JPS6171359A - Measurement of biotin and measuring agent therefor - Google Patents

Abstract

PURPOSE:To enable measurement of biotin quantity with a small quantity of testing liquid agent, by adding avidin and bonding carrier of protein biotinide and comparing thus aggregated matter with the standard solution of biotin. CONSTITUTION:0.01M phosphoric acid buffered physiologic salin solution (PBS) of pH 5-8 is introduced into each hole of U-bottomed of N-hydroxyplate 96 with a quantity 25-100mul. Next, lesting liquid agent or biotin standard solution of 25-100mul is introduced into the holes of the 1st row and the liquid is subjected to dilution by half concentration. Next, all the holes are filled with 5-50ng/ml of 25-100mul of avidin PBS solution and 25-100mul of 0.1-0.5% PBS floating liquid of rea blood cells aggregated with protein biotinide and the mixture is provided with through agitation. After 1-24hr the liquid is tested for the aggrogation condition, inhibition condition of standard liquid and testing agent is compared and an biotin concentration of the test agent is obtained. In the present invention, in order to prevent a non-singular reoction, it is preferable that the PBS is added with 0.05-1% serum albumin or protein of animum serum, etc. and 0.5-5% polyethylene glycol 2000-20000, etc.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for measuring biotin and a measuring reagent used therein, and more specifically, the present invention relates to a method for measuring biotin and a measuring reagent used therein. The present invention relates to a method for measuring biotin that utilizes the fact that free biotin inhibits agglutination reactions, 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-calorie infusions, so biotin levels should be measured in these patients. There is a need. 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 i of biotin in the blood of normal people determined by microbiological quantitative methods differs in details such as 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 has drawbacks such as complicated operations, a long time (24 to 72 hours) before measurement results are available, and the need for a large amount of sample (1 to 2 grams of blood). was there. Therefore, as an alternative to microbiological quantitative methods,
There has been a demand for the development of a method for easily and accurately measuring the amount of biotin.

[Means for solving problems]

The present inventor conducted various studies regarding methods for measuring biotin, and as a result, discovered that the amount of biotin can be easily and accurately measured by using a specific binding reaction between biotin and avidin, and completed the present invention.

That is, the present invention provides a method for measuring biotin, which is characterized by adding avidin and a biotinylated protein-binding carrier to a biotin-containing test solution, and comparing the resulting aggregation with a biotin standard solution, and a measuring reagent used therefor. It provides:

The principle of the method of the present invention is that carriers bound with biotinylated proteins, such as animal red blood cells and plastic beads, agglutinate when avidin is added, but this agglutination reaction is inhibited by the presence of free biotin. This method is used to measure the amount of biotin.

Avidin used in the method of the present invention is a basic albumin-like crystal protein contained in chicken egg white and is commonly commercially available. In addition, biotinylated protein-bound carriers are obtained by biotinylating proteins and further bonding them to υ carriers using conventional methods.Proteins that can be biotinylated include common proteins such as albumin, globulin, and lysozyme. Examples of carriers include proteins;
Red blood cells, plastic beads (e.g. polystyrene,
Examples include latex such as polyvinyl toluene and polybutadiene), glass beads, and the like.

Among these carriers, any animal can be used as red blood cells, but especially sheep, chicken, human O
Red blood cells and the like are preferred.

Biotinylation of proteins is carried out by first reacting a biotin derivative such as N-hydroxysuccinimide biotin with the protein in a solvent, and then removing unreacted biotin derivative from the reaction mixture. The weight ratio of piotin derivative and protein in the reaction is 1.
; 0. I Sl : in the range of 100, especially from 1:1 to 1
: The range of 10 is preferable.

Furthermore, methods for removing unreacted biotin derivatives from the reaction mixture include centrifugation and dialysis.

Binding of the biotinylated protein and the carrier can be carried out by a known method. In this case, the usage ratio of the carrier and the biotinylated protein is, for example, 1rn9 when biotinylated bovine serum albumin and immobilized red blood cells are used. : 0.02Tn
lS17#: 2d, especially 1m1O, 1lAt~1■:
It is preferable to set it as 0.5m.

To perform the method of the present invention, add avidin and the biotinylated protein-bound carrier obtained by the above method to a test solution containing otin, and compare the resulting aggregation with a biotin standard solution.

Avidin added in the method of the present invention is 1 to 1100n.
/lri, biotinylated protein-bound carrier is 0.1-IV/
The width is preferably 10-50119/ml for avidin, and 0.2-o, s V/, es for the biotinylated protein-bound carrier.

Examples of embodiments of the method of the present invention include the following method. That is, each hole of the 96-hole U-bottom plate has 0. OIM! J acid buffered saline CPBS)
25~100μ! put in. Next, put 25 to 100 μl of the test solution or biotin standard solution into the first row of holes, and sequentially dilute it 2 times. Then apply 5 to 50 n, jil to all holes.
Add 25 to 100 μl of a 7 ml avidin PBS solution and 100 μl of a 0.1 to 0.5% PBS suspension of biotinylated protein-bound red blood cells 25S and mix well. 1-24
In this method, the biotin concentration of the test solution is determined by observing the agglutination reaction after a period of time and comparing the agglutination inhibition between the standard solution and the test solution.

In the method of the present invention, in order to prevent non-specific reactions, P
BS with 0.05-1% protein such as serum albumin or animal serum and 0.5-5% polyethylene glycol 2
It is desirable to add 000''-20000, etc.

〔Effect of the invention〕

According to the method of the present invention, the required amount of test solution is 25 to 100 μl.
Adashi, the blood volume before hydrolysis is actually IOμ! If the amount is above, it is possible to measure the amount of biotin. In addition, the time required for measurement can be shortened compared to microbiological quantitative methods, and the measurement operation is very simple (7 samples can be measured on one 96-well U-bottom plate, and the quantitative operation can be completed in about 5 minutes. do),
One person can process more than 100 samples within one hour after preparing the test solution. Therefore, the present invention is particularly effective when it is desired to simultaneously measure many specimens, 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 hyotinated live serum albumin (BSA)-conjugated red blood cells: (+) N-hydroxysuccinimide biotin (N
HS biotin (Funakoshi Pharmaceutical) 2. Dissolved in 20 μj of dimethyl sulfoxide, and added 1/M BSA to this.
0. 4 d of IM sodium hydrogen carbonate solution was added and reacted at room temperature for 4 hours to biotinylate BSA. Next, to remove unreacted NHS piotin, 5 times the volume of ethyl alcohol was added, centrifuged at 8000 rpm for 5 minutes, and the supernatant was chilled. The precipitate was dissolved in 4MOPBS (pH 7,2) to obtain biotinylated BSA.

(11) Next, after thoroughly washing the sheep red blood cells with PBS, glutaraldehyde was added to a final concentration of well over 0.5, and the mixture was treated at room temperature for about 1 hour, and the red blood cells were washed with PBS to obtain fixed red blood cells. Combine 2.5% fixed red blood cell PBS (pf(7,2) suspension 40- and biotinylated BSA solution 40-4, add 800 μg of tannin rR, mix well, and incubate at 4°C for 1 hour.
After being left overnight, the cells were thoroughly washed with PBS to obtain biotinylated BSA-conjugated red blood cells. Biotinylated BSA-conjugated red blood cells are 0.1%
PB containing IJum and 0,5 BSA
It can be stored as a two-width suspension at S(pi(6,0)) or lyophilized and diluted to a predetermined concentration before use.

Example 2 Measurement of biotin concentration in rabbit blood: 100 μg of rabbit blood was added with papain solution (Sigma P-312
5) 20μ/and 4.4mM citric acid-11°2mM
Mix 380 µd of NatHPO4 buffer and add 1 µd of toluene.
After adding drops and reacting overnight at 37°C, autoclave (12
The supernatant obtained by centrifugation at 8000 rpm for 5 minutes was used as the test solution.

50 μj of 0.1% BSA in each well of a 96-well U-bottom plate.
PBS (1) containing PBS (1) H6, O) was added, and 50 μl of the test solution or biotin standard solution (0.8 nm) was added to the first row of holes, and diluted 2 times sequentially. n
il/ml Avidin D (Funakoshi Pharmaceutical) 5% PEG6
000-containing PBS (pH 6,0) solution 50μ! , and further 50μ of the biotinylated BSA-conjugated red blood cell 0.25% suspension obtained in Example 1 (PBS containing 0.1% BSA; pH 6, o).
Add l and mix well. After 16 hours, the agglutination reaction was observed and the agglutination inhibition was compared between the biotin standard solution and the test solution, and the blood biotin concentration was determined to be 0.5 n, Si/mJ.

Two samples of the same blood were similarly hydrolyzed with papain and the blood biotin concentration was determined using a microbiological quantitative method.
55 n&/rnl. Using this method, almost the same measurement results as the conventional method were obtained using a small amount of blood.

Example 3 Measurement of biotin concentration in rat blood: (1) 100 μlt of rat blood - A test solution was obtained by processing in the same manner as in Example 2, and 5% PEG400 was added to the avidin solution.
The procedure was carried out in the same manner as in Example 2, except that PBS containing 0.0 (pH 6.0) was used, and the blood biotin concentration was determined by checking for inhibition of aggregation after 18 hours and found to be ing/m. When the same blood 2- was treated in the same manner and the blood biotin concentration was determined by microbiological quantitative method, it was 0.97 nji/111
4! Met.

(2) 600 μl of 0.05N sulfuric acid in 200 μl of rat blood
Add e and hydrolyze in an autoclave (121°C) for 1 hour, centrifuge at 8000 rpm for 5 minutes, and collect the supernatant. Add 2 portions of diethyl ether, shake well, remove the ether layer, add 5M ethyl alcohol to the aqueous layer, mix, and centrifuge at 8000 rpm for 5 minutes to collect the supernatant. This was sucked to dryness, and PBS containing 0.1 h BSA (6,0%) was added to the
, dissolve in tll and use as a test solution. 2.5 in avidin solution
The procedure was carried out in the same manner as in Example 2 except that PBS (p) containing % PEG6000 (p16.0) was used, and the blood biotin concentration was determined by observing the inhibition of aggregation after 6 hours, and it was found to be in, p/d. When 2M of the same blood was similarly hydrolyzed with sulfuric acid and the blood biotin concentration was determined using a microbiological quantitative method, it was found to be 0.9.
8 n, @/at. Both papain and sulfuric acid treatment methods obtained almost the same measurement results as the conventional method using illegally small amounts of blood.

Example 4 Measurement of biotin concentration in human urine: Human urine (2 samples) was dissolved in PBS(1)H containing 0.1% BSA.
Avidin solution K diluted 25 times with 6,0) and used as a test solution
The procedure was carried out in exactly the same manner as in Example 2 except for using PBS (pH 6,0), and the urine biotin concentration was determined by observing the inhibition of aggregation after 2 hours. , F/ml teh fc. The biotin concentration of the same two urine samples was measured using the microbiological quantitative method.
Specimen ■21. Onυ moat, sample ■10.1 n777m. Almost the same measurement results as the conventional method were obtained in a short time.

Example 5 Reagent for measuring biotin concentration: Reagents 1 to 4 shown below were prepared, freeze-dried according to a conventional method,
Each was filled into a vial.

Reagent 1 (biotin standard) Reagent 2 (avidin) Reagent 3 (biotinylated BSA-conjugated red blood cells) Reagent 4 (sample dilution solution) 1 Sodium chloride 438.3 ■ The above reagents are for 10 plates (96-well U-bottom plate) Before use, dissolve each in 50 mJ of distilled water, 5050-15Q. Note that if Reagent 2 is difficult to dissolve, it can be easily dissolved by heating it in a hot water bath.

that's all

Claims (1)

[Scope of Claims] 1. A method for measuring biotin, which comprises adding avidin and a biotinylated protein-binding carrier to a biotin-containing test solution, and comparing the resulting aggregation with a biotin standard solution. 2. A biotin measurement reagent containing avidin and a biotinylated protein-binding carrier.