Development and characterization of IgY antibodies against synthetic peptides from Brucella abortus OMP25 and BP26 proteins / Desarrollo y caracterización de anticuerpos IgY contra péptidos sintéticos de las proteínas OMP25 y BP26 de Brucella abortus

Brucellosis is a zoonose produced by bacterial species from the Brucella genus. Its isolation and identification in food using classical microbiological techniques is not practical due to its slow growth rate. Therefore, it is necessary to establish fast and specific methods for the detection of the bacteria in food. The goal of this work was the production and characterization of monospecific polyclonal antibodies in chicken (IgY) against synthetic peptides from Brucella abortus OMP25 and BP26 proteins, suitable for an antigen-capture assay. Conformational as well as antigenic predictions were performed using the ANTHEPROT package. Chemical synthesis was carried out by the multiple manual synthesis using the t-boc strategy. The peptides were used as antigens for the preparation of polyclonal antibodies in chicken. Experimental animals produced specific antibodies against the OMP25 and BP26 peptides constructs determined by ELISA and MABA assays showing correspondence between the predictive study and the immunogenicity obtained in chicken. The IgY proved to be able to recognize B. abortus by MABA assays. The binding activity and specificity of antibodies was determined by Western blot with cell extract from B. abortus. In this study, we demonstrated that OMP25 and BP26 peptides constructs are good candidates for production of specific IgY antipeptide antibodies capable of recognizing proteins from sonicated B. abortus strain S19, indicating the potential usefulness of the IgY antibody for development of immunoassays for detection of Brucella abortus.

La brucelosis es una zoonosis producida por especies del género Brucella. El aislamiento e identificación de la bacteria en alimentos usando las técnicas clásicas de microbiología no es práctico debido a su lenta tasa de crecimiento. Por lo tanto, es necesario establecer métodos rápidos para la detección de la bacteria en alimentos. En el presente trabajo se desarrollaron y caracterizaron anticuerpos policlonales monoespecíficos en gallinas (IgY) contra péptidos sintéticos de las proteínas OMP25 y BP26 de Brucella abortus, que puedan ser utilizados en un ensayo de captura. Para ello, se realizaron estudios conformacionales y de predicción de epítopes en la selección de los péptidos, los cuales se utilizaron como antígenos para la producción de las IgY. Los animales desarrollaron anticuerpos específicos contra los péptidos, mostrando correspondencia entre los estudios predictivos y la inmunogenicidad obtenida. Las IgY reconocieron a B. abortus en un ensayo de MABA y la actividad de unión y especificidad fue determinada por western blot con extracto celular de B. abortus. En este estudio, demostramos que los péptidos de las proteínas OMP25 y BP26 de B. abortus son buenos candidatos para la producción de anticuerpos IgY especificos capaces de reconocer proteínas de extracto de B. abortus cepa S19, indicando el potencial uso de anticuerpos IgY para el desarrollo de inmunoensayos para la detección de Brucella abortus.

IgY pharmacokinetics in rabbits: implications for IgY use as antivenoms.

This paper presents the first study of chicken IgY pharmacokinetics (PK) in rabbits. We measured IgY blood serum concentrations using a specific high sensitivity ELISA method. The fast initial component observed when studying horse Fab, F(ab')2 or IgG was absent from IgY PK. During the first 80 min of observation there was only a single slow exponential decay, which sped up afterward to the point that IgY became undetectable after 216 h of observation; due to this time course, PK parameters were determined with trapezoidal integration. The most significant IgY pharmacokinetic parameters determined were (all presented as medians and their 95% confidence interval): Area Under the Curve = 183.8 (135.2, 221.5) mg·h·L(-1); Distribution volume of the central compartment·[Body Weight (BW)](-1) = 46.0 (21.7, 70.3) mL·kg(-1); Distribution volume in steady state·BW(-1) = 56.8 (44.4, 68.5) mLkg(-1); Mean Residence Time = 40.1 (33.6, 48.5) h; Total plasma clearance·BW(-1) = 1.44 (1.15, 1.66) mL·h(-1)·kg(-1). Anti IgY IgG titers determined by ELISA increased steadily after 72 h, and reached 2560 (1920, 5760) dilution(-1) at 264 h; anti-chicken IgG concentrations rose up to 3.19 (2.31, 6.17) µg/mL in 264 h. Our results show that IgY PK lacks the fast initial decay observed in other PK studies using horse IgG, F(ab')2 or Fab, remains in the body 39.0 (28.7, 47.2) % much as IgG and is ≈3 times more immunogenic that horse IgG in rabbits.

Production and evaluation of chicken antibodies against a synthetic peptide from glial growth factor.

Neuregulins (NRG) are proteins that belong to the family of epidermal growth factors. It is well established that these factors are essential for the development and maintenance of the nervous system. Due to the difficulty of purifying enough quantities of these factors and the lack of specificity from commercially available antibodies, the aim of this work was to produce antibodies against a synthetic peptide capable to detect and identify neuregulin GGFbeta isoforms. To accomplish this goal, polyclonal antibodies were raised in hens against a synthetic peptide designed from the GGFbeta1 extracellular sequence. The sequence analysis was made using different epitope-predicting programs. Our results showed that the peptide sequence selected was immunogenic because it was capable of inducing a specific type B immune response in the experimental animal model. These antibodies were also capable of recognizing a recombinant GGF protein and GGF isoforms present in different samples. Our results suggest that the development of immunoglobulin Y (IgY) using synthetic peptides represents, a valuable tool for neuroscience research.

IgY antibodies anti-Tityus caripitensis venom: purification and neutralization efficacy.

Tityus caripitensis is responsible for most of scorpion stings related to human incidents in Northeastern Venezuela. The only treatment for scorpion envenomation is immunotherapy based on administration of scorpion anti-venom produced in horses. Avian antibodies (IgY) isolated from chicken egg yolks represent a new alternative to be applied as anti-venom therapy. For this reason, we produced IgY antibodies against T. caripitensis scorpion venom and evaluated its neutralizing capacity. The anti-scorpion venom antibodies were purified by precipitation techniques with polyethylene glycol and evaluated by Multiple Antigen Blot Assay (MABA), an indirect ELISA, and Western blot assays. The lethality neutralization was evaluated by preincubating the venom together with the anti-venom prior to testing. The IgY immunoreactivity was demonstrated by a dose-dependent inhibition in Western blot assays where antibodies pre-absorbed with the venom did not recognize the venom proteins from T. caripitensis. The anti-venom was effective in neutralizing 2LD50 doses of T. caripitensis venom (97.8 mg of IgY neutralized 1 mg of T. caripitensis venom). Our results support the future use of avian anti-scorpion venom as an alternative to conventional equine anti-venom therapy in our country.

Production and evaluation of chicken antibodies against a synthetic peptide from glial growth factor / Producción y evaluación de anticuerpos de gallinas contra un péptido sintético del factor de crecimiento glial

Neuregulins (NRG) are proteins that belong to the family of epidermal growth factors. It is well established that these factors are essential for the development and maintenance of the nervous system. Due to the difficulty of purifying enough quantities of these factors and the lack of specificity from commercially available antibodies, the aim of this work was to produce antibodies against a synthetic peptide capable to detect and identify neuregulin GGFb isoforms. To accomplish this goal, polyclonal antibodies were raised in hens against a synthetic peptide designed from the GGFb1 extracellular sequence. The sequence analysis was made using different epitope-predicting programs. Our results showed that the peptide sequence selected was immunogenic because it was capable of inducing a specific type B immune response in the experimental animal model. These antibodies were also capable of recognizing a recombinant GGF protein and GGF isoforms present in different samples. Our results suggest that the development of immunoglobulin Y (IgY) using synthetic peptides represents, a valuable tool for neuroscience research.

Las Neuregulinas (NRG) son proteínas que pertenecen a la familia de los factores de crecimiento epidermal. Se ha demostrado que estos factores son esenciales para el desarrollo y mantenimiento de la funcionalidad del sistema nervioso. Debido a la dificultad para purificar estas proteínas y la falta de especificidad de los anticuerpos disponibles comercialmente, el objetivo de este trabajo fue producir anticuerpos contra un péptido sintético capaz de detectar e identificar una isoforma de la Neuregulina (GGFb). Para lograr este objetivo, se desarrollaron anticuerpos en gallinas (IgY) contra un péptido sintético diseñado a partir de la secuencia aminoacídica de la región extracelular de GGFb, utilizando programas de predicción de epítopes. Los resultados demuestran que el péptido seleccionado fue immunogénico debido a que estimuló una respuesta inmune específica tipo B en el modelo utilizado. Estos anticuerpos fueron también capaces de reconocer una proteína recombinante e isoformas de GGF presentes en diferentes muestras biológicas. Nuestros resultados demuestran el potencial valor de las inmunoglobulinas Y (IgY) contra péptidos sintéticos como una herramienta de aplicación para la investigación en neurociencia.

Producción y evaluación in vitro de IgY contra Streptococcus mutans / In vitro production and evaluation of anti-Streptococcus mutans IgY

La caries dental es una enfermedad infecciosa de alcance mundial, producida por Streptococcus mutans. Una estrategia para combatir la bacteria es previniendo su adherencia al esmalte dental mediante el uso de inmunoglobulinas de yema de huevo (IgY). En este trabajo se desarrollaron IgY contra S. mutans y se determinó su reactividad a fin de evaluar su potencial para prevenir la caries dental. Los anticuerpos fueron producidos inmunizando gallinas con un liófilo de la bacteria. Se recolectaron los huevos pre y post inmunes y se purificaron las IgY por precipitación con PEG 6000-cloroformo. El mayor nivel de IgY se obtuvo en el día 42 postinmunización, medido por un ensayo ELISA y la reactividad se evaluó por Western Blot, observándose el reconocimiento de bandas específicas entre 41 y 150 kDa que corresponden a proteínas implicadas en la adherencia a la superficie dental. Por la prueba MABA, se observó reactividad cruzada con S. salivarius. Observamos la aglutinación e inhibición del crecimiento (MIC) de S. mutans in vitro por acción de las IgY. Los resultados demuestran el potencial de estos anticuerpos para ser aplicados en ensayos de inmunización pasiva en nuestro país como una alternativa para prevenir la caries dental.

Dental caries is a worldwide infectious disease produce by Streptococcus mutants. A strategy to combat this bacterium consists in preventing its adherence to tooth enamel through the use egg yolk immunoglobulin (IgY). In this study we developed anti-S.mutans IgY and determined its reactivity to evaluate its potential for preventing dental caries. The antibodies were produced by immunizing hens with lyophilized bacteria. Pre and post immunization eggs were collected and their IgY was purified by precipitation with PEG 6000-chloroform. The highest IgY level was obtained on the 42nd day post-immunization, as measured by an ELISA test. Reactivity was determined by Western Blot, showing the recognition of specific bands between 41 and 150 kDa, which correspond to proteins involved in the adherence to dental surfaces. A MABA test showed cross-reactivity with S. salivarum. IgY activity was shown by in vitro agglutination and growth inhibition (MIC) of S. mutans. These results show the potential of these antibodies for application in passive immunization trials as an alternative to prevent dental caries.

Trypanosoma cruzi calmodulin: cloning, expression and characterization.

We have cloned and expressed calmodulin (CaM) from Trypanosoma cruzi, for the first time, to obtain large amounts of protein. CaM is a very well conserved protein throughout evolution, sharing 100% amino acid sequence identity between different vertebrates and 99% between trypanosomatids. However, there is 89% amino acid sequence identity between T. cruzi and vertebrate CaMs. The results demonstrate significant differences between calmodulin from T. cruzi and mammals. First, a polyclonal antibody developed in an egg-yolk system to the T. cruzi CaM recognizes the autologous CaM but not the CaM from rat. Second, it undergoes a larger increase in the alpha-helix content upon binding with Ca(2+), when compared to CaM from vertebrates. Finally, two classic CaM antagonists, calmidazolium and trifluoperazine, capable of inhibiting the action of CaM in mammals when assayed on the plasma membrane Ca(2+) pump, showed a significant loss of activity when assayed upon stimulation with the T. cruzi CaM.

Neuregulin-1 isoform induces mitogenesis, KCa and Ca2+ currents in PC12 cells. A comparison with sciatic nerve conditioned medium.

Neuregulin-1 (NRG-1) is an active component found in sciatic nerve conditioned medium (CM). NRG-1 is a growth and differentiation factor shown to have an effect on neuritogenesis and survival of neural cells. PC12 cells chronically treated with NRG-1 (beta1 isoform) show an increase in proliferation under low-serum condition (2.5% fetal bovine serum and 1.25% horse serum) and serum deprivation, without visible morphological changes. NRG-1 and CM treatments of PC12 cells induced an increase of voltage-activated Ca2+ currents and large-conductance calcium-activated K+ currents (KCa). AG825, a specific inhibitor for erbB2 receptor, abolishes KCa current, though Ca2+ currents were not inhibited. These results showed that NRG-1 is capable of inducing functional changes but is not sufficient on its own to have an effect on cell morphology.

Role of glypican-1 in the trophic activity on PC12 cells induced by cultured sciatic nerve conditioned medium: identification of a glypican-1-neuregulin complex.

Glypican-1 is an extracellular matrix component found by microsequencing in a medium conditioned by cultured rat-sciatic nerves (CM). This CM was concentrated by ultrafiltration and fractionated by quaternary ammonium chromatography, followed by Hi-Trap blue affinity chromatography to obtain the active fraction B1.2. Previously, we have reported a 54 kDa neuregulin (NRG) in the same B1.2 fraction [Villegas et al., Brain Res. 852 (2001) 304]. The effect of Glypican-1 on the neuron-like differentiation of PC12 cells was investigated by immunoprecipitation, Western blot and cellular image analysis. Removal of glypican-1 by immunoprecipitation with increasing concentrations of specific antibodies revealed a gradual decrease of the differentiation activity of fraction B1.2, which paralleled the results obtained by removal of the 54 kDa NRG protein. Colorless native electrophoresis and Western blot analysis was used to identify a glypican-1-NRG protein complex, which could be afterwards separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis into its individual components. Additionally, it was demonstrated that glypican-1, in cooperation with the 54 kDa NRG, is involved in the neuronal-like differentiation of PC12 cells and could play an important role on the regeneration responses of peripheral nerves.

Anticuerpos monoclonales aplicados al estudio de la leishmaniasis cutanea americana (LCA) / Monoclonal antibodies for the study of the leishmaniasis cutanea americana

Mediante la técnica de hibridoma pueden prepararse anticuerpos monoclonales específicos contra leishmaniasis, sin la expresión de reacciones cruzadas con otros protozoarios como el trypanosoma plasmadium o las microbacterias. En este estudio, según nos muestran las tablas incluidas se presentan las lesiones cutáneas localizadas, criterios para distinguir los complejos L. mexicana y L. braziliensis, protocolo de producción de anticuerpos de hibridoma e hibridomas anti-leishmaniasis