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UNIVERSIDADE FEDERAL DE PELOTAS

Programa de Pós-Graduação em Biotecnologia

TESE

Desenvolvimento de ensaio imunoquímico para

detecção de doping com eritropoetina

Thaís Farias Collares

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THAÍS FARIAS COLLARES

Desenvolvimento de ensaio imunoquímico para detecção de doping com eritropoetina

Tese apresentada ao Programa de Pós-Graduação em Biotecnologia da Universidade Federal de Pelotas, como requisito parcial à obtenção do título de Doutora em Ciências (área do conhecimento: Biotecnologia).

Orientador: Prof. Cláudia Pinho Hartleben, Dra.

Co-orientadores: Prof. Fabiana Kömmling Seixas, Dra. Leonardo Garcia Monte, Dr.

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Banca examinadora:

Prof. Dr. Airton José Rombaldi, Universidade Federal de Pelotas

Prof. Dr. Leonardo Garcia Monte, Universidade Federal de Pelotas

Prof. Dr. Vinicius Farias Campos, Universidade Federal de Pelotas

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AGRADECIMENTOS

À Universidade Federal de Pelotas pela oportunidade de realizar um curso de Pós - Graduação de qualidade.

Á Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) pela concessão da bolsa de estudos.

Á minha orientadora e amiga, Cláudia Hartleben, pelo incentivo e incansável apoio e dedicação na realização deste trabalho, contribuindo sempre na minha formação pessoal e profissional.

Aos meus co-orientadores, Fabiana Seixas e Odir Dellagostin pela dedicação e ensinamentos.

Aos amigos e colegas Vinicius, Leonardo e Eliza pela colaboração nesse trabalho e principalmente pela amizade que ganhei durante todo esse tempo.

Aos colegas do Laboratório de Imunodiagnóstico.

Aos demais professores, colegas e estagiários do Programa de Pós-Graduação em Biotecnologia pela amizade e convívio agradável.

Aos meus pais e irmãos pelo incentivo, carinho e confiança, por estarem do meu lado em todos os momentos da minha vida, sempre com palavras de apoio. Eles são os grandes responsáveis pela minha formação pessoal.

E a todos que direta ou indiretamente contribuíram de alguma forma para a realização deste trabalho.

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RESUMO

COLLARES, Thaís Farias. Desenvolvimento de ensaio imunoquímico para detecção de doping com eritropoetina. 2013. 59f. Tese (Doutorado) – Programa de Pós-Graduação em Biotecnologia. Universidade Federal de Pelotas. Pelotas.

A história da competição esportiva sempre esteve relacionada à utilização de metodologias de treinamento físico associadas a métodos de incremento fisiológico do atleta, visando sua máxima performance. Questões éticas envolvendo o esporte de alto rendimento e o doping se confundem com a própria história do esporte competitivo. A Agência Mundial Anti-Doping (WADA) proíbe o uso de substâncias ou métodos capazes de aumentar artificialmente o desempenho esportivo. Atualmente, 258 substâncias estão na lista da WADA e atletas do mundo inteiro são submetidos a testes comprobatórios da não utilização do doping. Entre essas substâncias ilícitas para uso por atletas destaca-se a eritropoetina (EPO). A EPO é um hormônio glicoproteico que possui como principal efeito fisiológico a indução da eritropoiese e consequente melhoria da capacidade de transporte de oxigênio no sangue. A diferenciação analítica da eritropoetina endógena produzida a partir de sua contraparte recombinante usando focalização isoelétrica e duplo blotting é um marco na detecção do doping com eritropoetina recombinante. Anticorpos monoclonais e policlonais específicos anti-EPO foram obtidos e utilizados em várias técnicas para a detecção da EPO em amostras biológicas a fim de melhorar os métodos atuais de detecção. Contudo, a especificidade destes anticorpos tem sido bastante controversa e discutida. Portanto, a obtenção de anticorpos específicos capazes de reagir especificamente com a EPO faz-se necessária. Neste estudo, a rHuEPO foi utilizada para imunizar coelhos New Zealand para gerar um anticorpo policlonal (pAb anti-rHuEPO). O pAb foi caracterizado quanto ao seu potencial na detecção da rHuEPO usando diferentes metodologias. O pAb anti-rHuEPO identificou a expressão da proteína recombinante em células eucarióticas e foi capaz de detectar rHuEPO em suspensão até 0,1 µg/mL, comprovando seu potencial como ferramenta para detecção do doping por rHuEPO.

Palavras-chave: rHuEPO. Eritropoetina. Anti-doping. Anticorpos. Imunoensaios. pAb.

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ABSTRACT

COLLARES, Thaís Farias. Development of immunochemical assay for detection of doping with erythropoietin. 2013. 59f. Tese (Doutorado) - Programa de Pós-Graduação em Biotecnologia. Universidade Federal de Pelotas. Pelotas.

The history of sports competition has been related to the use of methods for physical training associated with physiological methods to increase athlete performance. Ethical issues involving the high performance sport and doping are confused with the history of competitive sports. The World Anti - Doping Agency (WADA) prohibits the use of substances or methods to artificially increase sports performance. Currently, 258 substances are listed by WADA and athletes around the world are subjected to tests proving the non-use of doping. EPO is a glycoprotein hormone that has as its main physiological effect induction of erythropoiesis and thereby improving the capacity to transport oxygen in the blood. For these reason EPO has been included in WADA list. The analytical differentiation of endogenous erythropoietin from its recombinant counterpart, using isoelectric focusing and double blotting is a milestone in the detection of doping with recombinant erythropoietin. However, several analogs of the original recombinant EPO are not easily detectable by standard IEF method, requiring the development of alternatives for the detection of doping. In order to improve the current methods of EPO detection, monoclonal and polyclonal antibodies against EPO were obtained and used in various techniques for detection of EPO in biological samples. However, the specificity of these antibodies has been quite controversial and discussed. Therefore, it is necessary to obtain antibodies capable of reacting specifically with the EPO. In this study, rHuEPO was inoculated in New Zealand rabbits to generate a polyclonal antibody (pAb anti-rHuEPO). The pAb was characterized for its potential in detecting rHuEPO using different approaches. The pAb anti- rHuEPO identified the expression of recombinant protein in eukaryotic cells and was able to detect rHuEPO in a suspension at 0.1 µg/mL, showing its potential as a tool for detection of doping by rHuEPO.

Key words: rHuEPO. Erythropoietin. Doping control. Antibodies. Immunoassays. pAb.

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LISTA DE FIGURAS

ARTIGO 1 - EPO: advances in doping detection

Figura 1 Current detection methods of EPO isoforms ………...… 39 ARTIGO 2 - Development of polyclonal antibodies for the detection of

recombinant human erythropoietin

Figura 1 rHuEPO detection by anti-recombinant human erythropoietin

polyclonal antibody (rHuEPO pAb) in an immunoblotting assay... 49

Figura 2 Immunofluorescence analysis of anti-rHuEPO pAb in CHO cells

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LISTA DE ABREVIATURAS E SIGLAS

AIDS - Síndrome da Imunodeficência Adquirida BHK - Rim de Filhote de Hamster

cDNA - Ácido Desoxiribonucleico Complementar

CERA - Ativador Contínuo do Receptor da Eritropoetina CHO - Ovário De Hamster Chinês

DNA - Ácido Desoxirribonucleico

ELISA -Enzyme-linked immunosorbent assay EMP - Peptídeo Mimético da eritropoetina EPO - Eritropoetina

ESAs - Agentes Estimuladores da Eritropoiese

FDA - Administração Federal de Alimentos e Medicamentos FITC - Isotiocianato de Fluoresceína

HIV - Vírus da Imunodeficência Humana IEF - Focalização Isoelétrica

IEF-PAGE - Eletroforese em Gel de poliacrilamida com Focalização Isoelétrica IOC - Comitê Olímpico Internacional

MAII - Membrane-Assisted Isoform Immunoassay NESP - Nova Proteína Estimuladora da Eritropoiese pAb - Anticorpo Policlonal

PBS- Tampão Fosfato-Salino PEG - Polietilenoglicol

rHuEPO - Eritropoetina Recombinante Humana RNA - Ácido Ribonucléico

RT-qPCR - Reação em Cadeia da Polimerase Quantitativa em Tempo Real SAR-PAGE - Eletroforese em Gel de Poliacrilamida com Sarcosil

SDS-PAGE - Eletroforese em Gel de Poliacrilamida com Dodecil-Sulfato de Sódio SEP - Proteína Eritropoiética Sintética

sTFr - Concentração de Receptor de Transferrina Solúvel WADA - Agência Mundial Anti-Doping

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SUMÁRIO 1. Introdução geral... 9 2. Objetivos... 13 3. Artigo1... 14 Abstract... 16 Introduction……….………... 17

Recombinant erythropoietins, analogues and mimetics……….…..….…….. 17

Doping control for recombinant erythropoietin and analogues...……… 21

EPO detection methods………...……….……….... 22

Isoelectric focusing (ief) ……….….………...…..…..……. 23

IEF - Protease tests……….………..…………... 25

SDS-PAGE…….………...….…... 27

SAR-PAGE…..……….……….…..…….. 27

Membrane-assisted isoform immunoassay - EPO WGA MAIIA..………..… 28

Perspectives of EPO doping control..……….………... 29

Fusion protein detection……… 29

Blood cells RNA biomarkers…..………. 29

Conclusion……….……….. 29 Acknowledgements….……….……….. 30 References……..……….……... 30 4. Artigo 2... 40 Abstract... 42 Introduction………..……….…... 43

Materials and methods………...…………..………...……. 43

Results and discussion...………...…….. 45

Acknowledgements………... 46 References………... 46 5. Conclusões.……….………... 51 6. Referências... 52 Anexos... 55 Anexo A... 55 Anexo B... 56

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1 INTRODUÇÃO GERAL

A história da competição esportiva sempre esteve relacionada à utilização de metodologias de treinamento físico associadas a métodos de incremento fisiológico do atleta, visando sua máxima performance. Para tal, desde suplementos alimentares até a administração de potencializadores fisiológicos são utilizados como ferramentas associadas ao treinamento físico. Assim, muitos atletas acabam utilizando drogas e métodos ilícitos, os quais podem ter importantes efeitos adversos, que acabam por colocar em risco a integridade física. Questões éticas envolvendo o esporte de alto rendimento e o doping se confundem com a própria história do esporte competitivo. Seriam possíveis as quebras de recordes sem a utilização de substâncias proibidas que ajudem a potencializar a performance esportiva? E, será que treinadores e atletas estariam dispostos a abrir mão de tais recursos?

A Agência Mundial Anti-Doping (WADA) proíbe o uso de substâncias ou métodos capazes de aumentar artificialmente o desempenho esportivo, visando à proteção da saúde dos atletas e a ética esportiva. Atualmente, 258 substâncias estão na lista da WADA (WADA, 2013) e atletas do mundo inteiro são submetidos a testes comprobatórios da não utilização do doping. Entre uma das principais substâncias não lícitas para uso por atletas está a eritropoetina (EPO).

A EPO é um hormônio glicoproteico que possui como principal efeito fisiológico a indução da eritropoiese e consequente melhoria da capacidade de transporte de oxigênio no sangue (FISHER, 2003). Ela é produzida em quantidades correspondendo à concentração de O2 no sangue e é sintetizada primariamente no

rim, mas também em níveis mais baixos em outros tecidos, tais como fígado e cérebro (ELLIOTT, 2008).

A evolução das técnicas de engenharia genética permitiu a produção de uma forma análoga da EPO endógena.A clonagem bem sucedida do gene da EPO humana (LIN et al., 1985) possibilitou a produção da eritropoetina recombinante humana (rHuEPO) e, posteriormente, a aprovação para ser comercializada como forma de auxiliar nos tratamentos de quadros de anemias severas devido a insuficiência renal crônica. O uso desta também é indicado para pacientes que se

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submetem a sessões de quimioterapia, no tratamento de doenças auto imunes como o AIDS (Síndrome da Imunodeficência Adquirida), casos de transfusões de sangue devido à ocorrências de intervenções cirúrgicas, entre outras (MANCINI et al., 2003). Os análogos da EPO, tal como a rHuEPO, podem ser substitutos da eritropoetina endógena, através da ligação ao seu receptor, provocando sinalização intracelular de uma forma idêntica a do hormônio natural (LAMON et al., 2010). Devido ao fato do aumento do número de hemácias melhorar o desempenho de atletas em esportes de resistência, o uso de tais formas sintéticas da EPO são proibidos pela WADA.

Antes mesmo da comercialização da rHuEPO, suspeitava-se da possibilidade de uso abusivo por parte de atletas na busca artificial de melhor desempenho. Várias reportagens relataram a utilização de hormônios peptídicos, como a epoetina alfa, mas provas conclusivas de sua manipulação não foram geradas. A desidratação, frequente durante e após o esforço físico intenso de uma competição, aliada a hemoconcentração, poderia acarretar risco iminente à saúde, por aumento da viscosidade sanguínea e redução do débito cardíaco, levando a quadros de hipertensão e possíveis eventos trombóticos (HASSAN et al., 2005). Estudos recentes demonstraram que aparentemente a rHuEPO não afeta parâmetros reprodutivos em animais (COLLARES et al, 2012)

No esporte de alto rendimento, especula-se que a rHuEPO passou a ser utilizada de forma rotineira como meio artificial de produção de glóbulos vermelhos, devido a vantagem adicional da difícil detecção de sua presença na matriz biológica através dos métodos analíticos convencionais, além do efetivo ganho no desempenho esportivo (PASCUAL et al., 2004).

Durante vários anos foram pesquisadas maneiras de contornar o problema da dopagem por rHuEPO devido a impossibilidade de sua detecção e diferenciação, por se tratar de uma substância estruturalmente complexa de elevada massa molecular, presente em baixas concentrações nos fluidos biológicos e bastante semelhante a sua forma endógena. Além disso, a mesma pode ser obtida e administrada sem supervisão médica, o que aumenta o risco de seu abuso (KAZLAUSKAS et al., 2002;SHARPE et al., 2002).

A evolução dos métodos moleculares e imunoquímicos resultou em estratégias de detecção do doping com rHuEPO tanto indiretamente, com

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marcadores da eritropoiese aumentada ou reduzida, bem como a detecção direta das isoformas recombinantes (EKBLOM, 2000).

A epoetina alfa foi a primeira eritropoetina recombinante comercialmente produzida a partir de 1989 e está disponível mundialmente sob diferentes nomes, como Epogen, Eprex, Erypo (JELKMANN, 2008). Já a darbepoetina alfa foi desenvolvida pelo sitio de mutagênese dirigida à sequência original do gene da EPO. Cinco aminoácidos foram trocados - o que conduz a dois outros sítios de N-glicosilação e devido a isto a meia vida dela no soro pode ser aumentada cerca de três a quatro vezes em comparação com a epoetina alfa. A darbepoetina alfa (NESP, nova proteína estimulante da eritropoiese) é comercializada sob o nome de Aranesp e Nespo e devido à glicosilação alterada ela pode ser facilmente diferenciada de todas epoetinas por focalização isoelétrica (IEF) e SDS-PAGE(LAMON et al., 2007;MORKEBERG et al., 2007).

A diferenciação analítica da eritropoetina endógena produzida a partir de sua contraparte recombinante usando focalização isoelétrica e duplo blotting é um marco na detecção do doping com eritropoetina recombinante. No entanto, vários análogos dos produtos recombinantes iniciais, nem sempre são facilmente detectáveis pelo método padrão-IEF, exigindo o desenvolvimento de alternativas para a detecção do doping (REICHEL et al., 2010).

Até agora, anticorpos monoclonais e policlonais específicos anti-EPO humana foram obtidos e utilizados em várias técnicas para a detecção da EPO em amostras biológicas de pacientes com doença renal crônica, bem como no método utilizado para o controle de doping. Além disso, os anticorpos anti-EPO foram utilizados em eletroforese capilar para detectar e pré-concentrar a EPO, a fim de melhorar os métodos atuais de detecção (GIMENEZ et al., 2007).

O anticorpo monoclonal utilizado para a detecção da EPO nos testes antidoping (clone AE7A5) é dirigido contra os primeiros 26 aminoácidos da extremidade N-terminal da sequência de aminoácidos processados (SYTKOWSKI et al., 1985). Desde 2005, a especificidade deste anticorpo tem sido bastante controversa, discutida em público e em várias publicações revisadas (BEULLENS et al., 2006;FRANKE et al., 2006;KHAN et al., 2005;KHAN et al., 2007). O problema mais preocupante é a comprovada interação não-específica do anticorpo monoclonal anti-EPO utilizado, com outras proteínas, que não a EPO. Khan et al. (2005) identificaram uma reação cruzada com proteínas urinárias, onde cada uma das

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proteínas urinárias abundantes com reação cruzada mostrou alguma homologia com o epítopo da EPO, que provavelmente explica a afinidade do anticorpo contra a EPO com estas proteínas (DELANGHE et al., 2008).

Devido à existência de novas tecnologias, avanços foram feitos durante a última década para a detecção desses agentes de dopagem na urina. No entanto, ainda há muito a ser feito, em particular para a detecção desses agentes em fluidos biológicos como o soro ou plasma, e no desenvolvimento de métodos de rastreio rápido e barato, que permite detectar o abuso destas substâncias, em todas as amostras recolhidas. Esta tese tem o intuito de dar uma contribuição nesta área.

Neste contexto, a hipótese deste trabalho foi de que a eritropoietina recombinante humana pode ser utilizada para gerar anticorpos capazes de reconhecer esta proteína em solução ou quando expressa por células eucarióticas.

Sendo assim, os objetivos deste trabalho foram produzir um anticorpo policlonal contra rHuEPO; caracterizar o anticorpo obtido através de imunofluorescência indireta, western blotting e ELISA; avaliar a utilização do anticorpo obtido para detecção da expressão de rHuEPO por células eucarióticas e estabelecer o limite de detecção da proteína em suspensão utilizando o anticorpo policlonal.

Ainda neste processo, um anticorpo monoclonal contra a eritropoetina recombinante humana foi produzido. Obtivemos um clone secretor e este segue em fase de testes e avaliações. Para proteger este produto realizamos pedido de depósito de patente (Anexo A).

Os dados gerados nesta tese estão apresentados na forma de artigos científicos. Estes artigos estão formatados de acordo com a revista que foram ou serão submetidos. O primeiro artigo é uma revisão que relata os principais métodos de detecção do doping com eritropoetina e os avanços e perspectivas nessa área. Este trabalho será submetido ao periódico International Journal of Current Research. O segundo artigo aborda o desenvolvimento de um anticorpo policlonal contra a eritropoetina recombinante humana para uso em imunoensaios. Esse trabalho foi publicado no periódico African Journal of Biotechnology.

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2 OBJETIVOS

Os objetivos deste trabalho foram produzir um anticorpo policlonal contra rHuEPO; caracterizar o anticorpo obtido através de imunofluorescência indireta,

western blotting e ELISA; avaliar a utilização do anticorpo obtido para detecção da

expressão de rHuEPO por células eucarióticas e estabelecer o limite de detecção da proteína em suspensão utilizando o anticorpo policlonal.

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3 ARTIGO 1

EPO: Advances in doping detection

(Formatado nas normas do periódico International Journal of Current Research) (Manuscrito a ser submetido)

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EPO: ADVANCES IN DOPING DETECTION

Collares, Thaís Farias1; Xavier, Marina Amaral1; Hartleben Cláudia Pinho1*

1

Laboratório de Imunodiagnóstico, Núcleo de Biotecnologia, Centro de

Desenvolvimento Tecnológico, Universidade Federal de Pelotas, RS, Brasil

Corresponding author. Mailing address: Laboratório de Imunodiagnóstico, Núcleo de

Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de

Pelotas, CEP 96010-900, Pelotas, RS, Brasil - P.O. Box 354. Phone: +55 53

32757517.

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Abstract

Erythropoietin (EPO) is a glycoprotein hormone that promotes the production of red

blood cells and as consequence it increases tissue oxygenation. Recombinant

human erythropoietin (rHuEPO) is illicitly used to improve performance in endurance

sports, so it is prohibited by the World Anti-Doping Agency (WADA). Both direct tests

(indicating the presence of exogenous EPO isoforms) and indirect tests (indicating

hematological changes induced by exogenous EPO administration) can be used for

EPO detection. The analytical differentiation of endogenously produced

erythropoietin from its recombinant counterpart by using isoelectric focusing (IEF)

and double blotting is a milestone in the detection of doping with recombinant

erythropoietin. The constant development of new erythropoiesis stimulating agents

(ESAs) has also required constant development of methods for detecting the abuse

of these substances, since it is not always easily detectable by a standard method of

IEF. The article summarizes the various forms of recombinant erythropoietin and the

main strategies currently used in EPO anti-doping testing, with special focus on new

developments.

Key-words: rHuEPO; erythropoietin; doping control; isoelectric focusing; methods; recombinant proteins

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Introduction

Erythropoietin (EPO) is a glycoprotein hormone (34 kDa) substantially

glycosylated with 165 amino acids (Juul and Felderhoff-Mueser, 2007) produced

mainly in the mammalian kidney and the fetal liver. About 40% of its total molecular

mass corresponds to three N-linked (Asn24, Asn38, Asn83) and one O-linked

(Ser126) carbohydrate chains attached to the polypeptide backbone (Fried, 1995).

EPO plays an important role in red blood cell production through the promotion of

survival, proliferation and differentiation of the erythroid progenitors, acting

synergistically with other cytokines (Diamanti-Kandarakis et al., 2005). Therefore, the

oxygen transport from the lungs to the tissues is improved since it is main function of

red cells. Tissue hypoxia is the main stimulus of EPO production and secretion;

however, EPO is not only produced when oxygen capacity of the blood decreases,

but also when arterial oxygen pressure (pO2) decreases (Lacombe and Mayeux,

1998). Since oxygen levels are detected by peritubular interstitial cells through prolyl

hydroxylase - which hydroxylates the hypoxia inducible factor 1-α (HIF-1α), in order to inhibit the activity of erythropoietin gene - the enzyme activity leads to

erythropoietin synthesis (Jelkmann, 2011). Inadequate erythropoietin production, as

observed in patients with end-stage renal disease, results in anemia (Cazzola et al.,

1997).

Recombinant erythropoietins, analogues and mimetics

In 1985, the human erythropoietin gene was cloned (Lin et al., 1985) and in

1989 Recombinant Human Erythropoietin (rHuEPO) was approved as a therapeutic

protein for the treatment of anemia associated with chronic renal failure by the Food

and Drug Administration (FDA). The introduction of rHuEPO revolutionized the

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Moreover, clinical studies have demonstrated that it is also useful in uremic

conditions such as hematological and oncological disorders, prematurity, HIV

infection (Ng et al., 2003), myelodysplastic syndromes, bone marrow transplantation,

hepatitis C (Delanghe et al., 2008) and can be used to minimize allogeneic blood

transfusions after major surgical procedures (Diamanti-Kandarakis et al., 2005). The epoetin-α and epoetin-β, produced in transformed Chinese Hamster Ovary (CHO) cell cultures, and epoetin-ω, engineered in Baby Hamster Kidney (BHK) cells were the first-generation recombinant EPO (Lin et al., 1985) and carry

complementary DNA-encoding human erythropoietin. These transformed cells

secreting epoetins with identical amino acid sequence and the difference between

these products are in their glycosylation pattern, which is dependent of the

expression system. Some products contain more acidic and others more basic EPO isoforms (Macdougall and Ashenden, 2009). Epoetin-δ is special, as it was engineered by homologous recombination in human fibrosarcoma cells (HT-1080),

thus lacking N-glycolylneuraminic acid like native human EPO (Jelkmann, 2009).

In order to improve EPO treatment approach new drugs were developed with

complementary advantages. In the last decade, synthetic EPO analogs, known as

erythropoiesis-stimulating agents (ESAs), have been developed to substitute

endogenous EPO by the activation of EPO receptors in a manner identical to that of

the native hormone. Among these drugs, the novel erythropoiesis-stimulating protein

(NESP), the continuous erythropoietin receptor activator (CERA), synthetic

erythropoietin protein (SEP), erythropoietin-mimetic peptides and nonpeptides,

erythropoietin oligomers and fusion protein have been reported. Furthermore,

preclinical trials or clinical use have been performed with small orally active drugs

that stimulate endogenous EPO production by activating the EPO promoter

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NESP is produced in CHO, but differs from rHuEPO by five amino acid

residues (Ala30Asn, His32Thr, Pro87Val, Trp88Asn and Pro90Thr). This

erythropoietin analogue has two new asparagine in the middle of the consensus

sequence Asn-Xxx-Ser/Thr which results in additional attachment of two extra

N-linked oligosaccharide chains, each containing up to four terminal sialic acid residues

(Egrie and Browne, 2001, Macdougall et al., 1999).

CERA is the active ingredient of a new drug synthesized by integration of a

single large polyethylene glycol (PEG) chain into the epoetin molecule, which

increases the molecular weight to twice that of epoetin (∼60 kDa) (Macdougall, 2005); this analogue is commercially available as MIRCERA®.

The SEP is an erythropoietic polymer of 51 kDa similar to the EPO sequence

with 166-amino-acids polypeptide chain and two covalently attached polymer

moieties; this polypeptide stimulates erythropoiesis through activation of the

erythropoietin receptor (Chen et al., 2005, Kochendoerfer et al., 2003).

Other approaches in finding substitutes of EPO are EPO-mimetics that are

known as peptide and nonpeptides molecules. The EPO-mimetic peptides (EMP)

were obtained from screening random peptide-phage libraries in the search for an

agonist peptide (Barbone et al., 1999), and the EPO-mimetic nonpeptides were

obtained by selecting the residues involved in EMP1 and rHuEPO binding, which act

in the same way as EPO by dimerization of the EPO receptor.

The erythropoietin oligomers and fusion protein are derived from a cDNA

encoding fusion protein of two complete human erythropoietin domains linked by a

17-aminoacid flexible peptide (Weich et al., 1993).

Another compound proposed EPO-like properties is a fusion of EPO with

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mimetics of recombinant erythropoietin are the use of lower dose of drugs and higher

interval between administrations.

NESP has a greater biological activity and offers chronic renal failure patient a

lower dose of hormones to maintain normal hemoglobin levels (Egrie et al., 2003);

the PEG molecule integration in CERA result in a prolonged half-life, increasing

biologic activity in vivo when compared with epoetin (Lamon et al., 2009) and patients

treated once a month. The molecules from non-peptide libraries have been screened

to identify a molecule capable to bind to the erythropoietin receptor and several

constituents had been selected, but their biological activity and EPO receptor affinity

are much lower than EPO (Qureshi et al., 1999); however, Hematide, an

EPO-mimetic peptide linked to polyethylene glycol, has a long circulating half-life and

extended duration of erythropoietic effect (Nissenson et al., 2002). The oligomers and

fusion protein constructions improves treatment of anemia since after a single dose

of EPO-EPO fusion proteins results in a significant increase of hematocrit and the

fusion of EPO with immunoglobulin Fc portion could prolongs EPO in vivo half-time

(Schriebl et al., 2006).

As the expression of the erythropoietin gene is negatively controlled by the

transcription factor GATA-binding protein and is under the control of

hypoxia-inducible stimulatory transcription factor [HIF]-2, orally active compounds capable of

stimulating endogenous EPO production are in preclinical or clinical trials for

treatment of anemia. These agents include stabilizers of the HIFs that bind to the

EPO enhancer and GATA inhibitors which prevent GATA from suppressing the EPO

promoter (Jelkmann, 2007, Jelkmann, 2011).

A recent study of Bernhardt et al.(2010) using PHD-I FG-2216, a phase 1 drug

which inhibits prolyl hydroxylase, has proved that the oral administration of this drug

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patients. It is not yet known, but it can be assumed that long-term administration of

this kind of drug would also increase nHb, [Hb] and thus exercise performance. For

an anti-doping point of view, this is a setback, because it will become virtually

impossible to develop specific tests for drugs that inhibit HIF or stimulate the EPO

receptor (Lundby et al., 2012).

To examine alterations in levels of plasma protein after administration of GATA

inhibitors, Horie et al. (2011) conducted a study of proteomic analyses on mouse

plasma samples treated with GATA inhibitor K-11706. The results showed that the

expression of fetuin-B in mice plasma was increased by K-11706, but not by

recombinant human erythropoietin or hypoxia. These results suggest the potential of

proteomic-based approaches as tools to identify biomarkers for the illegal use of

novel drugs. Also, fetuin-B could be a sensitive marker for the detection of abuse of

GATA inhibitors.

Doping Control for Recombinant erythropoietin and analogues

The ESAs quickly became misused as a doping agent for endurance athletes

to improve aerobic performances, and the International Olympic Committee (IOC)

officially prohibited it in 1990 (Lasne et al., 2002). The rHuEPO is the preferred drug

for athletes seeking to artificially enhance their endurance performance because has

advantages compared to blood transfusions (autologous blood doping), including no

need for blood withdrawal, storage, transport, and reinfusion (Borrione et al., 2008). The World Anti-Doping Agency (WADA) defines blood doping as “the misuse of certain techniques and/or substances to increase one's red blood cell mass, which

allows the body to transport more O2 to muscles and therefore increase stamina and

performance” (WADA, 2013a). The excessive use of EPO can causes serious adverse side-effects as headaches and hypertension, and an increased rate of

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thrombotic events as a result of an EPO-induced rise in the hematocrit (Barroso et

al., 2008). EPO misuse could cause death and the use of doping substances in many

sports around the world has become a major public health issue. As rHuEPO have

been illicitly used for enhancement of sport performance (Diamanti-Kandarakis et al.,

2005), methods for detection of misused EPO have been established. A summary of

current methods for detection of EPO isoforms are shown in figure 1.

EPO detection methods

The detection of rHuEPO is based on two strategies: the indirect detection by

blood markers and the direct detection in serum and urine. Presently, the indirect

tests for rHuEPO detection are serum EPO concentration, serum soluble transferrin

receptor concentration (sTFr), hematocrit, percentage of reticulocytes, and a

percentage macrocytes (Delanghe et al., 2008). Based on the different behavior of

each of the parameters during and after rHuEPO treatment, two different models

were built: the “ON” model, fitting the data during treatment or shortly after, and the “OFF” model, fitting the data weeks after stopping treatment (Pascual et al., 2004). The ON model has high sensitivity during the first part and for a few days after the

end of rHuEPO administration. The OFF model becomes effective in the latter stages

of the period of performance enhancement.

At present, some federations are routinely using blood sampling. Parameters

measured are hematocrit, hemoglobin concentration, and percentage of

reticulocytes, and these are used to decide whether a nonstart ruling on a health risk

basis is issued while triggering a further confirmatory test for EPO (Pascual et al.,

2004).

Although the indirect test seems to be low cost and easier to perform, some

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instead of urine, since this one is the regular specimen obtained from athletes.

Because of rHuEPO is highly glycosylated and its carbohydrate structure is almost

identical to the natural protein, its detection is hampered (Breymann, 2000) because

a slight difference in the sugar profile between recombinant human EPO and

endogenous EPO is observed (Macdougall and Ashenden, 2009). For that reason,

an effort has been made to develop direct tests to detect the injected drug or its

metabolites using urine as specimen.

Direct tests are used to differentiate recombinant and endogenous

erythropoietin. WADA-accredited laboratories for detection of ESA doping with the

following techniques: gel electrophoretic methods (IEF-PAGE, SDS-PAGE,

SAR-PAGE), and protease tests associated with IEF-PAGE. Moreover, new approaches

have been developed to improve EPO direct detection methods, e.g.

membrane-assisted isoform immunoassay.

Gel electrophoretic methods use separation of EPO molecules and

immunological detection; and are similar to immunoassay, but in addition, allow a

visualization of the antibody-bound molecules regarding charge heterogeneity

(IEF-PAGE) or molecular mass (SDS-PAGE, SAR-(IEF-PAGE) (Reichel, 2011).

Isoelectric focusing (IEF)

A method for detecting rHuEPO in urine by electrophoresis was first described

in 1995 by Wide et al.(1995) and in 2000, Lasne and Ceaurriz (2000) introduced an

isoelectric focusing (IEF) method coupled with a technique that reduced the

nonspecific binding that accompanies immunoblotting. This is the first EPO

anti-doping approach approved by WADA and has been associated with a

blood-property-based indirect test (Reichel, 2011). In summary, this method consists of four

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chemiluminescence detection. Primarily urine aliquot is concentrated by ultrafiltration

and the sample is then applied onto an IEF gel with a pH gradient from 2 to 6. The

different isoforms of EPO are so separated in the applied electrical field on the basis

of their respective isoelectric points. Subsequently, the different EPO isoforms are

visualized by immunoblotting with monoclonal anti-EPO antibodies (Lasne, 2003),

adapted the normal blotting procedure by a (patented) double-immunoblotting

technique, to avoid the nonspecific interaction of the used secondary antibody with

urinary proteins. The primary monoclonal antibody to EPO recommended by WADA

(clone AE7A5, R&D Systems) is raised against the N-terminal 26 amino acids of

human EPO (Sue and Sytkowski, 1983). Chemiluminescence is used for the

visualization of EPO on the blot.

The evolution of criteria for the interpretation of EPO IEF profiles is directly

associated with the appearance of new EPO pharmaceuticals. When the IEF method

was published in 2000 only two types of rHuEPO (epoetin alfa and beta) were

commercially available; these could easily be differentiated from uHuEPO(Segura et

al., 2007). In 2001 darbepoetin alfa was approved and due of its higher content of

sialic acids, NESP is focused in the acidic region of the IEF gel (Catlin et al., 2002).

To adjust the detection of these three forms of non-endogenous epoetins by

IEF-PAGE the World Anti-Doping Agency issued a technical document in 2004

(TD2004EPO). A new version of the technical document was issued by WADA in

2007 (TE2007EPO). It explicitly mentioned immunoaffinity purification as an

acceptable additional step during sample preparation and switched from visual or

densitometric band intensity assessment to evaluation by densitometry only (Reichel,

2011). In 2007 epoetin delta (Dynepo) entered the European market and also

MIRCERA® (CERA) and several biosimilar epoetins were approved in Europe. Although all these EPO preparations were detectable in the pH range 2–6 of the

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PAGE method (Belalcazar et al., 2008, Lasne et al., 2009). Then, in 2009, a new

technical document was released by WADA (TD2009EPO); specifically, identification

criteria for CERA and other epoetins were added. SDS-PAGE procedure or

equivalent can be used complementarily to the IEF method for the purpose of helping

to confirm the exogenous or endogenous origin of the finding; this method has

proved especially useful for the prolonged detection of Dynepo and some biosimilar

or copy epoetins.

Sample integrity issues may pose additional complications to the detection of

recombinant proteins. For example, there is a need to protect EPO against proteases

in urine samples used in IEF. Another problem is presented by the time and

temperature dependence of blood samples to be analyzed for rHuEPO abuse. Such

samples have to be analyzed as soon as possible, preferably on-site of competition,

to avoid decline of red blood cell analytes with time and temperature (Azzazy et al.,

2005, Robinson et al., 2004).

Due the EPO IEF-PAGE method for anti-doping testing have been challenged because it is ‘low-throughput’ (Pascual et al., 2004), recently Reichel (2012a) demonstrated an improvement of the technique in which IEF-PAGE is able to run up

to 120 samples and standards on a single gel and electrophoretic chamber within the

same time-frame. Only minimal modifications of the established methodology were

necessary, i.e. using a third electrode and casting a double-sized gel. Despite IEF is

expensive and time consuming, it seems to be reproducible and reliable and,

therefore, worthwhile in the anti-doping context (Lamon et al., 2010).

IEF - Protease tests

The determination of protein- or peptide-based performance-enhancing

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decades. Adding proteases inhibitors, like pepstatin-A, is a common procedure in

routine IEF-PAGE analysis of EPO for a stability test, and so any alterations on EPO

profiles are indicative of an unstable sample (urine). In general, the instability is due

the presence of proteases, glycosidases, sulfatases or neuramidases, caused by the

proliferation of bacteria in the urine during the transportation of the samples. A high

enzymatic activity would completely empty IEF profiles in the pH range 2–6, because EPO degradation products had higher pI values.

However, difficulties in the procedure of IEF-PAGE may be caused by

purposeful addition of enzymes during the sample collection by the athlete (Reichel,

2011), because proteases can digest endogenous and recombinant EPO, as well as

other peptide hormones that are excreted by the kidneys. Therefore, a procedure

able to detect that manipulation by the athlete was developed (Thomas et al., 2009).

On Thevis et al.(2007) study, 1-D gel electrophoresis and mass spectrometric

methods were used to analyses proteases specimens in urine, and concluded that a

proteases concentration greater than 15 µg/mL is enough to detect its misuse by

athletes. However, the autolysis of proteolytic enzymes might result in inconclusive

data, showing significantly reduced urinary protein contents without remaining

proteases and, thus, an elimination of the evidence of protease addition.

Nevertheless, extra studies still remain to determine whether very low urinary protein

concentrations are indicative for protease manipulation or if such conditions are likely

under normal physiological conditions. The adulteration of urine with proteases is a

prohibited method (WADA, 2013b) and techniques have been developed for the

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SDS-PAGE

In contrast to isoelectric focusing, SDS-PAGE separates proteins according to

their apparent molecular mass. EPO glycoforms consists of weakly different

molecular masses; thus EPO bands on SDS-PAGE are usually broader than bands

of non-glycosylated proteins, e.g., urine human EPO (uHuEPO) and serum human

EPO (sHuEPO) showed a slightly lower molecular mass compared to most rHuEPOs

(such as epoetins alpha, beta, and delta). Therefore, this slight difference in migration

can be used as additional evidence to differentiate endogenous and exogenous

erythropoietins. However, in some cases, the SDS-PAGE method should be

associated with additional evidence when routine results are inconclusive (Reichel et

al., 2009b, Reichel and Gmeiner, 2010). By combining SDS-PAGE with upstream

immunoaffinity purification step the abuse of various forms of recombinant EPO can

be detected in urine (Kohler et al., 2008, Reichel et al., 2009b). Thus, since 2009,

SDS-PAGE has also been part of the WADA technical document on EPO analysis

(TD2009EPO) (Reichel, 2011).

SAR-PAGE

This method was created because of problems with detection of MIRCERA®

using SDS-PAGE, due to interference with the primary antibody used in Western Blot

and the large PEG group of the EPO analogue.

As MIRCERA® has a prolonged serum half-life and a limited excretion in urine,

the technique to be used should detect its presence in serum or plasma. The

alternative was to replace SDS with sarcosyl (SAR, sodium N-lauroylsarcosinate),

which is also an anionic detergent, but barely interacts with PEG group of

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same sensitivity as the other epoetins. This methodology can also be applied to other

analogues and rHuEpo (Reichel et al., 2009a, Reichel, 2011, Reichel, 2012b).

Membrane-assisted isoform immunoassay - EPO WGA MAIIA

The EPO WGA MAIIA method was developed by Lönnberg et al. (2012a), and

uses a test strip (dipstick) that can rapidly identify the presence of very little amounts

of rHuEPO with altered glycosylation (Lonnberg et al., 2012c). The principle of the

technique consists in the use of lectin, known as wheat germ agglutin (WGA),

bounded in a dipstick, where the interaction with erythropoietin isoforms occurs. This

specificity exists due lectins has at least one domain that possesses a highly specific

carbohydrate binding characteristics, allowing the detection of subtle differences in

the glycosylation pattern of glycoproteins. Furthermore, the method is also sensitive.

If a sample containing a mixture of endogenous and recombinant EPO, rHuEPO will

preferably bound to lectin and the endogenous isoforms will be easily released when

a solution of a specific sugar is added to compete with and elute the erythropoietin

transiently bound to the WGA zone (Ashenden et al., 2012).

According Lonnberg et al. (2012b), this technique enables to distinguish

several EPO forms in plasma and urine and only requires a few picograms of EPO,

and the detection sensitivity is superior to the current accredited IEF method.

Thereby, EPO WGA MAIIA technique shows rapid and sensitive detection of doping

with ESA, however, the test is allowed only for research purposes (Ashenden et al.,

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Perspectives of EPO doping control Fusion protein detection

In order to investigate the detectability of EPO-Fc in human blood, different

strategies were tested and developed. The detectability of EPO-Fc in human blood

have been investigated and two strategies reported presented accuracy: a couple

method consisted of enhances EPO-Fc from human serum using protein A beads

followed by a commercial EPO ELISA kit; and an immunopurification approach

followed by SDS-PAGE or SAR-PAGE and Western double-blotting with

chemiluminescence detection. The latter strategy allows the detection of EPO-Fc in

serum together with all other recombinant erythropoietins (Reichel and Thevis, 2012).

Blood cells RNA biomarkers

Since the accuracy of direct methods for EPO detection decreases after 48

hours of EPO administration, an alternative strategy for identification of doping is the

use of blood cell biomarkers to identify erythropoiesis induction by recombinant EPO

up to 60 days after use. The method is based on modified expression pattern of

blood cells detectable by RT-qPCR (Bailly-Chouriberry et al., 2010).

Conclusion

The sports world has taken significant steps in the last few years to fight off

the doping. However, the availability of recombinant products similar to endogenous

hormones, the generation of biosimilars and biological generics, analogs, and

releasing factors of currently detectable substances, and the appearance of new

designer drugs, can all lead to new doping practices against which appropriate

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need to promote a debate on an important question: to what extend developments in

the pharmaceutical industry are for therapeutic use or for deceive anti-doping?

Acknowledgements

T. F. Collares is a student of Graduate Program in Biotechnology at Federal

University of Pelotas. T. F. Collares are supported by Brazilian Capes. The authors

have no conflicts of interest that are directly relevant to the content of this review.

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Thomas, A, Kohler, M, Walpurgis, K, Schanzer, W and Thevis, M. 2009. Proteolysis

and autolysis of proteases and the detection of degradation products in doping

control. Drug Test. Anal., 1: 81-86.

WADA, World Anti-Doping Agency. Blood Doping. http://www.wada-ama.org.

Acessed 20-9-2013a.

WADA, World Anti-Doping Agency. The 2012 Prohibited List.

http://www.wada-ama.org. Acessed 20-9-2013b.

Weich, NS, Tullai, J, Guido, E, McMahon, M, Jolliffe, LK, Lopez, AF, Vadas, MA,

Lowry, PA, Quesenberry, PJ and Rosen, J. 1993. Interleukin-3/erythropoietin fusion

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Wide, L, Bengtsson, C, Berglund, B and Ekblom, B. 1995. Detection in blood and

urine of recombinant erythropoietin administered to healthy men. Med. Sci. Sports

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Figures

Figure 1 - Current detection methods of EPO isoforms.

COMPOUND NAME GROUP SYNTHESIS DIRECT DETECTION METHOD

Epoetin α/β rHuEPO CHO IEF, double immunoblotting;

Epoetin δ rHuEPO CMV promoter transfected HT-1080

cells IEF, SDS-PAGE

Epoetin ω rHuEPO BHK IEF, double immunoblotting

Darbepoetin (NESP) rHuEPO Mutated EPO transfected CHO cells IEF, double immunoblotting

CERA rHuEPO chemical synthesis IEF, SAR-PAGE

SEPs rHuEPO chemical synthesis IEF, double immunoblotting

EPO fusion proteins

(EPO-EPO, EPO-Fc, EPOβHCG) rHuEPO cDNA transfected cells IEF, double immunoblotting Hematide (peptidic) EPO mimetics (EMP) chemical synthesis ELISA, Western double blotting; DBS/MS

Non-peptidic EPO mimetics

(EMP) chemical synthesis LC/MS-MS

GATA inhibitors EPO gene activators chemical synthesis LC/MS-MS

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4 ARTIGO 2

Development of polyclonal antibodies for the detection of recombinant human erythropoietin

(Publicado no periódico African Journal of Biotechnology, v.12, p.5595-5598, 2013.) (Anexo B)

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Development of polyclonal antibodies for the detection of recombinant human erythropoietin

Collares, Thaís Farias1; Monte, Leonardo Garcia1; Campos, Vinicius Farias2; Seixas, Fabiana Kömmiling3; Collares, Tiago Veiras2; Dellagostin, Odir4; Hartleben, Cláudia

Pinho1*

1

Laboratório de Imunodiagnóstico, 2Laboratório de Embriologia Molecular e Transgênese, 3Laboratório de Genômica Funcional, 4Laboratório de Vacinologia, Núcleo de Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade

Federal de Pelotas, Brazil, 96010-900, Pelotas, RS, Brazil - P.O. Box 354.

*Corresponding author (Fax +55 53 3275-7351; E-mail, hartlebenclaudia@gmail.com)

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Abstract

Recombinant human erythropoietin (rHuEPO) is detected by using direct pharmacological assays and indirect haematological assays. However, both methods have several limitations including technical challenges and cost-related issues. The aim of this study was to develop polyclonal antibodies against rHuEPO (anti-rHuEPO pAb) that can be used in immunoassays. In this study, we purified anti-rHuEPO pAb that could be used in immunoblotting assays to efficiently detect rHuEPO. Furthermore, these anti-rHuEPO pAb could also detect rHuEPO that was expressed in a eukaryotic expression system (CHO cells). Thus, the anti-rHuEPO pAb developed in this study may be useful for rHuEPO detection.

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INTRODUCTION

Erythropoietin (EPO) is a glycoprotein hormone that is responsible for the homeostatic regulation of red blood cell production, and consequently increases tissue oxygenation (Lacombe and Mayeux, 1998). Human recombinant erythropoietin (rHuEPO) has been successfully produced in mammalian cells cultures since the late 1980s and has several therapeutic applications such as the treatment of anaemia and polycythemia in patients having chronic kidney disease, AIDS and cancer (Macdougall and Ashenden, 2009). rHuEPO was used as a performance enhancement drug by athletes participating in endurance sports, has therefore been

banned by the World Anti-Doping Agency since 1990 (Reichel and Gmeiner, 2010).

Thus, rHuEPO detection methods are used for evaluating blood doping and for disease diagnosis. The increased availability of biosimilars and uncontrolled drugs has resulted in a need for the development of reliable rHuEPO detection methods (Girard et al., 2012).

The rHuEPO detection assays either use a direct pharmacological approach, or an indirect haematological approach. However, both methods have several limitations (Diamanti-Kandarakis et al., 2005) including technical challenges and cost-related issues (Azzazy et al., 2005). Recently, several rHuEPO assays were developed for studying the pathophysiology of anaemia and polycythemia (Lonnberg et al., 2012). Immunoassays that use antibodies against rHuEPO have been immensely useful for studying the structure/function of EPO and for the sensitive detection of EPO in biological fluids (Bornemann et al., 2003; Mi et al., 2005; Sytkowski and Fisher, 1985; Wang et al., 2003).

Cell lines such as the Chinese Hamster Ovary (CHO) cell line are commonly used for producing recombinant human glycoproteins (Ghaderi et al., 2012). CHO cells have become a popular alternative to animals as an expression system for the efficient production of high quality recombinant proteins. Moreover, the glycosylation machinery of CHO cells is similar to that of human cells (Jeong et al., 2008; Stanley et al., 1996).The objective of this study was to develop polyclonal antibodies for rHuEPO detection.

MATERIALS AND METHODS

Two 6-month-old male New Zealand rabbits were immunized using rHuEPO following a 30-day adaptation period. Five subcutaneous injections were

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administered in the scapular area of each rabbit, alternating between the right and left sides. The first immunization dose contained 84 µg rHuEPO and complete Freund’s adjuvant (Sigma-Aldrich, USA). Subsequent immunizations were performed after 7, 14, 21 and 28 days using rHuEPO (84 µg) and incomplete Freund’s adjuvant (Sigma-Aldrich, USA). Prior to immunization, blood was collected to determine the antibody titres. After the last immunization, indirect ELISA was used to determine the rHuEPO antibody titres. Hyperimmune sera were obtained from animals with high antibody titre. The hyperimmune serum was stored at -20°C until required for further processing and purification. The antibodies were purified by affinity chromatography using a protein A-Sepharose CL-4B column (GE Healthcare Company, USA) according to the manufacturer’s instructions. The animals used in this study were treated in accordance with the guidelines recommended by Colégio Brasileiro de Experimentação Animal. The rHuEPO used in all experiments was EPREX® by Janssen Cilag (Issy-les-Moulinaux, France).

For performing the rHuEPO ELISA, polystyrene ELISA microtitre plates (NuncMaxiSorp®, NalgeNunc International, USA) were coated with rHuEPO (50 ng/well) and incubated overnight at 4°C. Next, the plates were washed with phosphate buffer saline with 0.05% Tween 20 (PBS-T). Then, the wells were treated with blocking buffer (PBS containing 5% skim milk). Serial dilutions of the purified anti-rHuEPO pAb were incubated with rHuEPO-coated wells. To detect the rHuEPO pAb complex, goat anti-rabbit antibody labelled with Ig-peroxidase conjugate (Sigma-Aldrich, USA) was added to the well. A substrate solution containing o-phenylenediamine (0.4 mg/mL in 0.1 M citrate buffer, pH 5.0) and 0.03% H2O2 was

added to the ELISA plate and incubated for 15 min. The reaction was stopped by adding H2SO4 (3N), and the optical densities of the solutions were measured at 492

nm using the VICTORTM X5 Multilabel Plate Reader (Perkin Elmer, USA).

Immunoblotting was performed to evaluate the specificity and sensitivity of the polyclonal antibodies. Rabbit preimmune sera and anti-EPO rabbit antibody (Sigma-Aldrich, USA) were used as the negative and the positive controls respectively. rHuEPO solutions with concentrations of 0.05-0.3 µg per well were loaded onto SDS-PAGE gels. For positive and negative controls, rHuEPO concentration of 1 µg per well was used. After electrophoresis, the rHuEPO proteins were transferred onto a nitrocellulose membrane (Millipore, USA) and then incubated at 37ºC for 1 h with anti-rHuEPO pAb that was diluted 1:10,000. Next, goat anti-rabbit Ig-peroxidase

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