JP2750600B2 - Method for measuring biological activity of erythropoietin - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for measuring the biological activity of erythropoietin (EPO).

Conventional technology EPO is a hematopoietic hormone that acts on immature erythroid progenitor cells and induces differentiation and proliferation.
It is a glycoprotein consisting of 166. EPO plays a very important role in maintaining a constant red blood cell count. That is,
EPO is usually produced mainly by the kidneys in response to tissue oxygen demand, and its production is promoted in hypoxia and conversely low in hyperoxia.

Measuring blood EPO can provide a great deal of useful information clinically. For example, polycythemia vera and 2
Secondary polycythemia presents clinically similar symptoms, but has completely different causes and different treatments. In these diseases, the former shows very low levels of blood EPO, while the latter shows high levels of EPO, and thus
By measuring the amount, both can be easily diagnosed and appropriate treatment can be performed. In addition, in the case of administering EPO to an anemia patient and performing treatment, tracking the amount of EPO in the blood of the patient is very important for proper administration of EPO.

Conventionally, immunological techniques have been used to measure EPO. U.S. Pat. No. 4,590,168 discloses a radioimmunoassay method in which an antibody against 26 peptide sequences from the N-terminal of the EPO amino acid sequence is obtained and used. In addition, the present inventors have obtained a monoclonal antibody against EPO, completed a sandwich immunoassay method using the same, and filed a patent application (JP-A-62-235564,
No. 62-235566). However, in the immunological measurement method, since an antibody recognizing the peptide portion of EPO is used, an erroneous measurement in response to EPO having no biological activity such as an EPO fragment, a polymer, and inactivity may be performed. Had the danger of doing so. Furthermore, in the case of radioimmunoassay, the use of radioisotopes makes
The need for a dedicated facility and the problem of waste disposal were not negligible.

On the other hand, as a method for measuring the biological activity of EPO, a method using small animals such as a mouse and a rat (Nozomi Kosakai et al., Clinical Laboratory Techniques, Vol. 3, p. 222, 1972, Medical Shoin) is widely known. The operation is complicated, the measurement sensitivity is poor, and it has not been put to practical use.

In recent years, with the development of cell culture technology, methods using bone marrow cells (E. Goldwasser et al., Methods Enzymolgy 37, 109
Pp. 1975), a method using splenocytes of phenylhydrazine-administered animal (NC Brandan et al., Br. J. Haematol. 47, 461).
P. 1975), a method using mouse fetal hepatocytes (G. Kry
stal, Exp. Haematol. 11, 649, 1983)
Bioassays have been developed to allow for the desired measurement sensitivity.

However, all of these methods require keeping animals in the required state at all times, and are susceptible to contaminants in the sample, and lack reliability in accuracy. . In addition, the operation is complicated, and it is necessary to use radioactive isotopes, which requires specialized facilities,
There were safety and waste disposal issues.

Problems to be Solved by the Invention In order to determine a method for measuring the true biological activity of EPO, it is necessary to develop a system that specifically reacts with EPO. Therefore, the present inventors have conducted intensive studies on the relationship between EPO and cell proliferation, and as a result, have developed a measurement method using a cultured cell line having high reactivity to EPO.

AB Janowska-Wieczorek et al., Human EPO and human G-CSF
(Exp. Haematol. 16, 489, 1988). However, these cells also have high reactivity to G-CSF, and therefore, G-CSF and EPO always coexist, such as blood and urine. .

The present inventors proceeded with the search for cells that are reactive to EPO and do not respond to other cell growth factors.As a result, mouse-derived EP-FDC-P2 cell line is suitable for this purpose, The present inventors have found that growth can be accurately reflected in biological activity, and have accomplished the present invention.

An object of the present invention is to provide a simple and highly accurate method for measuring the biological activity of EPO using an EP-FDC-P2 cell line.

Means for Solving the Problems The feature of the present invention is that by using a culture cell line that survives or grows in dependence on EPO, animal breeding and radioisotope It is where use has become useless. In addition, in the measurement, the cells grown in the microwell plate used for cell growth can be directly measured by coloring the soluble tetrazolium salt (MTT), and the EPO concentration can be easily measured. Is also a feature.

The EPO-dependent cells used in the present invention include mouse-derived EPO.
Although DC-P2 or EP-FDC-P2 cells are suitable for use, in particular EP-FDC-P2 cells only respond to EPO and mouse IL-3 and are not subject to other growth factors or human cell growth factors. It is particularly suitable for the practice of the invention because it does not react. In addition, EP
-FDC-P2 has been deposited with the Research Institute for Microbial Industry as Microorganisms Bacteria No. 10519.

This cell is contained in the culture medium under certain culture conditions
The cell number increases depending on the EPO concentration. By measuring this increase in cell number, the EPO concentration can be measured.
For cell measurement, it is possible to directly observe and count the culture wells with a stereomicroscope, but for easier measurement, add a soluble tetrazolium salt (MTT) to each well after culturing the cells. The color of MTT may be colored by dehydrogenase in the MTT, and the amount of coloring of the dye corresponding to the number of cells may be colorimetrically determined using an absorptiometer. Thus, the EPO-biological activity in the specimen added to the culture solution can be measured.

Generally, measurement can be performed by the following procedure. EP-
FDC-P2 cells were suspended in RPMI1640 medium containing 10% FCS,
Adjust the cell density to 10 ⁴ -5 × 10 ⁴ cells / 100μ, and add 100 cells per well to a 96-well microwell plate.
Seed by μ. At this time, a sample containing EPO to be measured or a standard EPO coexist, and in a CO ₂ incubator
Incubate for 12-24 hours. After cultivation, 5 mg / ml of the coloring dye MTT
10 μl of MTT solution dissolved in PBS to each well
After reacting for 3 to 5 hours, the cells are solubilized. For solubilization, an SDS solution or an isopropanol solution containing hydrochloric acid can be used. After solubilization, 54 of each well
The absorbance at 0 nm to 570 nm is measured with a microwell absorption spectrophotometer, and the absorbance at 620 nm to 630 nm is further measured.
The difference between the two is read and taken as the absorbance. The absorbance increases in proportion to the amount of EPO. EPO-biological activity can be measured by a calibration curve created by standard EPO.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

Example 1 Standard EPO-dependent cell EP-FDC-P2 in synthetic medium RPMI1640 50.mu. including measurement 20% FCS of EPO was 10 ⁴ dispersed, were seeded into each well of a 96-well flat-bottomed microplate. Subsequently, 50 μ of various concentrations (16-8200 mU / ml) of standard EPO was added to the wells,
The cells were cultured in a CO ₂ incubator for 20 hours. 10μ after culture
MTT reagent solution [3- (4,5
-Dimethylthiazol-2-yl) -2,5 diphenyl tetrazol
ium bromide (manufactured by Sigma) dissolved in PBS: prepared at the time of use] was added to each well, and the cells were further cultured in a CO ₂ incubator at 37 ° C for 4 hours. After the culture, 100 μl of isopropanol containing 0.04 M HCl was added to each well to solubilize the cells. After solubilization, using an immunoleader (manufactured by Nippon Intermed Co., Ltd., immunoleader NJ-2000),
Using 620 nm as a control, the absorbance at 540 nm was measured.

The result is shown in FIG. A dose-dependent increase in absorbance was observed for 8-250 mU / ml EPO, and the biological activity of EPO could be measured well. That is, the EPO biological activity in a sample containing EPO of 16 mU / ml or more could be measured.

Example 2 Effect of Human Hematopoietic Factors Other than EPO on the Method of Measuring the Activity: The same method as in Example 1 was used to examine the effect of various human hematopoietic factors on the method of measuring the activity of the present invention.

 The results are shown in Table 1.

This assay does not respond to various human hematopoietic factors,
The biological activity of O could be measured accurately.

Example 3 Measurement of EPO in Serum and Urine As in Example 1, EPO activity was measured in serum of healthy individuals, serum of dialysis patients, serum of iron deficiency anemia patients, and urine of aplastic anemia patients.

For urine, a sample from which low-molecular substances had been removed using Sephadex G-25 in advance was used. For solubilization of cells, 10% SDS solution dissolved in 10 mM NH ₄ Cl was used instead of isopropanol containing 0.04 M HCl.
The solution was kept overnight at room temperature for solubilization.

The results are shown in Table 2. The same result as the conventional ³ H-TdR method was obtained.

Effect of the Invention By the practice of the present invention, without being affected by other coexisting components,
EPO measurement can be performed accurately and easily. In addition, the breeding of animals and the necessity of radioactive isotopes, which have been conventionally required for the measurement of biological activity, are eliminated, so that a very effective measurement can be performed both from a safety viewpoint and from a facility.

[Brief description of the drawings]

The attached figure shows the measurement result (absorbance) of the standard EPO shown in Example 1.

Claims (2)

(57) [Claims] 1. A specimen is mixed with a culture solution in which cells that survive or proliferate only depending on erythropoietin and mouse interleukin III are mixed, and after culturing, a soluble tetrazolium salt is added to cause a reaction, and the cells are further solubilized. ,
A method for measuring the biological activity of erythropoietin, which comprises measuring pigment coloring. 2. The method for measuring the biological activity of erythropoietin according to claim 1, wherein the cells that survive or proliferate only depending on erythropoietin and mouse interleukin III are mouse myeloid cell line EP-EDC-P2.