutes was measured at a main wavelength of 546 nm and a subwavelength of 700 nm. Prior to the experiment, an absorbance difference was measured under the same conditions using a calibrator 2010 manufactured by Kudosei Co., Ltd. as a standard hemoglobin, and a calibration curve showing the relationship between the hemoglobin concentration and the absorbance difference was created. From this calibration curve, the hemoglobin concentration in the sample was calculated.
As Comparative Example 1, the first reagent to which polyoxyethylene octyl phenyl ether (addition mole number 10) was not added was used. In order to confirm the quantification of the present invention, a human erythrocyte suspension was diluted with distilled water at the same magnification, and a completely hemolyzed sample was also measured. The results are shown in FIG.
When the reagent of the present invention was used, it was possible to accurately analyze the total hemoglobin concentration of the sample in which red blood cells were mixed. On the other hand, when a reagent to which polyoxyethylene octylphenyl ether (addition mole number 10) was not used was used, the measured value of hemoglobin was extremely low and could not be quantified.

When using the 1st reagent which added 0.2 weight% sodium lauryl sulfate of the surfactant widely used as a hemolytic agent to the test paper instead of polyoxyethylene octyl phenyl ether (addition mole number 10) No reaction was observed and measurement could not be performed.

(Preparation of reagent) 0.02M containing 0.05 wt% polyoxyethylene nonylphenyl ether (addition mole number 10), 3 wt% polyethylene glycol 20000 (average molecular weight 20,000), 0.1 wt% sodium azide Piperazine-N, N′-bis-2-ethanesulfonic acid buffer (pH 6.8) was used as the first reagent. The same second reagent as in Example 1 was used.

(Measurement) The measurement was repeated 10 times using urine mixed with red blood cells as a sample. As in Example 2, a general-purpose automatic analyzer (7070 type automatic analyzer manufactured by Hitachi, Ltd.) was used for the measurement. The reaction system was sample: 5 μl, first reagent: 130 μl, second reagent: 140 μl, and the difference in absorbance immediately after the start of the reaction and after 5 minutes was measured at a main wavelength of 546 nm and a subwavelength of 700 nm. From the calibration curve prepared with standard hemoglobin, the hemoglobin concentration (ng / mL) in the sample was calculated.
The measurement result using the first reagent not added with polyoxyethylene nonylphenyl ether (addition mole number 10) is Comparative Example 2, and the measurement result using the first reagent not added with polyethylene glycol 20000 is Comparative Example 3. Table 1 shows the results.

When the reagent of the present invention was used, the total hemoglobin concentration could be analyzed with good reproducibility even when analyzing a urine sample in which red blood cells were actually mixed. On the other hand, in Comparative Example 2 using a reagent to which no surfactant was added, the hemoglobin measurement value was extremely low and there was no quantitative property. Further, in Comparative Example 3 in which no polymer was added, the difference in absorbance due to the reaction was remarkably reduced, and the variation rate indicating the variation in the measured hemoglobin value was increased.

Since this reagent can be applied to a general-purpose automatic analyzer, when a large amount of samples are processed such as a mass examination, the processing capability is improved, and labor saving and efficiency can be expected.

Effects of the present invention (Example 2, Comparative Example 1)

Claims (3)

A quantitative reagent for urinary total hemoglobin, which comprises a polyoxyethylene nonionic surfactant and a polymer in a quantitative immunoassay using a colloidal gold particle aggregation method.
The polyoxyethylene-based nonionic surfactant is polyoxyethylene octylphenyl ether (addition mole number 5-20) or polyoxyethylene nonylphenyl ether (addition mole number 5-20), and the concentration is 0. The reagent for quantifying total hemoglobin in urine according to claim 1, which is 0.01 to 0.5% by weight.
The urine total hemoglobin determination reagent according to claim 1, wherein the high molecular weight polymer is polyethylene glycol having an average molecular weight of 4,000 to 25,000, and the concentration thereof is 1 to 10% by weight.

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