JP3355416B2 - Method for measuring D-amino acid oxidase for determining degree of kidney damage using polyclonal antibody - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION This invention is, D for clinical examination
-Method for measuring amino acid oxidase, more specifically, kidney
Measurement of D-amino acid oxidase in urine to determine degree of injury
About the method .

[0002]

2. Description of the Related Art A renal function test, particularly a method for measuring tubular function and elucidation of pathophysiology, are not yet sufficient, and an appropriate test for obtaining information on the degree of cytotoxicity in renal tissue, particularly nephron of great clinical significance. The method is scarce.

[0003] In the examination of renal injury, when the renal tubular lysosomal site is damaged, the lysosome is disrupted, and N-acetyl-β-D-glucosidase (NAG), which is abundant in the lysosome, escapes into the urine. Therefore, a method for examining the degree of renal tubular lysosomal damage by measuring NGA in urine is known, but a method for measuring the degree of peroxisomal damage to the proximal tubule of the kidney has not yet been established. There was a need for a measurement method that would be an indicator of proximal tubule injury in children.

[0004] We report that D-amino acid oxidase (DAO), which is widely present in mammalian organs, is abundant in humans, particularly in peroxisomal granules of the proximal tubule of the kidney, and is highly active. We paid attention to being recognized. DAO is a flavin enzyme that uses FAD as a coenzyme and catalyzes the oxidative deamination reaction of D-amino acids, and is described in General Literature Methods in Enzymol, 17B,
It is an enzyme whose purification method and various properties have already been elucidated in pages 608 to 622 (1971).

[0005] The measurement of DAO was conducted in 1955 by Metho.
ds in Enzymol. vol. 2, p199 ~
205, Burton et al., And the 1968 Bio
chim. Biophys Acta. vol. 16
7, p9-22, keto acid colorimetry, 1978, Ana
l. Biochem. vol. 86, p310-315
Hydrogen peroxide measurement method, also described in J. Bioc
hem. vol. 60, pp. 219-221, etc., and these methods are methods for measuring generated hydrogen peroxide and ammonia.
It is considerably affected by coexisting substances such as ammonia in urine and its measurement sensitivity is low.It is not enough to measure low concentration of DAO activity, and it can be used for measurement of urinary DAO in clinical tests. Did not.

[0006] We also describe clinical chemistry, Vol.
In 360 to 364, ¹⁴ produced by using a radioisotope using D- [U- ^14C ] alanine as a substrate is used.
A method for measuring urinary DAO in which the concentration of C-pyruvic acid is measured by a counting rate using a liquid scintillation counter was reported. Although this method was highly sensitive and less susceptible to the effects of coexisting substances, in general, the use of radioisotopes and special facilities and equipment were required to spread it as a clinical test method for urinary DAO. There are drawbacks such as the necessity of being necessary and the inconvenience of measuring a large amount of a sample.

[0007] Therefore, as a result of our intensive research,
A simple urine D for measuring the degree of damage to the kidney without the use of radioisotopes, high sensitivity and less affected by coexisting substances
An AO measurement method could be provided .

[0008]

That is, the present invention provides an enzyme immunoassay (Enzyme-Linked Immu).
This is a method for measuring urinary DAO using a nonsorbent assay (ELISA), and a method using a polyclonal antibody against DAO in mammals, for example, rabbits, rats, dogs, and the like.

[0009] After administering DAO as an antigen to mammals such as rabbits, rats and dogs and boosting it several times,
Serum is collected, and normal protein purification methods such as molecular sieving chromatography, affinity chromatography, and electrophoresis can be used from the collected serum. IgG ₃ containing protein G as a ligand is commercially available as a kit.
A method of isolating and purifying a DAO polyclonal antibody by a selective filtration method using MAbTrap (registered trademark) (manufactured by Pharmacia) or Centricon (manufactured by Grace Japan ) having simple binding characteristics is preferred because it is simple and convenient. Less than,
These isolated DAO polyclonal antibodies are referred to as first antibodies, and biotin-labeled antibodies obtained by binding biotin to the first antibodies are referred to as second antibodies.

[0010] The measurement principle of the ELISA method of the present invention is as follows. First, using the obtained first antibody and second antibody, a sample is added to a support in which the first antibody is bound to a support. DAO in the sample is captured by the first antibody. When the second antibody is further added in that state, the second antibody further binds to DAO, which is an antigen captured by the first antibody, by reaction with the same antigen / antibody, and as a result, biotin is produced in proportion to the amount of DAO. Will be retained. In the biotin that is held,
When streptavidin peroxidase is added,
The same number of streptavidin peroxidases binds to biotin of the second antibody, and a synthetic substrate such as 2,2 '
Upon addition of -azino-di- (3-ethylbenzthiazolidinesulfonic acid) diammonium salt, the synthetic substrate is oxidized and colored by streptavidin peroxidase bound to biotin of the second antibody. By measuring the intensity of this coloring, the coloring intensity varies depending on the amount of streptavidin peroxidase, that is, the amount of DAO.
The amount of O can be determined.

The obtained DAO polyclonal antibody and DAO polyclonal biotin-labeled second antibody can be stored after lyophilization and dissolved in a buffer solution as required before use.

[0012] The measuring method of the present invention is 1 to 300 ng / m
It is possible to measure DAO at a concentration of 1 l, which is much more sensitive than conventional methods, and because it is an immunological assay for antigens and antibodies, it has high specificity and The effect of coexisting substances is small, and special facilities and equipment are not required unlike the measurement method using radioisotopes, and it is possible to process a large amount of samples at a time. Since it is a quantification method, it is a measurement method that does not need to be affected by the inactivation of the enzyme as in the method of measuring from enzyme activity.

[0013]

EFFECT OF THE INVENTION DAO and N in the case of performing extracorporeal shock lithotripsy (ESWL) on 6 patients with renal calculi
The amount of AG leaked into urine was measured using the present measurement method and a known NAG measurement method. The kidney of a patient who has undergone extracorporeal shock lithotripsy (ESWL),
Although a part of the cell tissue inside the kidney was damaged, the destruction of the tissue inside the kidney could not be grasped by the result of measurement using the NAG measurement method. The amount was measured to be rising.

[0014]

[Table 1]

As a result of the DAO addition and recovery test, 10 to 30
High recovery was obtained in the concentration range of 0 ng / ml.

[0016]

[Table 2]

As a result of the simultaneous reproducibility test, S.P.
D value or C.I. The V value was 1.870 and 3.05, and the S.V. D value or C.I. The V values were 0.461 and 9.70, showing excellent values.

[0018]

[Table 3]

As a result of the daily fluctuation test, the high value sample was found to be S. cerevisiae. D
Value or C.I. The V value was 2.709 and 4.47, and the S.V. D value or C.I. The V value was 1.380 and 9.78, showing excellent values.

[0020]

[Table 4]

L-amino acid oxidase (LDO), which is structurally closest and is likely to give an error to the measured value, is 0 to 5
In a concentration range of 00 ng / ml, and in a low concentration DAO 2.50 ng / ml solution where the measured value is most likely to be affected, LDO was added and the effect of LDO on the measured DAO value was confirmed. No effect was observed.

[0022]

[Table 5]

[0023]

Example 1 An example of a method for preparing a DAO polyclonal primary antibody of the present invention includes the following preparation method. A 2 mg / ml adiband suspension of pig kidney-derived DAO is intradermally administered to rabbits, rats or dogs, and thereafter, every two weeks, the intradermal administration of the 1 mg / ml suspension of DAO is similarly repeated four times. Ten days after the last intradermal administration of the 1 mg / ml DAO suspension, blood is collected and serum is obtained by centrifugation. The obtained serum was used for MAbTrap (registered trademark).
Name) Affinity chromatography using (Pharmacia) and Centricon-100 (Grace Japan)
To obtain a DAO polyclonal antibody.

[0024] DAO polyclonal primary antibody. Molecular weight: 100,000 or more Number of subunits: 4 Antigen / antibody, double diffusion method: One sharp precipitation line Antigen / antibody with L-amino acid oxidase, cross-reaction: negative Ninhydrin reaction: positive Maurish reaction: Positive

[0025]

Example 2 A 20 μg / ml solution was prepared using the DAO polyclonal primary antibody obtained in Example 1,
50 μl of this first antibody solution is dropped onto a protein adsorption support, for example, a microtiter plate, and shaken with a shaker for 30 minutes. Next, 0.1% of a surfactant such as Twe
After washing 5 times with 200 μl of 0.9% physiological saline containing en-20 (hereinafter referred to as washing solution), 1% (W / V)
200 μl of an egg albumin solution of the above was dropped and shaken for 30 minutes to apply a post-coating for adsorbing albumin to the remaining protein-bound portion of the support, and this was washed with a washing solution.

Next, urine, which is a sample, is diluted if necessary, 50 μl of the diluted urine is added, and the mixture is incubated with shaking for 30 minutes to allow DAO in the sample to be captured by an antibody. Wash. Next, 50 μl of a solution of 20 μg / ml of the second antibody labeled with biotin is added thereto, and the mixture is incubated with shaking for 30 minutes to further adsorb the second antibody on DAO captured by the first antibody. After washing the plate 5 times with 200 μl of a washing solution, 50 μl of a streptavidin peroxidase test solution (manufactured by Nippon Turner Co., Ltd.) was added, and the mixture was incubated with shaking for 30 minutes, so that streptavidin.
The peroxidase is bound and washed with a washing solution.

Next, the synthetic substrate 2,2'-azino-
After adding di- (3-ethylbenzthiazolidinesulfonic acid) diammonium salt solution and incubating with shaking for 15 minutes, 10 μl of 50 mmol / l acetate buffer having a catalase content of 150 U / ml or more and a pH of 5.5 or more was added.
Is added to stop the coloring reaction, and the sample absorbance As is measured at a wavelength of 415 nm. As a control, by measuring the absorbance A _B but was operated in the same manner without using the specimen from the difference between the control absorbance A _B and Sample absorbance A _S, determine the amount of DAO in the sample.

[0028]

[Brief description of the drawings]

FIG. 1 Calibration curve by the measurement method of the present application

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-60-250257 (JP, A) Masanao Katagiri, COMP. BIOCHEM. PHYSI OL. , VOL. 99B NO. 2,345-350 masanao katagiri, biomedical research h, vol. 5 NO. 2,125-134 (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 33/573 C12Q 1/26 C12Q 1/28 G01N 33/53

Claims (2)

(57) [Claims] (1) D used to determine the degree of kidney damage
- A method for measuring amino acid oxidase, (1) the protein-adsorbing support by binding D- amino acid oxidase Porikuro <br/> monoclonal antibody of the first antibody, urine added as specimen (2) Urine
(3) causing the first antibody to capture the D-amino acid oxidase of
Next, a second antibody labeled with biotin is added to the first antibody to allow the D-amino acid oxidase captured by the first antibody to adsorb the second antibody, and (4) streptavidin The peroxidase is bound to biotin of the second antibody by adding an oxidase solution, and (5) the synthetic substrate solution is colored with the peroxidase to measure the absorbance, and the amount of D-amino acid oxidase in urine is determined from the color intensity. D-
Method for measuring amino acid oxidase. 2. The method according to claim 1, wherein the synthetic substrate is 2,2′-azino-di- (3
- ethylbenzamide thiazolidine sulfonic acid) or the measuring method according to claim 1, wherein a salt thereof.