JPS62235564A - Immunological measurement of erythropoietin - Google Patents

Abstract

PURPOSE:To measure the density of erythropoietin (EPO) at a low cost in a short time with a high sensitivity in an oridinary inspection chamber, by using a labelled antibody comprising a specified antibody and a label substance. CONSTITUTION:An insoluble support to which is coupled a first antibody tended to react with EPO specifically is caused to react with a second antibody that is tended to react specifically with a liquid to be inspected containing EPO and EPO itself while obtained by immunization on an animal different from the first antibody and then, with a labelled antibody to measure the amount of the labelled antibody coupled onto the insoluble support or the amount of the labelled antibody not coupled thereonto. The labelled antibody is formed by bonding a label substance such as enzyme to a third antibody which will specifically react with immuno globulin of the animal from which the second antibody was prepared. With the bonding of the label substance onto the support proportional to the amount of EPO coupled, the EPO concentration of the liquid being detected is determined by comparison with a calibration curve prepared beforehand.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an immunoassay method for erythropoietin (hereinafter sometimes abbreviated as EPO). [Prior Art] EPO is a hormone with a molecular weight of 40,000 that is mainly produced in the kidney and controls the differentiation of red blood cells. It is known that the amount of EPO in body fluids increases or decreases in various anemias and polycythemias, and accurate measurement of the amount of EPO in body fluids is extremely important for the diagnosis of these blood diseases.

The conventional method for measuring EPO is the bioassay method (clinical test vol. 1.22.) using small animals such as mice and rats.
No. 2.1987), 89F to mouse bone marrow cells.
A method of measuring e uptake (J, Lab, Cl1n.

Med, Vol. 97. No, 2.158-169
Page, 1981), method using mouse fetal cells (Journal of the Japanese Society of Hematology, Vol. 44. No. 6. 1981)
etc. are known. Among these, the bioassay method is said to be the most reliable, but this method has the drawbacks of low detection sensitivity, long measurement times, and high cost due to the use of a large number of animals. are doing.

[Problems to be solved by the invention] In recent years, immunoassay methods have been used as a method for quantifying trace substances in body fluids.
Although there has been remarkable progress in enzyme-linked immunosorbent assays, the expansion of their use is limited due to lower detection sensitivity than radioimmunoassays. In particular, the normal blood concentration of EPO is 10-20 ml/mj! (0,1~
Since the amount is extremely small (0.3 ng/mft), it was thought that it would be difficult to detect using known enzyme immunoassay methods.

For this reason, the present inventors developed an antibody-bound insoluble support, in which an antibody that specifically reacts with EPO was bound to an insoluble support,
After reacting a sample solution containing PO with a label obtained by labeling an antibody that specifically reacts with EPO with an enzyme to form an antibody-EPO-label complex on the antibody-bound insoluble support, A patent application has already been filed for an enzyme immunoassay method for EPO, which involves measuring the amount of labeled substance bound to an insoluble support (Japanese Patent Application No. 60-39687).

However, in this method, the titer of the antibody that specifically reacts with EPO is low, so the above-mentioned normal blood concentration of EPO is not necessarily achieved.
It was not always possible to measure PO with high precision. The reason for the low titer of antibodies that specifically react with EPO is that
When animals other than humans are immunized with O, it is thought that the metabolism of EPO is rapid and that the animal reacts physiologically to the administered EPO, causing symptoms of extreme anemia.

In view of the current situation, the present inventors have conducted intensive research into a method for measuring EPO that can be carried out in a normal laboratory, can be carried out in an extremely short period of time at low cost, and has an extremely high detection sensitivity, and have completed the present invention. did.

[Means for Solving the Problems] The present invention provides an antibody-bonded insoluble support in which a first antibody that specifically reacts with erythropoietin is bound to an insoluble support, a test solution containing erythropoietin, and a first antibody that specifically reacts with erythropoietin. After reacting with a second antibody obtained by immunizing an animal species different from the first antibody and which reacts with the second antibody, the second antibody
A third antibody that specifically reacts with the immunoglobulin of the animal species in which the antibody was prepared is reacted with a labeled antibody that is bound to a labeling substance, and the amount of the labeled antibody bound to the antibody-bound insoluble support is measured. This is an immunoassay method for EPO, the gist of which is to measure the amount of erythropoietin in the test liquid by measuring the amount of unbound labeled antibody.

[Function] The present invention is configured as described above, but the point is that even when the titer of the second antibody that specifically reacts with EPO is low, the second antibody that specifically reacts with EPO
By using a labeled antibody in which a labeling substance is bound to a third antibody that specifically reacts with the immunoglobulin of the animal species for which the antibody was created, sensitivity can be amplified and good EP can be achieved.
A method for immunologically measuring O is provided. That is, the third antibody can be easily obtained by immunizing an animal other than the animal species in which the second antibody was produced with immunoglobulin, but the titer of the third antibody obtained is higher than that of the second antibody. It is much larger than its titer.

The first antibody and second antibody used in the present invention are EP
EPO purified from the urine of aplastic anemia patients who produce relatively large amounts of O is used to immunize different animal species selected from non-human mammals, such as rabbits, cows, sheep, and guinea pigs. It can be obtained by Further, either one of the first antibody and the second antibody may be a monoclonal antibody. In a more preferred embodiment of the present invention, the first antibody is an antibody obtained by immunizing an animal such as a rabbit or a cow, the second antibody is a monoclonal antibody derived from a mouse, and the third antibody is an antibody obtained by immunizing an animal such as a rabbit or a cow. This is the case when the antibody is obtained by immunizing an animal other than a mouse with globulin.

EPO is purified using affinity chromatography.

Although it can be carried out in combination with known purification means such as gel filtration,
In order to obtain highly specific antibodies, it is preferable to purify EPO as much as possible. EP purified in this way
The first antibody or second antibody is prepared by subcutaneously immunizing a rabbit, cow, or the like with O. In addition, the third antibody that specifically reacts with the immunoglobulin of the animal species in which the second antibody was created is
It is produced by immunizing an animal other than the animal species in which the second antibody was produced with a purified product of immunoglobulin. Monoclonal antibodies that specifically react with EPO include, for example, purified EP
BAL B/C mice were immunized with O to obtain tile cells.
Fused with myeloma cells using polyethylene glycol,
It can be obtained by repeated cloning using HAT medium.

Next, the first antibody thus obtained is bound to an insoluble support. Examples of the insoluble support include polystyrene tubes, polystyrene spheres, silicone pieces, microplates, and polystyrene spheres are particularly preferred. The bonding method to these insoluble supports may be any known chemical method or physical method. For example, as a physical method, the antibody is dissolved in a suitable buffer solution, the above-mentioned insoluble support is added, and the antibody is incubated at 0°C to room temperature for several hours to overnight.
An example of a method is to obtain an antibody-binding insoluble support (hereinafter simply referred to as antibody-binding support) by leaving it for 4 to 16 hours preferably at 4 to 20°C and washing with saline or the like. Antibody-conjugated supports are stable for at least one year when stored with a stabilizer such as bovine serum albumin.

Next, a labeled antibody (
(hereinafter simply referred to as labeled antibody).

The third antibody is an antibody against the immunoglobulin of the animal species in which the second antibody that specifically reacts with EPO was prepared, as described above. For example, if the second antibody is a mouse-derived monoclonal antibody, the third antibody is an antibody against mouse immunoglobulin, and if the second antibody is an antibody produced in a rabbit, the third antibody is an antibody against rabbit immunoglobulin. It is an antibody against globulin. Examples of the labeling substance include enzymes, isotopes, fluorescent substances, metals, etc., and enzymes are particularly preferred. Further enzymes include horseradish peroxidase and alkaline phosphatase.

β-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase.

Glucose dehydrogenase and the like are preferred, and horseradish peroxidase is particularly advantageous. These enzymes can be bound to the third antibody using known methods. For example, the sugar chain of the enzyme is oxidized with periodic acid and the generated aldehyde group is bonded to the amino group of the third antibody, or a maleimide group is introduced into the enzyme and the thiol group present in the third antibody is bonded to the enzyme. Examples include a method of combining the two.

EPO is measured using the antibody-bound support and labeled antibody thus obtained as follows. First, a test solution containing EPO and a second antibody that specifically reacts with EPO are applied to the antibody-binding support at 0°C to 50°C for 1 hour to 1 night, preferably at 15 to 37°C for 2 to 4 hours. Make it react. Here, the test liquid containing EPO refers to serum, plasma, urine, etc. Furthermore, the first antibody and the second antibody bound to the antibody-binding support must be antibodies produced in different animal species. That is, if the first and second antibodies are prepared using the same species of animal, the labeled antibody, which is labeled with a labeling substance, will bind to the third antibody against the animal's immunoglobulin, regardless of the amount of EPO present, making it virtually impossible to measure it. Because it will disappear.

Next, since the second antibody is bound to the antibody-binding support reacted as described above in proportion to the amount of EPO bound, the labeled antibody is further added at 0 to 50°C for 1 hour to 1 hour. When the reaction is carried out at night, preferably at 15 to 37° C. for 2 to 4 hours, the labeled antibody will bind to the antibody-binding support in proportion to the amount of EPO bound. Therefore, the amount of the labeled substance of this labeled antibody is measured, and the E
PO can be measured. If an enzyme is used as the labeling substance, the enzyme activity may be measured, and if it is an isotope, the amount of radioactivity may be counted.

[Examples] The present invention will be explained below with reference to Examples, but the present invention is not limited to these Examples.

Example 1 (a) Preparation of antibody-binding support Preparation of polyclonal antibody - Preparation of polyclonal specific antibody against eryboetin was carried out by immunizing rabbits three times as follows using purified EPO (derived from urine of an anemic patient) as an antigen. An antiserum was prepared by performing the following steps, and IgG was purified according to a conventional method.

1st immunization - 400 μg/mj of purified EPO in PBS
1.5nj of emulsion obtained by dissolving it to become Z and mixing it with an equal amount of Freund's complete adjuvant! It was administered to rabbits (approximately 2 kg) by subcutaneous injection at 15 to 20 locations along the vertebrae.

Second immunization - Two weeks later, emulsion tmi was administered to 10 to 15 locations in the same manner as the first immunization.

3rd immunization - after another 2 weeks, 200μ
Emulsion 1 all obtained in the same manner was administered to 10 sites using the antigen dissolved at 1 g/mjl.

The day before the third immunization, a small amount of blood was collected for testing, and the anti-EPO activity contained in the serum was examined. One week after the third immunization (final immunization), all the blood was collected, and the serum was separated using the usual method. anti-EPO serum was obtained. Anti-EPO-1gG was prepared from anti-EPO serum using a conventional method, including 50% ammonium sulfate precipitation,
This was done by obtaining the DEAE-cellulose non-adsorbed fraction.

Next, the first antibody dissolved in 0.1 M-NaHCOs was added to 100 polystyrene spheres (diameter 6.4 mm) for 2 hours.
0 nl was added, and the mixture was left at 4°C for 16 hours and then washed to obtain antibody-bound polystyrene spheres.

(b) Preparation of peroxidase-labeled anti-mouse IgG 5 mg of peroxidase (manufactured by Toyobo) and 0.08 M N
0.2 mJl of aIO4 was added, reacted for 20 minutes at room temperature, and dialyzed against sodium acetate buffer (pH 4) overnight. After dialysis, the pH was adjusted to 9.5, 5 mg of anti-mouse IgG (immunized to rabbits, manufactured by DAKO) was added, the reaction was allowed to proceed at room temperature for 2 hours, sodium borohydride was added to reduce the reaction solution, and the entire reaction solution was transferred to Sephadex. Gel filtration was performed using G-200, and active fractions were collected to obtain peroxidase-labeled anti-mouse IgG.

(c) Measurement of EPO As the second antibody, a monoclonal anti-EPO antibody (immunized to mice, see Japanese Patent Application Laid-open No. 155395/1983) was used.

Add one antibody-conjugated polystyrene sphere prepared in (a) and 50 μm of sample containing EPO to a polystyrene tube (12 x 75 mm) and 0 μm of 0.01M phosphate buffer, react at 37°C for 2 hours, and add saline. Washed. Next, monoclonal anti-EPO antibody (1,000-fold dilution) was added to
50 μm was added, reacted at 37° C. for 2 hours, and washed with saline.

On the other hand, peroxidase-labeled anti-mouse IgG obtained in (b)
was diluted 700 times with 0.5% bovine serum albumin superphosphate buffer and added at 250 μm, reacted at 37° C. for 2 hours, and washed with saline. Next, 0-phenylenediamine 3
A citrate buffer containing mg/mJ2 and 0.02% H2O2 was added at 500 .mu.C, and the mixture was reacted for 1 hour at room temperature. Further, 2 fflft of 1N-H2SO4 was added to stop the reaction, and the absorbance of the reaction solution at 492 nm was measured.

FIG. 1 shows the standard curve of EPO obtained.

The detection sensitivity of EPO determined from Figure 1 is 10m1I/
mj! It was.

Example 2 (a) Preparation of antibody-binding support 100 polystyrene spheres (diameter 6.4 mm) were
．． Monoclonal anti-E dissolved in 1 M NaHCO,
20 nl of PO antibody (immunized to mice, see JP-A-59-155395) was added, left at 4°C for 16 hours, and then washed to obtain antibody-bound polystyrene spheres.

(b) Preparation of peroxidase-labeled anti-rabbit IgG Example 1. (b) Te anti-mouse IgG (manufactured by DAKO)
Instead of anti-rabbit IgG (goat immunization, Cappej
Example 1. Prepared in the same manner as (b).

(c) Measurement of EPO As the second antibody, Example 1. The rabbit-derived antibody obtained in (a) was used.

In a polystyrene tube (12 x 75 mm), sample 5 containing one antibody-conjugated polystyrene sphere obtained in (a) and EPO
0μm, 0. 3 by adding 0 μm OIM phosphate buffer.
The mixture was reacted at 7°C for 2 hours and washed with saline. Next, the second antibody was diluted 500 times with 0.01M phosphate buffer, and
0μf1. The mixture was added, reacted at 37°C for 2 hours, and washed with saline. Furthermore, 250 μL of the peroxidase-labeled anti-mouse IgG obtained in (b) was diluted 500 times with 0.5% bovine serum albumin superphosphate buffer, added, reacted at 37° C. for 2 hours, and washed with saline. Next, a citric acid buffer containing 3 mg/ifL of O-phnylene diamine and 2% of H2020 was added at 500 μC, and the mixture was reacted at room temperature for 1 hour. Next, 2 mft of 1N-H2SO4 was added to stop the reaction, and the absorbance of the reaction solution at 492rv was measured. This method allowed detection of E P O25 mU/ff1f.

Example 3 (a) Preparation of glucose oxidase-labeled anti-mouse IgG Add N-(
4-carboxycyclohexylmethyl)maleimide N
- Add 2 mg of hydroxy succinimidyl ester in several portions every 5 minutes, and transfer the reaction solution to Sephadex G-25.
The enzyme fractions were collected and concentrated. An IgG fraction obtained by reducing anti-mouse IgG (immunized to rabbits, manufactured by DAKO) was added in an amount equivalent to 10 mg, reacted overnight at 4°C, gel-filtered with Sephadex G-200, and the active fraction were collected to obtain glucose oxidase-labeled anti-mouse IgG.

(b) Measurement of EPO Example 1
．． One antibody-bound polystyrene sphere obtained in (a).

Add 50 μJZ of a sample containing EPO, 0 μm of 0.01M phosphate buffer, react at 37°C for 2 hours, and wash with saline. Next, monoclonal anti-EP was used as the second antibody.
O antibody (see JP-A-59-155395), 0.01M mal! I solution Nitet, ooo diluted twice and added to 250 μm, reacted at 37°C for 2 hours, and washed with saline. Next, the glucose oxidase-labeled anti-mouse IgG obtained in (a) was added to 0.5% bovine. Serum albumin was diluted 400 times with superphosphate buffer and 250 μl was added.
The mixture was reacted at 7°C for 2 hours and washed with saline. Next, 0.
1% p-hydroxyphenylacetic acid, 0.002% peroxidase.

Add 250μ℃ of substrate solution containing 2% glucose and incubate for 30t.
The reaction was stopped for 1 hour with a glycine-N a OH111 solution, and the excitation wavelength was 320 nm and the fluorescence wavelength was 45 nm.
Fluorescence intensity was measured at 0 nm. In this method, EPO
It was possible to detect 120 mu/mIL.

Example 4 (a) Glucose-6-phosphate dehydrogenase (G6P
DH) Preparation of labeled anti-mouse IgG 2IIIg of N-hydroxysucciimidyl ester of N-(4-carboxycyclohexylmethyl)maleimide was added to 5mg of G6PDH (manufactured by Toyobo) and reacted at 30°C for 1 hour. The reaction solution was subjected to gel filtration using Sephadex G-25, and the enzyme fractions were collected and concentrated. To this, reduced IgG obtained by reducing anti-mouse IgG (immunized to rabbits, manufactured by DAKO)
Add 5mg of Ultrogel A and let it react overnight at 4°C.
Gel filtrate with cA44, collect active fractions and collect G6PDH
Anti-mouse IgG was obtained.

(b) Measurement of EPO Add 5ot1x of the N1 antibody obtained in Example 1(a) to each well of a polystyrene microplate (Costar).
1 was added, reacted overnight at 4°C, and washed. Next, 50 μm of a sample containing EPO was added to each well, reacted at 37° C. for 2 hours, and washed.

Furthermore, using a monoclonal anti-EPO antibody (immunized to mice, see JP-A-59-155395) as a second antibody, diluted 1,000 times with O,OIM phosphate equilibration buffer, added to 50 μm, and incubated at 37°C. It was allowed to react for 2 hours and then washed. Next, add 06PDH-labeled anti-Max IgG obtained in (a) to
．． The mixture was diluted 750 times with a phosphate buffer containing 5% bovine serum albumin (50uf), added, reacted at 37°C for 2 hours, and washed. Then 30mM glucose-6-phosphate and 2
mM nicotinamide adenine dinucleotide (NAD)
Add 200 μC of substrate solution consisting of
After reacting at ℃ for 30 minutes, the reaction solution is added to a substrate for luminescence analysis consisting of luciferase (manufactured by Toyobo), NADH-FMN oxidoreductase (manufactured by Toyobo), flavin mononucleotide (FMN), and decanal to generate. Luminescence meter T D-4 from the amount of light emitted
Reduced NAD (NA
DH) was measured. As a result, EPO measurement of 5 mlI/nu was possible.

[Effects of the invention] When measuring the amount of EPO, the antibody titer is generally low;
Although the conventional method described above did not necessarily provide a satisfactory measurement sensitivity, the present invention makes it possible to measure EPO with high sensitivity by amplifying the amount of binding with a third antibody. That is, according to the present invention, even when the titer of the second antibody against EPO is low, the normal blood concentration is 10 to 20 mu/
It has become possible to measure EPO, which is an extremely small amount of mJZ (0.1 to 0.3 ng/raft), with high sensitivity.

[Brief explanation of drawings]

FIG. 1 is an EPO standard curve in the present invention.

Claims (1)

[Claims] A test solution containing erythropoietin is placed on an antibody-bound insoluble support in which a first antibody that specifically reacts with erythropoietin is bound to an insoluble support, and an animal species that specifically reacts with erythropoietin and is different from the first antibody. after reacting with a second antibody obtained by immunizing with a third antibody that specifically reacts with the immunoglobulin of the animal species in which the second antibody was produced, reacting a labeled antibody with a labeled substance bound to a third antibody, The amount of erythropoietin in the test liquid is measured by measuring the amount of the labeled antibody bound to the antibody-binding insoluble support or by measuring the amount of labeled antibody that is not bound. An immunoassay of erythropoietin.