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Characterization of an Igm-Like Immunoglobulin from Silver Catfish

Characterization of an Igm-Like Immunoglobulin from Silver Catfish

Pesq. Vet. Bras. 36(9):819-825, setembro 2016 DOI:10.1590/S0100-736X2016000900005

Characterization of an IgM-like immunoglobulin from silver (Rhamdia quelen) serum and its use for the production of polyclonal 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 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 et characterize al. 2004, Grove and et study al. 2006a, the ontogenyTian 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- 2009).reagents 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 overnight serum Ig. (4 First,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 to components mammalian of IgM the (IgM-like) acquired sodiumsame buffer. phosphate, The column pH 7.0), was filtered incubated (0.22μm) in an orbital and 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 (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 was loaded under onto reducing the gel conditions for comparison using 5% acrylamide stacking gel overlaid on 10% acrylamide resolving In recent , knowledge on fish immune system has beenbenefited crucial from for thedeveloping characterization immunological of Ig andtools Ig-bearing to evalu- purposes.molecular weight Gels were markers stained (Amresco). by Coomassie blue R-250 and the cells. Polyclonal and monoclonal antibodies to fish Ig have proteinsRabbit obtained immunization compared with to purified silver catfishhuman serumIgM (Sigma) Ig. Three and produced in response to inoculated antigen and to monitor theate totaloccurrence and specific and prevalence concentrations of pathogenic of serum microorgaantibodies- New Zealand rabbits were primed by subcutaneous injection of nisms (Crosbie & Nowak 2002, Beelen et al. 2004, Grove et tothe 10 putative injection silver sites. catfish Boosting Ig (100µg/rabbit) immunizations emulsified were carried in FCAout al. 2006b, Li et al. 2007, Rathore et al. 2008, Suresh Babu (ratio 1:1.2). The inoculum (1.0ml) was equally distributed in 8 et al. 2008, Lim et al. 2009, Santos et al. 2009, Sood et al. 2011, Boardman et al. 2012, Hedfors et al. 2012, Purcell et rabbitswith the prior same to amount inoculation of antigen and at emulsified the time of in antigen FIA at 14injections and 28 days following priming. Blood samples were collected from the

Pesq. Vet. Bras. 36(9):819-825, setembro 2016 Rhamdia quelen) serum 821

Characterization of an IgM-like immunoglobulin from silver catfish (

- nization.to monitor the production of specific antibodies. Final bleeding peroxidaseempty space labeled on the was wells. diluted Fish serum1:20.000. was Antibodies diluted 1:100; were rabbit dilu- wasFish performed immunization by cardiac with puncture BSA. 14 days after the last immu tedpolyclonal in PBS-T antiserum 1%SK and was allowed diluted to bind 1:1000, for 60 and min goat at anti-rabbitroom tem- 100g) of both sexes were acclimatized in two self-cleaning tanks perature. Four washes were performed with PBST to remove Thirty silver catfish (80 to unbound antibodies. The reaction was developed using ortho- - containing 1000 L of continuously running water (15 fish/tank). peroxide (0.002%) for 15 min in the dark. The reaction was sto- BSAFish mixedfrom each with tank phosphate were then buffered immunized saline by (PBS, intraperitoneal pH 7.2). Blood in -phenylenediamine (OPD; 1mg/ml, Sigma) containing hydrogen 2SO4. Color development jection of BSA (200µg/fish) emulsified in FCA (1:1.2 ratio), or with pped by adding an equal volume of 2 N H - samples were collected from the caudal vein prior to (day 0) and- rentwas measuredwells, rabbit at 492nm. serum Negativeharvested controls prior to were immunization always included with at 14, 28 and 42 days after immunization to monitor the presence- and consisted of serum from non-immunized fish and, in diffe of antibodies to BSA. These blood samples and the rabbit polyclo Ethical approval. All applicable national, and/or institutional nal serum to the IgM-like Ig were used to develop an enzyme guidelinesfish Ig. for the care and use of were followed. The ex- the-linked experiment immunosorbent and found assay within (ELISA) the expected to measure range silver (dissolved catfish humoral response. Water parameters were measured throughout ethical use of animals.

3/L and total ammonia was perimental design was approved by a Committee of welfare and oxygen was 5.2±0.4; pH ranged from 7.0 to 7.2; water hardness andpellets alkalinity (42% crude were bothprotein, 45±5mg Supra, CaCO Brazil). RESULTS belowDot 0.6mg/L. blot immunoassay. Fish were fed Bovine twice serumdaily with albumin commercial (8 ug) wasfish Purification of silver catfish serum Ig dropped to nitrocellulose membrane strips and used to react with Aliquots eluted from both columns had the protein were blocked with phosphate buffered saline (PBS, pH 7.4) con- tainingserum from 0.05% fish Tween-20 immunized (PBS-T) with andBSA. 3% Nitrocellulose skim milk (PBS-TSK) membranes for content determined by the Bradford method (results not 1h with constant shaking and then allowed to react for 1h with shown) and analyzed by standard SDS-PAGE. Two main wereproteins observed of approximately in several 75fractions kDa and (Fig.1A). 28 kDa, Additional similar in SK. Membranes were washed three times, 10 min each with PBST serum from fish immunized with BSA, diluted 1:100 in PBS-T 1% size to the heavy (H) and light (L) chain of human IgM, PBST, for 1h. Membranes were washed again and incubated with shown) were observed in aliquots eluted from the BSA goatand incubatedanti-rabbit with peroxidase rabbit hyperimmune conjugated (1:1000 serum indiluted PBS-T 1:200 1% SK, in slightly smaller than the putative H chain (not 1h). After washing, membranes were incubated with peroxidase L chain were pooled, concentrated and stored at -20ºC substrate (60mg of 4-chloro-1-naphtol + 20ml of cold methanol) affinity column. Aliquots containing the putative H and

for rabbit inoculation. Western blot analysis with rabbit mixed to PBS (100ml) containing hydrogen peroxide (0.018%).- polyclonal antiserum demonstrated reactivity with the bitsThe werereaction also was included stopped as bynegative rinsing controls. the membrane with distilled purified protein and proteins of similar size from whole water. Serum from non-immunized fish and non-immunized rab- Specificitysilver catfish of serum rabbit (Fig.1B). polyclonal antiserum to silver ca- After ascertain that rabbit polyclonal antibodies were direc tfish IgM-like Ig ted to silver catfish IgM-like, the heterologous reactivity of the- - polyclonal antibodies was tested with serum from several fish species, as indicated in the figure legend. In this assay, total pro- Rabbits immunized with silver catfish IgM-like Ig pro tein content of each fish serum was adjusted to 5mg/ml and 2µl- duced a strong immune response, as detected by a dot-blot- jugatewas spotted as indicated in nitrocellulose above. membrane strips, followed by incu assay (data not shown). The identity of the Ig isolated from bationWestern with rabbit Blot topolyclonal silver catfish antibodies IgM-like. and goat After anti-rabbit SDS-PAGE, con sil- silver catfish, and the specificity of rabbit polyclonal anti- - bodies, were ascertain by a dot blot using serum from fish - columnimmunized D and with E) BSAwhereas (Fig.2). no Here,reaction rabbit was polyclonal observed antiwith tocols.ver catfish Nitrocellulose purified Igs membranes were transferred were blocked to nitrocellulose with phosphate mem body specifically detected fish immunized with BSA (Fig. 2, bufferedbranes (0.45µM) saline (PBS, using pH 7.4) a semi-dry containing apparatus 0.05% Tween-20 by standard (PBS-T) pro and 3% skim milk (PBS-TSK) for 1 h with constant shaking and then allowed to react with rabbit hiperimmune serum (1:100 in serum from pre-immunized fish (Fig.2, column C), or rabbit serum collected prior to immunization with fish Ig (Fig.2, PBST and incubated with goat anti-rabbit peroxidase conjugated column B). In addition, no reactivity was observed when (1:1000PBS-T 1% in SK,PBS-T 1h) 1% followed SK, 1h). by The three reaction washes was of then10 min developed each with as Detectionfish serum ofwas fish not anti-BSA included antibodies in the assay by (Fig.2, ELISA column A). indicated above for dot-blot. An indirect ELISA was standardized using serum col- Enzyme-linked immunosorbent assay. - The reactivity and lected from fish prior to and after immunization with BSA. potential use of rabbit anti-catfish Ig for diagnosis was evaluated by indirect ELISA using serum collected prior to and at 14, 28 Optical density (OD) obtained with pre-immunization se usingand 42 a dayscheckerboard after immunizing method. fishFlat withbottom BSA. 96-well The concentration plates were rum (day 0) was similar to background levels. Anti-BSA- coatedof antigen with and BSA antibodies (5ug/well) were diluted optimized in carbonate-bicarbonate by preliminary assays antibodies were detected in both groups of fish at 14 days post immunization (Fig.3). At 28 and 42 days post immuni- buffer (150mM Na2CO3, 348mM NaHCO3, 30mM NaN3, pH 9.6) and incubated at 4oC overnight. PBS-T 3% SK was used to block zation a significantly higher (p<0.001) anti-BSA antibodies titers was detected in fish immunized with BSA+FCA com pared to fish immunized with BSA+PBS. In the BSA+FCA Pesq. Vet. Bras. 36(9):819-825, setembro 2016 822 Luiz C. Kreutz et al.

lineatus, Hypostomus regani and Hypostomus strigaticeps).

species (Ictalurus punctatus, Pseudoplatystoma corruscans andA weaker Salminus cross-reactivity brazilienses was observed with three fish with the remaining 5 species (Piaractus mesopotamicus, Brycon amazonicus, Brycon) andhilarii, faint Leporinus or no cross-reactivity friderici, Oreo- chromis niloticus

). The putative IgM-like Ig from these fish- species were slightly different in molecular size, mostly the- L chain, as observed by SDS-PAGE and western blot analy sis (Fig.4B,C). Cross reactivity and specificity of rabbit anti -silver catfish IgM wasDISCUSSION detected solely for the H chain. The humoral branch of the acquired immune response is - - aumediated et al. 2013). mostly Igs by have Igs beensecreted isolated by B andlymphocytes characterized in res in ponse to specific antigens (Magnadottir et al. 2005, Fillatre - several fish species and anti-Ig monoclonal and polyclonal antibodies are widely applied to study the humoral immu ne system and for developing immunoassays. In this study, an IgM-like Ig was purified from silver catfish serum and- polyclonal antibodies against it were evaluated in different antigens.immunoassay aiming to further our understanding of sil ver catfish immune system and its response to inoculated

proteinsA molecular similar weight in size analysisto the H ofand the L proteins chain of eludedhuman fromIgM. the affinity columns indicated the presence of two major and L (28 kDa) chains are within the range reported for se- The presumed molecular weight of silver catfish H (75 kDa) Santos et al. 2009, Purcell et al. 2012). Additional proteins veral teleost fish Igs (Grove et al. 2006b, Bag et al. 2009,

in the vicinity of the 75 kDa major band were also observed- in some aliquots eluted from the BSA-affinity column, or detected by western blot in the serum of several fish spe etcies al. used 2004, in thisSuresh study. Babu Evidence et al. 2008) for the and existence L chain of (Espelid distinct &forms Grøntvedt of H (Palenzuela 2003, Ishikawa et al. et1996, al. 2004, Watts Feng et al. et 2001, al. 2009) Jang

Fig.1. (A (B - within teleost fish has already been reported and our data ) Coomassie blue stained reduced SDS-PAGE analysis and inindicates the molecular that this weight could alsoof a beprotein true formight silver be catfish attributed and ) western blot detection of silver catfish IgM-like immuno the other fish evaluated here. However, slight differences- globulin. Panel A lane 1: whole silver catfish serum; lane M: te moieties might account for up to 10% of the molecular size of molecular weight marker as indicated on lane 2; lane- 3: purified silver catfish immunoglobulin; lane 4: human IgM. to different pattern of glycosylation. Because carbohydra Panel B lane 1: whole silver catfish serum; lane 2 and 3 cor al. 2006, Grove et al. 2006b, Mancia et al. 2007, Feng et al. positionrespond of to molecular purified silverweight catfish markers. purified immunoglobulin weight of fish IgM-like (Magnadóttir et al. 2002, Cheng et obtained from MBP and BSA affinity columns, respectively. M: - tion2009), of the sameadditional Ig protein. proteins bands observed in our study - could be the result of differences in amino acid glycosyla- mn was used to immunize rabbits. To assure that the ra- immunized group antibody titer peaked at 28 days decrea The IgM-like Ig eluted from the immuno-affinity colu Cross-reactivitysing slightly (p < 0.05)of IgM-like at 42 days. antiserum to other fish Igs protein with Ig characteristics and that it could be used to bbit polyclonal antiserum was indeed directed towards a - The cross-reactivity of polyclonal antibodies to silver evaluate fish humoral response, we tested their binding catfish IgM-like Ig was investigated using a dot blot as properties in a dot-blot targeting BSA. In this assay, rabbit- differentsay and whole species serum (Hypostomus from twelve albopunctatus fish species, Prochilodus (Fig.4A). A polyclonal antibodies reacted specifically with serum from- strong cross-reactivity was observed with serum from 4 fish immunized with BSA, indicating that the protein isola ted by affinity chromatography had indeed binding proper Pesq. Vet. Bras. 36(9):819-825, setembro 2016 Rhamdia quelen) serum 823

Characterization of an IgM-like immunoglobulin from silver catfish (

Fig.2.goat Dot anti-rabbit blot analysis peroxidase of silver conjugated, catfish immunoglobulin as indicated for binding each lane. properties (A) Fish and serum rabbit was polyclonal omitted and antibody. membranes Nitrocellulose were incubated membranes with were spotted with 2µl of BSA (4mg/ml) and reactedB with silver catfish serum, rabbit polyclonal antibody to silver catfish IgM-like and C rabbit polyclonal and goat anti-rabbit conjugate; ( ) rabbit polyclonalD,E antibody was omitted and membranes were incubated with fish anti-BSA serum and goat anti-rabbit conjugate; ( ) membranes were incubated with serum from fish prior to immunization with BSA followed by rabbit polyclonal and goat anti-rabbit conjugate; ( ) membranes were incubated with serum from fish immunized with BSA followed by rabbit polyclonal and goat anti-rabbit conjugate. ties and that rabbit polyclonal antibodies should be useful speciesfor developing was investigated immunoassay. using Next, dot-blot the cross-reactivityand western blot. of rabbit polyclonal antibodies to serum Ig from several fish IgM-like cross-reacted with serum Ig from several other We found that rabbit polyclonalI. punctatus antibodies, Pseudoplatystoma to silver catfish corruscans, Hypostomus albopunctatus, Hypostomus regain, Siluriformesand Hypostomus fish, strigaticeps namely and with serum Ig from a Cha- Prochilodus lineatus). A weaker cross-reac- raciformesPiaractus fish (mesopotamicus, Salminus brazilienses and Leporinustivity was alsofriderici observed with Igs from three Characiformes fish ( Brycon amazo- nicus, Brycon hilarii,). No Oreochromis cross-reactivity niloticus was detected with Igs from the other fish species analyzed ( - ). Interestingly,- Western blot analysis revealed a higher degree or cross pronounced-reactivity compared contrast tobetween dot-blot, dot-blot and indicated and western that cross blot -reactivity was targeted solely to the H chain. The most Brycon amazonicus - was observed comparing the reactivity of the polyclonal antibody to ; for this species, no cross- -reactivity was observed with the native protein (dot blot) variablecompared regions to a significantthat interact cross-reactivity with antigen and following are expo de- sednaturing on the and native western protein, blot whereas analysis. constant Ig H chain regions contains might et al. 2004, Rathore et al. 2008, Bag et al. 2009, Santos et contain shared epitopes across different species (Miyadai- - al. 2009). Because polyclonal antibodies were directed to - - ward the native silver catfish Ig, a less pronounced cross -reactivity was observed by dot-blot. However, the dena Fig.3. Indirect ELISA to detect antibodies in silver catfish im turing process, required to analyze proteins by SDS-PAGE munized with BSA. Serum was collected from silver catfish might have exposed hidden Ig epitopes present mostly in- prior to or at 14, 28 and 42 days post-immunizations with the constant region that are shared by a larger number of BSA (200µg/fish) adjuvanted with FCA or mixed to PBS. fish, even within different , resulting in a higher de Each point represents the mean ±SEM (n=15). One-way gree of cross-reactivity following denaturing. In contrast, ANOVA and Bonferroni’s posttest was used to compare the polyclonal antibody reacted weakly to silver catfish L chain, data. Significant difference (p<0.001) between groups on the- and no cross-reactivity was observed with the L chain from same day of sampling is indicated by asterisks. Difference fish species analyzed here, suggesting that the primary (p<0.05) on antibody titers within the same group is indi structures of L chains are less conserved amongst fish. cated by letters. Pesq. Vet. Bras. 36(9):819-825, setembro 2016 824 Luiz C. Kreutz et al.

- A Fig.4. Cross-reactivity of rabbit polyclonal antibodies toB silver catfish IgM with immunoglobulins from other Siluriformes and Characifor immunoglobulinmes fish. ( ) Fish H serum and L was chains. spotted (C onto nitrocellulose membranes and incubated with rabbit polyclonal antibody to silver catfish IgM-like followed by goat anti-rabbit conjugate. ( ) Denaturing SDS-PAGE analysisRhamdia of fish serum quelen indicating the putativeIctalurus location punc of- tatus Pseudoplatystoma) corruscansWestern blot analysis of Piaractusserum fish mesopotamicus detected using (Pacu) rabbit; 5:polyclonal Brycon amazonicus antibody to (Matrinxâ) silver catfish; 6: SalminusIgM-like and brazilienses goat anti-rabbit (Dourado) conjugate.; 7: Brycon In all hilarii panels (Piraputanga) the serum analyzed; 8: Hypostomus were: 1: albopunctatus (Cascudo (silver catfish); verdinho) 2: ; 9: Prochilodus lineatus (Catfish); (Curimbatá) 3: ; 10: Leporinus friderici (Piau)(Pintado);; 11: Oreochromis 4: niloticus (Tilápia); 12: Hypostomus regani (Cascudo pintado); 13: Hypostomus strigaticeps (Cascudo chato).

combinations of adjuvants and bacterial antigens (Zheng et - al. 2012). Finally, the usefulness of rabbit polyclonal antibodies to - tosilver PBS catfish or adjuvanted IgM like with was FCA)demonstrated had the Ig by titer indirect evaluated ELI priorSA. For to this, and serumafter immunization. from fish immunized There was with no BSA difference (mixed In conclusion, this is the first study describing the iso- lation of and IgM-like Ig from an indigenous Brazilian fish - species and its use to produce polyclonal anti-Ig antibo on anti-BSA antibody levels at 14 days post immunization. todies indicate for the the development existence of of common immunoassays. epitopes In in addition, several However, fish immunized with BSA+FCA had a sharp in polyclonal antibodies to silver catfish IgM-like were useful crease on anti-BSA antibodies titer determined at 28 days South American rivers and lakes and should be likewise reductionpost immunization in anti-BSA followed antibodies by atiters discrete was observed but significant at 28 usedeconomically to investigate important the humoral fish species immune commonly response found in these in reduction by 42 days. In contrast, a sharp and significant- species. antibodiesdays in fish to immunized soluble antigens. with BSA Knowing mixed the with kinetics PBS, streng and ti- Acknowledgements.- - thening that in fish adjuvants are fundamental to induce to vaccine development and vaccination strategies. In ge- 476317/2012-6, and from This the studySecretaria was de funded Desenvolvimento by Conselho Econômico, Nacio ming of antibody response to inoculated antigen is central Ciêncianal de Desenvolvimentoe Tecnologia (SDECT, Científico grant #481-2500/13-2). e Tecnológico (CNPq), CAPES Brazil, provided grant a Master fellowship to Raíssa Canova (01589073029). Undergraduate fello- wship was granted to Cristian O. Nied (CNPq n. 125852/2013-4) and to followingneral, the priming,antibody asresponse observed to here,inoculated and also antigens reported occurs for Márcia Bortoluzzi (Probic/FAPERGS). Sabrina Ribeiro de Almeida (FZEA- turbotwithin few(Scophthalmus days and reaches maximus maximum) inoculated levels with 4 to different5 weeks

-USP) kindly provided fish serum samples for analysis. Pesq. Vet. Bras. 36(9):819-825, setembro 2016 Rhamdia quelen) serum 825

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