JP3266079B2 - Method and reagent for measuring albumin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method and a reagent for measuring albumin in a biological sample such as serum or plasma, and more particularly to an improvement of the bromcresol purple method.

[0002]

2. Description of the Related Art As a method for measuring serum albumin, for example, an anti-albumin serum is used to measure a change in light scattering intensity derived from an antigen-antibody complex caused by a reaction with albumin present in a sample. Although the measurement method is complicated, the operation is complicated. As a routine inspection method, a dye binding method (pH error method) typified by the bromocresol green (BCG) method and the bromocresol purple (BCP) method has been conventionally used. Widespread. Both dyes are
Both are triphenylmethane-type pH indicators belonging to the sulfophthalein group, but have different specificities. BCG develops color not only in the presence of albumin but also in the presence of globulin.
It has the disadvantage of low specificity for albumin. That is, assuming that the color development due to the reaction with albumin is 100%, the color development due to the same weight of α ₂ -globulin is about 49%.
%, 23% for β-globulin and about 3% for γ-globulin. Therefore, when albumin in a serum sample is measured by the BCG method, the globulin coexisting in the sample and the standard is used. Has the disadvantage that the measured values are not stable.

[0003] On the other hand, BCP does not develop color depending on globulin, and has higher specificity for albumin than BCG.
However, since BCP is susceptible to other coexisting substances and is affected by SH compounds present in samples and standards, the BCP method, for example, shows a daily increase in measured values of stored serum. Have the disadvantage that measured values are not stable (Ryozo Muramoto et al., Clinical Chemistry, 20: 13,199
1). Therefore, both the BCG method and the BCP method
Although it is easier than immunoassay, it is not always sufficient in terms of accuracy, and each of them has a major problem in accurately measuring albumin.

[0004]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above situation, and uses BCP having high specificity for albumin and reproduces without being affected by coexisting substances in samples and standard products. It is an object of the present invention to provide a stable albumin measurement method and reagent which have good properties and dilution linearity, and whose measured values agree with the immunological measurement method.

[0005]

SUMMARY OF THE INVENTION The present invention provides urea, guanine,
Zin salts, thiocyanates or anionic surfactants
In the presence of a protein denaturant or / SH reagent selected from
An invention of a method for measuring albumin, characterized in that measurement is performed based on a change in absorbance of bromcresol purple. The present invention also relates to urea, guanidine salts, thione
The present invention relates to a reagent for measuring albumin, comprising a protein denaturant selected from cyanates or an anionic surfactant and / or an SH reagent and bromcresol purple.

That is, the present inventors have conducted intensive studies to improve the method of measuring albumin using BCP, and as a result, the measurement system was found to contain urea, guanidine salts, thiocyanates or negative ions.
Protein denaturants selected from on-active surfactants (the present invention
The protein denaturing agent according to the present invention ) and / or the presence of the SH reagent can prevent the coexisting substance in the sample or the standard from affecting the measured value and establish a measurement method having high specificity for albumin, The present invention has been completed.

The protein denaturant according to the present invention is not particularly limited as long as it has a protein denaturing action and does not affect the measured value of albumin. For example, urea such as guanidine hydrochloride, guanidine Guanidine salts such as sulfates , for example, salts such as thiocyanates such as ammonium thiocyanate, potassium thiocyanate and sodium thiocyanate , and anionic surfactants.
In particular, an anionic surfactant is preferably used. These may be used alone or in any combination.

Examples of the anionic surfactant include an alkyl sulfate ester surfactant such as sodium lauryl sulfate (SDS) and sodium cetyl sulfate, and a polyoxyethylene alkyl phenyl such as sodium polyoxyethylene alkyl phenyl ether sulfate. Ether sulfates, for example, polyoxyethylene alkyl ether sulfates such as sodium polyoxyethylene alkyl ether sulfate and triethanolamine sulfate, alkyl benzenes such as sodium laurylbenzenesulfonate and sodium cetylbenzenesulfonate Sulfonates are preferred. Among them, sodium lauryl sulfate (SDS), sodium laurylbenzenesulfonate and the like are preferably used. These may be used alone, or may be used in combination as appropriate. The concentration of these protein denaturants varies depending on the type of protein denaturant and the sample to be treated, but it does not affect the coexisting substances contained in the sample and the standard, and affects the measured value of albumin. The concentration can be selected without particular limitation as long as it is not present, and is usually 0.001 to 10 w / w%, preferably 0.01 to 1 w / w%, more preferably 0.02 to 0.3 w / w% as the concentration at the time of albumin measurement. It is appropriately selected and used so that

The SH reagent according to the present invention includes, for example, 5,
5'-dithiobis (2-nitrobenzoic acid) (DTNB), 2,2'-dithiobis (5-nitropyridine) (NPDS), 2,2'-dithiodipyridine (2-PDS), 4,4'- Dithiodipyridine (4-PDS), 4,
Disulfides such as 4′-dithiobis (1-azidobenzene) (DTBPA) and oxidized glutathione, for example, oxidizing agents such as iodine, ferricyanide, iodosobenzoic acid, iodate, chlorites, mercury, and zinc; For example, alkylating agents such as iodoacetic acid, chloroacetic acid, iodoacetamide, and chloroacetophenone; maleimides; maleimide derivatives such as N-methylmaleimide, N-ethylmaleimide, N, N'-p-phenylenedimaleimide;thiophthalimide; No. Among these, disulfides such as DTNB, 2-PDS and 4-PDS, and maleimide derivatives such as maleimide and N-ethylmaleimide are particularly preferably used. These may be used alone or in any combination. The concentration used depends on the type of SH reagent and the sample to be treated, and is not particularly limited as long as it does not affect the coexisting substances contained in the sample and the standard and does not affect the measured value of albumin. Can be set without being measured, but the concentration is usually 0.
It is appropriately selected and used so as to be 001 to 1 mM, preferably 0.01 to 0.5 mM.

[0010] As the BCP according to the present invention, it is sufficient to use a commercially available BCP.
The concentration at the time of albumin measurement is appropriately selected and used so as to be usually 0.02 to 0.20 mM, preferably 0.03 to 0.10 mM.

When albumin in a biological sample such as serum or plasma is measured by the method of the present invention , BCP, the protein denaturing agent and / or SH reagent of the present invention are contained in the solution at the time of measurement. In addition, reagents usually used in this field, such as buffers, preservatives, surfactants having no protein denaturing action, and the like may be contained.The concentration of these used is usually used in this field. It suffices to select appropriately from the given concentration range.

The protein denaturant and / or SH according to the present invention
Buffers that can be used in solutions containing reagents include, for example, acetate, glycine, citrate, phosphate, veronal,
Good such as borate, succinate, tris (hydroxymethyl) aminomethane (Tris) such as 3- (N-morpholino) ethanesulfonic acid (MES) and 3- (N-morpholino) propanesulfonic acid (MOPS) Any buffer usually used in this field, such as a buffer, can be used without limitation. When the protein denaturant and / or SH reagent of the present invention coexists with BCP, the pH of the solution is usually 4.5 to 5.7.
And when not coexisting with BCP, it is usually appropriately selected from the range of 3 to 9. Examples of the sample applied to the present invention include biological samples such as serum and plasma.

The measurement of albumin by the method of the present invention may be performed by a one-part method or by a two-part method. It should be done as follows. That is, the sample, the BCP, and the present invention
A protein denaturant or / and an SH reagent according to the present invention, if necessary, a buffer, a preservative, a surfactant having no protein denaturing effect, a pH of 4.5 to 5.7 containing a stabilizer and the like at the above-described concentrations;
Preferably, the mixture is mixed with a reagent solution (measurement reagent solution) having a pH of 5.0 to 5.5 to form a color, and after an appropriate time, the absorbance at a wavelength appropriately selected from the range of 570 to 660 nm is measured. By applying the obtained absorbance to an albumin solution having a known concentration in advance as a sample, and applying the calibration curve representing the relationship between the albumin concentration and the absorbance obtained by the same operation using the same reagent for measurement as described above, Can be determined. The temperature during the measurement operation is usually 15 to 37
° C, preferably 37 ° C.

In the case of performing by the two-liquid method, for example, it may be performed as follows. That is, first, the sample and the present invention are related.
And a test solution (first test solution) which may contain a protein denaturant, an SH reagent, a buffer, a preservative, a surfactant having no protein denaturing action, and a stabilizing agent. The absorbance (absorbance 1) at a wavelength appropriately selected from the range is measured. Then, the mixing solution, BCP, if necessary the
A protein denaturant, an SH reagent, a buffer, a preservative according to the invention,
Mix with a reagent solution (second reagent solution) that may contain a surfactant, stabilizer, etc. that does not have a protein denaturing effect to develop color, and after an appropriate time, absorbance at the wavelength selected previously (Absorbance 2) is measured. However, the protein denaturant and / or SH reagent according to the present invention must be contained in at least one of the first reagent solution and the second reagent solution. Next, the absorbance difference obtained from the absorbance 2 and the absorbance 1 was determined using the albumin concentration of known concentration in advance as a sample, and the albumin concentration and the absorbance difference obtained by the same operation using the same first and second test solutions as described above. The albumin concentration in the sample can be determined by fitting to a calibration curve representing the relationship

When albumin in a sample is measured by the method of the present invention, it is preferable to use the protein denaturing agent of the present invention and an SH reagent in combination in consideration of effects such as improvement of measurement accuracy. The albumin measurement of the present invention using BCP is usually performed using an automatic analyzer or the like.

The albumin measuring reagent of the present invention is BCP
And a protein denaturant and / or SH reagent according to the present invention . More specifically, BCP and book
Reagent for one-pack method comprising protein denaturant and / or SH reagent according to the invention, first reagent without BCP and BC
A reagent for two-pack method, which comprises a second reagent containing P, and wherein at least one of the protein denaturant and the SH reagent according to the present invention is contained in at least one of the first reagent and the second reagent. Can be The preferred embodiments and specific examples of the components are as described in the description of the one-component method and the two-component method. Also, the albumin measuring reagent includes BCP, which is related to the present invention.
In addition to the protein denaturant and / or SH reagent, other reagents such as stabilizers, buffers, surfactants having no protein denaturing action, preservatives and the like usually used in this field are used. It may be contained, and its concentration may be appropriately selected from the concentration range usually used in this field. Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

[0017]

【Example】

Example 1 Using a Hitachi 7070 type automatic analyzer (manufactured by Hitachi, Ltd.), a test solution containing a protein denaturant and an SH reagent described in Table 1 was prepared (R-1), and the albumin was measured. Was measured for the degree of negative error. [Sample] Human pooled sera 1 and 2 were used as samples. [Albumin standard solution] Human protein standard serum (Wako Pure Chemical Industries, Ltd.)
(Designated value: 43 g / l) was dissolved in a specified amount of purified water to obtain a standard solution. [Reagent] Reagent 1 (R-1): Tris-HCl buffer (pH 8.0) 25 mM Predetermined protein denaturant 0.03% Predetermined SH reagent 0.1 mM Reagent 2 (R-2): Succinate buffer (pH 5. 5) 250 mM BCP 0.15 mM Triton X-100 (polyoxyethylene (10) octylphenyl ether) 0.3% [Measurement parameters (measurement conditions)] Measurement method: 2-point end method [15]-[31] Sample volume: 4 μl R -1: 240 μl R-2: 240 μl Measurement wavelength: 700/600 nm Measurement temperature: 37 ° C. The measured value of albumin in the sample obtained by the method of the present invention and N-antiserum albumin (manufactured by Hoechst Japan) were used. Tables 1 and 2 also show the relative values (%) of the measured values obtained by the method of the present invention when the measured value obtained by the immunological measurement method is taken as 100%.

[0018]

[Table 1]

[0019]

[Table 2]

From the results shown in Tables 1 and 2, it was found that BC was obtained without adding any of the protein denaturant of the present invention and the SH reagent.
When the P measurement is performed, the measured value becomes lower than that of the immunological measurement method (a negative error occurs), whereas the protein denaturant and / or SH reagent according to the present invention coexist Then, the measured values agree with the immunological measurement method, and the negative error of the measured values is improved.

Example 2 A sample of 69 patients' sera was used as a sample, using a Hitachi 7070 automatic analyzer (manufactured by Hitachi, Ltd.), sodium lauryl sulfate as a protein denaturant and DTNB as an SH reagent. Albumin was measured by the method. For comparison, the same sample was subjected to immunoassay [N
-A method using antiserum albumin (Hoechst Japan). Measuring equipment: Bering nepherometer BN100
(Manufactured by Behringberge)]. 1. Measurement method of the present invention [albumin standard solution] human protein standard serum (Wako Pure Chemical Industries, Ltd.)
(Designated value: 43 g / l) was dissolved in a specified amount of purified water to obtain a standard solution. [Reagent] Reagent 1 (R-1): Tris-HCl buffer (pH 8.0) 25 mM SDS 0.03% DTNB 0.1 mM Reagent 2 (R-2): Succinate buffer (pH 5.5) 250 mM BCP 0.15 mM Triton X-100 0.3% [Measurement parameters (measurement conditions)] Measurement method: 2-point end method [15]-[31] Sample amount: 4 μl R-1: 240 μl R-2: 240 μl Measurement wavelength: 700/600 nm Measurement temperature: 37 ℃ 2. Immunological measurement method [Reagent] [N-antiserum albumin (manufactured by Hoechst Japan)] [Measurement parameters (measurement conditions)] Sample volume: 15 µl (patient serum diluted 400 times with physiological saline) : 30 μl Buffer solution (1): 100 μl Buffer solution (2): 130 μl Measurement temperature: room temperature (25 ° C.) Measurement method: Measure the scattering intensity after 10 seconds and 6 minutes and 10 seconds, determine the difference, and determine the concentration. The albumin concentration in the sample was calculated in comparison with the scattering intensity obtained using the albumin solution (standard). FIG. 1 shows a correlation diagram between the measurement values obtained by the measurement method of the present invention and the measurement values obtained by the immunological measurement method.

[0022]

FIG.

(R = 0.982, y = 0.978x + 1.8, average value of x (immunological assay) = 36.0 (g / l), average value of y (assay of the present invention) = 36.9 (g / l) l), n = 69) FIG. 1 shows that the assay of the present invention has a high correlation with the conventional immunoassay.

[0024]

As described above, the present invention relates to a method for measuring albumin in a biological sample such as serum or plasma using BCP, and an improved technique of a reagent.
According to the measurement method of the present invention, albumin can be measured with good reproducibility without being affected by coexisting substances in a sample or a standard, and the correlation with an immunological measurement method is good. It is a very large invention that contributes to the industry.

[0025]

[Brief description of the drawings]

FIG. 1 is a correlation diagram between a measurement value obtained by an albumin measurement method of the present invention obtained in Example 2 and a measurement value obtained by a conventional immunological measurement method.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 33/68 G01N 33/52

Claims (12)

(57) [Claims] 1. Urea, guanidine salts, thiocyanates
Alternatively , a method for measuring albumin, wherein the measurement is performed based on a change in absorbance of bromcresol purple in the presence of a protein denaturing agent selected from an anionic surfactant and / or an SH reagent. 2. The method according to claim 1, wherein the protein denaturant is an anionic surfactant. 3. An SH reagent comprising a disulfide compound, an oxidizing agent,
Killing agent, maleimide, maleimide derivative or thiophthal
2. The method for measuring albumin according to claim 1, wherein the albumin is imide.
Law. 4. The method according to claim 1, wherein the SH reagent is a disulfide compound, maleimide or
The albumin according to claim 1, wherein is a maleimide derivative.
Measurement method. 5. The method according to claim 5, wherein the combination of the protein denaturant and the SH reagent is a negative
An on-surfactant and a disulfide, maleimide or
The albumin according to claim 1, which is a reimide derivative.
Measuring method. 6. The method according to claim 1, wherein the protein denaturant is an anionic surfactant,
The al reagent according to claim 1, wherein the H reagent is a disulfide form.
How to measure Bumin. 7. Urea, guanidine salts, thiocyanates
Alternatively , a reagent for measuring albumin, comprising a protein denaturant selected from an anionic surfactant and / or an SH reagent and bromcresol purple. 8. The protein denaturant is an anionic surfactant.
An albumin measuring reagent according to claim 7. 9. The SH reagent comprises a disulfide compound, an oxidizing agent,
A killing agent, a maleimide derivative or a thiophthalimide.
The reagent for albumin measurement according to claim 7. 10. The SH reagent is a disulfide form or maleimi.
8. A test for albumin measurement according to claim 7, which is a chloride derivative.
medicine. 11. The combination of a protein denaturing agent and an SH reagent,
An ionic surfactant, a disulfide form, maleimide or
The albumin according to claim 7, which is a maleimide derivative.
Measurement reagent. 12. The protein denaturant is an anionic surfactant,
The method according to claim 7, wherein the SH reagent is a disulfide form.
Reagent for measuring albumin.