TW201213807A

TW201213807A - Bioassay for the measurement of CEPO concentration and activity

Info

Publication number: TW201213807A
Application number: TW100122114A
Authority: TW
Inventors: Thomas Nikolaj Sager; Michael Chopp
Original assignee: Lundbeck & Co As H
Priority date: 2010-07-19
Filing date: 2011-06-24
Publication date: 2012-04-01
Also published as: WO2012010176A1; AR082229A1

Abstract

The present invention relates to a bioassay for detecting the effective concentration of carbamylated erythropoietin (CEPO) and estimating the specific activity using neural progenitor cells, as well as the use of these neural progenitor cells for determining the concentration or activity of CEPO in a sample.

Description

201213807 六、發明說明：【發明所屬之技術領域】本發明係關於一種使用神經祖細胞偵測胺甲醯化紅血球生成素之有效濃度且估計比活性之生物分析。【先前技術】幾十年來已知激素紅血球生成素（EP0)能夠刺激骨趙及脾臟中之紅血球系細胞增殖及存活。存在各種形式之試管内及活體内生物分析以測量生物體液以及醫藥製劑中 EPO之含量。EPO之生物活性係藉由使用測量含有Ep〇之樣本刺激紅血球系細胞之能力的活體内生物分析與使用5 微莫耳鈷之刺激相比較來測量，以習知為每毫升毫單位 (mU/mL )之國際單位表示。近來，已知EPO亦具有其他生物功能。已發現Ep〇在例如心臟及腦組織中具有組織保護特性。已研發出保留Ep〇之保護特性但不具有EPO之紅血球生成效應的新穎EPO衍生物。作為一實例’已描述藉由胺甲醯化修飾EPO之離胺酸殘基，可獲得對造血系統不具有影響之EPO衍生物（Leist 等人，Derivatives of Erythropoietin That Are Tissue Protective But Not Erythropoietic. Science. 2004.第 3〇5 卷，第5 68 1期，第23 9-242頁）。此胺曱醯化EPO ( CEPO)仍能介導例如神經組織中之細胞保護，即使CEPO不結合至典型 EPO 受體（EPOR ) ( Leist 等人，DeHwm.va 〇/ Erythropoietin1 That Are Tissue Protective But Not 201213807 五Science· 2004·第 305 卷，第 5681 期，第 23 9-242頁）。更特定言之，近來已顯示CEPO增強來源於成年小鼠腦室下區之神經祖細胞的增殖及其向神經元之分八t ( Wang 等尺，The Sonic Hedgehog Pathway Mediates Carbamylated Erythropoietin-enhanced Proliferation and201213807 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to a biological analysis for detecting the effective concentration of amine methotrexate erythropoietin using neural progenitor cells and estimating specific activity. [Prior Art] It has been known for decades that hormone erythropoietin (EP0) can stimulate the proliferation and survival of red blood cell cells in the bone and spleen. There are various forms of in-tube and in vivo bioanalysis to measure the amount of EPO in biological fluids and pharmaceutical preparations. The biological activity of EPO is measured by in vivo bioassay using the ability to measure red blood cell lines in samples containing Ep〇 compared to stimulation using 5 micromolar cobalt, conventionally known as milliunits per milliliter (mU/ The international unit of mL) is indicated. Recently, EPO is also known to have other biological functions. Ep〇 has been found to have tissue protective properties in, for example, heart and brain tissue. Novel EPO derivatives that retain the protective properties of Ep〇 but do not have the erythrocyte-forming effect of EPO have been developed. As an example, it has been described that an EPO derivative having no effect on the hematopoietic system can be obtained by modifying the lyophilic acid residue of EPO by amine methylation (Leist et al., Derivatives of Erythropoietin That Are Tissue Protective But Not Erythropoietic. Science 2004. Vol. 3, Vol. 5, No. 5, 68, pp. 23-242. This aminated deuterated EPO (CEPO) still mediates, for example, cellular protection in neural tissue, even if CEPO does not bind to a typical EPO receptor (EPOR) (Leist et al, DeHwm.va 〇/ Erythropoietin1 That Are Tissue Protective But Not 201213807 Five Science·2004·Vol. 305, No. 5681, pp. 23-242). More specifically, it has recently been shown that CEPO enhances the proliferation of neural progenitor cells derived from the subventricular zone of adult mice and their differentiation into neurons. The Sonic Hedgehog Pathway Mediates Carbamylated Erythropoietin-enhanced Proliferation and

Differentiation of Adult 】它(2. 200Ί. 2X2, 3>2Α61-37ΛΊ O')。詳言之，本發明之發明者已發現CEPO介導對來源於大鼠SVZ ( subventricular zone，室下區）之募樹突神經膠質細胞的增殖及成熟反應之劑量依賴性作用。本發明描述適於測疋基於券樹突神經膠質細胞之試管内系統中CEp〇之生物活性的有效濃度、條件及可能讀數。因此顯示可使用此研究結果作為測定指定樣本中CEPO之有效濃度及活性的生物分析。 CEPO目前正處於臨床研發中用於中風及弗里德賴希氏共濟失調（Friedreich's ataxia)，然而，目前不存在用於 CEPO之生物分析。因4 CEp〇 #紅血球系細胞無任何作用，所以不能使用EPO之現存生物分析。因為最終產品之生物活性可能由於生產製程中之小變化而改變，所以重要的是對待投入市場之新生物產品就地進行生物分析。本發明揭示一種用於CEP〇之新穎生物分析。【發明内容】本發明係關於一種測定樣本中胺甲醯化紅血球生成素 (CEPO)之濃度或活性之方法，該方法包含以下步驟： 201213807 1)使包含CEPO之樣本與諸如齧齒動物之哺乳動物物種之神經祖細胞接觸， Π)添加基本上不含外加生長因子的分化培養基， 111 )測疋對於寡樹突神經膠質細胞新生之特定標記的表現，及 iv )建立該表現與CEP〇標準或參考者的相關性。本發明亦關於該等神經祖細胞用於測定樣本中之濃度或活性之用途。【實施方式】中樞神經系統（CNS )由許多類型之神經元及膠質細胞構成。匕等細胞係自位於例如室下區之多能共同前驅體產生。作為分化過程之一部分’ SVZ中之袓細胞在一系列漸進式抉擇令受到其分化潛能限制，最終形成單一細胞系 (Levinson 及 Goldman 施"㈣灿α/ ^ ⑹響 restricted precursors coexist in the mammalian perinatal j. Neurosci Res 48 1997 (2) 83_94)。多能祖細胞之分化為一種極複雜的受調節過程，其與發育 -文化對壓力情丨兄（例如損傷）之反應及在營養因子及細胞因子存在下之一般變化有關。因而多能祖細胞可視外界環境而定發育成神經元或神經膠質表型。在袓細胞之漸進式發育中，神經膠質祖細胞可成熟長成星形膠質細胞或寡樹突神經膠質細胞表型。分化成例如寡樹突神經膠質細胞之部分可藉由特性化特定細胞類型之蛋白質之特定標記的 201213807 寡樹突神經膠質細胞為涉及軸突之髓鞘形成的CNS神經膠質細胞之主要種類。成人CNS中之成熟寡樹突神經膠質細胞係源自胼胝體及紋狀體中存在之無髓鞘寡樹突神經膠質細胞袓細胞（OPC )。側腦室之室下區（s vz )中之神經祖細胞亦產生分散遍佈於胼胝體及紋狀體中之〇PC。〇pc 之標記包括神經節苷脂GD3、NG2硫酸軟骨素蛋白聚糖、 04及血小板衍生生長因子α受體次單元PDGFRa。成熟寡樹突神經膠質細胞表現MBP及CNPasee本發明之發明者已發現CEPO以劑量依賴性方式刺激神經袓細胞增殖及分化成寡樹突神經膠質細胞系細胞且促進〇pc成熟成為有髓鞘寡樹突神經膠質細胞’且因此可用於生物分析中以測定樣本（諸如醫藥組成物）之效能。試官内神經祖細胞可自例如哺乳動物之各物種（例如齧齒動物’諸如小鼠、大鼠、松鼠、豪豬、海狸、花栗鼠、天竺鼠及田鼠）之腦室下區收集。特別較佳為小鼠、大鼠或兔子。預計所培養之幹細胞亦可原樣使用。Differentiation of Adult] It (2. 200Ί. 2X2, 3> 2Α61-37ΛΊ O'). In particular, the inventors of the present invention have found that CEPO mediates a dose-dependent effect on proliferation and maturation of dendritic glial cells derived from the SVZ (subventricular zone) of rats. The present invention describes effective concentrations, conditions, and possible readings suitable for measuring the biological activity of CEp(R) in an in vitro system based on v. dendritic glial cells. It is therefore shown that this study can be used as a bioassay for determining the effective concentration and activity of CEPO in a given sample. CEPO is currently in clinical development for stroke and Friedreich's ataxia, however, there is currently no bioanalysis for CEPO. Because 4 CEp〇 #erythrocyte cells have no effect, existing biopsies of EPO cannot be used. Because the biological activity of the final product may change due to small changes in the manufacturing process, it is important to conduct bioanalysis in situ on new bioproducts that are placed on the market. The present invention discloses a novel biological assay for CEP(R). SUMMARY OF THE INVENTION The present invention relates to a method for determining the concentration or activity of a methotrexate erythropoietin (CEPO) in a sample, the method comprising the steps of: 201213807 1) making a sample comprising CEPO with a mammal such as a rodent Contact with neural progenitor cells of the species, Π) adding a differentiation medium that is substantially free of added growth factors, 111) measuring the performance of specific markers for oligodendrocyte glial cell renewal, and iv) establishing the performance and CEP standards or The relevance of the reference. The invention also relates to the use of such neural progenitor cells for determining the concentration or activity in a sample. [Embodiment] The central nervous system (CNS) is composed of many types of neurons and glial cells. Cell lines such as sputum are produced from pluripotent common precursors located, for example, in the subventricular zone. As part of the differentiation process, 'SVZ cells in the SVZ are limited by their differentiation potential in a series of progressive selections, eventually forming a single cell line (Levinson and Goldman Shi) (4)can α/ ^ (6) ringing restricted precursors coexist in the mammalian perinatal j. Neurosci Res 48 1997 (2) 83_94). The differentiation of pluripotent progenitor cells is a highly complex regulated process that is associated with developmental-culture responses to stressful brothers (eg, injury) and general changes in the presence of trophic factors and cytokines. Thus, multipotent progenitor cells develop into a neuronal or glial phenotype depending on the external environment. In the progressive development of sputum cells, glial progenitor cells can mature into astrocyte or oligodendrocyte glial cell phenotypes. The 201213807 oligodendrocyte glial cells differentiated into, for example, oligodendrocyte glial cells by specific proteins that characterize specific cell types are the major species of CNS neuroglial cells involved in myelination of axons. Mature oligodendrocyte glia cell lines in adult CNS are derived from unmyelinated oligodendrocyte glial cells (OPC) present in the corpus callosum and striatum. The progenitor cells in the subventricular zone (s vz ) of the lateral ventricle also produce 〇PC dispersed throughout the corpus callosum and striatum. The markers of 〇pc include ganglioside GD3, NG2 chondroitin proteoglycan, 04, and platelet-derived growth factor alpha receptor subunit PDGFRa. Mature oligodendrocyte glia cells exhibit MBP and CNPasee The inventors of the present invention have found that CEPO stimulates proliferation and differentiation of neural crest cells into oligodendrocyte glial cell line cells in a dose-dependent manner and promotes maturation of 〇pc into myelinated oligos. Dendritic glial cells' and thus can be used in biological assays to determine the efficacy of a sample, such as a pharmaceutical composition. The progenitor neural progenitor cells can be collected from the subventricular zone of various species such as mammals (e.g., rodents' such as mice, rats, squirrels, porcupines, beavers, chipmunks, guinea pigs, and voles). Particularly preferred is a mouse, a rat or a rabbit. It is expected that the cultured stem cells can also be used as they are.

6 201213807 為研究指定樣本中CEPO之存在（例如濃度或活性形式），可將CEPO添加至生長培養基中。為建立參考或標準，所用CEPO之濃度可在約0至約1〇〇 ng/mi之範圍内。根據一具體實例，可添加以下濃度之CEP〇 ;約〇 ng/ml、約丄 ng/m卜約5 ng/m卜約7.5 ng/m卜約1〇 ng/ml及約1〇〇叫/⑹。將細胞靜置約5至約1 〇天，諸如約7天。接著可將細胞轉移至包含約2%至約5%胎牛企清 (FBS)但無生長因子之分化培養基中。根據一具體實例， FBS用量為約2%。接著將細胞靜置約10至約2〇天，諸如約14天，隨後偵測特定標記。預計可使用顯示CEp〇對神經祖細胞分化成成熟寡樹突神經膠質細胞（稱為寡樹突神經膠質細胞新生）之劑量依賴性作用的任何特定標記。根據一具體實例，標記可選自04、NG2、MBP及CNpase中之一或多者或全部且可如實施例1中所示使用免疫染色定量。根據一態樣，本發明因此係關於一種測定樣本中胺曱醯化紅血球生成素（CEPO)之濃度或活性之方法，特定言之試管内方法，該方法包含以下步驟： 1)使包含CEPO之樣本與來自諸如齧齒動物之哺乳動物物種之神經祖細胞接觸， 11 )添加基本上不含外加生長因子的分化培養基， ⑴）測定對於募樹突神經膠f細胞新生之特定標記的表現，及 W)建立該表現與CEPQ標準或參考者的相關性。 201213807 根據另—態樣，本發明亦關於用於測定樣本中CEPO之濃度或活性之此等神經祖細胞。本發明之其他具體實例可見於申請專利範圍及實施例中〇在本發明中’術語CEPO意欲包括胺曱醯化EPO之任何變異體或衍生物（例如US 2004157293或Science，第35 卷，第23 9-242頁或W0 2〇〇6/〇5〇819中所述，該等文獻以全文引用的方式併入本文中），胺曱醯化Ep〇為其中該蛋白質之至少一個一級胺基（離胺酸及N末端基團）經胺曱醯化的Ερ〇變異體或衍生物。詳言之，本發明係關於具有如下表1中所述之胺基酸序列（SEq ID NO 2 )、或在C末端中包含另一個精胺酸（SEQ ID NO i )或與SEQ ID N〇 i或 2 95°/。、98°/。或 99%—致之序列的 CEPO。兩個胺基酸序列之間的一致性百分比為該等序列所共有之相同位置數的函數（亦即，同源性百分比=相同位置數 /位置總數X 1 〇〇 )，其考慮到為兩個序列之最佳對準所需要引入的間隙數及各間隙之長度。序列之比較及兩個序列之間的一致性百分比之測定可使用諸如BLAST程式之數學演算法實現（例如使用標準設定BLOSUM62經由NCBI猶π 设传之 BLAST 2.2_8，開放間隙=π且擴展間隙=1)。 201213807 表16 201213807 To study the presence of CEPO (eg concentration or activity pattern) in a given sample, CEPO can be added to the growth medium. To establish a reference or standard, the concentration of CEPO used can range from about 0 to about 1 ng/mi. According to a specific example, the following concentrations of CEP can be added; about ng/ml, about ng/m, about 5 ng/m, about 7.5 ng/m, about 1 ng/ml, and about 1 〇〇/ (6). The cells are allowed to stand for about 5 to about 1 day, such as about 7 days. The cells can then be transferred to a differentiation medium containing from about 2% to about 5% fetal bovine clear (FBS) but no growth factors. According to a specific example, the amount of FBS is about 2%. The cells are then allowed to stand for about 10 to about 2 days, such as about 14 days, after which specific markers are detected. Any specific marker showing the dose-dependent effect of CEp〇 on the differentiation of neural progenitor cells into mature oligodendrocyte glial cells, known as oligodendrocyte glial cells, is expected to be used. According to a specific example, the marker can be selected from one or more or all of 04, NG2, MBP, and CNpase and can be quantified using immunostaining as shown in Example 1. According to one aspect, the invention is therefore directed to a method of determining the concentration or activity of an amine deuterated erythropoietin (CEPO) in a sample, in particular an in-vitro method, comprising the steps of: 1) including CEPO The sample is contacted with neural progenitor cells from a mammalian species such as a rodent, 11) a differentiation medium substantially free of additional growth factors is added, (1)) the performance of the specific marker for dendritic neutrophil f-cell regeneration is determined, and Establish a correlation between this performance and the CEPQ standard or reference. 201213807 According to another aspect, the invention also relates to such neural progenitor cells for determining the concentration or activity of CEPO in a sample. Further specific examples of the invention can be found in the scope of the patent application and in the examples. In the present invention, the term CEPO is intended to include any variant or derivative of an amine deuterated EPO (for example US 2004157293 or Science, Vol. 35, No. 23 U.S. Patent Nos. 9-242, or W0 2〇〇6/〇5〇 819, incorporated herein by reference in its entirety, the entire disclosure of which is incorporated herein by reference. An amine deuterated Ερ〇 variant or derivative of an amine acid and an N-terminal group. In particular, the present invention relates to an amino acid sequence (SEq ID NO 2 ) as described in Table 1 below, or another arginine (SEQ ID NO) or SEQ ID N in the C-terminus. i or 2 95°/. , 98°/. Or 99% of the sequence of CEPO. The percent identity between the two amino acid sequences is a function of the number of identical positions shared by the sequences (i.e., percent homology = number of identical positions / total number of positions X 1 〇〇), which takes into account two The number of gaps to be introduced and the length of each gap for optimal alignment of the sequences. The comparison of the sequences and the determination of the percent identity between the two sequences can be achieved using a mathematical algorithm such as the BLAST program (eg BLAST 2.2_8 via NCBI using the standard setting BLOSUM62, open gap = π and extended gap = 1). 201213807 Table 1

1APPRLICDSRVLERYLLEAK 21EAENITTGCAEHCSLNENIT 41VPDTKVNFYAWKRMEVGQQA 61 VEVWQG LALLSEA VLRGQ AL 81LVNSSQPWEPLQLHVDKAVS 101GLRSLTTLLRALGAQKEAIS 121PPDAASAAPLRTITADTFRK 141LFRVYSNFLRGKLKLYTGEA 161 CRTGD 表1 :可能之胺曱醯化位點以粗體顯示，且習知胺基酸以 arial字體顯示。如表1中所示存在9個可能之胺甲醯化位點：位置！之丙胺酸及位置 20、45、52 ' 97、116、140、152 及 154 之離胺酸。因此，本發明係關於CEPO，其中至少一或多個，例如至少1個、至少2個、至少3個、至少4個、至少5 個、至少6個、至少7個或所有9個選自包含位置1之丙胺酸及位置20、45、52、97、116、140、152及154之離胺酸（如表1中所示）之群的胺基酸經胺曱醯化。 CEPO可藉由胺甲醯基化epo產生，例如 W〇2〇06/0〇2646所揭示。簡言之，純化人類Epo (或者重組人類EPO或經生物學或化學修飾之人類Epo )可與大致等體積之1 Μ氣酸钟/0.25 Μ四爛酸鉀（pH值約9.0)混合，且在約29 C下培育約24至48小時。反應可藉由冷卻至室溫、添加3 Μ硫酸銨/1 50 mM Tris-HCI ( pH 7.5 )及疏水性相互作用層析（HIC )來中止。 201213807 實施例實施例1 材料：初級SVZ神經祖細胞自成年Wistar大鼠分離而得。方法：為研究CEPO是否增強神經祖細胞之增殖及分化，使用 SVZ神經袓細胞。 SVZ細胞之分離：簡言之’藉由斷頭術殺死麻醉成年Wistar大鼠（3至4 月齡）且自切斷之頭部快速移出腦。在手術顯微鏡下，解剖SVZ組織，沿側腦室之外側壁、在胼胝體下直至側腦室之腹面頂端得到厚度約1.5至2 ·0 mm之旁矢狀切片。切除. 之組織收集於漢克氏緩衝鹽水溶液（Hanks' buffered saline solution ’ HBSS ; GIBCO)中且藉由在0.25%胰蛋白酶令培育解離成單細胞懸浮液’且用一系列遞減之火焰磨光玻璃吸液管滴定。將細胞洗蘇若干次且再懸浮於補充有1 〇 %胎牛血清（FBS )、5%馬血清、1 %抗生素抗真菌劑及bFGF 20 ng/ml ( R & D System，Minneapolis)之伊斯科夫氏改良達爾伯克氏培養基（Iscove's Modified Dulbecco's Medium， IMDM)中。實驗方案： 10 201213807 、肊在έ有達爾伯克氏改良伊格爾培養基-F-12 (麵心.12 )培養基、2G 表皮生長因子及2㈣基本纖維母細胞生長因子之生長培養基存在下以每毫㈣ 2x1〇4個細胞之密度接種。添加〇、卜75、i(^i〇〇ng/mi 遭度之CEPO且靜置7天。在第6天將漠脫氧尿皆（Brdu， 2〇 gg/ml )添加至培養基中，以標記增殖細胞。隨後將細胞轉移至含有2%胎牛血清（FBS )但排除生長因子之分化培養基中。再培養細胞i 4天。免疫活性細胞之免疫染色及定量：對所培養細胞執行免疫螢光染色。在本發明之研究中使用以下一次抗體：兔抗NG2 ( 1:200，Chemicon，CA )、小鼠抗 04( 1:1〇〇, Chemic〇n，CA)、小鼠抗 CNpase( ι:4〇〇， Chemicon，CA )及兔抗MBp (丨：4〇〇，Dak〇 )。所培養細胞在室溫下於4%三聚曱醛中固定15至20分鐘。在室溫下用 5%正常山羊金清阻斷非特異性結合位點6〇分鐘。細胞接著與以上所列舉之一次抗體及CY3結合或FITC結合之二次抗體一起培育。細胞核用4，，6，-二甲脒基-2-苯基吲哚（DAPI) (Vector Laboratories，Burlingame, CA)對比染色。在隨機選擇之5個顯微鏡視野中在20倍物鏡下計數陽性細胞數及總DAPI細胞數，且定量免疫活性細胞佔dapi陽性細胞總數之百分比。結果：CEP0對SVZ神經祖細胞之增殖及分化之作用 201213807 ίο及 100 ne/rnl 夕 as ^1APPRLICDSRVLERYLLEAK 21EAENITTGCAEHCSLNENIT 41VPDTKVNFYAWKRMEVGQQA 61 VEVWQG LALLSEA VLRGQ AL 81LVNSSQPWEPLQLHVDKAVS 101GLRSLTTLLRALGAQKEAIS 121PPDAASAAPLRTITADTFRK 141LFRVYSNFLRGKLKLYTGEA 161 CRTGD Table 1: Possible amine deuteration sites are shown in bold, and conventional amino acids are shown in arial font. There are 9 possible amine methylation sites as shown in Table 1: Location! Alanine and lysine at positions 20, 45, 52 '97, 116, 140, 152 and 154. Accordingly, the present invention relates to CEPO, wherein at least one or more, such as at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or all 9 are selected from the group consisting of The amino acid of the group 1 alanine and the group of amino acids (as shown in Table 1) at positions 20, 45, 52, 97, 116, 140, 152 and 154 were amine deuterated. CEPO can be produced by amine-mercapto-epo, as disclosed in, for example, W〇2〇06/0〇2646. Briefly, purified human Epo (or recombinant human EPO or biologically or chemically modified human Epo) can be mixed with approximately equal volumes of 1 xenon acid clock / 0.25 Μ potassium sulphate (pH 9.0), and Incubate at about 29 C for about 24 to 48 hours. The reaction can be stopped by cooling to room temperature, adding 3 Μ ammonium sulfate / 1 50 mM Tris-HCI (pH 7.5 ) and hydrophobic interaction chromatography (HIC). 201213807 EXAMPLES Example 1 Materials: Primary SVZ neural progenitor cells were isolated from adult Wistar rats. Methods: To investigate whether CEPO enhances proliferation and differentiation of neural progenitor cells, SVZ neural crest cells were used. Isolation of SVZ cells: Briefly, anesthetized adult Wistar rats (3 to 4 months old) were killed by decapitation and the brain was quickly removed from the severed head. Under the operating microscope, the SVZ tissue was dissected, along the lateral wall of the lateral ventricle, under the corpus callosum to the ventral apex of the lateral ventricle to obtain a parasagittal section with a thickness of about 1.5 to 2.0 mm. The excised tissue was collected in Hanks' buffered saline solution 'HBSCO; GIBCO and disintegrated into a single cell suspension by 0.25% trypsin incubation' and polished with a series of decreasing flames. Glass pipette titration. The cells were washed several times and resuspended in supplemented with 1% fetal calf serum (FBS), 5% horse serum, 1% antibiotic antifungal agent and bFGF 20 ng/ml (R & D System, Minneapolis). Scob's Modified Dulbecco's Medium (IMDM). Experimental protocol: 10 201213807, in the presence of a growth medium containing Dalberg's modified Eagle's medium-F-12 (face center.12) medium, 2G epidermal growth factor and 2 (four) basic fibroblast growth factor Density inoculation of milli (4) 2x1〇4 cells. Add 〇, 卜 75, i (^i〇〇ng/mi CEPO and rest for 7 days. On the 6th day, add deoxyurethane (Brdu, 2〇gg/ml) to the medium to mark Proliferating cells. The cells are then transferred to a differentiation medium containing 2% fetal bovine serum (FBS) but excluding growth factors. The cells are further cultured for 4 days. Immunostaining and quantification of immunocompetent cells: Immunofluorescence is performed on the cultured cells. Staining. The following primary antibodies were used in the study of the present invention: rabbit anti-NG2 (1:200, Chemicon, CA), mouse anti-04 (1:1, Chemic〇n, CA), mouse anti-CNpase ( : 4〇〇, Chemicon, CA ) and rabbit anti-MBp (丨: 4〇〇, Dak〇). The cultured cells were fixed in 4% trimeric furfural for 15 to 20 minutes at room temperature. 5% normal goat Jinqing blocked the non-specific binding site for 6 min. The cells were then incubated with the primary antibody and CY3 binding or FITC-conjugated secondary antibody listed above. The nuclei were 4,6,-dimethyl decyl. Comparative staining with -2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA). 5 microscopes randomly selected In the wild, the number of positive cells and the total number of DAPI cells were counted under a 20-fold objective lens, and the percentage of immunoreactive cells in the total number of dapi-positive cells was quantified.Results: The effect of CEP0 on the proliferation and differentiation of SVZ neural progenitor cells 201213807 ίο and 100 ne/rnl Eve as ^

ng/ml下達到峰值（圖1E)。例大作现膠買細胞數目，其中在1 〇 1Ε )。然而’儘管在1〇 ng/mi下谓測到MBP陽性有髓勒寡樹突神經膠質細胞增加（圖1C )，但未達到統計顯著性（圖1C)。實施例2 為研究CEPO是否增強0PC成熟成為成熟寡樹突神經膠質細胞，使用經SV40T抗原不朽化之小鼠寡樹突神經膠質細胞N20.1細胞系，其係由Dr Anthony Campagnoni， University of California，Los Angeles 慷慨提供。在 37°Ct 在含有10% FBS(胎牛血清）及G418( 100 μΐ/ml)之DMEM/ 漢姆氏 F12 培養基（DMEM/Ham’s F12 medium)中培養 N20 1 細胞。在胰蛋白酶處理（0.05%胰蛋白酶-乙二胺四乙酸； Life Technology)之後，細胞每週繼代一次。實驗方案： N20.1 細胞在 39°C下用含 CEPO( 0、卜 10 及 1〇〇 ng/ml) 12 201213807 之DMEM/漢姆氏F12處理9天，該培養基含有FBS但不含 G418。西方墨點分析（Western blot analysis): 簡s之’將小鼠募樹突神經膠質細胞溶解且將等量蛋白質裝載於10% SDS-聚丙烯醯胺凝膠上。電泳之後，將蛋白質轉移至確化纖維薄膜上且隨後用以下一次抗體探測墨點：小鼠多株抗CNPase ( 1:1000, Millipore)及大鼠多株抗 ^0?(1:1〇〇〇’^^114〇1^)。偵測時，使用辣根過氧化酶（1^1)_) 結合之二次抗體（1:2000 )，之後進行增強化學發光（E(：L ) 顯衫（Pierce Biotechnology公司）。以β肌動蛋白抗體用作内部對照物、藉由西方墨點並行移動來破保結果校正。使用景W象處理及分析程式（Scion image，Eder-ick, ΜΑ )定量光學密度。結果：CEPO對N20.1細胞之寡樹突神經膠質細胞成熟之作用 1、10及100 ng/ml之CEPO實質上升高CNPase蛋白質含量，而1及1〇ng/m丨之CEP0升高Μβρ蛋白質含量（圖 2 )。成熟寡樹突神經膠質細胞表現MBP及CNPase基因。因此，此等資料指示CEP0促進寡樹突神經膠質細胞成熟。【圖式簡單說明】圖1顯示當添加不同濃度之⑽時神經祖細胞之劑量 13 201213807 依賴性反應。媒劑組中之編號表示不同媒劑體積，此等體積與CEPO在相應濃度下的使用體積相同’ *P<0·05 ’相對 .於對照組。圖2以西方墨點顯示用1 ng/ml CEPO ( CE1 )、10 ng/ml CEP〇 ( CE10)及 100 ng/ml CEP〇 ( CE100)處理之 N20.1 細胞中MBP、CNPase及β肌動蛋白之含量。p肌動蛋白用作内部對照物^ C =對照組。【主要元件符號說明】無 14 201213807 序列表 <110> Η.朗徳貝克公司 <120>用於測量CEPO濃度與活性的生物分析The peak was reached at ng/ml (Fig. 1E). For example, the number of cells purchased in the gums is 1 〇 1Ε). However, although an increase in MBP-positive oligodendrocyte glial cells was detected at 1 ng/mi (Fig. 1C), statistical significance was not reached (Fig. 1C). Example 2 To investigate whether CEPO enhances the maturation of 0PC into mature oligodendrocyte glial cells, the mouse oligodendrocyte glial cell line N20.1, which is immortalized with SV40T antigen, is used by Dr Anthony Campagnoni, University of California. , Los Angeles is generously available. N20 1 cells were cultured in DMEM/Ham's F12 medium (DMEM/Ham's F12 medium) containing 10% FBS (fetal calf serum) and G418 (100 μΐ/ml) at 37 °C. After trypsin treatment (0.05% trypsin-ethylenediaminetetraacetic acid; Life Technology), the cells were subcultured once a week. Experimental protocol: N20.1 cells were treated with CEPO (0, Bu 10 and 1 ng/ml) 12 201213807 in DMEM/Ham's F12 for 9 days at 39 ° C. The medium contained FBS but no G418. Western blot analysis: The mouse was used to solubilize dendritic glial cells and load equal amounts of protein onto a 10% SDS-polyacrylamide gel. After electrophoresis, the protein was transferred to a definitive fiber membrane and the dots were subsequently probed with the following primary antibodies: multiple anti-CNPase (1,1000, Millipore) mice and multiple anti-zero (1 〇〇〇) rats. '^^114〇1^). At the time of detection, horseradish peroxidase (1^1)_) was used to bind the secondary antibody (1:2000), followed by enhanced chemiluminescence (E(:L) shirt (Pierce Biotechnology). The kinesin antibody was used as an internal control, and the results were corrected by parallel movement of western ink dots. The optical density was quantified using the image processing and analysis program (Scion image, Eder-ick, ΜΑ). Results: CEPO vs. N20. The role of 1 cell oligodendrocyte glial cell maturation 1, 10 and 100 ng / ml of CEPO substantially increased the CNPase protein content, while 1 and 1 ng / m 丨 CEP0 increased Μβρ protein content (Figure 2). Mature oligodendrocyte glial cells express MBP and CNPase genes. Therefore, these data indicate that CEP0 promotes maturation of oligodendrocyte glial cells. [Simplified schematic] Figure 1 shows the dose of neural progenitor cells when different concentrations (10) are added. 13 201213807 Dependent reaction. The number in the vehicle group indicates the volume of different vehicles. These volumes are the same as the volume used by CEPO at the corresponding concentration '*P<0·05'. In the control group. Point display with 1 ng/ MBP, CNPase and β-actin content in N20.1 cells treated with ml CEPO (CE1), 10 ng/ml CEP〇 (CE10) and 100 ng/ml CEP〇 (CE100). p actin was used as internal Control ^ C = control group [Explanation of main component symbols] No 14 201213807 Sequence Listing <110> 徳.Landmark Company <120> Bioassay for measuring CEPO concentration and activity

<130> 0754-WO-PCT <160> 2 <170〉 Patentln第3.5版 <210> 1 <211> 166 <212> PRT <213>智慧人 <220> <221>胜肽 <222> (1)-.(165) <223〉紅血球生成素 <400> 1<130> 0754-WO-PCT <160> 2 <170> Patentln 3.5th edition <210> 1 <211> 166 <212> PRT <213> wise man <220><221> Peptide <222> (1)-.(165) <223>erythropoietin<400> 1

Ala Pro Pro Arg Leu lie Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu 15 10 15Ala Pro Pro Arg Leu lie Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu 15 10 15

Leu Glu Ala Lys Glu Ala Glu Asn lie Thr Thr Gly Cys Ala Glu His 20 25 30Leu Glu Ala Lys Glu Ala Glu Asn lie Thr Thr Gly Cys Ala Glu His 20 25 30

Cys Ser Leu Asn Glu Asn lie Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45Cys Ser Leu Asn Glu Asn lie Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45

Tyr Ala Trp Lys Arg Met Glu Val Gly Gin Gin Ala Val Glu Val Trp 50 55 60Tyr Ala Trp Lys Arg Met Glu Val Gly Gin Gin Ala Val Glu Val Trp 50 55 60

Gin Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gin Ala Leu 65 70 75 80Gin Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gin Ala Leu 65 70 75 80

Leu Val Asn Ser Ser Gin Pro Trp Glu Pro Leu Gin Leu His Val Asp 85 90 95Leu Val Asn Ser Ser Gin Pro Trp Glu Pro Leu Gin Leu His Val Asp 85 90 95

Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 100 105 110Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 100 105 110

Gly Ala Gin Lys Glu Ala lie Ser Pro Pro Asp Ala Ala Ser Ala Ala 115 120 125Gly Ala Gin Lys Glu Ala lie Ser Pro Pro Asp Ala Ala Ser Ala Ala 115 120 125

Pro Leu Arg Thr lie Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val 130 135 140Pro Leu Arg Thr lie Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val 130 135 140

Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 201213807Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 201213807

Cys Arg Thr Gly Asp Arg 165 <210〉 2 <211> 165 <212> PRT <213>智慧人 <220> <221> EPO <222> (1)..(165)Cys Arg Thr Gly Asp Arg 165 <210> 2 <211> 165 <212> PRT <213> Wisdom <220><221> EPO <222> (1)..(165)

<223> _R166 之截短 EPO <400> 2<223> _R166 truncation EPO <400> 2

Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160

Cys Arg Thr Gly Asp 165 2Cys Arg Thr Gly Asp 165 2

Claims

201213807 七、申請專利範圍： 1 · 一種測定樣本中胺曱醯化紅血球生成素（CEPO )之濃度或活性之方法，其包含以下步驟： 1)使包含CEPO之樣本與諸如齧齒動物之哺乳動物物種之神經祖細胞接觸， II) 添加基本上不含外加生長因子的分化培養基， III) 測定對於寡樹突神輯質細胞m定標記的表現 π)建立該表現與CEPO標準或參考者的相關性。 2 ·如申明專利範圍第1項之方法，其中步驟i)中之該等神經祖細胞係自大鼠或小鼠分離而得且連同該包含 CEP〇之樣本一起於生長培養基中培育約5天至約10天，之後進行步驟ii )。 3·如申請專利範圍第1項或第2項中任一項之方法，其中步驟1 )中所用之該等神經祖細胞之密度為約i 〇4個細胞/毫升。 4·如申請專利範圍第2項之方法，其中該生長培養基包含約20ng/m丨基本纖維母細胞生長因子及約2〇ng/mi表皮生長因子。 5.如甲睛專利範圍前述任一項之该#細胞係培育約1 〇天至約2 〇天之時期。 6. 如申請專利範圍前述任一 1 孭之方法，其中步驟Η)中之該分化培養基包含約2 %至約^ Α .主 _ ❶芏4 5/。血清，諸如胎牛血清。 7. 如申請專利範圍前述任一項貝之方法，其中步驟iii ) 201213807 中之該等標記係選自NG2、MBP、〇4及CNPase。 8 ·如申凊專利範圍第1項之方法，其中步驟iv )包含將該表現與CEPO標準或參考曲線進行比較。 9.如申請專利範圍前述任_項之方法，其中步驟iv)中之-玄CEPCM不準或參考係使用約〇至約⑽ng/ml CEp〇劑量範圍製得。 10·—種來自諸如窨啬衝齒動物之哺乳動物物種之神經祖細胞的用途，其係用於測定心様·本中CEPO之濃度或活性。 11.如申請專利筋圍笛，λ 弟1 〇項之神經祖細胞之用途，其係用於如申晴專利範圍第乐項至第9項中任一項之方法中。八、圖式： (如次頁） 2201213807 VII. Patent Application Range: 1 · A method for determining the concentration or activity of amidinated erythropoietin (CEPO) in a sample, comprising the following steps: 1) making a sample containing CEPO with a mammalian species such as a rodent Neural progenitor cell contact, II) addition of differentiation medium substantially free of added growth factors, III) determination of the expression of m-labeled markers for oligodendrocyte cells π) establishment of correlation of this performance with CEPO standards or reference . 2. The method of claim 1, wherein the neural progenitor cell lines in step i) are isolated from rats or mice and are incubated in a growth medium for about 5 days together with the sample comprising CEP(R). After about 10 days, proceed to step ii). 3. The method of any one of claims 1 or 2, wherein the density of the neural progenitor cells used in step 1) is about i 〇 4 cells/ml. 4. The method of claim 2, wherein the growth medium comprises about 20 ng/m 丨 basic fibroblast growth factor and about 2 ng/mi epidermal growth factor. 5. The period of the # cell line according to any one of the above-mentioned items of the nail-like patent is cultivated for a period of from about 1 day to about 2 days. 6. The method according to any one of the preceding claims, wherein the differentiation medium in step Η) comprises from about 2% to about Α. Main _ ❶芏 4 5/. Serum, such as fetal bovine serum. 7. The method according to any one of the preceding claims, wherein the markers in step iii) 201213807 are selected from the group consisting of NG2, MBP, 〇4 and CNPase. 8. The method of claim 1, wherein step iv) comprises comparing the performance to a CEPO standard or a reference curve. 9. The method of any of the preceding claims, wherein in the step iv), the sinusoidal CEPCM is misaligned or the reference system is prepared using a range of from about 〇 to about (10) ng/ml of CEp 〇. 10. Use of a neural progenitor cell from a mammalian species such as a sputum toothing animal for determining the concentration or activity of CEPO in the heart palpitations. 11. The use of a neural progenitor cell of the ribs of the ribs, for example, in the method of any one of the items of the ninth aspect of the ninth. Eight, the pattern: (such as the next page) 2