Pesq. Vet. Bras. 36(9):819-825, setembro 2016 DOI:10.1590/S0100-736X2016000900005 Characterization of an IgM-like immunoglobulin from silver catfish (Rhamdia quelen) serum and its use for the production of polyclonal antibodies and development of immunoassays1 Luiz C. Kreutz2*, Raíssa Canova3, Cristian O. Nied4, Márcia Bortoluzzi5 and Rafael Frandoloso2 ABSTRACT.- Kreutz L.C., Canova R., Nied C.O., Bortoluzzi M. & Frandoloso R. 2016. Cha- racterization of an IgM-like immunoglobulin from silver catfish (Rhamdia quelen) serum and its use for the production of polyclonal antibodies and development of immunoassays. Pesquisa Veterinária Brasileira 36(9):819-825. Universidade de Passo Fundo, Campus I, Bairro São José, BR-282 Km 171, Passo Fundo, RS 99052-900, Brazil. E-mail: [email protected] - Knowledge on fish immunoglobulin (Ig) characteristics and the availability of mono clonal or polyclonal antibodies to fish Igs are essential to evaluate the humoral immune response and the Ig distribution on leukocyte cells. We demonstrated that silver catfish serum Ig is composed of one immunodominant H chain with approximately 75k Da and one L chain with approximately 28 kDa, similar to human IgM. Rabbit polyclonal antibodies to the catfish IgM-like Ig recognized both the H and L chain and were useful in developing an indirect ELISA to measure the production of antibodies in fish immunized with bovine serum albumin. Dot blot and western blot cross-reactivity studies indicated a wide degree chain.of epitope The sharing results amongst presented Ig fromhere severalare central Siluriformes to the development and Characiformes of tools fish and indigenous strategies to Brazilian rivers. In these fish species, polyclonal antibodies reacted mostly with the H to investigate the antibody production to inoculated antigens and tissue distribution of Ig molecules in native fish species. Furthermore, because of the wide range of cross-reactivity, polyclonal antibodies to silver catfish IgM-like Ig might be used to develop immunoassays Rhamdia quelen, - to measure the humoral immune response in other fish species. INDEX TERMS: IgM, antibody production, silver catfish, polyclonal antibodies, im RESUMO.- [Caracterizaçãomunoassay. de imunoglobulina simi- volvimento de imunoensaios.] Informações sobre as lar a IgM em soros de jundiás (Rhamdia quelen) e sua características das imunoglobulinas (Ig) de peixes e a dis- utilização para a produção de soro policlonal e desen- ponibilidade de anticorpos mono ou policlonais são essen- ciais para avaliar a resposta imune humoral e a distribuição 1 leucocitária de Igs. Nesse trabalho nós demonstramos que Accepted for publication on June 20, 2016. a Ig do soro de jundiás é composta por uma cadeia pesa- 2 LaboratórioReceived on deFebruary Microbiologia 15, 2016. e Imunologia Aplicada, Programa de Pós- da (H) imunodominante, de aproximadamente 75kDA e de -Graduação em Bioexperimentação, Universidade de Passo Fundo (UPF), uma cadeia leve (L) de aproximadamente 28 kDa, similar Campus I, Bairro São José, BR-282 Km 171, Passo Fundo, RS 99052-900, Brazil. *Corresponding author: [email protected] à IgM humana. Anticorpos policlonais produzidos contra 3 Médica Veterinária, Mestranda do Programa de Pós-Graduação em Bio- a Ig do jundiá reconheceram a cadeia H e L e permitiram experimentação, UPF, Campus I, Bairro São José, BR-282 Km 171, Passo o desenvolvimento de um ELISA indireto para mensurar a Fundo, RS 99052-900, Brazil. produção de anticorpos em peixes imunizados com albu- 4 Graduando do Curso de Medicina Veterinária, UPF, Campus I, Bairro São mina sérica bovina. Estudos de reatividade cruzada, por José, BR-282 Km 171, Passo Fundo, RS 99052-900, Brazil. Bolsista Pibic/CNPq. 5 Curso de Ciências Biológicas, UPF, Campus I, Bairro São José, BR-282 meio de Dot blot e western blot, indicaram um alto grau de Km 171, Passo Fundo, RS 99052-900, Brazil. Bolsista FAPERGS. compartilhamento de epitopos entre as Igs de diversos pei- 819 820 Luiz C. Kreutz et al. xes Siluriformes e Caraciformes nativos do Brazil. Nestas al. 2012). Anti-immunoglobulin antibodies have also been espécies de peixes, os anticorpos policlonais reconheceram principalmente a cadeia H. Os resultados deste estudo são distribution of cells subsets bearing membrane bound Ig fundamentais para o desenvolvimento de ferramentas e (Beelenused to etcharacterize al. 2004, Grove and et study al. 2006a, the ontogeny Tian et al. and2009) tissue and estratégias para investigar a produção de anticorpos sub- - sequente à imunização e a distribuição tecidual de Igs em netic relationship amongst different species (Swennes et peixes nativos. Além disso, devido ao compartilhamento de al.in cross2007, reactivity Rathore etstudies al. 2008, to gain Bag informationet al. 2009, Santoson phyloge et al. epitopos entre as espécies de peixes avaliadas, os anticor- - pos policlonais anti Ig do jundiá poderão ser usados para desenvolver ensaios imunoenzimáticos para avaliar a res- reagents2009). Studies for immunological on silver catfish studies Ig and hampers reacting furtheranti-Ig antistu- posta imune humoral nestas espécies. bodies have not been reported yet and the lack of relevant- luation of the humoral immune response following immu- TERMOS DE INDEXAÇÃO: IgM, jundiá, Rhamdia quelen, produção anticorpos, produção de anticorpos, ensaio imunoenzimático. nization,dies on this and fish the species, functional mainly characterization those concerning of Ig-bearing the eva INTRODUCTION Here, our main goals were to isolate and characterize the subsets of leukocytes. Rhamdia quelen) is an indigenous species en- demic to South American rivers and lakes and considered a major serum Ig of silver catfish and to produce and test the Silver catfish ( use of anti-Ig polyclonal antibodies in immunological assays temperate climates (Schulz & Leuchtenberger 2006) either and their reactivityMATERIALS with serum AND Ig METHODSfrom other fish species. good candidate for intensive husbandry in neotropical and Purification and analysis of silver catfish Ig. Two different alone or commingled with other fish species (Da Silva et al. challenges2008). However, like outbreaks the continuous of infectious growth ofdiseases fish husbandry and the strategiesfrom the caudal were vein,applied pooled to isolate and allowed silver catfishto clot overnightserum Ig. (4First,oC). needand intensive to monitor production exposure systems to microorganism comes along (Secombes with new blood samples from five adult healthy silver catfish were collected stored at -20o 2008). In this scenario, knowledge of effective defense me- Serum was separated by centrifugation at 1000g for 20 min and- chanisms and developing immunobiological reagents for C until use. Igs were purified from serum using MBP affinity columns (ImmunoPure-IgM, Pierce, Rockford, Il) follo - wing manufactures instruction. Second, five adult healthy silver nentsimmunoassays are essential are much for pathogen needed. recognition and to trig- catfish were immunized with bovine serum albumin (BSA, 200µg/ incomplete adjuvant (FIA), at the same ratio. Blood was collec- The innate immune system and corresponding compo fish) mixed to Freund’s complete adjuvant (FCA, 1:1.2 ratio) and boosted at 21 and 42 days with BSA (200µg/fish) mixed to Freund 2006, Iwasaki & Medzhitov 2010). Although innate immu- ger cells responsible for acquired immunity (Magnadóttir ted from the caudal vein 14 days after the last boosting injection of infecting microorganism relies on a robust response andBSA processedcross-linked as toindicated CNBr-Activated above. Anti-BSA Sepharose fish 4B Ig (GE was Healthca purified- ne mechanisms mediate first line of defense, elimination- by an immunoaffinity chromatography column (5ml) containing re), prepared according to the manufacturer’s instructions. Silver of cells, e.g. lymphocytes, and the production of antigen catfish serum was diluted 10 times with binding buffer (20mM a-specific single tetrameric Ig which areIg similar major tocomponents mammalian of IgM the (IgM-like) acquired sodiumsame buffer. phosphate, The column pH 7.0), was filtered incubated (0.22μm) in an orbitaland then shaker applied for immune response (Magnadottir et al. 2005). In teleost fish,- by2h atgravity room into temperature. the BSA-column, Non-binding previously immunoglobulins equilibrated were with wathe- shed out with 10 columns volumes of binding buffer. Anti-BSA Ig membranehas been described bound molecule. as the predominant In addition, Ig chimeric isotype (FillatreIg genes au et al. 2013) found soluble in serum or as a lymphocyte was eluted with 10ml of elution buffer (0.1 M glycine-HCl, pH 2.7).- Fractionsds, the protein (1ml) concentration were collected of and eluted immediately fractions neutralizedwas determined with 60μl of neutralizing buffer (1 M Tris-HCl, pH 9.0). In both metho with similarity to other mammalian isotypes, namely IgD on PBS (pH 7.2) at 4oC, concentrated (Amicon Ultra-10 K, Millipo- Althoughand IgT/IgZ, recent have reports already point been outfound for in a apossible few fish involve species- byre) the and Bradford stored at method -20oC. (Bradford 1976). Purified Ig was dialyzed (Zhang et al. 2011, Fillatreau et al. 2013, Sunyer 2013). Sodium dodecyl sulfate polyacrylamide gel electrophore- sis (SDS-PAGE). matterment in of mucosal debate. immunity (Zhang et al. 2010, Salinas et Aliquots eluted from both affinity columns were al. 2011), the role of these isotypes in protection is still a analyzedgel. Human by IgMstandard (Sigma) SDS-PAGE