Production and evaluation of egg derived hot start antibodies

BACKGROUND: Hot start can greatly improve specificity, sensitivity and yield of PCR. Non-specific amplification can occur in PCR when reaction mixture is prepared at room temperature, because Taq DNA polymerase is active and the primers can hybridize non-specifically. Hot start Taq DNA polymerases remain inactive at room temperature and are activated after heating at 95°C preventing non-specific amplification. Monoclonal antibodies against Taq DNA polymerase is the first line of reagents used for turn on regular Taq DNA polymerase into Hot start one. The goal of this research was to produce and evaluate Hot Start antibodies derived from chicken eggs. RESULTS: We performed affinity purification of yolk immunoglobulin (IgY) and obtained polyclonal Hot Start antibodies. The yield of specific antibodies was 0.36 mg per egg or 0.2% of total yolk antibodies. The protocol for real time measurement and Hot start IgY activity assessment was developed. We found that Hot start IgY can reversibly block Taq DNA polymerase activity at 50°C and have no negative impact neither on the Taq DNA polymerase activity after denaturation nor on the reverse transcriptase. We estimated that 1.0 µg of Hot start IgY effectively blocks 5 U activity of Taq DNA polymerase. CONCLUSIONS: Egg derived Hot Start polyclonal antibodies are the cheapest source of Hot start antibodies, from one immune egg we can isolate 0.36 mg IgY, this quantity is enough for producing 1800 U activity of Hot start Taq DNA Polymerase.

CORAL SNAKE ANTIVENOM PRODUCED IN CHICKENS (Gallus domesticus) / Antiveneno de serpiente coral producido en gallinas (Gallus domesticus)

The production of anti-snake venom from large mammal's blood has been found to be low-yielding and arduous, consequently, antivenom immunoglobulins for treatment are achieved regularly as polyvalent serum. We have standardized an undemanding technique for making purified immunoglobulin IgY antivenom consisting of polyclonal antibodies against coral snake venom in the egg yolk of immunized hens. We have adapted a reported process of antibody purification from egg yolks, and achieved 90% antibody purity. The customized technique consisted of the removal of lipids from distilled water-diluted egg yolks by a freeze–thaw sequence. The specific immunoglobulins were present in the egg yolk for up to 180 days postimmunization. Therefore, by means of small venom quantities, a significant amount of immunoglobulins were found in an adequately purified state (The obtained material contained about 90% pure IgY). The antigen binding of the immunoglobulins was detected by a double immunodiffusion test. Titers of antibodies in the yolk were estimated with a serum protection assay (Median effective dose = ED50) (ED50= 477 mg/kg). Given that breeding hens is economically feasible, egg gathering is noninvasive and the purification of IgY antibodies is quick and easy, chicken immunization is an excellent alternative for the production of polyclonal antibodies. To the best of our knowledge, this is the first coral snake antivenom prepared in birds.

La producción de antiveneno de serpiente usando sangre de grandes mamíferos se ha encontrado que es de bajo rendimiento y de trabajo arduo, en consecuencia, las inmunoglobulinas antiveneno para el tratamiento se obtienen generalmente, como suero polivalente. Hemos estandarizado una técnica poco exigente para la fabricación de inmunoglobulina purificada IgY, que consistió en generar anticuerpos policlonales contra el veneno de la serpiente coral en huevos de gallinas inmunizadas. La técnica consistió en la eliminación de lípidos de las yemas del huevo, diluidas en agua y en una secuencia de congelación-descongelación. Las inmunoglobulinas específicas estuvieron presentes en la yema de huevo hasta 180 días después de la inmunización. La unión del antígeno a las inmunoglobulinas se detectó mediante un ensayo de inmunodifusión doble. Los títulos de anticuerpos en la yema fueron estimados con un ensayo de protección (dosis efectiva media = ED50). Dado que las gallinas reproductoras son económicamente viables, la recolección de huevos es no invasiva y la purificación de anticuerpos IgY es rápida y fácil, la inmunización de la gallina es una excelente alternativa para la producción de anticuerpos policlonales. A nuestro entender, esta es el primer anti-veneno contra serpiente de coral preparado en aves.

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.

Growth inhibition of Staphylococcus aureus and escherichia coli strains by neutralizing IgY antibodies from ostrich egg yolk

Ostrich raising around the world have some key factors and farming profit depend largely on information and ability of farmers to rear these animals. Non fertilized eggs from ostriches are discharged in the reproduction season. Staphylococcus aureus and Escherichia coli are microorganisms involved in animal and human diseases. In order to optimize the use of sub products of ostrich raising, non fertilized eggs of four selected birds were utilized for development of polyclonal IgY antibodies. The birds were immunized (200ug/animal) with purified recombinant staphylococcal enterotoxin C (recSEC) and synthetic recRAP, both derived from S. aureus, and recBFPA and recEspB involved in E. coli pathogenicity, diluted in FCA injected in the braquial muscle. Two subsequent immunization steps with 21 days intervals were repeated in 0,85% saline in FIA. Blood and eggs samples were collected before and after immunization steps. Egg yolk immunoglobulins were purified by precipitation with 19% sodium sulfate and 20% ammonium sulphate methodologies. Purified IgY 50µL aliquots were incubated in 850µL BHI broth containing 50µL inoculums of five strains of S. aureus and five strains of E.coli during four hours at 37ºC. Growth inhibition was evaluated followed by photometry reading (DO550nm). Egg yolk IgY preparation from hiperimmunized birds contained antibodies that inhibited significantly (p<0,05) growth of strains tested. Potential use of ostrich IgY polyclonal antibodies as a diagnostic and therapeutic tool is proposed for diseased animals.

Analysis of isotype-specific antibody responses to bovine herpesviruses 1.1 and 1.2a allows to estimate the stage of infection

Specific IgM, IgA, IgG1, IgG2, as well as neutralizing antibody responses were evaluated in sera of calves experimentally infected with two isolates of bovine herpesvirus type 1 (BoHV1) of distinct subtypes (subtype 1, BoHV1.1; subtype 2a, BoHV-1.2a). No significant differences were observed in the antibody responses induced by each BoHV-1 subtype. The antibody responses following primary acute infection were characterized by an increase in specific IgM and IgA levels between days 2 and 14 post inoculation (pi). IgG1 was detected from days 11 to 30 pi. IgG2 was detected on the sample taken on day 30 pi. Reactivation of infection following dexamethasone administration induced a significant rise in IgA levels, whereas IgG1 and IgG2 levels, which were at high levels from the beginning of the reactivation process, showed a slight alteration after corticosteroid treatment. These results suggest that it is possible to estimate the dynamics of BoHV-1 infections with basis on the analysis of class- and subclass-specific antibody responses. Such information may be particularly useful for the study of the kinetics of the infection in a herd and to aid in the adoption of appropriate control measures..

Factors affecting immunoglobulin concentration in colostrum of healthy Holstein cows immediately after delivery / Fatores que afetam o nível de imunoglobulina no colostro de vacas holandesas sadias imediatamente após o parto

This study analyzed the influence of the number of milkings, number of births, and udder quarter in immunoglobulin (Ig) concentration in the colostrum of healthy Holstein cows. It was collected two samples of colostrum by manual milking, getting the first jets to completion of bacteriological examination and immunoglobulin levels by radial immunodiffusion test in agar gel. Positive samples for bacteriological examination were excluded from this investigation. Medians of immunoglobulin's G, A and M in the colostrum collected before the first and second milking were respectively 9,200 and 6,400mg/dL (p=0.0029); 400 and 200mg/dL (p=0.0018); 800 and 400mg/dL (p=0.0001). Median immunoglobulin concentration in animals that calved once, twice or three times or in cows that calved 4 to 6 times were 6,400; 6,400; 3,200 and 11,200mg/dL IgG; 100, 200, 100 and 800mg/dL IgA ; and 400, 400, 100 and 800mg/dL IgM, respectively. Concentrations of IgG, IgA and IgM were greater in animals that calved more than 4 times (p<0.05). Medians of IgG, IgA and IgM in the right fore quarter (RF), right hind quarter (RH), left fore quarter (LF) and left hind quarter (LH) were, respectively, 7,800; 6,400; 7,800 and 6,400mg/dL; 200, 200, 200 and 200mg/dL; and 400, 400, 400 and 400mg/dL. Ig concentrations in the colostrum of Holstein cows were influenced by the number of milkings after delivery and number of lactations. These variations may be considered risk factors to passive immunity transfer to newborn calves, predisposing them to diseases and causing economic losses to dairy production.

A pesquisa avaliou a influência do número de ordenhas, número de parições e quarto mamário na concentração de imunoglobulinas (Ig) do colostro de vacas hígidas da raça Holandesa. Foram colhidas duas amostras de colostro por ordenha manual, obtendo-se os primeiros jatos para a realização do exame bacteriológico e determinação dos níveis de imunoglobulinas pelo teste de imunodifusão em gel de ágar. As amostras positivas ao exame bacteriológico foram eliminadas desta investigação. Os valores medianos obtidos para a concentração de imunoglobulinas das classes G, A e M do colostro colhido antes da primeira e segunda ordenha foram, respectivamente de 9.200 e 6.400mg/dL (p=0,0029); 400 e 200mg/ dL (p=0,0018); 800 e 400mg/dL (p=0,0001), respectivamente. Os valores medianos da concentração de imunglobulinas, nos animais com apenas 1 parto, 2, 3 ou nas vacas com 4 a 6 partos foram de 6.400, 6.400, 3.200 e 11.200mg/ dL para a IgG; 100, 200, 100 e 800mg/dL para a IgA; e 400, 400, 100 e 800mg/dL para a IgM, respectivamente. As concentrações de IgG, IgA e IgM foram superiores nos animais com mais de 4 partos (p<0,05). Os valores medianos de IgG, IgA e IgM obtidos nos quartos mamários anterior direito (AD), posterior direito (PD), anterior esquerdo (AE) e posterior esquerdo (PE) foram respectivamente 7.800, 6.400, 7.800, 6.400mg/dL; 200, 200, 200, 200mg/dL; e 400, 400, 400 e 400mg/dL, não observando-se diferenças estatísticas (p>0,05) entre os quartos mamários. Os teores de Igs do colostro de vacas Holandesas sofrem influência do número de ordenhas pós-parto e número de lactações. Estas variações podem ser consideradas fatores de risco associados à falha na transferência de imunidade passiva em bezerros neonatos, predispondo-os às doenças e ocasionando perdas produtivas á pecuária.

Anticuerpos aviares: alternativa en producción y diagnóstico / Avian antibodies: an alternative in production and diagnosis

El uso de anticuerpos para investigación y diagnóstico se realiza desde hace varias décadas en todo el mundo. Normalmente, estos anticuerpos se obtienen a partir del suero de mamíferos (roedores, caprinos, equinos, etc.) De acuerdo con el tamaño de huésped, se pueden producir pequeñas o grandes cantidades de suero, haciendo siempre sangrías regulares para su recolección. En los últimos años, se han utilizado cada vez con mayor frecuencia anticuerpos purificados a partir de huevos de gallina inmunizadas, los cuales presentan diferencias con los anticuerpos producidos en mamíferos en su estructura y características fisicoquímicas, pero además, son una alternativa que disminuye el estrés e injuria al huésped y tiene alta productividad y facilidad para su recolección. Se ha informado el uso de estos anticuerpos aviares en ensayos inmunoquímicos, producción de conjugados y en terapéutica con un éxito similar al de los anticuerpos en mamíferos y a un costo menor. En este trabajo, se hace una revisión del tema y se plantean sus posibles usos tanto en investigación y diagnóstico, como en terapia

Isolation of egg yolk immunoglobulins [IgY] by chloroform polyethylene glycol technique and assaying of antibodies against avian infectious bronchitis

The present research study was conducted to isolate the chicken egg yolk immunoglobulins [IgY] by chloroform polyethylene glycol [CPEG] technique and to measure antibody titers against avian infection bronchitis [AIB]. For this purpose 300 eggs of broiler breeder were procured from 10 different flocks [30 eggs from each], at least 3 to 4 weeks post-vaccination against avian infectious bronchitis [AIB]. The yolk of three eggs was pooled and subjected to the isolation of IgY by SPEG technique. The concentration of purified IgY was quantified by spectrophotometer and antibody titers against AIB were measured through hemagglutination inhibition [HI] test. Mean concentration of IgY in different flocks ranged from 2.65 to 3.7 g/dl with overall mean of 3.24 g/dl. The geomean antibody titers [GMT] against AIB ranged from 147 to 388 with cumulative GMT of 256. Correlation between concentration of IgY and AIB antibody titers was computed to be 0.88

Detection of immunoglobulins on bacterial surface by laser flow cytometry: analysis between Haemoplhilus influenzae type b and Vibrio cholerae 01 of healthy mother-full term newborn sera

The identification of human IgG immunoglobulins on the surface of Vibrio cholerae O1, and Haemophilus influenzae type b microorganisms was assessed via a flow cytometric technique. A group of 31 healthy mother-full term nerwborn duo sera from a non-endemic cholera area was assayed. The sera of mothers and full-term newborns against both microorganisms were compared. The mean fluorescent intensity of the samples was not different at the 0.05 significance level by paired t-test. On the other hand, the immunoglobulins of newborn and mothers for V. cholerae O1 was notably lower when compared with H. influenzae type b microorganisms (p<0.05 by paried t-test, t=-5.570 for mother' sera, and t=-7.496 for the sera of the newborns). These data provide circumstantial evidence that LFC techique would be useful on bacteria-related serology

A study of immune profile in human hydatid diseases.

A clinico haematological and immunological study was undertaken in 90 patients clinically suspected to be suffering from hydatid disease over a period of 1 year. The parameters studied included age of presentation, site of cyst localisation, haematological profile, total immunoglobins of different classes (IgG, IgM, IgA & IgE) and complement component C3, rosette forming lymphocytes, blast cell formation in response to phytohaemagglutinin (PHA-P) and concanavalin A (Con-A) and casoni test using standard methods. Twenty two out of 90 (22.44%) clinically suspected patients were surgically confirmed as hydatid disease cases. Hydatid disease occured in all age groups. Youngest case was 8 years and oldest 70 years. In 17/22 cases the cyst localised in the liver followed by lungs (3) neck (1) and kidney (1). Majority of patients (63.65%) belonged to blood group B. The mean total leucocyte and eosinophill counts were raised significantly (p < 0.001 and p < 0.05, respectively) in confirmed patients. The mean ESR value was raised in hydatid patients though, not significantly (P > 0.08). All the four classes of immunoglobulins viz. IgG, IgA, IgM, IgE and complement C3 were significantly raised in patients of hydatid disease compared to controls (P < 0.001, P < 0.002, p < 0.01 and p < 0.01, P < 0.02 respectively). The percentage of lymphocytes in peripheral blood in hydatid patients was reduced though, not significantly (P > 0.2). The absolute lymphocyte count was raised and mean percentage of T cells was reduced in patients with hydatid disease (P < 0.001 and P < 0.001, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of immunoglobulin G from Kala-Azar patients

The immunoglobulin G [lgG] of kala-azar patients was purified by ammonium sulfate precipitation and ion-exchange chromatography on DEAE-cellulose column. The purified IgG was found to be homogeneous as judge by polyacrylamide disc gel electrophoresis on 7.5% gel and SDS polyacrylamide gel electrophoresis. The molecular weight of IgG was estimated to be 158,489. The precipitation reaction was observed when the purified IgG was mixed with the serum of kala-azar patient [i.e., antigen]. Maximum precipitation was observed when IgG-antigen reaction at pH 7.0 and at 37°C. The specificity of the purified IgG was also confirmed from other serological tests such as haemagglutination and double diffusion tests

Gérmenes de transmisión sexual e inmunoglobulinas en el líquido amniótico, en madres portadoras de enfermedades de transmisión sexual / Sexually transmited germs and immunoglobulins in the amniotic fluid of mothers with sexually transmited diseases

Se desconoce el comportamiento inmunológico fetal como paciente intrauterino. El diagnóstico de estas enfermedades y el criterio de curación se realizan sólo en la madre dejando al feto fuera de éste. La presencia de inmunoglobulina M en el líquido amniótico es un indicador de infección fetal y permitiría un diagnóstico preciso de enfermedad o curación fetal correlacionando dicha medición con la presencia o ausencia de gérmenes en el líquido amniótico, estudiando así el comportamiento inmunológico fetal frente a estos microorganismos. El objetivo de este trabajo fue determinar la frecuencia de estas enfermedades de transmisión sexual en el líquido amniótico de embarazadas portadoras de gérmenes de transmisión sexual y evaluar la prevalencia y evolución de estas patologías en el feto. En las 13 amniocentesis realizadas (8 pre y post-tratamiento) no encontramos agentes de transmisión sexual, ni presencia de inmunoglobulina M. No pudimos observar lo opuesto, vale decir la presencia de gérmenes y su relación con inmunoglobulina. Se necesitan más casos para llegar a alguna conclusión acerca de la inmunología fetal y a un mejor entendimiento de las propiedades antibacterianas del líquido amniótico. Concluimos que no encontramos contaminación del líquido amniótico ni contaminación o infección fetal en embarazadas portadoras de enfermedades de transmisión sexual

Purificación de inmunoglobulina M humana / Purification of human immunoglobulin M

Utilizando un procedimiento combinado de precipitación con polietilenglicol y cromatografía de intercambio iónico se pudo obtener en forma simple IgM a partir de suero humano en buena cantidd y con un alto grado de pureza. El método es fácil y permite por tanto purificar esta IgM como antígeno en la producción de suero homólogo tan útil en inmunología clínica

Manifestaciones cutáneas en pacientes infectados con H.I.V. / Cutaneous manifestations in infected patient with H.V.I

La infección por H.I.V., constituye una enfermedad infectocontagiosa, causada por un agente viral perteneciente a la familia Retroviridae y a la subfamilia Lentivirinae, la cual presenta una significativa morbimortalidad que va en rápido ascenso. Se estudian las manifestaciones mucocutáneas de esta infección en 23 pacientes evaluados en el Servicio de Dermatología del Hospital Militar "Dr Carlos Arvelo"