CN1107870C - Method for in vitro determination of erythropoietin bioactivity - Google Patents

Abstract

The present invention relates to a method for the in vitro assay of the in vivo EPO activity of EPO containing samples, more specifically, the method comprises processing the EPO containing samples and measuring the in vitro EPO activity of the processed samples under the conditions of removing desialylated EPO. In a preferred embodiment, the desialylated EPO is removed from the samples by incubating the samples with cells of the human hepatoma cell system HepG2, the processed samples are incubated with the cells of an EPO reactivity cell system, and then the proliferation or the activity of the EPO reactivity cells is measured so as to measure the in vitro EPO activity. For example, the present invention is useful to measure the bioactivity EPO in various quantitative samples.

Description

The method and the kit thereof that are used for the external test erythropoietin bioactivity

Technical field

Erythropoietin(EPO) (EPO) is a kind of glycoprotein hormones that stimulates mammalian erythropoietin class precursor maturation and therefore regulate the red blood cell generation.The purifying of people EPO (hEPO) and the clone of this EPO gene cause commercially producing of recombinant human epo, and it successfully is used for having because anaemia patient's treatment due to the kidney failure, and also is useful to treating other anaemia clinically.At present quantitative sample, for example the method clumsiness of biologically active EPO, costliness and consuming time in the high amount of drug preparation.The invention provides and measure biologically active safety in the EPO body, quick and relatively inexpensive method and be used to measure the bioactive kit of EPO.

Background technology

EPO is a kind of red blood cell class precursor propagation, differentiation and ripe glycoprotein hormones to ripe red blood cell of stimulating.EPO is purified (Miyake etc., (1977) Journal of biological chemistry 252:5558) and by molecular cloning (Lin etc., (1985) Institute of U.S. natural science institute newspaper 82: 7580), and reorganization hEPO successfully is used for the treatment by anaemia due to the end-stage renal disease.With the treatment of AIDS, rheumatoid arthritis, leukemia and precocious relevant anaemia in and in order to collect the clinical use of also existing report EPO in the autohemic output before increasing art.

Reorganization hEPO has the molecular weight of 30.4KD, and it 40% is a carbohydrates.The most of oligonucleotide chain that studies show that hEPO to EPO glycosylation character and function is four days line style oligosaccharides that contain fucose, sialylated (sialylated).The glycosylation similar of EPO and the recombinant epo that in Chinese hamster ovary (CHO) cell, young hamster kidney (BHK) cell and people B-lymphoblast, prepares in the people urine but inequality.

As if glycosylation play a role in the internal metabolism of EPO dissolubility, biosynthesizing and secretion and EPO.Metabolism and particularly sialic existence in vivo studied to glycosylation.Confirmed that sialic acid EPO is taken off in urine and reorganization because the quick removing of liver and loss of activity in vivo.Covered the galactose residue of penultimate and therefore protected EPO to avoid the removing of liver asialoglycoprotein (galactosyl) acceptor from these and the similar sialic acid that studies show that.Therefore, terminal the losing of sialic acid residues of EPO oligosaccharides exposes galactose residue, causes taking off sialic acid (desialylated) (being also referred to as asialoglycoprotein (asialylated)) EPO and removes fast from blood plasma owing to be bonded to liver receptor.

Essentially no in vivo biologically active can be kept its biologically active in the external test of having set up because it is eliminated fast though take off sialic acid EPO.Be used for the bioactive standard test method of external test EPO and comprise that measuring phenylhydrazine handles anemia mice spleen hemocytoblast (Krystal (1983) Experimental hematology. 20: 649) or people's multipotency leukaemia system (Lewis etc., (1989) Experimental hematology. 17: 102) to containing mixing, measuring of tritium thymidine ⁵⁹Fe to cultivate the mixing of bone marrow cell (Goldwasser etc. (1975) Endocrinology 97: 315) and the growth (Kitamura etc. (1989) that measure the reactive clone of EPO- Blood 73: 375).

Because external test can not be distinguished sialylated and asialylated EPO, be otiose so be determined at quantitative expection and have in vivo in the quantity of active EPO.For example, if contain that the sample of EPO also contains significant quantity take off sialic acid EPO, external test will provide the too high estimation of this activity in vivo so.Therefore, measure the activity in vivo that is used to measure EPO in the body.Specifically, by inciting somebody to action ⁵⁹Fe is incorporated into polycythemia mouse (Cotes etc. (1961) Nature 191: 1065) or hungry rat (Gold wasser etc. (1975) Zymetology Method 37: measure the EPO activity in the hemocytoblast 109).

Current used be in the mensuration of method modification such as Cotes, female mice was exposed to low pressure following 14 days.The endogenous red blood cell forms and is subjected to through being exposed to the inhibition of the decompression polycythemia that produces.Because the polycythemia state continues to keep behind the hypoxemia, any new red blood cell forms can return just using in exogenous EPO.Then with specimen and the hypodermic injection of EPO standard items to the condition mouse.The EPO injection was measured after 48 hours ⁵⁹Fe.Blood sampling after 48 hours, and quantitative radioactive activity.The amount of radioactivity is directly proportional with the standard EPO dosage of injection; Calculate the activity in vivo of unknown sample from typical curve.

Biologicall test is extensively regarded as the unique real mensuration of biologic activity in the body in this body, because it not only measures the cycle life of EPO but also measure its proliferation activity.Yet measuring in this body has obvious defects because its effort, costliness, consuming time and be easy to animal to animal and make a variation.

The invention provides the method for the external quantitative EPO activity in vivo that overcomes background technology method defective.

Summary of the invention

The present invention relates to be used for the method that external test contains the interior EPO activity of body of EPO sample.More especially, present invention resides in to remove and handle the sample that contains EPO and external test under the condition take off sialic acid EPO this obtains handling the EPO activity of sample.In preferred embodiments, remove from this sample by sample and human hepatoma cell line HepG2's cell is hatched and to take off sialic acid EPO, and hatch and measure the propagation of this EPO-reactivity cell or survival and the activity of external test EPO by the cell of the sample that will handle and EPO-reactivity clone.

Another aspect of the present invention provides the kit that comprises 2 containers, wherein contains the cell of expressing the asialoglycoprotein acceptor in first container, and second container contains the reactive cell of EPO-.In another embodiment, this kit further comprises the 3rd container, and it contains vigor and shows dyestuff, as 3-[4, and 5-dimethylthiazole-2-yl]-2,5-diphenyl bromination tetrazolium (MTT).

The present invention relates to be used for the method that external test contains the interior EPO activity of body of EPO sample.More particularly, this method is included in to remove and handles the sample that contains EPO and external test under the condition take off sialic acid EPO this obtains handling the EPO activity of sample.The activity in vivo that the present invention desires to be used for clinical application EPO preparation in mensuration is particularly useful.

External EPO activity is defined as in the stimulated in vitro expression function EPO-recipient cell, for example the ability of red blood cell class precursor propagation or survival.As described below, sialylated and take off sialic acid EPO and activity all arranged external.The EPO activity is defined as the ability that stimulates red blood cell class precursor propagation in vivo in the body.Here, because hepatic clearance fast, so think and take off the essentially no activity in vivo of sialic acid EPO.

According to the present invention, the sample that contains EPO can be graininess or liquid, and preferably serum or blood plasma, cell culture medium, purifying or partially purified recombinant epo, or desires to be used for the EPO preparation of clinical application, stability or preparation research.

According to the present invention, take off in removal and handle the sample that contains EPO under the condition of sialic acid EPO.EPO takes off the exposure that sialic acid causes EPO oligosaccharides time terminal galactose residues.Therefore, by with this sample agglutinin that galactose is had compatibility, for example AbrinA carries out affinity chromatography or by removing taking off sialic acid EPO with the affinity chromatography to the specific sessile antibody of galactose from the sample that contains EPO.The method that is used to carry out lectin affinity chromatography is known to those skilled in the art.

In another embodiment, take off sialic acid EPO by hatching from the sample that contains EPO, to remove with expression asialoglycoprotein recipient cell.This cell can natural expression asialoglycoprotein acceptor, maybe can recombinate to be processed into and express this acceptor.For example, express the acceptor that function is arranged with the fibroblast of the cDNA transfection of coding asialoglycoprotein acceptor HL-1 and HL-2 subunit.Can be according to (1989) methods such as Shia U.S.'s natural science Institute of institute newspaper 86: 1158 obtain to express the transfection fibroblast of this reorganization asialoglycoprotein acceptor.

In preferred embodiments, by with known have the human hepatoma cell line HepG2's (ATCC No.HB 8065) of function asialoglycoprotein acceptor cell to hatch from the sample that contains EPO to remove with the natural expression of high concentration take off sialic acid EPO and (see, for example, Lodish, (1991) Biochemical scientific advance 16: 374).HepG2 clone is by (1980) such as Knowles Science 209: 497 and United States Patent (USP) 4,393,133 such as Knowles described in.

The appropraite condition that is used to hatch the sample that contains EPO and the cell of expressing the asialoglycoprotein acceptor can be measured and can be changed according to cell type and sample source by those skilled in the art.In one embodiment, with cellular incubation to inferior-converge, washing also joins cell suspension in tissue culturing plate's aperture.Each hole of containing attached cell is washed several times with damping fluid, removes damping fluid, and EPO standard items or specimen are joined in each aperture.With this cell with contain the sample of EPO in 37 ℃ of 5%CO ₂With overnight incubation (about 15-20 hour) in the wet environment of 95% air.Be transferred to the analysis that is used for the EPO proliferation activity in the container subsequently from being called " sample of handling " according to the present invention and taking off the supernatant that sialic acid EPO is adsorbed to mixtures incubated wherein.

In order to confirm that taking off sialic acid EPO is removed by affinity chromatography or with expressing hatching of asialoglycoprotein recipient cell, can be applied to EPO the sialic acid condition of taking off.Being used for asialylated chemistry and Enzymology method is known to those skilled in the art.Briefly, EPO can be by 80 ℃ of heating in 0.1mol/L HCl 60 minutes and sloughed sialic acid by chemistry.Taking off sialic acid also can be finished by EPO is hatched with fixing neuraminidase.Be used for EPO and take off sialic actual conditions by Spivak etc., (1989) Blood 73: 90 descriptions.For example, by isoelectric focusing the analysis that EPO takes off the sialic acid front and back be can be used for measuring whether sialic acid is removed.

Containing by above-mentioned processing after the EPO sample takes off sialic EPO with absorption, analyze the EPO proliferation activity that this handled sample.As above-mentioned, term " sample of handling " refers to processedly take off the sample of sialic acid EPO with removal, for example, and by lectin affinity chromatography or by having the cell of function asialoglycoprotein acceptor to hatch with expression.For example, the sample of this processing can be a cell culture supernatant.

For example, can be by measuring the reactive cell of EPO-pair and handling the variation that cell number after the sample incubation reaction or DNA synthesize and measure proliferation activity.According to the present invention, the reactive cell of EPO is defined as breeding or keep the cell of survival after the EPO reaction.The reactive cell of EPO that can be used for measuring EPO in-vitro multiplication activity comprises from the establishment clone of the fresh red blood cell class precursor that shifts out of blood forming organ, natural expression EPO acceptor and is processed to express the establishment clone of EPO reporter gene.The clone of natural expression EPO acceptor is generally from the malignant cell with tumour animal.For example, the muroid erythron of setting up from the erythroleukemia mouse that infects with Friend leukemia virus compound or Rauscher leukemia virus compound (erythroid cell line).From leukemia patient, set up the human cell line.Other clone has been processed into a large amount of EPO acceptors of generation and has needed EPO to be used for survival by importing and express the EPO acceptor gene.The reactive cell system of EPO is known and by (1992) such as Koury to those skilled in the art The biochemical magazine in Europe 210: 649 summarized.In preferred embodiments, the reactive cell of EPO is the cell of B6SUtA for the multipotency hemopoietic forebody cell of reporting 80:2931 to describe by institutes of (1983) U.S. natural science institute such as Greenberger.B6SUtA clone is from the long-term marrow culture of B6.S mouse.

The propagation of EPO reactivity cell can synthesize by the DNA that quantitatively EPO reaction back is increased and be measured.The DNA that increases is synthetic generally to be incorporated in external mensuration by measuring tritium-labeled thymidine.The suitable this area that is determined at is known and by, Krystal etc. (1983) for example Experimental hematology. 11: 649 and Lewis etc. (1989) Experimental hematology. 17: 102 described.In brief, with the reactive cell product of EPO at 5% CO ₂With generally incubated 22 hours with the specimen that contains EPO in the microlitre plate well in 37 ℃ in the wet environment of 95% air.Add the final concentration of tritium-labeled thymidine to about 0.1 μ Ci/ hole.After hatching 2 hours once more, the aperture content is collected on the glass fiber filter and measures the radioactivity that reclaims by scintillation spectrum counting device.For standardization, be incorporated among the DNA radioactive amount EPO activity (mU/ml) is mapped.

Avoiding using in radioactive preferred embodiment, the propagation by using dyestuff MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl bromination tetrazolium) the reactive cell of spectrophotometry EPO (Mossman, 1983, Immunological method 65, 55).MTT is light yellow in solution, and becomes the water-insoluble MTT-first of black-and-blue (dark blue)/purple derivant by the mitochondrial dehydrogenase of living cells.With blue this crystal of acid isopropyl alcohol dissolving and in 570 and 690nm place colorimetric estimation intensity.Therefore, the amount that converts the MTT of painted formazin to is the direct measurement of the cell number of on cell proliferation indicator.Unknown optical density to standard relatively is used for the calculation biology activity.In preferred embodiments, the reactive cell of sample that this was handled and EPO in the titer plate aperture in moist 5% CO ₂With hatched about 46-50 hour in 37 ℃ in the environment of 95% air.MTT is joined in each aperture and then and hatched 2 hours in 37 ℃.After blue colored crystal dissolves, measure optical density also by comparing by the cell number of unknown sample generation and the activity of EPO standard curve determination EPO.MTT commercial be available, it is the kit (MTT kit, Promega company) that contains MTT dyestuff and solvent soln.Except MTT, other indicator dye such as XTT (3,3 '-(1-((phenylamino) carbonyl)-3, the 4-tetrazolium)-two (4-methoxyl-6-nitro) benzene sulfonic acid, sodium salt) and MTS (3-(4,5-dimethylthiazole-2-yl)-and 5-(3-carboxylic anisyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) can be used for identical purpose.

In the preferred embodiment of the inventive method, to take off sialic acid EPO from then on remove in the sample by hatching this sample and human hepatoma cell line HepG2, and external EPO activity is by hatching the sample of this processing, promptly measured from the cell of the supernatant of HepG2 cell and the reactive clone of EPO and propagation or the survival of measuring the reactive cell of this EPO-.In the embodiment of giving an example in highly preferred the following examples 1, the reactive cell of EPO-is that B6SUtA and cell proliferation or survival are measured with spectrophotometric method through using MTT.

Found that according to the present invention HepG2 cell asialoglycoprotein acceptor takes off the function that sialic acid EPO removes fast in the parody effectively to the external compatibility of taking off sialic acid EPO-.In addition, the cell culture medium of having found to contain EPO can be induced the propagation of the reactive cell of EPO.

Another aspect of the present invention is provided for measuring the kit that contains EPO activity in the EPO sample body.In one embodiment, kit is cut apart with admittance and contained first container of expressing the asialoglycoprotein recipient cell and second container that contains the reactive cell of EPO-.In preferred embodiments, the cell of expression asialoglycoprotein acceptor is that the reactive cell of HepG2 cell and EPO-is the B6SUtA cell.In another embodiment, this kit further comprises the 3rd container that contains MTT.

Embodiment

The following examples further illustrate the present invention.The present invention should not think and limited by these embodiment, but limited by appended claims.

Embodiment 1

The HepG2 cellular incubation is paved with until reaching in the 100mm tissue culture plate.Thereby this moment with phosphate buffered saline (PBS) wash cell and with trypsin treatment with its from flat board separate and the preparation cell culture medium 0.25 * 10 ⁶The suspension of individual cell/ml.This suspension of 1ml is joined in each aperture of 24-hole tissue culture plate.The HepG2 cell can be ready for use on EPO mensuration after three days.

Each aperture that contains the HepG2 cell is washed 5 times with phosphate buffered saline (PBS) (PBS).After removing PBS fully, (the DulbeccoShi MEM that contains 10% hyclone and glutamine) joins in each aperture with 100 μ l nutrient culture media.By the EPO reference standard of employing 10U/ml and by the every kind of solution of 100 μ l that is mixed to join in each aperture this dilution is begun the EPO typical curve for 2 times.This cell is added that EPO solution is in 5%CO ₂The high humility incubator in 37 ℃ of overnight incubation.After hatching, by 5 times of the 5U/ml standards handled of dilution HepG2-in nutrient culture media to reach 1U/ml solution the preparation standard curve.Each sample of handling is diluted 10 times to reach the solution that is estimated as 0.5U/ml in nutrient culture media.Then this standard and sample are ready for use on proliferation assay.Greatly when this standard and sample joined in the HepG2 cell, preparation B6SUtA cell was used for second day proliferation assay that will begin.With these cellular incubation in nutrient culture media (by above-mentioned) and replenish IL-3 with 20ng/ml; Go down to posterity weekly 2 times with 1: 20 dilution.Wash with nutrient culture media and once to prepare this cell and to return to original volume again with the nutrient culture media of no IL-3.With this cell in the 100mm flat board (37 ℃, 5% CO ₂) overnight incubation.After hatching, cell washed in nutrient culture media once more and with 1 * 10 ⁶The final concentration of individual cell/ml is resuspended.With 96-hole tissue culture plate, with 50 μ l B6SUtA cells and every kind of EPO standard of 50 μ l and sample mix.With flat board at 5%CO ₂Hatched 48 hours in 37 ℃ in the incubator.

Propagation or survival with the MTT kit measurement cell of Promega company.The amount of MTT that is transformed into painted first is directly related with the number of cell.This method is summarized as follows.20 μ lMTT dyestuffs (Promega company) are added in each aperture and in 37 ℃ hatched 2 hours.Then 100 μ l dissolution solvents (Promega company) are added to every hole and in incubated at room to all blue-blacks/the violet precipitate dissolving, 1-4 hour usually.In plate reader, this flat board is carried out reading then in O.D.570nm and 690nm place.Measure the EPO activity by the cell number and the EPO typical curve that relatively produce by unknown sample.By with activity calculated divided by 0.5U/ml and multiply by 100% and calculate and to tire.

Table 1 has been listed representational optical density of measuring under these conditions and the EPO standard value that calculates in the 0-1000mU/ml concentration range.As can be seen from the value that calculates, the inventive method provides to be measured accurately to the EPO standard.

When not having HepG2 cell preincubate, measuring the proliferation activity of identical standard, obtain similar result, as listed in Table 2.These results show that the processing of HepG2 cell does not influence the EPO typical curve significantly.

By this method and also not with HepG2 cell preincubate the time, measure and have the sample that known and variable quantity takes off sialic acid EPO.As can from seen in the table 3 to, contain sample that significant quantity takes off sialic acid EPO with HepG2 cell (R ₂) hatch to hatch and adsorb with the HepG2 cell before and take off sialic acid EPO (R ₁) induce bigger breeder reaction afterwards.Table 3 also provides the amount and the value from calculating according to the opacimeter of the inventive method acquisition of the expection of sialylated (complete) EPO.As can from table 3, seeing, be pin-point accuracy in the amount of this method sialylated (being activated in the body) EPO in quantitative sample.

This paper has described the present invention with reference to some embodiment preferred and embodiment.Because obvious variation is conspicuous for those skilled in the art, not limited by it so the present invention should not think, and only should be limited by following claims.

Table 1

EPO activity with standard behind the HEPG2 cell preincubate

The value that standard standard value OD mean value standard deviation CV calculates

STD00 1000 mU/ml. 1.304 1.336 0.029 2.151 942.9

1.359 1080

1.346 1044

STD01 900.0 mU/ml. 1.244 1.271 0.057 4.494 824.6

1.233 805.6

1.337 - 1021

STD02 800.0 mU/ml. 1.223 1.240 0.037 2.951 788.9

1.215 775.9

1.282 896.4

STD03 700.0 mU/ml. 1.133 1.161 0.040 3.410 659.9

1.143 672.6

1.206 761.7

STD04 600.0 mU/ml. 1.114 1.109 0.019 1.713 636.6

1.088 606.6

1.125 649.9

STD05 500.0 mU/ml. 0.961 0.965 0.034 3.488 483.5

0.933 460.5

1.000 517.8

STD06 400.0 mU/ml. 0.827 0.846 0.025 2.950 383.0

0.874 415.6

0.836 389.0

STD07 300.0 mU/ml. 0.691 0.689 0.017 2.414 300.1

0.704 307.4

0.671 289.0

STD08 200.0 mU/ml. 0.477 0.496 0.016 3.261 192.6

0.505 205.6

0.505 205.6

STD09 100.0 mU/ml. 0.269 0.270 0.002 0.565 99.79

0.270 100.2

0.272 101.1

STD10 0.000 mU/ml. 0.093 0.096 0.006 6.720 <<<<<

0.091 <<<<<

0.103 10.30

Table 2

Not with HEPG2 cell preincubate after the EPO activity of standard

The value that standard standard value OD mean value standard deviation CV calculates

STD00 1000 mU/ml. 1.439 1.517 0.069 4.566 923.1

1.539 1141

1.572 1236

STD01 900.0 mU/ml. 1.362 1.425 0.074 5.227 800.7

1.405 865.4

1.507 1062

STD02 800.0 mU/ml. 1.264 1.364 0.097 7.122 679.2

1.371 813.5

1.458 958.4

STD03 700.0 mU/ml. 1.251 1.278 0.054 4.240 665.2

1.242 655.7

1.340 770.6

STD04 600.0 mU/ml. 1.177 1.185 0.007 0.599 592.8

1.186 601.0

1.191 605.6

STD05 500.0 mU/ml. 1.071 1.083 0.011 1.028 505.9

1.085 516.4

1.093 522.6

STD06 400.0 mU/ml. 0.917 0.902 0.019 2.061 403.7

0.907 397.8

0.881 382.8

STD07 300.0 mU/ml. 0.724 0.744 0.035 4.732 300.8

0.785 331.1

0.724 300.8

STD08 200.0 mU/ml. 0.435 0.455 0.017 3.813 173.6

0.464 185.7

0.466 186.5

STD09 100.0 mU/ml. 0.271 0.281 0.009 3.225 104.6

0.288 112.0

0.285 110.7

STD10 0.000 mU/ml. 0.096 0.097 0.003 2.728 <<<<<

0.095 <<<<<

0.100 4.484

Table 3

Complete (sialylated) EPO's is quantitative

% takes off saliva R1 w/ R2 w/o/ R2/R1 K=0.15 K=0.15 and expects complete %

Liquid acid HepG-2 HepG-2 ratiometer is calculated calculate the EPO accuracy

The complete EPO% mU/ml of the complete EPO of EPO mU/ml mU/ml

mU/ml

0.00 289.10 303.60 1.05 286.54 1.00 300.00 0.96

10.00 268.60 352.80 1.31 253.74 0.89 270.00 0.94

20.00 261.70 424.20 1.62 233.02 0.81 240.00 0.97

30.00 254.70 494.20 1.94 212.44 0.74 210.00 1.01

40.00 217.60 555.40 2.55 157.99 0.55 180.00 0.88

50.00 214.60 628.30 2.93 141.59 0.49 150.00 0.94

60.00 213.40 693.60 3.25 128.66 0.45 120.00 1.07

70.00 192.30 755.50 3.93 92.91 0.32 90.00 1.03

80.00 196.10 959.90 4.89 61.31 0.21 60.00 1.02

90.00 178.60 999.20 5.59 33.79 0.12 30.00 1.13

100.0 150.80 1029.00 6.82 -4.18 -0.01 0.00 #DIV/

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Claims (19)

1, is used for external test and contains the active method of erythropoietin(EPO) (EPO) in the EPO sample body, be included in to remove and handle this sample under the conditions in vitro of taking off sialic acid EPO, and measure the external EPO activity that this handled sample.

2, the process of claim 1 wherein that the conditions in vitro that sialic acid EPO is taken off in described removal comprises lectin affinity chromatography.

3, the process of claim 1 wherein that conditions in vitro that sialic acid EPO is taken off in described removal comprises with the cell of expressing the asialoglycoprotein acceptor hatches.

4, the method for claim 3, the natural or recombinant expressed described asialoglycoprotein acceptor of wherein said cell.

5, the method for claim 3, wherein said cell are the HepG2 cell.

6, the process of claim 1 wherein that described external EPO activity measures by processing sample of hatching the reactive cell of the described EPO of having and propagation or the survival of measuring the reactive cell of described EPO.

7, the method for claim 6, the reactive cell of wherein said EPO-is the B6SUtA cell.

8, the method for claim 6, the propagation of the reactive cell of wherein said EPO is measured by measuring the synthetic increase of DNA.

9, the method for claim 8, the synthetic increase of wherein said DNA is measured by measuring mixing of tritiate thymidine.

10, the method for claim 6, the propagation of the reactive cell of wherein said EPO is measured by spectrophotometric method.

11, the method for claim 6, the propagation of the reactive cell of wherein said EPO is passed through described cell and 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl bromination tetrazolium (be called for short MTT) is hatched and is measured MTT and measured to the conversion of MTT first derivant.

12, be used for the method that external test contains the interior erythropoietin(EPO) EPO activity of body of EPO sample, comprise: (a) described sample and HepG2 cell are hatched; (b) from described HepG2 cell, remove supernatant; (c) described supernatant and B6SUtA cell are hatched; (d) propagation of the described B6SUtA cell of mensuration; (e) calculate the EPO activity.

13, the method for claim 12, the wherein said sample of EPO that contains is at 5% CO ₂With hatched about 15 to 20 hours with described HepG2 cell in 37 ℃ in the humidification atmosphere of 95% air.

14, the method for claim 12, wherein said supernatant was hatched about 46 to 50 hours with described B6SUtA cell in 37 ℃.

15, the method for claim 12, the propagation of wherein said B6SUtA cell is passed through described cell and 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl bromination tetrazolium (be called for short MTT) is hatched and is measured MTT and measured to the conversion of MTT-first derivant.

16, the method for claim 12, wherein said EPO activity is by relatively calculating by containing EPO sample propagation that is produced and the propagation that is produced by the EPO standard.

17, a kind of kit of subregionization comprises second container that contains first container of expressing the asialoglycoprotein recipient cell and contain the reactive cell of EPO-.

18, the subregion kit of claim 17, the cell of wherein said expression asialoglycoprotein acceptor is the HepG2 cell.

19, the subregion kit of claim 17, the reactive cell of wherein said EPO is the B6SUtA cell.