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WO2008108007A1 - Procédé de dosage d'une protéine dans un échantillon, et réactif de dosage pour celui-ci - Google Patents

Procédé de dosage d'une protéine dans un échantillon, et réactif de dosage pour celui-ci Download PDF

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Publication number
WO2008108007A1
WO2008108007A1 PCT/JP2007/055021 JP2007055021W WO2008108007A1 WO 2008108007 A1 WO2008108007 A1 WO 2008108007A1 JP 2007055021 W JP2007055021 W JP 2007055021W WO 2008108007 A1 WO2008108007 A1 WO 2008108007A1
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WIPO (PCT)
Prior art keywords
sample
protein
reagent
surfactant
measurement
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PCT/JP2007/055021
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English (en)
Japanese (ja)
Inventor
Emiko Kaneko
Junichi Isoe
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Shino Test Corp
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Shino Test Corp
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Priority to PCT/JP2007/055021 priority Critical patent/WO2008108007A1/fr
Publication of WO2008108007A1 publication Critical patent/WO2008108007A1/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6827—Total protein determination, e.g. albumin in urine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour

Definitions

  • 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.
  • Serum or ⁇ protein levels are high in dehydration, reticuloendothelial disease, or chronic sensation.
  • plasma protein leakage, malnutrition or liver function ⁇ $ ⁇ is low.
  • the serum or bell protein belly changes with each morbidity or transformation, its t-rule is emphasized in the diagnosis and treatment of symptoms.
  • urinary protein appears in kidney disease, heart disease, manic, jaundice or high fever.
  • 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.
  • 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.
  • 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.
  • 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.
  • bromcresol green binds to albumin in the presence of citrate acid and exhibits a blue color, and measures the increase in ⁇ due to this color development.
  • BCG bromcresol green
  • 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.
  • 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 tJ3 ⁇ 4l3 ⁇ 4 drugs have been used (measurement sensitivity: 5 to 30 mg / L).
  • 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.
  • 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 ⁇ degree.
  • 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.
  • 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.
  • 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 surfactant is a nonionic surfactant and / or a cationic surfactant (8) to (10) in the sample according to any one of (10) Protein measurement.
  • 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.
  • 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.
  • the sample in the present invention is not particularly limited as long as it may contain a protein.
  • 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, 3 ⁇ (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.
  • 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 # 3 ⁇ 4. Just do it.
  • shelves or dredging may be performed as necessary.
  • the protein in the present invention is a protein contained in (or possibly contained in) a sample and is intended to be measured.
  • the total protein can be exemplified by albumin.
  • this protein is a protein that can only be leaked into a sample. Is preferred.
  • urinary albumin (urinary albumin), Bens-Johns protein, and the like as proteins that can be expressed only in f ⁇ .
  • the anion I ′ biocompound having a fluorescein skeleton is a fluorescein skeleton.
  • it is an anionic compound having, it can be used without particular limitation.
  • anionic compound having the fluorescein skeleton examples include xanthene-based pigments.
  • xanthene dye examples include Ellis Mouth Shin B, Yersin Sin, Yeosin, Phloxin, and Rose Benga! / ⁇ .
  • 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.
  • 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 0 ⁇ or less, more preferably 10 0 ⁇ or less, and particularly preferably 50 0 ⁇ or less.
  • the surfactant in the present invention is not particularly limited as long as it is a surfactant.
  • this surfactant examples include nonionic surfactants, cationic surfactants, and sexual boundary surfactants.
  • 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.
  • the worm with this surfactant provides the ability to measure i M protein in the sample.
  • the degree of purity obtained in the measurement of the sample is increased because the surfactant is a nonionic I ′ raw surfactant, a cation ⁇ ′ raw surfactant or This is especially true when the mixture is a mixture of a biosurfactant and a cationic surfactant.
  • 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.
  • 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.
  • the liquid it is preferably 0.1 M or more, more preferably 20 xM or more, particularly preferably 40 or more.
  • 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.
  • concentration is less than degree (CMC).
  • nonionic surfactants and cationic surfactants their compound names and their critical micelle concentration (CMC) are shown in Table 1, Table 2, and Table 3. .
  • CMC Critical micelle concentration
  • 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.
  • Triton; X—100 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.
  • the soot in the Kamami mixture is preferably less than 0.35 mM, which is a critical micelle 3 ⁇ 4, more preferably 0.15 mM or less, Particularly preferred is 0.1 ImM or less.
  • 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.
  • the sample is mixed with a mixture of an anion 1 ′ biocompound having a fluorescein skeleton and a surfactant (for example, measurement M drug).
  • a surfactant for example, measurement M drug
  • 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.
  • anion I 'biocompound with fluorescein skeleton, and surfactant are worms?
  • 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. . .
  • 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.
  • the pH of the mixture of humility should be ⁇ 5.0 or less after reversion, but the ability to further increase the value of Kamiki by making this pH ⁇ 4.0 or less? It is more preferable, and it is particularly preferable to set this pH to 3.5 or less for the same reason.
  • the ⁇ of the mixed solution containing at least the sample and the anionic compound having the fluorescein skeleton is ⁇ ⁇ 5.0 or less. (More preferably ⁇ ⁇ 4.0 or less, particularly preferably pH 3.5 or less) Any method may be used as long as this is difficult.
  • 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).
  • 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 »
  • citrate starch syrup for example, citrate starch syrup, vinegar syrup solution, salt solution, slag aqueous solution, or swelled ⁇ slag can be cited.
  • 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.
  • 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).
  • 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.
  • This sample, absorption of a mixed solution containing an anionic compound having a fluorescein skeleton and a surfactant, is measured at 1 ⁇ m, and the absorption is measured at a wavelength at or near the absorption maximum of the mixed solution. .
  • 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.
  • 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.
  • 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.
  • reaction cuvette 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.
  • 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.
  • the reagent blank (reagent blind tree) was obtained from the 3 ⁇ 4 example value obtained in S [6]. Calculate the difference in absorbance by subtracting the subtracted value.
  • the reagent for measuring protein in the sample of the present invention contains an anionic compound having a fluorescein skeleton and a surfactant.
  • sample in the present invention is as described in “2. Sample” of “[1]. Method for measuring protein in sample” above.
  • the protein in the present invention is as described in “3. Protein” in “[I]. Method for measuring protein in sample” above.
  • 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.
  • 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. .
  • the drug is preferably composed of two reagents or three or more reagents.
  • it is preferably composed of two reagents.
  • 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.
  • First reagent anionic compound having fluorescein skeleton
  • surfactant Second reagent: surfactant, buffer, etc.
  • 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.
  • 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.
  • 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.
  • a first reagent comprising at least an anionic compound having a fluorescein skeleton
  • a second reagent comprising an acidic solution.
  • the following can be cited as the constitution of the components contained in each of these two reagents.
  • Other ingredients
  • 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.
  • 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.
  • a buffer having an ability in the pH range necessary for measurement is preferable to have a buffer having an ability in the pH range necessary for measurement as a buffer for a person.
  • 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.
  • MES MES
  • B Tris
  • B Tris Propon
  • ADA PI PES
  • ACES MOPS
  • MOPS MOPS
  • BES TES
  • HE PES DI PSO
  • TAP SO POP SO
  • HEPPSO HEPPSO
  • 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.
  • 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 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.
  • quenic acid (Wako Pure Chemical Industries, Ltd.) was dissolved in / i to prepare 500 mM quencher.
  • Pure water was prepared as a surfactant-free reagent containing no surfactant.
  • ⁇ Lye X—1 0 0 As a reagent, ⁇ 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.
  • Tween 20 As a Tween 20 reagent, Tween 20 was dissolved in zK to prepare a 0.4 mM Tween 20 aqueous solution.
  • zephyramine As a zephyramine reagent, zephyramine is dissolved in pure water and a 4 mM zephyramine aqueous solution is used. Prepared.
  • human serum albumin [HSA] (Sigma 3) was dissolved in pure water to prepare 2. Omg / L human serum albumin aqueous solution.
  • 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.
  • 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.
  • the second reagent (d) in Zekai (3) in (d) is the second 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.
  • triton X—100 a nonionic surfactant
  • erythrosine synthase B which is an anionic compound having a fluorescein skeleton
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Tween 20 which is a nonionic surfactant
  • the fluorescein skeleton is an anionic compound Eris Mouth Syn B and a worm.
  • 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.
  • Tween 20 as a nonionic surfactant when 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.
  • zephyramine a cationic surfactant
  • erythrincin B an anionic compound that cleaves the fluorescein skeleton
  • zephiramine is used as a measurement M drug.
  • 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.
  • the value of the crane obtained by the measurement (the force at the time of measuring the sample ⁇ minus the 3 ⁇ 4 ⁇ of the reagent blank) is greater when the 3 ⁇ 4M 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.
  • Ellis Mouth Syn B (Wagaku Kogyo Neko: fc) was dissolved in pure water to prepare an aqueous solution of 102 / LLM erythrosine B.
  • Pure water was prepared as a surfactant-free reagent containing no surfactant.
  • Triton X-100 (Nacalati Skene ⁇ ) ⁇ ⁇ ⁇ 625] in ⁇ 7_ ⁇ , and then add 3 mM Triton X-100100 Prepared.
  • Triton X—100 (Nacalati Skene ⁇ ) [ ⁇ ? Amount: 625] was dissolved in pure water to prepare a 4.8 mM Triton X-100 aqueous solution.
  • Triton X-100 (Nakaraitesk 3 ⁇ 4) ⁇ ⁇ ⁇ : 625] was immersed in water to prepare a 6.4 mM Lyton X-100 aqueous solution.
  • Triton X-100 (Nacalai Tesque 3 ⁇ 4) [ ⁇ amount: 625] was dissolved in / J ⁇ to prepare an 8 mM Triton X-100 solution.
  • Triton X-100 (Nacalati Skene ⁇ ) [ ⁇ amount: 625] was dissolved in thread water to prepare a 9.6 mM Triton X-1007 solution.
  • Triton X-100 (Nacalai Tesque Co., Ltd.) KJ ⁇ ?
  • 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.
  • human serum albumin [HSA] (Sigma 3) was dissolved in pure water to prepare 1.5 mg / L human serum albumin water night.
  • Pure water was prepared as a sample that does not contain the protein.
  • Figure 5 shows the following. .
  • 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)?
  • CMC critical micelle contribution
  • 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.
  • mmen of citrate was dissolved in pure water to prepare soomM Quen Night.
  • Pure water was prepared as a surfactant-free reagent containing no surfactant.
  • Zephiramine As a 4 mM Zephiramine ornamental drug, Zephiramine was dissolved in pure water to prepare a 4 mM Zephiramine aqueous solution.
  • HSA human serum albumin
  • Pure water was prepared as a protein-free sample containing no protein.
  • 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.
  • an anionic compound that has a fluorescein skeleton, and a surfactant (Zephiramine)
  • Zephiramine a surfactant that increases
  • Both the absorption of the mixed solution and the mixed solution when measuring the protein-free sample are increased.
  • the value obtained by 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.
  • CMC critical micelle concentration
  • 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.
  • the following reagents were prepared.
  • Pure water was prepared as a citrate anti-oxidant containing no citrate.
  • citrate As a 125 mM aging reagent, citrate (Wagaku Industrial Co., Ltd.) was dissolved in 7j ⁇ to prepare a 125 mM citrate aqueous solution.
  • citrate As 50 OmM citrate drug, citrate (Wako Pure Chemical Industries, Ltd .: h) was dissolved in Itosui, and 50 OmM citrate water was prepared.
  • human serum albumin [HSA] (Sigma 3) was dissolved in pure water to prepare a 1.5 mg / L human serum albumin aqueous solution.
  • Pure water was prepared as a protein-free sample containing no protein.
  • 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. .
  • 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)
  • an anionic compound having a fluorescein skeleton Ellis mouthcin B
  • Triton X 1 100 Triton X 1 100
  • 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 e3 ⁇ 43 ⁇ 4 obtained by the measurement (from the sample measurement concentration based on the concentration at the time of sample measurement)
  • the sample, and the fluorescein skeleton are combined.
  • 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)
  • kenic acid (Wako Pure Chemical Industries, Ltd.) was dissolved in J to prepare 50 OmM Quench Night.
  • Triton X-1100 (Nacalai Tesque) C H3 ⁇ 4: 6 2 5] was dissolved in pure water to prepare a 6.4 mM Triton X-10 Oz solution.
  • human serum albumin [HSA] (Sigma) was dissolved in pure water to prepare a 0.25 mg / L human serum albumin aqueous solution.
  • human serum albumin [HSA] (Sigma 3) was dissolved in zf to prepare a 0.5 Omg / L human serum albumin aqueous solution.
  • human serum albumin [HSA] (Sigma ⁇ ) was dissolved in pure water to prepare a 0.75 mg ZL human serum albumin aqueous solution.
  • HSA human serum albumin
  • Human serum albumin [HSA] (Sigma 3) was dissolved in ⁇ as a 1.5 Omg / L protein solution to prepare a 1.5 Omg / L human serum albumin aqueous solution.
  • HSA Human serum albumin
  • Pure water was prepared as a protein-free sample containing no protein.
  • 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.
  • 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.
  • 3 ⁇ 4 ⁇ is divided by M3 ⁇ 4 of the standard solution obtained in (4), and then multiplied by human serum albumin m of this standard solution, so that Asked.
  • 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.
  • Protein Assay Rapid Kit (Wako Pure Chemical Industries, Ltd.), a texture measuring agent for proteins in samples by the PR method, was used.
  • the sample of Kamii 2 was measured by the microplate method, and the separation of each human serum albumin sample was obtained.
  • “Sulfosalicyl (Wagaku Industrial Co., Ltd.)” was used as a reagent for measuring proteins in samples by the S S ⁇ method.
  • 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.
  • 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. .

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Abstract

L'invention vise à proposer un procédé de dosage extrêmement sensible d'une protéine dans un échantillon, lequel procédé peut être conduit à un coût faible, est applicable non seulement à un appareil pour une utilisation exclusive mais encore à des analyseurs automatiques de type courant qui ont été employés dans un grand nombre d'hôpitaux, et permet même le dosage d'une protéine contenue en quantité mineure dans un échantillon ; et l'invention vise également à proposer un réactif de dosage pour celui-ci. Les objectifs décrits ci-dessus peuvent être atteints par le procédé suivant de dosage d'une protéine dans un échantillon et par un réactif de dosage pour celui-ci. Notamment, le procédé décrit ci-dessus de dosage d'une protéine dans un échantillon comprend la mise en contact de l'échantillon avec un composé anionique ayant un squelette de fluorescéine et un agent tensio-actif, et la mesure de l'absorbance du mélange liquide contenant l'échantillon, le composé anionique ayant un squelette de fluorescéine et l'agent tensio-actif. Le réactif pour doser une protéine dans un échantillon comme décrit ci-dessus contient un composant anionique ayant un squelette de fluorescéine et un agent tensio-actif.
PCT/JP2007/055021 2007-03-07 2007-03-07 Procédé de dosage d'une protéine dans un échantillon, et réactif de dosage pour celui-ci Ceased WO2008108007A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8353393B2 (en) 2006-12-22 2013-01-15 Lord Corporation Operator interface controllable brake with field responsive material

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005055417A (ja) * 2003-08-03 2005-03-03 Shino Test Corp タンパク質の測定方法及び測定キット

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005055417A (ja) * 2003-08-03 2005-03-03 Shino Test Corp タンパク質の測定方法及び測定キット

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ISOE J: "A New Spectrophotometric Method for Determination of Urinary Protein Using Erythrosin B", CHEM LETT, vol. 35, no. 8, pages 922 - 923, XP003023247 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8353393B2 (en) 2006-12-22 2013-01-15 Lord Corporation Operator interface controllable brake with field responsive material

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