JPS61149863A - Measuring method of biotin and reagent for measurement to be used therein - Google Patents

Abstract

PURPOSE:To measure easily and exactly the quantity of biotin by adding a biotin-contg. liquid to be examined and labeling avidin to immobilized-biotin substd protein and comparing the conjugated labeling body activity with the activity of a standard liquid. CONSTITUTION:The liquid to be examined prepd. of a physiological salt soln. of phosphoric acid buffer (PBS) or biotin standard liquid is put into each hole of a U-bottom plate immobilized with the biotin substd, protein. The labeling aviding PBS soln. is added thereto and is thoroughly and intimately mixed therewith. The liquid rested for about 0.5-4hr to allow the reaction to be completed and thereafter the supernatant is discarded. A solute soln. for the labeling body is then added to the liquid to allow the liquid to color. The respective light absorptivities are measured and the labeling body activity is measured. The labeling body activities of the liquid to be examined and the standard liquid are thereafter compared and the quantity of the biotin in the liquid to be examined is calculated.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for measuring biotin and a measuring reagent used therefor.

[Conventional technology]

Biotin is a vitamin that is involved in carbonic acid fixation and carbonic acid transfer reactions in the carbohydrate and lipid metabolic systems in living organisms.
Deficiency symptoms such as itchy dermatitis, glossitis, and depression have been reported. Biotin deficiency in humans is said to rarely occur except when large amounts of raw eggs are ingested, but biotin deficiency has also been reported in patients who have undergone long-term high-strength aliquot infusions, and these patients have a low biotin level. need to be measured. A common method for measuring the amount of biotin is to measure biotin in blood or urine, and microbiological quantitative methods are currently used.

Normally, most biotin in blood is tightly bound to proteins, and in order to measure the blood concentration, biotin must first be liberated by hydrolyzing proteins. As a hydrolysis method for this purpose, a method using sulfuric acid, a method using papain, etc. are used.

[Problem that the invention seeks to solve]

However, it has been known that the amount of biotin in the blood of normal people determined by microbiological quantitative methods varies considerably depending on the hydrolysis method and the bacterial species used for the quantitative determination. This is because, depending on the bacterial species, bound biotin or biotin-like substances can also be used, and unsaturated fatty acids such as oleic acid and linoleic acid can be substituted for biotin.

In addition, the microbiological quantitative method itself is complicated to operate, takes a long time (24 to 72 hours) to produce measurement results, and requires a large amount of sample (1 to 21 E/blood). was there. Therefore, there has been a demand for the development of a method for easily and accurately measuring the amount of biotin in place of microbiological quantitative methods.

[Means for solving problems]

Under these circumstances, the present inventor conducted intensive research to overcome the above-mentioned drawbacks, and found that the binding reaction of avidin to biotinylated proteins is inhibited by free biotin, and that this specific binding reaction is inhibited by free biotin. The present invention was completed based on the discovery that the amount of biotin in a test solution can be easily and accurately measured by using the method. The present invention provides a method for measuring biotin, which is characterized by adding avidin, measuring the activity of a label bound to the biotinylated protein, and comparing this with that of a standard solution.

Furthermore, the present invention provides a biotin measurement reagent containing labeled avidin and immobilized biotinylated protein used in this measurement.

Examples of the labeled avidin used in the method of the present invention include labeled avidin, which is a basic albumin-like crystal protein contained in chicken egg white. The sign is
For example, peroxidase, beta-galactosidase,
Labeling with an enzyme such as alkaline phosphatase is preferred for ease of measurement.

Further, as the immobilized-biotinylated protein, a protein obtained by biotinylating and further immobilizing this by a conventional method is used. Proteins that are biotinylated include albumin,
General proteins such as globulin, lysozyme, and polyamino acids having amino groups in their side chains can be mentioned, and plastic containers such as polystyrene, polyvinyl toluene, polybutadiene, etc. are used for immobilization.

Biotinylation of proteins is carried out by reacting the protein with a biotin derivative such as N-hydroxysuccinimide biotin in a solvent.

The weight ratio of the biotin derivative to the protein in the reaction is preferably in the range of 1:1 to 1:20, particularly around 1:5 when polylysine is used.

Immobilization of biotinylated proteins can be carried out by known methods; for example, when biotinylated polylysine is used, it is possible to
It is preferable to carry out the reaction at 0°C for 30 to 60 minutes.

To carry out the method of the present invention, the immobilization obtained by the above method -
A test solution containing labeled avidin and biotin may be added to the biotinylated protein, and the activity of the labeled substance bound to the immobilized biotinylated protein may be compared with that of a biotin standard solution.

The amount of labeled avidin added in the method of the present invention is 10 to 10
00 nt/114, especially 20-100 ny/at
It is preferable that

Preferred embodiments of the present invention are as follows.

■ A test solution prepared with 0.01-0.2M phosphate buffered saline (hereinafter abbreviated as PBS) with pH 5-8 or biotin was added to each well of a 96-well U-bottom plate on which biotinylated protein was immobilized. 25-100 μ of standard solution! put in.

(2) Add 20 = 100 ny/d standard R7 Vidine PBS solution 25~I Q Oplt- to this and mix well.

(2) Allow to stand for 0.5 to 4 hours to complete the reaction, then discard the supernatant.

(2) Add a substrate solution for the label to develop color, and measure the absorbance of each to measure the activity of the label.

■ Calculate the amount of biotin in the test solution by comparing the labeled activity in the test solution and the standard solution.

In addition, in the method of the present invention, in order to prevent non-specific reactions,
PR8K0.05~1. Proteins such as Ots serum albumin and animal serum, and 0.01-0.5s polyoxyethylene sorbitan monolaurate (Tween-2
0) etc. is desirable.

〔Effect of the invention〕

According to the method of the present invention, the amount of test liquid required is approximately 115° for microbial quantification, and the time required for measurement is shortened to 5 hours, compared to 24 to 72 hours for microbial quantification. It was done. Furthermore, the measurement operation is extremely simple, making it possible to measure a large number of samples at once (96 holes on one sheet).
Approximately 20 samples can be measured on the bottom plate and more than 200 samples per person can be processed for 91 days). Therefore, the present invention is particularly advantageous when many specimens need to be measured simultaneously, such as when measuring biotin in blood or urine when monitoring the nutritional status of a patient undergoing infusion.

〔Example〕

Next, the present invention will be explained in detail with reference to Examples.

Example 1 Preparation of immobilization-biotinylated polylysine: 1) Dissolve 1 kg of N-hydroquine succinimide biotin (NHS biotin; Funakoshi Pharmaceutical) in 50μe of dimethylformamide, and add 0.1M of polylysine at 1~/ml to this. Sodium bicarbonate solution 5M (poly L-lysine; Sigma P-26
36), react at room temperature for 4 hours, store at 4°C, or freeze-dry and store, and dilute to a predetermined concentration before use.

it) Next, biotinylated polylysine 0,0 obtained in 1)
1-chi solution (0.01MPBS, p) 17.2) 10
Pour 0 μl into a 96-well U-bottom plate and incubate for 60 minutes at 45
Keep at ℃. From now on, 0.01MPBS (pH7,2)
Wash with 0.5% casein at 0.0°C MPH8 (7.
2) Add 250 μg't of the solution and treat at 25° C. for 60 minutes. After finishing, first add 0.01MPBS (pH 7,2
), followed by 0.05% Tween-20.
Wash thoroughly with 1MPR8 (pH 7,2).

Example 2 Measurement of biotin concentration in rat blood: (1) 50 μg of rat blood hemolyzed by freezing and thawing
10 μl of papain solution (Sigma P-3125) and 4
．． Mix 4mM 7-acid-190μ of 2mM Nat HPO4 buffer, add 1 drop of toluene and
After overnight reaction at °C, the mixture was autoclaved (121'Cio min), centrifuged at 8000 rpm for 5 minutes, and the pH of the supernatant 1 was adjusted to 6.0. Next, add 0.25 m of diethyl ether, shake well, remove the ether layer,
Add 1 cup of ethyl alcohol to water and mix, then add 800
Collect the supernatant after centrifugation at 0 rpm for 5 minutes. This was suctioned to dryness and filtered with 200μ6km of 0.1MPBS (pH 7,2) to obtain a test solution.

In each well of a 96-well U-bottom plate immobilized with biotinylated polylysine, 0.4 μl of bovine serum Alpumi 7CBSA) and 0
．． 14Tween-20-containing 0.1MPBS (pH 7,
Add 50 μlt of the 80 ny/nl noavidin peroxidase (Funakoshi Pharmaceutical) solution prepared in 2),
Furthermore, test solution or biotin standard solution (0, 0, 125,
0,25,0,5 or 1.0 ny/ml) 507
Add 16 and mix well.

After reacting at 25°C for 120 minutes, remove the supernatant and add 0.05%
0.1 MPBS (p) f7. containing Tvreen-20.
After thorough washing with 2), add 0.01 MPB of 3q/ld ortho-phenylenediamine containing 0.01 v/v% hydrogen peroxide.
S (pH 6,0) interrogation 250pe k each hole with lc 37
React for 30 minutes at °C. After finishing, add 200 μe of supernatant to
-2yt of sulfuric acid- is added and its absorbance at 492 nm is measured. The blood biotin concentration was determined from the calibration curve obtained from the biotin standard solution and was 2.14±0.08 ny.
It was 74 tl. 2ml of the same blood was similarly hydrolyzed with papain, and the blood biotin concentration was determined by microbiological quantitative method, and it was found to be 2.46 nf/R1.

(2) U1) Blood 50μJK0.05N sulfuric acid 150μ/
was added and hydrolyzed in an autoclave (121°C) for 60 minutes, and the supernatant was centrifuged at 8000 rpm for 5 minutes. Add 200μg of diethyl ether to this, shake well,
Remove the ether layer, add 1.0-ethyl alcohol to the aqueous layer, mix, and centrifuge at 8000 rpm for 5 minutes to collect the supernatant. Strain and evaporate to dryness, 0.1 M P B S
CpH7,2) was dissolved in 200 μg to prepare a test solution.

The blood biotin concentration was determined by carrying out all the same quantitative operations as in Example 2 (II) and found to be 1.91±0.07 ny
/ml. The same blood 2rILl was similarly hydrolyzed with sulfuric acid and the blood biotin concentration was determined by a microbiological quantitative method and was found to be 1.64 ny/at. With this method, almost the same measurement results as conventional methods can be obtained with both papain treatment and sulfuric acid treatment using a small amount of blood.

Example 3 Measurement of biotin concentration in rabbit blood: (1) 10 μl of papain solution (Sigma P-3125) was added to 50 pl of rabbit blood hemolyzed by freeze-thawing. (A test solution was prepared in the same manner as in 11, and the blood biotin concentration was determined in the same manner as in 1.
58±0.16np/-. Two samples of the same blood were similarly hydrolyzed with papain, and the blood biotin concentration was determined by a microbiological quantitative method and was found to be 1.46 ny/ml.

+21 f blood 50 filVc 0.05N (ff
Add l acid 150/j/all and autoclave (121℃)
The following example 2. (A test solution was prepared in the same manner as in Example 2.21, and the blood biotin concentration was determined in the same manner as in Example 2.+I+.
．． It was 18±0.17 ny7ml. Same blood 2
ml? t- When the blood biotin concentration was similarly determined by the microbiological quantitative method after hydrolysis with sulfuric acid, it was found to be 0.95n.
It was t/go. With this method, almost the same measurement results as the conventional method were obtained for both papain treatment and sulfuric acid treatment using a small amount of blood.

Example 4 Measurement of biotin concentration in human urine: Human urine was diluted 30 times with 0.1 MPBS (pH 7,2) to prepare a test solution. Example 2. (The urinary biotin concentration was determined by the p operation exactly as in 11, and was found to be 16.7 ± 2.
．． It was 5 np/ml. When the sybiotin concentration of the same urine was measured using a microbiological quantitative method, it was found to be 15.8 n
It was y/ld. Using this method, almost the same measurement results as the conventional method were obtained in a short time.

Example 5゜1, Biotin standard a, Biotin 1.0ny Sodium chloride 8.8 Kirin Sodium (dihydrate) 4.411F Disodium phosphate (1decahydrate) 25.811fb, Biotin
0.5nt sodium chloride
8.8q Sodium phosphate (dihydrate) 4.4
ff Disodium phosphate (12 hydrate) 25.8ηC, biotin 0.25ny Sodium chloride 8.8 ~ Sodium phosphate (2
Water 4.4 Misaki Disodium Phosphate (Decahydrate)
2&8+1Wd, Piochi y O
, 125nf Sodium chloride 8.8~
Sodium phosphate (dihydrate) 4.4111P disodium phosphate (dodecahydrate) Both 25.8ff and -d are freeze-dried according to a conventional method and filled into vials. When each is used, it is dissolved in distilled water and used as 1d.

2. Specimen dilution solution Sodium chloride 438.3~Phosphoric acid-
Sodium (dihydrate) 218.4 nwi Phosphorus disodium (12 hydrate) 1290.6++v Lyophilize according to the conventional method for the above composition and fill into vials. Before use, dissolve in distilled water and use as 50wt1.

3. Orthophenylenediamine Orthophenylenediamine 150. Ov phosphoric acid-
Sodium (dihydrate) 68.4Wq Disodium phosphate (decahydrate) 22. OWqThe above composition is freeze-dried according to a conventional method and filled into vials. When used: 0. O
Dissolve in IV/v96 hydrogen peroxide solution and use as 5Qd.

4. Avidin peroxidase Avidino peroxidase 800. Ony sodium chloride 87.6■ Sodium phosphate (dihydrate) 43.7q Disodium phosphate (
12 Water [) 258.1ηBSA
40.0 ■ The above composition is lyophilized according to a conventional method and filled into a vial, or the above composition is dissolved in a 0.1% Tween-20 solution and used as 10M.

& Immobilization - Biotinylated polylysine Biotinylated polylysine 1 d (Example 1
．． 1)) Sodium chloride 87.6 #9 Sodium phosphate (dihydrate) 4.11F Disodium phosphate (1decahydrate) 25.8F The above composition was freeze-dried according to a conventional method and placed in a vial. Fill it with a running material or dissolve the above composition as it is in distilled water at the time of use to make 10 m,
100 μI in each hole of a 96-well U-bottom plate? : Put, Example 1. All immobilization of biotinylated polylysine is prepared by the method II).

that's all

Claims (1)

[Claims] 1. Immobilization - Adding a biotin-containing test solution and labeled avidin to a biotinylated protein, measuring the activity of the labeled substance bound to the biotinylated protein, and comparing this with that of a standard solution. A biotin measurement method characterized by: 2. A reagent for measuring biotin containing labeled avidin and an immobilized biotinylated protein.