WO2008108007A1 - Method of assaying protein in sample and assay reagent therefor - Google Patents

Abstract

It is intended to provide a method of highly sensitively assaying a protein in a sample which can be conducted at a low cost, is applicable not only to an apparatus for exclusive use but also to automatic analyzers of common type that have been employed in a large number of hospitals, and enables the assay of even a protein contained in a minor amount in a sample; and an assay reagent therefor. The above-described objects can be achieved by the following method of assaying a protein in a sample and an assay reagent therefor. Namely, the above-described method of assaying a protein in a sample comprises bringing the sample into contact with an anionic compound having a fluorescein skeleton and a surfactant, and measuring the absorbance of the liquid mixture containing the sample, the anionic compound having a fluorescein skeleton and the surfactant. The reagent for assaying a protein in a sample as described above contains an anionic compound having a fluorescein skeleton and a surfactant.

Description

 Measurement method and regulation reagent of protein in samples Sickle field

 The present invention relates to a measurement method and a drug capable of measuring a protein contained in a sample * in a sample with very high sensitivity. The present invention is useful in the fields of analytical chemistry, sacrifice and is therapy, and particularly useful in the field of clinical examination.

 Background thigh

 Serum or β protein levels are high in dehydration, reticuloendothelial disease, or chronic sensation. In addition, plasma protein leakage, malnutrition or liver function Ρ $ ^ is low. Thus, since the serum or bell protein belly changes with each morbidity or transformation, its t-rule is emphasized in the diagnosis and treatment of symptoms.

 In addition, urinary protein appears in kidney disease, heart disease, manic, jaundice or high fever.

 Measuring the urinary protein in the urine is also important for the diagnosis and treatment of autism. In addition, albumin, which is a kind of protein, becomes the serum or bell level in the blood stool state. Serum or plasma iim is low in congenital analbuminemia, fl dry parenchymal disorder, ingestion of sputum, burns, bleeding, shock, protein-losing gastroenteritis, malabsorption syndrome or exhaustion It becomes.

 Measuring albumin in serum or plasma is important for diagnosis and treatment of the above-mentioned diseases.

Urinary albumin (urinary albumin) is extremely poor in healthy subjects, and its reference reference value is less than 6.5 ± 5. l mg / L (urgent urine). This urinary albumin appears in diabetic nephropathy, non-diabetic kidney disease such as itt glomerulonephritis shelf or benign nephrosclerosis, urinary sensation, high I & L £ E, or congestive heart failure To do. Measuring albumin in urine (urinary sputum albumin) is important for the diagnosis and treatment of Kamiki disease.

 As a method for measuring the total protein of koji (as well as measuring agents and measurement kits), there is first a method of using a test piece in which a carrier is impregnated with a dye that changes its color tone when combined with a protein. However, the method using this test piece has a detection limit of 100 to 20 Omg / L, and the protein contained in the sample cannot be measured. In addition, this method sometimes caused measurement ^^ due to the influence of other substances in the sample and unbound dyes.

 In addition, the B. iuret method, which uses a reaction in which four peptide bonds in the protein bind to divalent copper ions in the alkaline solution and form a purple color, has a detection limit of 1 0 Omg / L The regular sensitivity was low.

 The Lowry method using divalent copper ions and phenol reagent still has a detection limit of 25 mgZL, which is more sensitive than the Biuret method, but is still not sensitive to the measurement of thigh protein in the sample. It was insufficient.

 In addition, the pyrogallol red-molybdenum complex coloring method, which uses a reaction that turns amber by binding to proteins under the conditions of the strength of the pyrogallol red-molybdenum complex, has a detection limit of 2 OmgZL and Low ry Although it is more sensitive than the method, it was still inadequate for measuring the thigh protein in the sample.

 As described above, the total protein measurement method and the weaving medicine and rule determination kit have a detection limit, that is, a low sensitivity of the measurement, and the sensitivity is not good when measuring a very small amount of protein in the sample. It was enough.

«Measurement of albumin ^ As a method and rule glaze, bromcresol green (BCG) binds to albumin in the presence of citrate acid and exhibits a blue color, and measures the increase in ^ due to this color development. Thus, the BCG method for determining albumin bacteria in samples was known. In addition, the BCP method is known in which promocresol purple (BCP) binds to albumin and exhibits a blue color, and the increase in translocation due to this color development is measured to determine albumin separation in the sample.

 The BCG method and BCP method can be applied to general-purpose automatic analyzers used in many hospitals, and can be measured easily and within a short time. As described above, in the measurement of albumin which does not exert a very high strength in the sample, the measurement sensitivity was not sufficient.

 Therefore, for the measurement of albumin in urine (urine albumin in urine), a more sensitive radioimmunoassay, enzyme immunoassay, immunoturbidimetric assay, or latex »S response immunoassay is used. Conventional measurement methods and tJ¾l¾ drugs have been used (measurement sensitivity: 5 to 30 mg / L). However, these immunoassay reagents are commonly used because the antibody that specifically binds to albumin is prepared from an animal and used as a raw material, and the cost of the assay reagent is high.

 Furthermore, since this antibody is prepared from an animal or the like, there may be a difference in sensitivity depending on the lot of the antibody, and it may be difficult to provide a drug of 1 β degree.

 In addition, these immunological measurement methods and general pharmaceuticals are not capable of performing measurement with a general-purpose automatic analyzer, and can only be performed with a dedicated measuring device, take time to measure, or Some have the disadvantage that operation is tricky.

 [Tanano Reference 1] “Clinical Enclosure Needs” (Revised 3rd 2nd M), 1 17 1-1 77 7 Kanehara Publishing, 2005

 [Non-patent document 2] “Clinical envelopment requirement” (Revised 3 2, 47 3 to 4 81 1, Kanehara Publishing, 2000

Problems to be solved by the invention The present invention has a low cost for measurement, and can be applied to a general-purpose automatic analyzer used in many hospitals instead of a dedicated device, and the protein contained in the thigh in the sample can be applied. It is to provide a highly sensitive method for measuring protein in a sample and a regular reagent. Means for solving the problem

 The present invention includes the following inventions.

 (1) Insect a sample, an anionic compound having a fluorescein skeleton and a surfactant, and measure the concentration of the sample, a mixture containing an anionic compound having a fluorescein skeleton and a surfactant. A method of measuring protein in a sample consisting of:

 (2) After the sample and the anionic compound having the full-year-old resin skeleton are prepared, the pH of the mixed solution containing at least this sample and the full-year-old resin skeleton: pH 5.0 is adjusted to pH 5.0. The method for measuring a protein in a sample as described in (1), characterized by:

(3) that the _{Ρ Η} 5 · 0 following the pH of the mixed solution, the measurement of protein in a sample according to (2) to Norishiki to be performed by Ru ¾¾ an acidic solution to the mixed solution Method.

(1) The protein in the sample according to any one of (1) to (3), wherein the surfactant is a nonionic surfactant and / or a cationic surfactant. Method of measurement.

 (5) Disgusting in the sample according to any one of (1) to (4), assuming that the surfactant is in the mixed solution with a critical micelle IS * of the surfactant. Protein measurement.

 (6) The method for measuring a protein in a sample according to any one of (1) to (5), wherein the anionic compound having a fluorescein skeleton is Ellis mouth syn-B.

(7) The method for measuring a protein in a sample as described in any one of (1) to (6) above, wherein the protein is albumin. (8) A reagent for measuring protein in a sample, which contains an anion I 'biocompound and a U-surfactant that has a fluorescein skeleton.

 (9) A combination of a first reagent comprising a solution containing at least an anionic compound having a fluorescein skeleton and a second reagent comprising an acidic solution, wherein the surfactant is the first reagent and / or the first reagent. (2) The drug for measuring the protein in the sample described in (8) above, which is said to be contained in the reagent.

 (10) The acidic reversal, the second reagent may add the second reagent to the mixed liquid in which the sample and the first reagent are mixed, so that the pH of the mixed liquid becomes pH 5.0 or lower. The measurement of the protein in the sample as described in (9), characterized in that it is possible.

 (11) Disgusting that the surfactant is a nonionic surfactant and / or a cationic surfactant (8) to (10) in the sample according to any one of (10) Protein measurement.

 (12) Any one of the above (8) to (11), wherein the surfactant is set to a concentration that satisfies a critical micelle of the surfactant in the mixed solution. The reagent for measuring the protein in the sample according to one.

 (13) The method for measuring protein in a sample according to any one of (8) to (12) above, wherein the anionic compound having a fluorescein skeleton is Ellis mouth syn-B.

 (14) The reagent for measuring a protein in a sample according to any one of (8) to (13), wherein the protein is albumin. Brief Description of Drawings

 FIG. 1 is a graph showing an absorption curve when a surfactant is not removed or contained in a drug according to the present invention.

FIG. 2 shows a method for measuring a protein in a sample of the present invention and a nonsense ion boundary. FIG. 6 is a graph showing an absorption curve when Triton X—100, which is a surface active agent, is included in a worm.

 FIG. 3 is a diagram showing an absorption curve when tween 20 which is a nonionic surfactant is brought into contact with and contained in the protein measurement and drug of the present invention. FIG. 4 is a graph showing an absorption curve when the method and rule drug for protein in the sample of the present invention contains zephiramine, which is a cationic surfactant, as an insect. FIG. 5 is a diagram showing the relationship between the concentration of Triton X-1100, which is a nonionic 1 biosurfactant, and its effect in the measurement of protein in the sample of the present invention and in the case of a medicinal drug. . FIG. 6 is a graph showing the relationship between the concentration of zephyramine, a p-easy ionic surfactant, and its effect in the method for measuring protein in the sample of the present invention and the rule glaze.

 FIG. 7 is a graph showing the relationship between the effect and pH of a protein in a sample of the present invention.

 FIG. 8 is a diagram showing a line in the method of measuring protein in the sample of the present invention and the rule drug. BEST MODE FOR CARRYING OUT THE INVENTION

 [I]. Measurement of protein in sample

 1. Method for measuring protein in the sample of the present invention

 The protein in the sample of the present invention is obtained by letting a sample, an anionic compound having a fluorescein skeleton and a surfactant, and an anionic compound having a fluorescein skeleton and a surface activity. It consists of measuring the degree of the liquid mixture containing the agent.

2. 麵

The sample in the present invention is not particularly limited as long as it may contain a protein. For example, a sample derived from a human or an animal, a sample derived from a plant, a sample derived from a microorganism, Examples include food, beverages, drugs, reagents, and environmental samples. Samples derived from humans or animals are not particularly Mg, for example human, animal blood, serum, urine, stool, semen, spinal fluid, saliva, sweat, tears, ascites, or amniotic fluid; brain, 3β (such as heart, kidney, or liver); fine wrinkles such as hair follicles, skin, nails, muscles, or nerves; or cells.

 The food is not particularly limited, and examples thereof include meat, vegetables, grains, eggs, marine products, processed foods, and the like.

 Beverages are not particularly brewed and can include, for example, juice, milk, tea, coffee, or awakening.

 The drug is not particularly! ^ And can include, for example, infusion, injection, powder, or the like.

 Since the sample used for the measurement needs to be liquid, if the sample is not liquid, pre-treatment such as extraction or solubilization is performed according to a known method to obtain liquid # ¾. Just do it.

 In addition, shelves or dredging may be performed as necessary.

 3. Protein

 The protein in the present invention is a protein contained in (or possibly contained in) a sample and is intended to be measured.

 As this protein, various proteins can be used as examples. For example, the total protein can be exemplified by albumin. In the present invention, this protein is a protein that can only be leaked into a sample. Is preferred.

 In this sample, it is possible to mention urinary albumin (urinary albumin), Bens-Johns protein, and the like as proteins that can be expressed only in f 疆.

 In particular, it is progressing to the measurement of albumin in urine (urine «returned albumin}.

 4. Anionic compounds ^ τ on the fluorescein skeleton

In the present invention, the anion I ′ biocompound having a fluorescein skeleton is a fluorescein skeleton. As long as it is an anionic compound having, it can be used without particular limitation.

 Examples of the anionic compound having the fluorescein skeleton include xanthene-based pigments.

 Examples of the xanthene dye include Ellis Mouth Shin B, Yersin Sin, Yeosin, Phloxin, and Rose Benga! / ^.

 As an anionic compound having a fluorescein skeleton, Ellis Mouth Shin is particularly preferred.

 If the anionic compound having a fluorescein skeleton is too low, the sensitivity of the measurement can be obtained and the protein in the t¾ material cannot be measured. Mi in the mixed solution containing is preferably 0.1 or more, more preferably 0.5 M or more, and particularly preferably 0.7 M or more.

 In addition, if the anionic compound habit of the fluorescein skeleton is too high, the reagent blank (the reagent blind tree may also become a high level), so at least the sample and the anion having the fluorescein skeleton The separation in the liquid mixture containing the active compound is preferably 1,0 0 0μΜ or less, more preferably 10 0 ΟΜ or less, and particularly preferably 50 0 χΜ or less.

5. Surfactant

 The surfactant in the present invention is not particularly limited as long as it is a surfactant.

 Examples of this surfactant include nonionic surfactants, cationic surfactants, and sexual boundary surfactants.

When a sample and an anionic compound that has a fluorescein skeleton are combined with a surfactant, it is obtained by measurement compared to the case where the surfactant is not removed. Can be made. In addition, reversal with this surfactant reduces the reagent blank (reagent blind tree value m).

 The value obtained by the measurement (by subtracting the value of the reagent blank from the time of sample measurement) is obtained as a result of measurement of this sample, and the reagent blank (reduction of the value of the reagent blind tree) Since it becomes large, that is, a larger value can be obtained, the sensitivity of the measurement becomes high, and high sensitivity is achieved.

 Therefore, the worm with this surfactant provides the ability to measure i M protein in the sample.

 Due to transversal with this surfactant, the degree of purity obtained in the measurement of the sample is increased because the surfactant is a nonionic I ′ raw surfactant, a cation ί ′ raw surfactant or This is especially true when the mixture is a mixture of a biosurfactant and a cationic surfactant.

 Further, the decrease in the reagent blank (reagent blindness value (degree)) due to this surfactant is remarkable when the surfactant is a nonionic surfactant.

 Therefore, in the present invention, the surfactant is a nonionic surfactant, a cationic I ′ raw surface active agent, or a mixture of a nonionic surfactant and a cationic surfactant. More preferably, a nonionic surfactant is particularly preferable.

 The key to this surfactant is that if it is too low, the effect of reversing with Kamai's surfactant cannot be obtained sufficiently, so a sample, a mixture containing an anionic compound having a fluorescein skeleton and a surfactant is included. In the liquid, it is preferably 0.1 M or more, more preferably 20 xM or more, particularly preferably 40 or more.

 If this surfactant is too high, the effect of repelling the surfactant with the selfish surfactant may be weakened.

Thus, in the present invention, the surfactant has a concentration in the sample, a mixed solution containing the anionic compound having the fluorescein skeleton and the surfactant, and the critical micelle concentration of the surfactant. Preferably, the concentration is less than degree (CMC).

As specific examples of nonionic surfactants and cationic surfactants, their compound names and their critical micelle concentration (CMC) are shown in Table 1, Table 2, and Table 3. .

〔table 1〕

(: * POE: Polyoxyethylene) (Table 2)

! : * POE: Polyoxyethylene)

(Table 3)

 Critical micelle concentration (CMC) in aqueous cationic surfactant solution

 C M C Measurement temperature Compound name

 (m)

Octylamine hydrochloride 175 25 Decylamine hydrochloride 48

Dodecylamine Dodecylamine hydrochloride 14 30 Tetradecylamine hydrochloride 3.1 1 40 Hexadecylamine hydrochloride 0. 85 55 Octadecylamine hydrochloride 0.555 60 Dodecylethylenediamine acetate 5.7 25 Dodecyl Ethylene diamine lactate 4.4 4 // dodecyl ethylene diamine propionate 9.2 II tetradecyl ethylene diamine acetate 3.5 5 II tetradecylethylenediamine lactate 2. 7 II tetradecyl Ethylene diamine propionate 3.4 4 11 Hexadecyl ethylene diamine acetate 3.4 4 II Hexadecylethylenediamine propionate 2.3 3 II Octyl trimethylammonium promide 137 II Decyl Trimethylammonium bromide 65 II Decyltrimethylammonium chloride 65 II Dodecyltrimethylammonium bromide 16 II Dodecyltrimethylammonium bromide 16 30 Tetradecyltrimethylammonium chloride 4.0 0 40 Hexadecyltrimethylammonium chloride 1. 0 60 Hexadecyltrimethylammonium chloride 1. 3 30 Octadecyltrimethylammonium chloride 0. 34 25 Dodecylpyridinyl chloride 1. 5 30 Teradecyl pyridinyl chloride 3. 6 40 Hexadecyl pyridinic chloride 0. 9 30 Octadecyl pyridinyl chloride 0. 8 To II Xyldimethylbenzylammonium chloride 43. 4

Decyldimethylbenzylammonium chloride 6.1

Dodecyldimethylbenzylammonium chloride 2.8

Teradecyldimethylbenzylammonium chloride 0. 37

Hexadecyldimethylbenzyl ammonium chloride 0.04

Age Kutadecyl dimethylbenzyl ammonium chloride 0. 0071

Dioctyldimethylammonium chloride 26. 6

Didecyldimethylammonium chloride 2. 0

Didodecyldimethylammonium chloride 0. 18 As the surfactant in the present invention, “Triton; X—100 (Triton X-100)”, which is a nonionic surfactant, [polyoxyethylene (1 0) octylphenol] (critical micellar 0.3) 5mM) and "Tween 20" [Polyxylene (20) sorbitan monolaurate] (critical micelle boat: 3.2mM; 18 ° C) and a cationic surfactant "Zephiramine" [Benzyldimethyltetradecylammonium hydrochloride; ^ (critical micelle: 0.37 mM) is particularly preferred. For example, in this Triton X-100, the soot in the Kamami mixture is preferably less than 0.35 mM, which is a critical micelle ¾, more preferably 0.15 mM or less, Particularly preferred is 0.1 ImM or less.

6. Measurement of protein in sample

 The measurement of protein in a sample in the present invention will be described below.

 (1) Sample, anion I 'biocompound with fluorescein skeleton and surfactant worm sample, anionic compound with fluorescein skeleton and surfactant are maleated, but selfish sample, hate Any method can be used as long as it removes the anion I 'biocompound having its own fluorescein skeleton and its own surfactant.

 Each of the anionic compound having a fluorescein skeleton at the time of this worm and a surfactant? About hate.

 The removal of the sample, the anionic compound having a fluorescein skeleton and the surfactant may be carried out in one step or in two or more steps.

 When the worm is performed in one step, the sample is mixed with a mixture of an anion 1 ′ biocompound having a fluorescein skeleton and a surfactant (for example, measurement M drug).

 If you hate yourself ^ [in two or more stages, you can list the following as the order.

(a) A sample and an anionic compound having a fluorescein skeleton are used, and then a surfactant is wormed. (b) Invert the sample and surfactant, and then incubate an anionic compound with a fluorescein skeleton.

 (c) Insect an anion 1 ′ biocompound having a fluorescein skeleton and a surfactant, and then remove the sample.

 The time for the sample, the anion I ′ biocompound having a fluorescein skeleton, and the surfactant to disappear is preferably 30 seconds or more, more preferably 1 minute or more. Particularly preferred is 3 minutes or more.

 This sample, anion I 'biocompound with fluorescein skeleton, and surfactant are worms? However, if the temperature is 60 ° C or higher, the protein may be denatured. Therefore, 0 to 50 ° C is preferable, 5 to 45 ° C is more preferable, and 15 to 40 ° C is particularly preferable. . .

 (2) pH

 In the present invention, after the sample and the anionic compound having the fluorescein skeleton are added, the pH of the mixed solution containing at least the sample and the anionic compound having the fluorescein skeleton is set to pH 5.0. The following is preferable.

After removing the worms, reducing the pH of the mixture of hate to pH 5.0 or less will increase the absorbance obtained by the measurement, and the value of the reagent blank (reagent blind) (absorption) ¹ ^) can be reduced.

¾ ^ Obtained by measuring this sample, the force, the value of the reagent blank (reagent blind) {m decreases, the value of p ¹ ^ obtained by the measurement (from the time of sample measurement) This is preferable because it gives a larger value, that is, a higher measurement sensitivity can be obtained.

It is preferable that the pH of the mixture of humility should be ρΗ5.0 or less after reversion, but the ability to further increase the value of Kamiki by making this pH ρΗ4.0 or less? It is more preferable, and it is particularly preferable to set this pH to 3.5 or less for the same reason.

 After removing the sample and the anionic compound having the fluorescein skeleton, the ρΗ of the mixed solution containing at least the sample and the anionic compound having the fluorescein skeleton is ρ Η 5.0 or less. (More preferably ρ Η 4.0 or less, particularly preferably pH 3.5 or less) Any method may be used as long as this is difficult.

 For example, by adding an acidic solution that is an acidic solution to a mixed solution containing at least a sample and an anionic compound having a fluorescein skeleton, the pH of the mixed solution is adjusted to pH 5.0 or less (more preferably pH 4). 0 or less, particularly preferably pH 3.5 or less). Of course, during this acidic period, the pH of the mixture is reduced to pH 5.0 or less (more preferably pH 4.0 or less, particularly preferably pH 3.5) by applying force to the mixture of selfishness. The following must be of the acidic substance content, pH or addition »

 As this acidic solution, for example, citrate starch syrup, vinegar syrup solution, salt solution, slag aqueous solution, or swelled κ slag can be cited.

 After the sample and an anionic compound having a fluorescein skeleton are wormed, the pH of the mixed solution containing at least the sample and the anionic compound having a fluorescein skeleton is adjusted to pH 5.0 or less (more preferably In the present invention, the following may be mentioned as the order in which pH is 4.0 or less, particularly preferably pH 3.5 or less.

 (A) Mixture of sample, anion compound with fluorescein skeleton and surfactant

The pH of the mixture is adjusted to pH 5.0 or less (more preferably pH 4.0 or less, particularly preferably pH 3 5 or less).

(B) The sample and the anion I ′ biocompound having a fluorescein skeleton are wormed, then a surfactant is transferred to this, and then the pH of the mixed solution is adjusted by, for example, stimulating an acidic solution. , PH 5.0 or less (more preferably pH 4.0 or less, particularly preferably pH 3.5 or less). (C) A sample and a surfactant are prepared, and then an anionic compound having a fluorescein skeleton is added thereto, and then an acidic solution is applied thereto to apply ITT, etc. ρΗ

5.0 or less (more preferably ρΗ4.0 or less, particularly preferably ρΗ3.5 or less). (D) Insect an anionic compound having a fluorescein skeleton and a surfactant, then remove the sample, and then add an acidic solution to the mixture so that

5.0 or less (more preferably ρΗ4.0 or less, particularly preferably ρΗ3.5 or less). (Ε) The sample and the anionic compound having a fluorescein skeleton are stored, and then the acid of the mixed solution is added to the mixture by adding an acidic solution, etc., ρ Η 5.0 or less (more preferably ΡΗ4 0 or less, particularly preferably ρΗ 3.5 or less), and then the surfactant is removed from the mixture. -(F) Insect ft compound with a sample and a fluorescein skeleton is added to the worm, then an acidic solution containing a surfactant is added thereto, and the pH of the mixture is adjusted to pH 5.0 or less (more preferably Is pH 4.0 or less, particularly preferably pH 3.5 or less.

 (3) Temperature measurement

In the measurement method of the present invention, a sample, an anionic compound having a fluorescein skeleton and a surfactant are wormed, and a mixed solution containing the sample, an anion I biocompound having a fluorescein skeleton and a surfactant is prepared. ¾ Measure ^ to determine the protein concentration in the sample. This sample, absorption of a mixed solution containing an anionic compound having a fluorescein skeleton and a surfactant, is measured at ¹ μm, and the absorption is measured at a wavelength at or near the absorption maximum of the mixed solution. .

Then, 3 ^ (measured value) when measuring this sample is divided by the absorbance (measured value) when measuring the sample (standard solution, standard substance) whose concentration of the protein to be measured is known, By multiplying the key value of the protein in the standard solution, the concentration of the protein contained in the sample can be calculated. Incidentally, intake) ¹ when measuring the sample (measured value), and concentrations known samples of proteins to make a measurement (standard solution (measured value when measuring the standard substance)) makes measurements Measurements were made using saline or physiology that contained no protein at all, and the absorbance of the obtained reagent blank (reagent blind test) was subtracted.

 (4) Measurement operation example

 An example of the operation when obtaining the method for measuring the protein in the sample of the present invention using a general-purpose automatic analyzer is shown below.

 [I] Anion I with fluorescein skeleton I Bios, Eris Mouth Syn B, M-on surfactant Triton X—100 Place each of the second reagent of the protein powder glaze that consists of acid water nights into a container that is compatible with the general-purpose automatic dispensing device to be turned over. -[2] Place each of the containers containing these reagents in the specified position on the automatic self-analyzer. [3] Also, place the sample for protein measurement in a container suitable for the self-discipline automatic analyzer, and place it in the specified position.

 [4] Input the measurement reagent to be used and the measurement conditions (measurement parameters) for the test to be ruled into the self-resolving automatic analyzer.

 [5] Then, start measuring the sample.

 Usually, each of the sample and the first reagent of the measurement reagent is dispensed into a reaction cell (reaction cuvette) with a pipette (probe) or tube, and mixed and leaked to form the first stage reaction system. -Keep under certain conditions and allow the first stage reaction to take place.

 [6] After a certain period of time (end of first stage reaction ^), the reaction between the sample in this reaction cell (reaction cuvette) and the first reagent of the measurement drug (first stage reaction) Measure the angle value at a preset wavelength.

[7] Next, on the anti-J¾ night in the reaction cell (reaction cuvette), dispense the second reagent of Kamiki's measurement with a pipette (pro force or a tube, mix, invert, Second stage reaction system The second stage reaction is carried out under certain conditions.

 [8] After a certain period of time (end of reaction in the second stage, reaction between the sample in the reaction cell (reaction cuvette) and the first and second reagents of the drug (in the second stage) For the reaction system), the absorbance value at the wavelength of Kamiki [6] is measured.

 [9] For the pure water instead of the sample, perform the operation of Kamami [5] to [8] and measure the value of the reagent blank (reagent blindness).

 [10]. From the value obtained by subtracting the absorbance value of the reagent blank (reagent blind) from the absorbance value obtained in [8] above, the reagent blank (reagent blind tree) was obtained from the ¾ example value obtained in S [6]. Calculate the difference in absorbance by subtracting the subtracted value.

 [1 1] Kamiki [10] The difference in absorbance calculated in [1 0] is compared with the value of [absorption) in a sample (standard solution) with a known concentration of the protein to be measured [calibration curve]. It is obtained by calculating the value of the protein contained in it.

 W. Reagent for measuring protein in sample

 1. Reagent for measuring protein in the sample of the present invention

 The reagent for measuring protein in the sample of the present invention contains an anionic compound having a fluorescein skeleton and a surfactant.

 2. Sample

 The sample in the present invention is as described in “2. Sample” of “[1]. Method for measuring protein in sample” above.

 3. Protein

 The protein in the present invention is as described in “3. Protein” in “[I]. Method for measuring protein in sample” above.

4. Anion I biocompound with fluorescein skeleton

The anion I biocompound having a fluorescein skeleton in the present invention is described in “4. Anionic compound having a fluorescein skeleton” in the above “[1]. Method for measuring protein in sample”. As described above, the concentration described here is achieved in the mixture at the time of helminth between the protein contained in the sample and the anionic compound having a fluorescein skeleton. It is preferable to include an anionic compound that has the fluorescein skeleton in the drug.

5. Surfactant

 The surfactant in the present invention is as described in “5. Surfactant” in “[1]. Method for measuring protein in sample” above.

 This surfactant is such that the critical micelle of the surfactant is filled in the mixture upon reversal of the protein contained in the sample and an anionic compound having a fluorescein skeleton. This surfactant is preferably contained in the measurement drug. .

 6. Composition of dentistry

 (1) Configuration

 Measurement of protein in the sample of the present invention: The drug is preferably composed of two reagents or three or more reagents.

 In particular, it is preferably composed of two reagents.

 When the measurement reagent consists of two reagents, the first reagent and the second reagent, the following can be cited as the constitution of the components contained in each of the two reagents.

 (a) First reagent Anionic compound with fluorescein skeleton

 Second reagent Surfactant

 (b) First reagent Anionic compound having a full-year-old resin skeleton, surfactant

 2 pi surfactant

 (c) First reagent Surfactant

 Second reagent Anion 1 'biocompound with a fluorescein skeleton

(d) First reagent Surfactant Second reagent Anionic compounds, surfactants with fluorescein skeleton

 (e) First reagent Anionic compound having fluorescein skeleton and surfactant Second reagent

 (f) First reagent: anionic compound having fluorescein skeleton, and surfactant Second reagent: surfactant, buffer, etc.

 (2) Acidic solution

 The protein measurement in the sample of the present invention comprises a combination of a first reagent that contains at least an anionic compound having a fluorescein skeleton and a second reagent that consists of an acidic solution. It is preferably added to the first reagent and Z or the second reagent.

 Note that the second reagent consisting of the acidic solution is added to the mixed solution in which the sample and the first reagent are mixed, so that the pH of the mixed solution becomes pH 5.0 or less. It is something that can be done.

 Details of this acidic solution pH are as described in “(2) pHJ” in “6. Measurement of protein in sample” in “[1]. Method for measuring protein in sample” above. The protein measurement reagent in the sample of the present invention comprises a combination of a first reagent comprising at least an anionic compound having a fluorescein skeleton and a second reagent comprising an acidic solution. In this case, the following can be cited as the constitution of the components contained in each of these two reagents.

 (Ii) First reagent Anionic compound with fluorescein skeleton, and surface active agent

 (Ii) First reagent Anionic compound having a fluorescein skeleton, and surfactant An acidic solution containing a surfactant

 (C) First Reagent: A ionic compound with a Fresen resin skeleton

Second reagent Acidic solution containing surfactant 7. Other ingredients

 In the measurement of the protein in the sample of the present invention, if necessary, a buffer, an ion such as alkali metal or alkaline earth metal, or a salt containing the same; a chelating agent; sodium azide, antibiotic or Preservatives such as synthetic antibacterials; Stabilizers such as saccharides or high ^^ compounds; Activators; Elimination of measurement hindrances contained in samples or substances related to suppression of effects; or other reagent components Can be applied as needed.

 In addition, it is preferable to have a buffer having an ability in the pH range necessary for measurement as a buffer for a person.

 Examples of such buffering agents include MES, B is—Tris, B is—Tris Propon, ADA, PI PES, ACES, MOPS 0, MOPS, BES, TES, HE PES, DI PSO, TAP SO, POP SO, HEPPSO, EPPS, Trieine, Bicine, TAPS, CHES, inorganic phosphate, inorganic phosphate, boric acid, borate, glycine, glycylglycine, imidazole, or tris ( Hydroxymethyl) aminomethane [Tris] and the like. The invention's effect

 The protein measurement method in the trial of the present invention has a low cost for measurement, and can be applied not only to a dedicated device but also to a general-purpose automatic analyzer used in many hospitals. It is possible to measure the protein contained in the sample with high sensitivity and to obtain the effect.

In addition, the protein measuring agent in the sample of the present invention is low in cost, and can be applied not only to a dedicated device but also to a general-purpose automatic analyzer used in many hospitals, and in the sample. It has the effect of being able to measure the proteins contained in the water with high sensitivity. Example

 Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited by the description of these examples.

 [Example 1] (Measurement method of the present invention and confirmation of the effect of the surfactant in the t ^ law drug) Measurement method of the protein in the sample of the present invention and the objective] Confirm the effect of the surfactant in the drug. I confirmed.

 1. Type B

 (1) First reagent

 As a first reagent, Ellis Mouth Syn B (Wako Pure Chemical Industries, Ltd.) was dissolved in pure water to prepare 10 erythrosin BzK?

 (2) Second reagent.

 As the second reagent, quenic acid (Wako Pure Chemical Industries, Ltd.) was dissolved in / i to prepare 500 mM quencher.

 (3) Third reagent

 The following reagents were prepared as third reagents.

 (a) Surfactant-free reagent

 Pure water was prepared as a surfactant-free reagent containing no surfactant.

 (b) 卜 Lighton X— 1 0 0 Reagent

 卜 Lye X—1 0 0 As a reagent, 卜 Lye X—loo (Nacalai Tesque; fc) im-amount: 6 2 5] is dissolved in pure water to give 6.4 mM Triton X— 1 0 07 Prepared the night.

 (c) Tween 20 Reagent

 As a Tween 20 reagent, Tween 20 was dissolved in zK to prepare a 0.4 mM Tween 20 aqueous solution.

 (d) Zephylamine reagent.

As a zephyramine reagent, zephyramine is dissolved in pure water and a 4 mM zephyramine aqueous solution is used. Prepared.

 2. Sample

 (I) Protein-containing sample

 As a protein wire containing protein, human serum albumin [HSA] (Sigma 3) was dissolved in pure water to prepare 2. Omg / L human serum albumin aqueous solution.

 (H) Protein-free sample

 Was prepared as a protein-free sample containing no protein.

 3. Measurement

 (I) To 3,660 L of the protein-containing sample of (2) above, 80 ^ L of the first reagent of (1) of (1), 160 L of the second reagent of (2) of sickle 31, and Sickle 31 (3) third reagent (a) surfactant-free reagent (100 L) was added and mixed to obtain a mixture.

 (2) Next, the liquid mixture of l self (1) was measured by measuring the wavelength from 400 nm to 700 nm, and the absorption curve of volume 3 liquid mixture was recorded.

 (3) Perform the procedure as described in (1) and (2) except that the protein-containing sample of (2) above is used instead of the protein-containing material of (2) above. An absorption curve was recorded.

 Fig. 1 shows the absorption curve when using a protein-containing sample as a sample, and the negative absorption curve when using a protein material as a sample.

 (4) Disgusting S1 (3) Second reagent (a) Surfactant inert drug, lift yourself 1 (3) Second reagent (b) Triton X-100 reagent Except for using as a second reagent, perform the operation as described in Tatsumi (1), (2) and (3), and use the protein polarizing material as the sample. Figure 2 shows the self-absorption curve when using a protein-free sample.

(5) Kamii 1's (3) 2nd reagent (a) surfactant inactive: Replace the medicine with (2) 2nd reagent (c) Tween 20 reagent Other than the second reagent, selfish (1), (2) and Figure 3 shows the absorption curve when the protein-containing sample was used as the sample, and the absorption curve was obtained when the protein material was used as the coating material, as described in (3). Got.

 (6) Instead of the surfactant reagent (a) of the second reagent in Kamiki 1 (3), the second reagent (d) in Zekai (3) in (d) is the second reagent. (2) Other than using the reagent, the absorption curve when using a protein-containing sample as a sample, operating as described in (1), (2) and (3), and as a protein as a sample Figure 4 shows the self-absorption curve when using the non-containing sample.

 4. Measurement results

 (1) From Fig. 1 and Fig. 2, the following can be understood.

 When triton X—100, a nonionic surfactant, is contained in the measuring agent and is mixed with erythrosine synthase B, which is an anionic compound having a fluorescein skeleton, and the sample, Compared to the case where the sample X-1100 is not contained in the measurement reagent and the insect is not used, the absorption maximum of the absorption curve of the mixed solution and the absorbance in the vicinity when the sample containing the protein is measured are significantly higher. .

 In addition, when this triton X-1 100 is contained in the measuring agent and brought into contact with Ellis mouth syn-B and the sample, the triton X-1 100 is not contained in the measurement reagent. In addition, the absorption pole of the absorption curve of the mixed solution [reagent blank (reagent blind tree)] when measuring a protein-free sample is markedly reduced.

Based on the above, Triton X—100, a nonionic surfactant, is included in the current IJ reagent, and is transferred to Ellis Mouth Syn B, an anionic compound having a fluorescein skeleton. In the case of SS, the value of SS) ¹ ^ obtained by measurement (the value obtained by subtracting reagent blank ^ i from when measuring the sample) will be larger, and high sensitivity will be obtained. I was reprimanded.

(2) From Fig. 1 and Fig. 3, the following can be understood. When the measurement reagent contains Tween 20 which is a nonionic surfactant, it is twisted 2 when the fluorescein skeleton is an anionic compound Eris Mouth Syn B and a worm. Compared to the case where 0 is not included in the drug and the insect is not included, the absorption pole of the absorption curve of the mixed solution when the protein material is measured and the absorption power in the vicinity are remarkably increased.

 In addition, when Tween 20 is contained in the measurement reagent and Eris Mouth Sin B and the sample are removed, compared to the case where Tween 20 is not contained in the measurement reagent and the insect is not exposed, protein-free. The value near the absorption pole of the absorption curve of the mixed solution [reagent blank (reagent blind tree)] when the sample is measured is clearly reduced.

 From these facts, when Tween 20 as a nonionic surfactant is included in the drug, it is transferred to Ellis Mouthsin B, an anionic compound with a full-year-old resin skeleton, and a sample. It was confirmed that the value obtained by the measurement (the value obtained by subtracting the reagent blank from the sample measurement) was larger, and high sensitivity was obtained.

 (3) From Fig. 1 and Fig. 4, the following can be seen.

 When zephyramine, a cationic surfactant, is included in the measurement reagent and it is mixed with erythrincin B, which is an anionic compound that cleaves the fluorescein skeleton, and zephiramine is used as a measurement M drug. Compared to the case where the protein-containing material is not contained, the absorption pole in the absorption curve of the mixed solution when measuring the protein-containing material and the absorption J in the vicinity thereof are remarkably high.

In addition, when this Zephyramine is contained in the measurement reagent and the sample i is added to Ellis Mouth Syn B and the sample, compared to the case where Zephiramine is contained in the measurement reagent and not allowed to be tested, the protein-free sample is measured. The absorption of the absorption curve of the mixed solution [reagent blank (reagent blind tree) and near absorption) ¹ ^ is high.

However, even in this: ^, the value of the crane obtained by the measurement (the force at the time of measuring the sample ^ minus the ¾ ^ of the reagent blank) is greater when the ¾M drug contains zephyramine and is contacted. Rather than letting Zephiramine be included in the measurement drug and not causing insects In this case, it was confirmed that high measurement sensitivity was obtained.

 [Example 2] (Confirmation of the relationship between the concentration and the effect of the surfactant in the measuring method and regular reagent of the present invention)

 The relationship between the concentration of triton X-100, a surfactant, and the effect in the measurement and measurement reagent of proteins in the sample of the present invention was HI.

1. Trial

 (1) First reagent

 As the first reagent, Ellis Mouth Syn B (Wagaku Kogyo Neko: fc) was dissolved in pure water to prepare an aqueous solution of 102 / LLM erythrosine B.

 (2) Second reagent-As the second reagent, quenic acid (Wako Pure Chemical Industries) was dissolved in pure water to prepare 50 OmM night.

 (3) Third reagent

 The following reagents were prepared as third reagents.

 (a) Surfactant-free reagent

 Pure water was prepared as a surfactant-free reagent containing no surfactant.

 (b) Blue Line X—100 Reagent

 (A) 1. Reagent containing 6mM 卜 Ryton X-100

 1. As a formula drug containing 6 mM Triton X-100, 卜 Lion X-100 (Nacalati Skene ±) [^? Amount: 625] was dissolved in pure water to prepare a 1.6 mM Triton X-100 aqueous solution. did.

 (B) 3.2mM Lyon X—100 containing ^ lit medicine

3. As a reagent containing 2 mM Triton X-100, dissolve Triton X-100 (Nacalati Skene ±) ΐ ^ Λ 625] in ίβ7_Κ, and then add 3 mM Triton X-100100 Prepared.

 (C) 4. 8mM Triton X-100 containing medicine

 4. 8mM Ryton X—100 as a glaze, Triton X—100 (Nacalati Skene ±) [^? Amount: 625] was dissolved in pure water to prepare a 4.8 mM Triton X-100 aqueous solution.

 (D) 6.4 Reagents containing 4 mM 卜 Line X-100

 6. As a reagent containing 4 mM Triton X-100, Triton X-100 (Nakaraitesk ¾) Ι ^ Λ: 625] was immersed in water to prepare a 6.4 mM Lyton X-100 aqueous solution.

 (E) 8mM Triton X—100 containing ¾ drug

 As a reagent containing 8 mM Triton X-100, Triton X-100 (Nacalai Tesque ¾) [^ amount: 625] was dissolved in / J <to prepare an 8 mM Triton X-100 solution.

 (F) 9.6 mM Triton X-100 containing medicine

 9. As a 6 mM Triton X-100 polar drug, Triton X-100 (Nacalati Skene ±) [^ amount: 625] was dissolved in thread water to prepare a 9.6 mM Triton X-1007 solution.

 (G) 12. 8 mM 卜 Line X—100 containing ¾ | ¾ medicine

 12. Sm 卜 line X—100 containing medicine, 卜 Lyton X—100 (Nacalai Teskne ±) {^ Λ: 625] is added to the water, 12.8 mM 卜 line X-100 water soluble A liquid was prepared.

 (H) 16mM Triton X-100 containing; medicine

 As a reagent containing 16 mM Triton X-100, Triton X-100 (Nacalai Tesque Co., Ltd.) KJ ^?

(I) 19. Reagents containing 2 mM Triton X-100 19. Reagent X—100 (Nacalai Teskne: h) (^ amount: 625) in pure water as a reagent containing 2 mM 卜 line X—100, 19. 2 mM Triton X—10 ΟτΚ solution Was prepared.

 (J) 25. Reagents containing 6 mM Triton X-100

 25. Triton X-100 (Nacalai Tesque) ^ amount: 625] was dissolved in pure water as a 6 mM Triton X-100 mechanical drug to prepare a 25.6 mM Triton X-100 aqueous solution.

 (K) 32mM 卜 Lighton X—100

 As a reagent containing 32 mM 卜 Ryton X-100, 卜 Lyen X-100 (Nacalai Tesque) ϊό ^ ': 625] was solved to φ7_Κ to prepare a 32 mM Triton X-100 aqueous solution. -

2. Sample

 (I) Protein-containing material

 As a protein texture containing protein, human serum albumin [HSA] (Sigma 3) was dissolved in pure water to prepare 1.5 mg / L human serum albumin water night.

 (Ii) Protein-free sample

 Pure water was prepared as a sample that does not contain the protein.

 3. Measurement

 (1) Kamii 2 (I) protein material 3, 660 zL, Kamii 1 (1) first reagent 80 L, knitting 1 (2) second reagent 160 L 100 parts of the (a) surfactant-free reagent of (3) in (1) and (3) were added and mixed to obtain a mixed solution.

 (2) Next, the concentration at a wavelength of 547 nm was measured using a ^^ meter for the mixture of Kamami (1).

(3) The procedures described in (1) and (2) are the same except that the protein-free sample in (2) above is used instead of the protein-containing sample in (2) above. And measure the degree did.

 (4) Next, instead of the surfactant-free reagent (a) of the third reagent (3) of sickle 31, (b) Triton X—1 of the third reagent (3) of Kamami 1 0 0 reagent (A) 1.6 6 mM Triton X— 1 0 0 containing reagent to (K) 3 2 mM Triton X— 1 0 0 The operation was performed as described in (1), (2) and (3), and the temperature was measured.

(5) sickle Yuki (1) to the case of using the third reagent in (4), intake ¹ ^ when the protein-containing sample was measured, and the absorbance when measuring protein-含試fee, 5 It was shown to.

4. Measurement results

 Figure 5 shows the following. .

 Sample, containing an anion with fluorescein skeleton 1 'biocompound (Ellis mouth cin B) and U ^ surface active chiral! I (trienne X— 1 0 0)? The key of Qi (卜 Lyn X—1 0 0) is the critical micelle S (CMC) of this surfactant (Triton X—1 0 0) 0.3. At 5 mM or more, ill is 0 · 3 Compared to the case of less than 5 mM, the degree of the mixed solution when measuring the protein-containing sample is reduced, and the absorption of the mixed solution [reagent blank (reagent blind tree)] when measuring the protein-free sample is reduced. ) ¾S is increasing.

That is, when the concentration of the surfactant (Triton X—100) in the mixed solution is 0.35 mM or more, which is the critical micelle contribution (CMC)? Compared to when に よ り is less than 0.35mM, the ^ value obtained by measurement (the value obtained by subtracting the reagent blank from ¾ ^ at the time of sample measurement) is smaller, and the sensitivity of the measurement is reduced. It can be seen that the effect of containing and reversing the activator (Triton X—1 0 0) is weakened. From this, the surfactant (Triton X—loo) in the mixed solution containing the sample, the anionic 1 ′ biocompound with fluorescein skeleton and the surfactant (Triton X—1 0 0) is Interfacial Tongue 'I Seiki U (Triton X— 1 0 0) Boundary Miserile? Is less than (CMC) It turns out that it is preferable.

 [Example 3] (Confirmation of the relationship between the concentration and the effect of the surfactant in the measuring method and regular reagent of the present invention)

 The relationship between the effect of zephylamine, a surfactant, in the measurement and regulation of proteins in the samples of the present invention was confirmed.

 1. Reagents ''

 (1) First reagent

 As the first reagent, Ellis Mouth Syn B (Wagaku Kogyo Kogyo Ne ±) was dissolved in pure water to prepare a 102β erythrosine 7_Κ solution.

 (2) Second reagent

 As the second reagent, mmen of citrate was dissolved in pure water to prepare soomM Quen Night.

 (3) Third reagent

 The following reagents were prepared as third reagents.

 (a) Surfactant-free reagent

 Pure water was prepared as a surfactant-free reagent containing no surfactant.

 (b) Zephyramine reagent

 (A) 4mM zephiramine

 As a 4 mM Zephiramine ornamental drug, Zephiramine was dissolved in pure water to prepare a 4 mM Zephiramine aqueous solution.

 (B) 8mM Zefirami

 As an 8 mM zephyramine-containing drug, zephyramine was thoroughly dissolved and prepared 8 mM zephyramine 7j ^ l night.

 (C) 15mM zephiramine

Contains 15 mM Zephyramine; As a medicine, dissolve Zephyramine in _ W

 32 An aqueous solution of iramine was prepared.

 (D) 20mM zephiramine

 As a reagent containing 2 OmM Zephyramine, Zephiramine was dissolved in pure water to prepare a 20 mM Zephylamine aqueous solution.

 (E) 3 OmM Zephyramine

 As a reagent containing 3 OmM Zephiramine, Zephyramine was dissolved in pure water to prepare a 3 OmM Zephyramine water night.

 2. Sample

 (1) Protein-containing flit charge

 As a protein adornment containing protein, human serum albumin [HSA] (Sigma 3) was dissolved in pure water, and 2. An OmgZL rabbit serum albumin aqueous solution was prepared.

 (Ii) Protein-free sample

 Pure water was prepared as a protein-free sample containing no protein.

 3. Measurement

 (1) To 3,660 L of the protein-containing sample of (2) above, add 80 L of the first reagent of (1) of (1) above, 160 L of the second reagent of (1) of (1) above, and One hundred (100) surfactant detergents in (1) (3) third reagent were leaked and mixed to obtain a mixed solution.

(2) Next, the concentration of the ffflB (1) mixture at a wavelength of 547 nm was measured with a ^ ¹ meter.

 (3) The procedure described in ttflB (1) and (2) is used except that the protein-free sample of (ii) is used instead of the protein-containing material of (2) above. The degree was measured.

(4) Next, instead of the surfactant-free reagent of (a) in the third reagent of (1) in the disgust 1 of (3) in the third reagent of (3) in Kamiji (b) (A) 4mM zephiramine wetting agent ~ (E) 3OmM zephirami ^ tM drugs The procedure was as described in (1), (2) and (3), and was measured.

 (5) Figure 6 shows the concentration when the protein-containing material was measured and the concentration when the protein-free sample was measured when each of the third reagents in (1) to (4) was used. Indicated.

4. Measurement results

 Figure 6 shows the following.

 In the mixed solution containing the sample, an anionic compound (Erysucin®) that has a fluorescein skeleton, and a surfactant (Zephiramine), when the amount of surfactant (Zephiramine) increases, the protein-containing sample is measured. Both the absorption of the mixed solution and the mixed solution when measuring the protein-free sample (reagent blank (reagent blind)) are increased.

 However, the value obtained by the measurement (the value obtained by adding the reagent blank from the ¾¾¾ at the time of measurement of the funnel), that is, the sensitivity of the measurement is the critical micelle concentration (CMC) of this surfactant (Zephiramine) At 0.37 mM or higher, the concentration is smaller than when the concentration is less than 0.37 mM.

 This indicates that the surfactant (zephiramine) in the mixed solution containing the sample, the anionic compound having a fluorescein skeleton, and the surfactant (zephylamine)

It can be seen that it is more preferable that the value is less than the critical micelle itS (CMC) of (zephylamine).

 [Example 4] (Confirmation of the relationship between the measurement method of the present invention and the effect of the regular reagent and PH) The measurement method of the protein in the sample of the present invention and the effect of the drug on the relationship with PH were perceived.

 Top, medicine

 (1) First reagent

As the first reagent, Ellis Mouth Syn B (Wako Pure Chemical Industries, Ltd.) was dissolved in 10 to prepare 10 Erythrosine B 7 solution. (2) Second reagent

 As the second reagent, the following reagents were prepared.

 (a) Chenic acid-free reagent

 Pure water was prepared as a citrate anti-oxidant containing no citrate.

 (b) 125 mM Quen medicine

 As a 125 mM aging reagent, citrate (Wagaku Industrial Co., Ltd.) was dissolved in 7j <to prepare a 125 mM citrate aqueous solution.

 (c) 50 OmM

 As 50 OmM citrate drug, citrate (Wako Pure Chemical Industries, Ltd .: h) was dissolved in Itosui, and 50 OmM citrate water was prepared.

 (d) 2, 50 OmM

As a 2,50 OmM citrate-containing li drug, quanic acid (Wakuyakue W :) was dissolved in pure water to prepare a 2,50 OmM citrate aqueous solution.

 (3) Third reagent

 As a third reagent, 卜 Lion X-100 (Nacalai Tesque) (-: 625) was dissolved in pure 7 to prepare a 6.4 mM Triton X-100 aqueous solution.

 2. Sample

 (I) M-containing protein

 As a protein adornment containing protein, human serum albumin [HSA] (Sigma 3) was dissolved in pure water to prepare a 1.5 mg / L human serum albumin aqueous solution.

 (Π) Protein free

 Pure water was prepared as a protein-free sample containing no protein.

 3. Measurement

(1) tif self 2 containing (I) protein; ¾ sample 3,660 / iL, Kamii 1 (1) first reagent 80 xL, 1 (2) second reagent (A) 160 L of citrate-free reagent, And 100 / L of the third reagent of (3) of Kamami 1 was added and mixed to obtain a mixed solution.

 (2) Next, the temperature at a wavelength of 547 nm was measured with a total of the mixed solution of (1).

 (3) Instead of using the protein-containing sample of (2) above, the sample containing the protein-free sample of (ii) above is used as the sample. The degree was measured.

 (4) Next, in the second reagent of Kamii (1) (2) (a), the succinic acid ^ :: In place of the drug, (2) Second reagent (b) 125mM Reagents ~ (d) 2, 50 OmM citrate-containing l ^ medicines A total of 3 types of second reagents are used, except that the procedures are as described in (1), (2) and (3). The absorbance was measured.

 (5) The pH of the mixture of the sample, the first reagent, the second reagent, and the third reagent when using each of the second reagents in Kamii 1 (2) is as follows: there were.

 (a) Cenoic acid-free reagent: pH 5.3

 (b) 125mM citrate-containing drug: pH3.5

 (c) 50 OmM Quenic acid ornamental agent: pH 2.3

 (d) 2, 50 OmM quencher: pH2.0

 (6) Figure 7 shows the Pmm when the protein-containing material was measured and the protein-free sample when each of the second reagents in (1) to (4) was used. .

4. Measurement results

 Figure 7 shows the following.

In a mixed solution in which the sample, the first reagent, the second reagent, and the third reagent are mixed, that is, the sample, containing an anionic compound having a fluorescein skeleton (Ellis mouthcin B) and a surfactant (Triton X 1 100) When the pH is 5.3 in the mixed solution, the value obtained by measurement (the value obtained by subtracting the reagent blank from the sample measurement) is small. The sensitivity of the measurement is not high.

However, as the pH in the selfish liquid mixture decreases to pH 5.0, pH 4.0, and pH 3.5, the concentration of the mixed liquid increases when protein-containing material is measured, and no protein is contained. The absorbance of the mixed solution [reagent blank (reagent blind test)] when measured was decreased. The value of ¹ e¾¾ obtained by the measurement (from the sample measurement concentration based on the concentration at the time of sample measurement) In other words, in the present invention, the sample, and the fluorescein skeleton are combined. After contacting the anion I 'biocompound with # τ, the pH of the mixed solution containing at least this sample and the anion 1' biocomposite containing the J-resin skeleton is adjusted to pH 5.0. It is found that the pH is preferably below, more preferably pH 4.0 or lower, and particularly preferably pH 3.5 or lower.

 Example 5] (Measurement method and rules of the present invention ^ Making of soot in medicine)

 The protein in the sample of the present invention was measured and the line of sight in the reagent was prepared. 1. Reagent

 (1) First reagent

 As a first reagent, Ellis Mouth Syn (Bym imf) was dissolved in pure water to prepare a solution of erythrosine B7JC.

 (2) Second reagent

 As a second reagent, kenic acid (Wako Pure Chemical Industries, Ltd.) was dissolved in J to prepare 50 OmM Quench Night.

 (3) Third reagent

 As the third reagent, Triton X-1100 (Nacalai Tesque) C H¾: 6 2 5] was dissolved in pure water to prepare a 6.4 mM Triton X-10 Oz solution.

2. Sample (A) 0.25mg / L protein content '

 As a 25 mg / L protein weaving agent, human serum albumin [HSA] (Sigma) was dissolved in pure water to prepare a 0.25 mg / L human serum albumin aqueous solution.

 (B) 0.50mg / L protein

 As a 50 mg / L protein weaving agent, human serum albumin [HSA] (Sigma 3) was dissolved in zf to prepare a 0.5 Omg / L human serum albumin aqueous solution.

 (C) 0.775 mg / L protein containing material

 As a 75 mg / L protein wire, human serum albumin [HSA] (Sigma ±) was dissolved in pure water to prepare a 0.75 mg ZL human serum albumin aqueous solution.

 (D) 1.0 Omg / L protein wire

 1. As an Omg / L protein weaving agent, human serum albumin [HSA] (Sigma: fc) was dissolved in pure water to prepare a 1.0 OmgZL aqueous solution of human serum albumin.

 (E) 1.5 Omg / L protein containing it charge

 Human serum albumin [HSA] (Sigma 3) was dissolved in τΚ as a 1.5 Omg / L protein solution to prepare a 1.5 Omg / L human serum albumin aqueous solution.

 (F) 2.0 Omg / L Protein #

 2.0 Human serum albumin [HSA] (Sigma) ± was dissolved in pure water as a 2.0 Omg / L protein adjuvant to prepare a 2.0 OmgZL human serum albumin aqueous solution.

 (G) Protein-free sample

 Pure water was prepared as a protein-free sample containing no protein.

3. Measurement

 (1) Kamii 2 (A) of 0.25 mg ZL protein-containing material (3, 660 L), 1 (1) (1) Reagent 1 (80 L), Homi 1 (2) 2 160 L of the reagent and 100 L of the third reagent of (1) (1) were mixed and mixed.

(2) Next, the liquid mixture of hate (1) is measured at a wavelength of 547 nm The degree was measured.

 (3) (B) 0. S OmgZL protein-containing material in (2) instead of (0) S. SmgZL protein-containing material in (A) above (F) 2. 0 OmgZL protein-containing material and (G ) Protein protein: The procedure was as described in (1) and (2) except that the sample was used as a sample.

 (4) In the above (1) to (3), the S line showing the relationship between the concentration when each sample is measured and the concentration of human serum albumin, which is a protein in the sample, is shown in FIG. It was shown to.

 4. Measurement results

 From Fig. 8 showing this calibration curve, it can be seen that good H spring properties are obtained even in the extremely thigh range where the concentration of human serum albumin in the sample is Omg / to 1.5 mg / L. .

 From this, it is possible to measure the protein in the sample of the present invention even for very small amount of albumin (urinary sputum albumin) whose fresh range is less than 6.5 ± 5. l mgZL (as occasional urine). In general, it can be seen that accurate measurement is a power factor. Difficulty 6] (Measurement method of the present invention and detection limit and ¾M limit of ¾M drug and other methods) Confirmation of detection method and regular reagent of protein in the sample of the present invention, and detection limit and ¾S limit of other methods It was.

 [I]. Method for measuring protein and ϋ¾Ι in the sample of the present invention

1. Reagent

 (1) First reagent

 As the first reagent, Ellis Mouth Shin Β (Wakuu Pharmaceutical Co., Ltd. ±) and Triton X—1 0 0 (Noraku Raitesk Co., Ltd.) [^ =: 6 2 5] should be dissolved in water. An aqueous solution containing 10 2 M Ellis Mouth Syn B and 0.37 3 mM Triton X—100 was prepared.

(2) Second reagent As a second reagent, queenic acid (Wako Pharmaceutical Co., Ltd .: t) was dissolved in pure water to prepare a 50 mM quen solution.

 2. Sample

 (A) Human serum albumin [HS A] (Sigmane ±) was dissolved in pure water and diluted stepwise with pure water to prepare a dilution series of human serum albumin aqueous solution.

 (B) Pure water was prepared as a protein-free material.

 (C) A human serum albumin aqueous solution with a known concentration was prepared as a standard solution.

3. Measurement

 (1) To 50 L of each m human serum albumin hydrolyzed material in (A) of 2 above, add 1st reagent of (1) of 1500 0 L and 1 of Ml 1 ( The second reagent from step 2) was calcined and mixed to obtain a mixed solution. -

(2) Next, the thickness of the mixed solution of B (1) at a wavelength of 5 47 nm was measured.

 (3) Next, the protein-free sample of selfish person (B) was prepared as described in (1) and (2) above, and the absorbance was measured (reagent blank).

 (4) Next, for the standard solution of hatred (C), operation was performed as described in hatred (1) and (2), and was measured.

 (5) For each of the human serum J-levamine aqueous solution samples of each concentration in (2) above, the concentration (reagents) in the protein-free sample obtained in hate (3) from the ¾ example obtained from the measurement. Blank) was subtracted.

 Next, ¾ ^ is divided by M¾ of the standard solution obtained in (4), and then multiplied by human serum albumin m of this standard solution, so that Asked.

 (6) The above operations (1) to (5) were repeated a total of 12 times, and a total of 12 measured values (conversions were determined) for each human serum albumin dehydrated material.

[Π]. Immunoturbidimetric method 1. Reagent

 We used “Mike Mouth Albumin-HA Test” (Wayu Pharmaceutical Co., Ltd.), a reagent for measuring albumin (thigh albumin) in urine samples by immunoturbidimetry.

 2. Sample

 The two samples of [I] were used.

 3. Measurement

 Hitachi 7 1 7 0 Using the S-type automatic and nebula device, measure the sample of hate 2 according to the method specified in the size document of this language, and measure each human serum albumin aqueous solution sample. I asked for a spear. In addition, the measurement of each said sample performed 12 measurement.

 [Π] .PR method

 1. Reagent-

“Protein Assay Rapid Kit” (Wako Pure Chemical Industries, Ltd.), a texture measuring agent for proteins in samples by the PR method, was used.

 2. Sample

 The second sample of ΙίίΗΒ C I] was used.

 3. Measurement

 In accordance with the method specified by the size of etc., the sample of Kamii 2 was measured by the microplate method, and the separation of each human serum albumin sample was obtained.

 In addition, the measurement of each said sample performed 12 measurement.

 [ΙΠ]. S S A method

 1. Reagent

 “Sulfosalicyl (Wagaku Industrial Co., Ltd.)” was used as a reagent for measuring proteins in samples by the S S Α method.

 2. Sample

Two samples of disgust [I] were used. 3. Measurement

 The size of this glaze: In accordance with the method specified in the above, etc., samples of hate 2 were measured by the microplate method, and the amount of each human serum albumin water withdrawal material was determined. Since the range of Nezumi spring is narrow, the sample was diluted with 2 to 10 brine and measured.

 The measurement of each of the above samples was performed by double measurement.

 [IV] Detection limit and quantification limit

 In each of the method of measuring the protein in the sample of the present invention of Kamami [I] and the medicine, the immunoturbidimetric method of [Π], the PR method of disgust [ΠΙ], and the SSA method of disliked SiW) The standard deviation was calculated from the measured values of the double measurement of each human serum albumin aqueous sample, and the detection limit (3σ) and the quantification limit (10) were obtained.

This is shown in Table 4. -

(Table 4)

4. Measurement results

 From Table 4, the detection limit and the quantification limit of the measurement reagent and the regular determination method for the protein in the sample of the present invention are far superior to those of the PR method and SSA method of the other methods. It turns out that it exceeds the detection limit and the limit of quantification.

 More than this, the method for measuring and measuring the protein in the sample of the present invention has a measurement sensitivity superior to the immunoturbidimetric method, while the cost required for the measurement is low, and the method for measuring the protein in the urine sample. It has been proved that it is possible to accurately measure a protein such as albumin which is present only in a minute amount in a sample. .

Claims

The scope of the claims 1. Specimen, fluorescein skeleton: Anionic compound and surfactant are wormed, and this sample, anion I ′ fluorescein skeleton, and a mixture containing surfactant and surfactant are measured. A method for measuring protein in a sample.  2. After allowing the sample and the anionic compound that exhibits the fluorescein skeleton to disappear, the pH of the mixture containing at least this sample and the anionic compound that exhibits the fluorescein skeleton should be pH 5.0 or less. The method for measuring a protein in a sample according to claim 1, characterized in that it is characterized by the following. 3. The method for measuring protein in a sample according to claim 2, wherein the pH of the mixed solution is adjusted to pH 5.0 or less by adding an acidic solution to the mixed solution. . 4. The surfactant according to any one of claims 1 to 3, wherein the surfactant is a nonionic surfactant and Z or a cationic surfactant. Measurement of protein in samples. 5. The surfactant according to any one of claims 1 to 4, wherein the surfactant is a critical micelle of the surfactant in the mixed solution ^ ¾ *. Measurement of protein in samples.  6. The method for measuring a protein in a sample according to any one of claims 1 to 5, wherein the anionic compound having a fluorescein skeleton is Ellis mouth syn-B. .  7. The method for measuring a protein in a sample according to any one of claims 1 to 6, wherein the protein is albumin.  8. A reagent for measuring protein in a sample, which contains an anion compound having a fluorescein skeleton and a surfactant. 9. A solution comprising at least an anionic compound having a fluorescein skeleton. 9. The method according to claim 8, comprising a combination of one reagent and a second reagent comprising an acidic solution, wherein a surfactant is added to the first reagent and Z or the second reagent. A measure of the protein in the sample.  1 0. When the second reagent consisting of an acidic solution is added to the mixed solution in which the sample and the first reagent are mixed, the pH of the mixed solution can be reduced to pH 5.0 or less. The reagent for measuring a protein in a sample according to claim 9, wherein the reagent is a tree. 1 1. Any one of claims 8 to 10 characterized in that the surface activity 'I' is a nonionic surfactant and / or a cationic surfactant 1 Measuring agents for proteins in samples as described in the section. 1 2. The surfactant according to claims 8 to 11 characterized in that the surfactant is set to it so as to be the key to the critical micelle fullness of the surfactant in the mixed solution. (1) The reagent for measuring the protein in the sample according to (1). 1 3. The protein in the sample according to any one of claims 8 to 12, characterized in that the anionic compound having a fluorescein skeleton is Ellis mouth syn-B. Mm ₀  1 4. The protein measuring agent for proteins in a sample according to any one of claims 8 to 13, wherein the protein is albumin.