Isolation and Characterization of IgM and IgY Antibodies from Plasma of Magellanic Penguins (Spheniscus magellanicus) Camila C. Bizelli, A Sandriana R. Silva, AB Jessica D. da Costa, A Ralph E. T. Vanstreels, C Marina V. Atzingen, A Marcelo L. Santoro, D Irene Fernandes, A Jose ´ L. Cata ˜o-Dias, C and Eliana L. Faquim-Mauro AE A Laborato ´rio de Imunopatologia, Instituto Butantan, Av. Vital Brasil 1500, 05503-900, Sa ˜o Paulo, SP, Brazil C Laborato ´rio de Patologia Comparada de Animais Selvagens, Departamento de Patologia, Faculdade de Medicina Veterina ´ria e Zootecnia, Universidade de Sa ˜o Paulo, Av. Prof. Orlando Marques de Paiva 87, 05508-270, Sa ˜o Paulo, SP, Brazil D Laborato ´rio de Fisiopatologia, Instituto Butantan, Av. Vital Brasil 1500, 05503-900, Sa ˜o Paulo, SP, Brazil Received 2 December 2013; Accepted 13 October 2014; published ahead of print 14 October 2014 SUMMARY. Infectious diseases such as aspergillosis, avian malaria, and viral infections are significant threats to the conservation of penguins, leading to morbidity and mortality of these birds both in captivity and in the wild. The immune response to such infectious diseases is dependent on different mechanisms mediated by cells and soluble components such as antibodies. Antibodies or immunoglobulins are glycoproteins that have many structural and functional features that mediate distinct effector immune functions. Three distinct classes of antibodies have been identified in birds: immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin Y (IgY). In this study we aim to establish an efficient laboratory method to obtain IgM and IgY antibodies from plasma samples of healthy adult Magellanic penguins (Spheniscus magellanicus). The protocol was developed combining plasma delipidation, sequential precipitation with caprylic acid and ammonium sulfate, and size-exclusion chromatography. The efficiency of the protocol and the identity of the purified IgM and IgY antibodies were confirmed through enzyme-linked immunosorbent assay, Western blotting, one-dimensional and two-dimensional polyacrylamide gel electrophoresis, and lectin binding assay. Structural and physicochemical properties of IgM and IgY from Magellanic penguins were consistent with those of other avian species. This purification protocol will allow for more detailed studies on the humoral immunity of penguins and for the development of high specificity serologic assays to test Magellanic penguins for infectious pathogens. RESUMEN. Aislamiento y caracterizacio ´n de anticuerpos del tipo IgM e IgY del plasma de pingu ¨inos de Magallanes (Spheniscus magellanicus). Las enfermedades infecciosas, como la aspergilosis, la malaria aviar, y las infecciones virales son amenazas significativas a la conservacio ´n de los pingu ¨inos y provocan morbilidad y mortalidad de estas aves, tanto en cautiverio como en vida silvestre. La respuesta inmune a este tipo de enfermedades infecciosas depende de diferentes mecanismos mediados por ce ´lulas y componentes solubles, tales como anticuerpos. Los anticuerpos o inmunoglobulinas son glicoproteı ´nas que tienen muchas caracterı ´sticas estructurales y funcionales que median diferentes funciones inmunes efectoras. Tres clases distintas de anticuerpos se han identificado en las aves: inmunoglobulina A (IgA), inmunoglobulina M (IgM) e inmunoglobulina Y (IgY). En este estudio se pretende establecer un me ´todo de laboratorio eficiente para obtener anticuerpos IgM e IgY de muestras de plasma de pingu ¨inos de Magallanes (Spheniscus magellanicus) adultos sanos. El protocolo fue desarrollado combinando la eliminacio ´n de lı ´pidos del plasma, la precipitacio ´n secuencial con a ´cido caprı ´lico y sulfato de amonio y por cromatografı ´a de exclusio ´n por taman ˜o. La eficacia del protocolo y la identidad de los anticuerpos IgM e IgY purificados fue confirmada a trave ´s de pruebas de inmunoensayo con enzimas ligadas, por inmunoelectrotransferencia, mediante electroforesis en geles de poliacrilamida unidimensionales y bidimensionales y por el ensayo de unio ´n a la lectina. Las propiedades estructurales y fisicoquı ´micas de las inmunoglobulinas IgM e IgY de los pingu ¨inos de Magallanes fueron consistentes con las de otras especies aviares. Este protocolo de purificacio ´n permitira ´ estudios ma ´s detallados sobre la inmunidad humoral de pingu ¨inos y para el desarrollo de ensayos serolo ´gicos de alta especificidad para el ana ´lisis de los pingu ¨inos de Magallanes para la deteccio ´n de agentes pato ´genos infecciosos. Key words: penguins, Sphenisciformes, antibody, humoral immunity, protein purification, size-exclusion chromatography, electrophoresis Abbreviations: 2D 5 two-dimensional; Abs 5 antibodies; EIA 5 enzyme immunoassay; HRP 5 horseradish peroxidase; IEF 5 isoelectric focusing; IgM 5 immunoglobulin M; Igs 5 immunoglobulins; IgY 5 immunoglobulin Y; IPG 5 immobilized pH gradient; MW 5 molecular weight; PAGE 5 polyacrylamide gel electrophoresis; PBS 5 phosphate-buffered saline; PBS- T 5 PBS with 0.05% Tween 20; pI 5 isoelectric point; PI 5 putative penguin IgM; PII 5 putative penguin IgY; PIII 5 low molecular weight proteins; Ppt 5 precipitate; RIA 5 radio immunoassay; RT 5 room temperature; SDS 5 sodium dodecyl sulfate; Sup 5 supernatant; TBS 5 Tris-buffered saline Infectious diseases are significant threats to the conservation of penguins, leading to the morbidity and mortality of these birds both in captivity and in the wild (10,20,21,38,42,49). Blood parasites (7,45), aspergillosis (16,77), and viral (36) and bacterial infections (33,43) pose a significant limitation to the rehabilitation efforts of penguins and to their maintenance in captivity. In particular, avian malaria (Plasmodium sp.) is considered one of the most significant potential threats to the conservation of penguins in tropical regions, since penguins are known to be more susceptible to this disease than are most birds (35,61,66,67). The immune system plays a critical role in the elimination of pathogens and then in the maintenance of individual health through distinct mechanisms mediated by soluble components and cells B Present address: Instituto Pasteur, Av. Paulista 393, 01311-000, Sa ˜o Paulo, SP, Brazil E Corresponding author. E-mail: eliana.faquim@butantan.gov.br AVIAN DISEASES 59:79–86, 2015 79