UNIVERSIDADE FEDERAL DE PELOTAS Thaís Farias Collares

UNIVERSIDADE FEDERAL DE PELOTAS
Programa de Pós-Graduação em Biotecnologia
TESE
Desenvolvimento de ensaio imunoquímico para
detecção de doping com eritropoetina
Thaís Farias Collares
Pelotas, 2013
THAÍS FARIAS COLLARES
Desenvolvimento de ensaio imunoquímico para detecção de doping com
eritropoetina
Tese apresentada ao Programa de PósGraduação
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.
Pelotas, 2013
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
Prof. Dra. Cláudia Pinho Hartleben, Universidade Federal de Pelotas
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 PósGraduação 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.
Muito Obrigada.
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.
ABSTRACT
COLLARES, Thaís Farias. Development of immunochemical assay for detection
of doping with erythropoietin. 2013. 59f. Tese (Doutorado) - Programa de PósGraduação 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.
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
Figura 2
rHuEPO detection by anti-recombinant human erythropoietin
polyclonal antibody (rHuEPO pAb) in an immunoblotting assay.......
Immunofluorescence analysis of anti-rHuEPO pAb in CHO cells
transfected with pTARGET/EPO ………………………………………
49
50
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
WGA - Aglutinina de Germe de Trigo
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
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 O 2 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
10
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
11
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 Nglicosilação 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
12
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.
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.
3 ARTIGO 1
EPO: Advances in doping detection
(Formatado nas normas do periódico International Journal of Current Research)
(Manuscrito a ser submetido)
15
EPO: ADVANCES IN DOPING DETECTION
Collares, Thaís Farias1; Xavier, Marina Amaral 1; 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.
E-mail: [email protected]
16
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
17
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
therapeutic approaches in treating patients with anemia of chronic renal disease.
18
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 ("GATAinhibitors") or enhancer ("HIF-stabilizers"), respectively.
19
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 Nlinked 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
immunoglobulin Fc portion (Schriebl et al., 2006). The aims of analogues and
20
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 EPOmimetic 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 hypoxiainducible 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
can increase endogenous EPO synthesis in healthy volunteers and hemodialysis
21
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 O 2 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
22
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
parameters measured in blood are instable. Furthermore, blood needs to be collected
23
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, SARPAGE), and protease tests associated with IEF-PAGE. Moreover, new approaches
have been developed to improve EPO direct detection methods, e.g. membraneassisted 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 (IEFPAGE) or molecular mass (SDS-PAGE, SAR-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 antidoping approach approved by WADA and has been associated with a bloodproperty-based indirect test (Reichel, 2011). In summary, this method consists of four
major
steps:
urine
concentration,
IEF
separation,
double
blotting
and
24
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 IEFPAGE 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 IEF-
25
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
pharmaceuticals has gained increasing attention in sports drug testing in recent
26
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
detection of their misuse.
27
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
MIRCERA®, just with the protein chain and is detect by the primary antibody with the
28
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.,
2012, Reichel, 2011).
29
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
doping control and detection methods must be developed. Meanwhile, specialists
30
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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39
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 (EPOEPO, EPO-Fc, EPOβHCG)
rHuEPO
cDNA transfected cells
IEF, double immunoblotting
chemical synthesis
ELISA, Western double blotting;
DBS/MS
chemical synthesis
LC/MS-MS
Hematide (peptidic)
Non-peptidic
EPO mimetics
(EMP)
EPO mimetics
(EMP)
GATA inhibitors
EPO gene activators
chemical synthesis
LC/MS-MS
HIF stabilizers
EPO gene activators
chemical synthesis
LC/MS-MS
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)
41
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,
[email protected])
42
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.
Keywords: antibodies, rHuEPO, immunoassays, pAb
43
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 costrelated 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
44
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 (SigmaAldrich, USA) was added to the well. A substrate solution containing ophenylenediamine (0.4 mg/mL in 0.1 M citrate buffer, pH 5.0) and 0.03% H 2O2 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 (SigmaAldrich, 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 SDSPAGE 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
45
conjugate (Sigma-Aldrich, USA) was added. The bands were visualized by using
substrate/chromogen solution (0.6 mg diaminobenzidine, 0.03% nickel sulfate, 50
mM Tris-HCl at pH 8.0, and 0.03% H2O2).
The Chinese Hamster Ovary (CHO)-K1 cell line was purchased from American
Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium
supplemented with 10% fetal bovine serum and grown at 37°C in 5% CO2. Prior to
transfection, the cells were seeded in 96-well plates (104 cells/well) and incubated
until 80% confluence was reached. pTarget/EPO plasmid construct (Collares et al.,
2012), pTARGET™ (Promega, USA) and pEGFP (Clontech, USA) were prepared for
transfection according to the manufacturer’s instructions (Lipofectamine® 2000
Transfection Reagent - Life Technologies,USA). The cells were washed twice with
PBS and incubated for 4 h with lipoplex mixture. After 24 h, the lipoplexes were
removed by aspiration and the cells were fixed using methanol. The cells were then
washed twice with PBS, blocked by using PBS with 10% fetal bovine serum and then
incubated with anti-rHuEPO pAb for 2 h. After washing, the cells were incubated with
goat anti-rabbit FITC conjugated antibodies (Invitrogen, USA) for 1 h. The fluorescent
labels were visualized by using a fluorescence microscope (Olympus BX 71) with an
excitation wavelength of 450 nm. CHO cells transfected with pEGFP and stained with
a commercially available anti-EPO antibody (Sigma-Aldrich, USA) were used as
positive controls; CHO cells transfected with pTARGET and stained with rabbit sera
collected before immunization were used as negative controls.
RESULTS AND DISCUSSION
Several manufacturers produce anti-rHuEPO. However, commercially
available anti-rHuEPO antibodies remain expensive, and the appearance of new
competing manufacturers still has not led to price reduction and solution doping.
Considering the high cost for production of monoclonal antibodies, the objective of
this study was to produce cost-effective anti-rHuEPO pAb that can be used for
immunoblotting and immunofluorescence assays. First, the anti-rHuEPO pAb was
purified and used to perform indirect ELISA. The ELISA data indicated that rHuEPO
could be efficiently detected using high antibody dilutions (1:10,000). To evaluate the
anti-rHuEPO pAb detection sensitivity, an immunoblotting assay was performed
using various concentrations of rHuEPO. The anti-rHuEPO pAb (1:10,000) detection
limit was determined to be 0.1 µg rHuEPO. Bands corresponding to the molecular
46
weight of rHuEPO (30 kDa) were visualized (Figure 1C). Similar results were
obtained with the commercial anti-EPO antibody (Figure 1B). In contrast, the rabbit
sera obtained before immunization failed to detect rHuEPO (Figure 1D). Since the
erythropoietin forms epoetin α and epoetin β are both produced in CHO cell lines and
the basis for detecting rHuEPO doping relies on the glycosylation pattern of the
protein (Franz, 2009; Girard et al., 2012; Reichel and Gmeiner, 2010), we
hypothesized that anti-rHuEPO pAb could detect rHuEPO obtained from a eukaryotic
expression system. To evaluate this hypothesis, pTARGET/EPO transfected CHO
cells were stained with either anti-rHuEPO pAb (Figure 2C) or the commercial antiEPO antibody (Figure 2B) to detect rHuEPO expression. The preimmunization sera
failed to detect rHuEPO in pTARGET/EPO-transfected CHO cells (Figure 2D) and on
pTARGET transfected CHO cells (data not shown).To evaluate the transfection
efficiency and rHuEPO expression capability of CHO cells, pEGFP was used as a
transfection control (Figure 2A).
The anti-rHuEPO pAb specifically detected rHuEPO by immunofluorescence
staining and immunoblotting, which are methods that are commonly used to measure
protein expression levels and to evaluate physiological roles of proteins (BenGedalya et al., 2011; Brown et al., 1998; Valdenaire et al., 1999). Polyclonal antibody
production offers a rapid and cost-effective alternative to commercially available
monoclonal antibodies. In addition, polyclonal antibodies are known to recognize
several epitopes on the same antigen and therefore detect antigens more efficiently
than monoclonal antibodies. In conclusion, the anti-rHuEPO pAb produced in this
study may be useful for various rHuEPO detection assays.
ACKNOWLEDGEMENTS
T.F. Collares is a graduate student in the Biotechnology Program at Federal
University of Pelotas and is supported by a grant from CAPES. F.K. Seixas, T.V.
Collares, O.A. Dellagostin are research fellows of Brazilian CNPq.
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49
FIGURES
Figure 1. rHuEPO detection by anti-recombinant human erythropoietin polyclonal
antibody (rHuEPO pAb) in an immunoblotting assay. A: molecular weight standard;
B: positive control; C: anti-rHuEPO pAb (1:10,000); D: negative control.
50
Figure 2. Immunofluorescence analysis of anti-rHuEPO pAb in CHO cells transfected
with pTARGET/EPO. Panels: A, transfection control (pEGFP); B, specific staining of
CHO with commercial anti-EPO antibody; C, specific staining of CHO with antirHuEPO pAb diluted 1:10,000; D: antibody negative control. Phase contrast (a, b, c
and d) microphotographs. Scale bars represent 100 µm.
5 CONCLUSÕES
Neste estudo foi possível concluir que:
- o anticorpo policlonal anti-rHuEPO obtido reage especificamente com a proteína
rHuEPO, o que foi demostrado através do western blotting;
-
a imunofluorescência demonstrou a capacidade do anticorpo policlonal
produzido em reconhecer determinantes antigênicos em cultivos celulares
eucarióticos.
-
o anticorpo policlonal anti-rHuEPO é capaz de detectar a proteína rHuEPO
em suspensão.
-
o anticorpo policlonal anti-rHuEPO pode ser utilizado como insumo para
detecção da rHuEPO em diferentes ensaios de detecção da EPO.
53
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55
ANEXOS
Anexo A - Deposito de pedido de patente
56
Anexo B - Artigo publicado no periódico African Journal of Biotechnology
57
58
59