JPH05296999A - Human lipocortin i as marker for discriminating effect of flucocorticoid and its determining method - Google Patents

Abstract

PURPOSE:To accurately measure the concentration of human lipocortin I contained in a blood serum or blood plasma (hereinafter omitted) by determining the human lipocortin I as a marker for discriminating the effect of glucocorticoid. CONSTITUTION:A human lipcortin I antibody and human lipocortin I labeled with a detectable substance are prepared by using human lipocortin I obtained from human placentas. Then a water insoluble carrier prepared by bonding a fixed amount of blood serum to an antibody is made to react to a fixed amount of a buffer solution containing albumine, etc., for 10 minutes to four days at, for example, 2-40 deg.C. The human lipocortin labeled with the detectable substance is added to the resulted solution of reaction and they are made to react to each other for the same period at the same temperature. After reaction, the water insoluble carrier is washed and the detectable substance bonded to the carrier is measured. The concentration of the human lipocortin I in the blood serum is determined by performing the same operation to a standard solution containing the human lipocortin at a known concentration and comparing measured values obtained from the standard and living body fluid with each other. The pharmacological effect of glucorticoid is discriminated from the quantity variation of the concentration.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to human lipocortin I as a marker for determining the effect of glucocorticoid and a method for quantifying the human lipocortin I. More specifically, human lipocortin I in plasma or serum was quantified, compared with a control level, the change in concentration was confirmed, and human lipocortin I as a marker for determining the effect of glucocorticoid and human lipocortin by immunological measurement. It relates to a method for quantifying rutin I.

[0002]

Glucocorticoids are corticosteroid hormones, and their pharmacological actions include anti-inflammatory action and
Cell growth inhibitory action and anabolic inhibitory action are known. In particular, it has a strong anti-inflammatory effect, and its mechanism of action is the inhibition of the synthesis of proinflammatory substances such as prostaglandin and leukotriene, and inhibits phospholipase A ₂ that releases arachidonic acid from phospholipids at the first stage of the arachidonic acid cascade It is believed that the protein is induced by glucocorticoids.

This induced protein was called macrocortin, lipomodulin or renocortin, but it is considered that they are derived from a common precursor because they cannot be immunologically separated, and their names are unified to lipocortin. [(Metabolism, Vol. 24, No. 3, 1987)].

As the lipocortin, recently, lipocortin I and lipocortin II are known at the base sequence level, and lipocortins III, IV, V and VI have been reported.

There are many reports on the biological action of lipocortin, such as action on inflammation, immunity, coagulation, cell proliferation and differentiation. However, most of the studies are in cells, and no report has been made on their kinetics outside the cells, especially in blood.

A number of methods for quantifying lipocortin have been reported so far [Biochemical and Biophysical Research Communications (Bioche
M. Biophys. Res. Commun.) 109, 223 (19)
82), Biochemical Society Transactions, Vol. 18, 1230.
(1990) and Journal of Immunological
Method (J. Immunol. Meth.) 131, 119 (1)
990)].

[0007]

However, the above-mentioned respective measurements only measure lipocortin in rat peritoneal fluid, macrophage culture medium, rat pituitary gland, hypothalamic tissue, and human monocytes, respectively. However, there is no report quantifying the concentration of human lipocortin in human serum or plasma.

Further, there is no report that lipocortin induced by administration of glucocorticoid was measured in serum or plasma, and the relationship between the pharmacological effect of glucocorticoid and the concentration in serum or plasma was found.

The anti-inflammatory effect of glucocorticoids is
In the mechanism, the inhibitory action of phospholipase A ₂ is induced in blood by administration of glucocorticoid,
Since it is considered to be performed by increased lipocortin, it is considered that the anti-inflammatory effect of glucocorticoid is reflected in the amount of induced lipocortin. Therefore, it is considered that the measurement of the lipocortin concentration in blood after administration of glucocorticoid serves as an index for determining the effect of glucocorticoid in the administered patient.

The inventors of the present invention purified human lipocortin I from human placenta, prepared a specific antibody against human lipocortin I, and developed an immunoassay using the obtained antibody and purified human lipocortin I. It has made it possible to accurately measure human lipocortin I concentrations in serum or plasma.

Further, by measuring the concentration of human lipocortin I in the blood of a glucocorticoid-administered patient, the blood human lipocortin I induced by the glucocorticoid was measured.
The present invention was completed by confirming the increase in the amount and finding the usefulness of human lipocortin I as an index for determining the effect of glucocorticoid.

[0012]

The present invention provides a method for determining the pharmacological effect of glucocorticoid by assaying human lipocortin I as a marker for determining the effect of glucocorticoid in plasma or serum. ..

That is, by quantifying human lipocortin I as a marker for determining the effect of glucocorticoid, it is possible to determine the effect of glucocorticoid administered in diagnosis or set an optimum dose for each patient.

The glucocorticoid as a drug to be evaluated for the effect of the present invention is a drug having an action of inducing human lipocortin having a phospholipase A ₂ inhibitory action as its mechanism of action. For example,
Cortisol, Cortison
e), dexamethasone (Dexamethasone), corticosterone (Corticosterone) and the like.

The present invention further provides the above-mentioned method for quantifying human lipocortin I. For example, an immunoassay using a specific antibody against human lipocortin I, which is a suitable quantification method, will be described in detail as an example.

Human lipocortin I can be prepared, for example, from human placenta according to a known method [Journal of
Biological Chemistry (J. Biol. Chem.) 264
Vol., 6948 (1989)].

Using the prepared human lipocortin I, a human lipocortin I antibody can be obtained. That is, human lipocortin I is injected as it is or after being mixed with an appropriate adjuvant, into the skin, abdominal cavity or the like of an animal. This operation is repeated at intervals of 1 to 2 weeks for immunization. By hybridizing cells of the immunized animal with myeloma cells, a hybridoma producing a human lipocortin I antibody can be obtained. A monoclonal antibody can be obtained from the obtained culture medium of the hybridoma. A polyclonal antibody can be obtained from the serum of an animal in which antibody production has been confirmed after immunizing an animal with human lipocortin I in the same manner as described above.

Although the antibody to be used can be used in the prepared immunoglobulin fraction itself, it is further used as a fraction such as F (ab ') ₂ , Fab', Fab or the like in which only the binding site for human lipocortin I is separated. You can also

On the other hand, highly purified human lipocortin I
Alternatively, an anti-human lipocortin I antibody is used to prepare labeled human lipocortin I or anti-human lipocortin I antibody bound to a detectable labeling substance. It is necessary to select, as the detectable labeling substance, one whose detection sensitivity is sufficient for the detection sensitivity required for the method for quantifying human lipocortin I. Specifically, there are various radioisotopes, various enzymes, fluorescent substances, metal ions and the like, more preferably ¹²⁵ I,
^Examples include ¹³¹ I, β-D-galactosidase, peroxidase, alkaline phosphatase, glucose oxidase, malate dehydrogenase, fluorescein, methylumbelliferone, magnesium and the like.

A known method is used for binding these detectable substances to human lipocortin I or anti-human lipocortin I antibody. For example, when a radioisotope is used, reagents such as chloramine T and iodogen (registered trademark by Pierce) are used, and in the case of an enzyme, a coupling method using a bifunctional reagent, a periodate oxidation method and the like are used. It can be applied.

The labeled human lipocortin I or anti-human lipocortin I antibody thus obtained can be detected in addition to the originally intended labeled human lipocortin I or labeled anti-human lipocortin I antibody. Different binding number of substance and human lipocortin I, detectable substance partially changed during binding procedure to human lipocortin I or anti-human lipocortin I antibody and lost original activity, binding reaction Or a human lipocortin I or anti-human lipocortin I antibody itself which did not bind to the human lipocortin I or anti-human lipocortin I antibody Sometimes Therefore, in order to increase the sensitivity of quantification, it is preferable to separate only the desired labeled human lipocortin I or labeled anti-human lipocortin I antibody from the above mixture. As a method for this, ion exchange chromatography,
Gel filtration chromatography, affinity chromatography and the like can be used.

In addition to the above, as a reagent necessary for quantifying human lipocortin I, there is a water-insoluble carrier to which an antibody is directly bound. Further, there is a factor that binds to an antibody that is insolubilized in a water-insoluble carrier. Since the antibody is an immunoglobulin, an anti-immunoglobulin antibody, that is, a second antibody can be used as the factor that binds. Other protein A and protein G derived from microorganisms that are immunoglobulin-binding proteins
Etc. can also be used. These binding factors are preferably highly purified by means such as affinity chromatography in order to increase the binding amount of the antibody. Examples of water-insoluble carriers for insolubilizing binding factors include various polysaccharide gels, particles made of synthetic resins such as polystyrene, beads, test tubes, other small containers, glass of the same form, metals, etc. Suitable are synthetic thin films such as nitrocellulose and nylon.

As a method of insolubilizing the factor that binds to these water-insoluble carriers, chemical adsorption such as physical adsorption or covalent binding can be used. For example, in the physical adsorption method, the polystyrene beads and the like can be physically insolubilized by putting the polystyrene beads and the like in a solution of the binding factor of a fixed concentration and leaving it for a fixed time. On this occasion,
By constantly moving the solution of the binding factor upon insolubilization, the factor binding to the surface of the water-insoluble carrier can be uniformly insolubilized. In addition, the solution of the binding factor is 15-40 ℃
It can be insolubilized in a short time by keeping it at. In the chemical coupling method, various coupling reagents such as cyanogen bromide, glutaraldehyde, sodium periodate, carbodiimide derivative, silane agent, maleimide derivative, succinimide ester, oxirane compound, tosyl chloride, carbonylimidazole, etc. are used. Be done.

It is not preferable to use the thus-prepared factor that binds to the antibody insolubilized in the water-insoluble carrier as it is when quantifying human lipocortin I.
That is, when used as it is, the surface of the water-insoluble carrier, or the chemically active reactive group used for insolubilizing the factor to be bound, nonspecifically adsorbs and binds the reaction solution component during the quantification, which deteriorates the measurement sensitivity. Will bring. In order to prevent this, it is preferable to treat the surface of the water-insoluble carrier after insolubilizing the binding factor. As this treatment method, a method of inactivating the surface of the water-insoluble carrier after thoroughly washing the water-insoluble carrier after insolubilizing the binding factor is preferable. In order to inactivate the surface of the water-insoluble carrier, a solution containing one or more kinds of albumin, gelatin, and other proteins, a solution of various amino acids, other compounds having an amino group, a reducing agent, an oxidizing agent, etc. are used alone. Alternatively, they can be used in combination.

Human lipocortin I is quantified using the reagent thus prepared. First, a fixed amount of serum or plasma is reacted with a fixed amount of a water-insolubilized carrier bound with an antibody and a buffer solution. The total reaction volume at this time is preferably 50 to 1000 μl. The buffer is preferably a buffer containing 0.01 to 1% of one or more proteins such as albumin and gelatin at pH 4 to pH 8.5, and the reaction temperature is preferably 2 ° C to 40 ° C.

The reaction time is an important factor influencing the measurement sensitivity, and considering the operability, it is preferably 10 minutes to 4 days. Then, human lipocortin I or anti-human lipocortin antibody labeled with a detectable substance is added to this reaction solution, and the reaction is further carried out. The reaction temperature and time are the same as above, 2 ℃ -40 ℃,
10 minutes to 4 days is preferable.

After the reaction, the water-insoluble carrier is washed with water or an appropriate buffer, and the detectable substance bound to the water-insoluble carrier is measured. The above operation is also performed on a standard solution containing a known concentration of human lipocortin I, and the concentration of human lipocortin I in plasma or serum can be quantified by comparing the measured value of the standard solution with the measured value of biological fluid. ..

It is, of course, possible to quantify human lipocortin I using a known immunological measuring method other than the above.

The amount of human lipocortin I in the biological fluid thus quantified can be used as an index for determining the pharmacological effect of glucocorticoid in humans or an index for setting the optimal dose. For that purpose, it is necessary to quantify a serum or plasma sample collected from a patient in need of clinically established administration of glucocorticoid by the method of the present invention and examine the human lipocortin I concentration obtained by these measurements. Is. The present inventors have found that when the concentration of human lipocortin I in the serum or plasma of a patient administered with glucocorticoid is measured, the concentration of human lipocortin I varies depending on the administered glucocorticoid. Therefore, it is possible to determine the effect of glucocorticoid by comparing the human lipocortin I concentration in serum or plasma before and after the administration of glucocorticoid or to set the optimal dose for each patient. is there.

Furthermore, by carrying out such an assay, it is possible to use it as data for judging the effectiveness of treatment with glucocorticoids, and for each patient of glucocorticoids which is a steroid drug with strong side effects. It is possible to set the optimal dose of

The embodiments of the present invention will be described in more detail below. The present invention is not limited to these. In addition, the antigen and antibody used in this example were prepared as follows.

Antigen Human lipocortin I is derived from human placenta [Journal of Biological Chemistry (J. Biol. Chem.) 264, 6948 (1989)].
Was prepared and purified according to.

Antibody As a polyclonal antibody, first, 0.5 mg of human placenta-derived lipocortin I was subcutaneously injected into a rabbit.
/ Head was administered once a week for 3 weeks, and 3 weeks after the final administration, it was collected from the ear vein every week to obtain antiserum.

Next, the obtained antiserum was salted out with 50% saturated ammonium sulfate,
Ion-exchange chromatography with DEAE-cellulose and CNBr-activated Sepharose 4B (Pharmacia)
What was purified by chromatography with an affinity column in which the antigen was bound to was used.

As the monoclonal antibody, 45% of a culture solution of hybridoma 4E4 obtained by fusing spleen cells taken from a mouse immunized with human placental cell-derived lipocortin I and mouse myeloma cells was saturated with ammonium sulfate and subjected to antigen binding. What was purified by chromatography using an affinity column was used.

[0036]

【Example】

Example 1 (1) Preparation of solid phase bound to antibody Polyclonal antibody or monoclonal antibody was added to pH 7.0.
It was dissolved in the phosphate buffer solution of and the polystyrene beads as a solid phase were immersed in the solution to bind to the surface thereof.

(2) Preparation of various labeled substance-bound antibodies and labeled substance-bound human lipocortin I As a labeling enzyme for the antibody or human lipocortin I, peroxidase, β-, which is generally used in enzyme immunoassay, is used. Labeled with D-galactosidase or alkaline phosphatase.

The antibody is obtained by treating IgG, F (ab ') ₂ obtained by treating IgG with pepsin, and further treating Fab', IgG obtained by reducing F (ab ') ₂ with a reducing agent, with papain. The Fab obtained by was used.

In the case of radioactivity labeling, ¹²⁵ I was labeled with antibody or human lipocortin I by a method using chloramine T.

(3) Labeling enzyme activity measuring method The enzyme activity was measured by a coloring method, a fluorescence method and a luminescence method. The labeling enzymes and substrates used are shown in Table 1.

[0041]

[Table 1]

In the table, AMPGD is 3- (4-Methoxyspiro [1,2
-dioxetane-3,2'-tricyclo [3.3.1. ^3,7 ] decan] -4-yl) phe
nyl-β-glactopyranoside, AMPPD is 3- (4-Methoxy
spiro [1,2-dioxetane-3,2'-tricyclo [3.3.1. ^3,7 ] decan]
-4-yl) phenyl-phosphate.

(4) Immunoassay Method As the immunoassay method, a sandwich method and a competitive method were carried out. Sandwich method 10 μl of specimen with antibody-bonded polystyrene beads (diameter 6.5 m
m) 1 pc, 10 mM sodium phosphate buffer 0.5 ml (0.1% B
SA, 0.5% gelatin, 1 mM MgCl ₂ , 1% NaN ₃ , 0.3M
NaCl) was added, and the mixture was reacted at 37 ° C. for 3 hours. After the reaction,
Wash the beads, add 0.5 ml of each labeled antibody solution, and 4 ℃
I made it react overnight.

After the reaction was completed, the beads were washed and the labeled substance bound to the beads was measured. Enzyme activity is measured at 37 ℃,
The color development method was measured in 60 minutes, the fluorescence method in 30 minutes, and the light emission method in 30 seconds. Radioactivity was measured by using a scintillation counter.

Competitive method 10 μl of sample was bound to antibody-bonded polystyrene beads (diameter 6.5 m
m) 1 unit, 10 mM sodium phosphate buffer solution (0.1% BSA, 0.1%) in which human lipocortin I labeled with various labels was dissolved.
5% gelatin, 1 mM MgCl ₂ , 1% NaN ₃ , 0.3M NaCl)
0.5 ml was reacted at 37 ° C. for 3 hours. After the reaction was completed, the beads were washed and the labeled substance bound to the beads was measured. Enzyme activity was measured at 37 ° C for 60 minutes for color development, 30 minutes for fluorescence, and 30 seconds for luminescence. Radioactivity was measured by using a scintillation counter.

(5) Measurement of standard human lipocortin I A rabbit anti-human lipocortin I polyclonal antibody F (ab ') ₂ was bound to 10 μl of a standard human lipocortin I solution, 6.5 mm diameter polystyrene beads, 10 mM phosphoric acid Sodium buffer (pH 7.0, 0.1% BSA, 0.5% gelatin, 0.1MN
aCl) 0.5 ml was added, and the mixture was reacted at 37 ° C. for 3 hours.

After completion of the reaction, the beads were washed and β-D-galactosidase-labeled mouse anti-human lipocortin I was added.
Add 0.5 ml of monoclonal antibody F (ab ') ₂ solution,
Let it react overnight. After the reaction, the chromogenic substrate as the substrate was chlorophenol red-β-D-galactopyranoside solution.
5 ml was added, and the fluorescent substrate was 4-methylumbelliferyl-
0.5 ml of β-D-galactopyranoside solution was added, and the enzyme reaction was carried out at 37 ° C for 1 hour and 30 minutes, respectively. As the enzyme reaction stop solution, 2 ml of 1% galactose solution was used for the coloring method and 0.1 M glycine for the fluorescence method. The enzymatic reaction was stopped by adding 2 ml of NaOH buffer (pH 10.3). The color development method measured the absorbance at 575 nm, and the fluorescence method measured the fluorescence intensity at an excitation wavelength of 390 nm and a measurement wavelength of 460 nm. The measurement results of standard human lipocortin I are shown in FIG. The measurement sensitivity of the colorimetric method is 10ng / Tube, and the fluorescence sensitivity is 1ng / Tube.
It was a Tube.

Example 2 Quantification of Human Lipocortin I Level in Serum Human lipocortin I level in serum of patients receiving glucocorticoid was quantified. The amount of serum used in the measurement system is 10 μl
Was measured by the same method as in (1) of Example 1, the enzyme activity was measured by a fluorescence method, and the fluorescence intensity of each sample was read from the fluorescence intensity of the standard line to obtain the amount of human lipocortin I.

An increase in the amount of human lipocortin I was observed in the serum of patients with nephrotic syndrome after administration of glucocorticoid. The result is shown in FIG.

Example 3 The amount of human lipocortin I in the serum of a glucocorticoid-administered patient was quantified. The amount of serum used in the assay system was 10 μl, and the enzyme activity was measured by the same method as in (5) of Example 1, the colorimetric method was used, and the absorbance of each sample was read from the absorbance of the standard line to obtain human lipocortin I. Calculated as quantity.

An increase in the amount of human lipocortin I was observed in the serum of the glucocorticoid-administered patients (patients A to J). The results are shown in Table 2.

[0052]

[Table 2]

[0053]

INDUSTRIAL APPLICABILITY According to the present invention, it becomes possible to accurately quantify human lipocortin I, and it becomes possible to judge the effect of glucocorticoid administered by the change in the amount of human lipocortin I in the quantified plasma or serum. It was

[Brief description of drawings]

FIG. 1 shows a calibration curve of standard human lipocortin I. In the figure, the black circles show the results of the fluorescence method and the white circles show the results of the color development method.

FIG. 2 is a graph showing changes in the amount of human lipocortin I in plasma after administration of glucocorticoid to patients with nephrotic syndrome.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Kuniyoshi Matsunaga Kunitsubo Nishijo Yashiki, Nishiharu Town, Nishikasugai-gun, Aichi Prefecture 51 Amano Pharmaceutical Co., Ltd. Central Research Laboratory

Claims (4)

[Claims] 1. Human lipocortin I in human plasma or serum
In vitro as a glucocorticoid effect determination marker. 2. A method for immunologically quantifying human lipocortin I in vitro by utilizing cross-reaction of a plasma or serum sample containing human lipocortin I with an antibody specific for human lipocortin I. And a method for quantifying human lipocortin I. 3. The method according to claim 2, wherein human lipocortin I is induced by a drug. 4. The quantification method according to claim 2, wherein the presence of human lipocortin I, the concentration of which varies depending on the administration of glucocorticoid from the control level, is a diagnostic instruction for determining the pharmacological effect of glucocorticoid.