Detection of Rubisco and mycotoxins as potential contaminants of a plantibody against the hepatitis B surface antigen purified from tobacco.

Antibodies have been one of the proteins widely expressed in tobacco plants for pharmaceutical purposes, which demand contaminant free preparations. Rubisco constitutes 40-60% of tobacco leaf soluble proteins; therefore it is the major potential protein contaminant of plantibodies, while mycotoxins are toxic compounds that could be introduced during the biomass production and post-harvest stages with important consequences to human health. The objective of this paper was to investigate whether Rubisco and mycotoxins are present in Plantibody HB-01 preparations used in the immunopurification of the hepatitis B surface antigen. Rubisco was purified from Nicotiana tabacum yielding 154 microg of protein per gram of leaves and purity over 95%. Among mouse monoclonal antibodies generated against this enzyme, the CBSS.Rub-2 was selected for its immunodetection. It recognizes a conserved sequential epitope of Rubisco large subunit with an affinity constant of 0.13 x 10(8)M(-1). Rubisco quantification limit was 1 microg spreading to the measurement of this contaminant less than 4% of plantibodies samples. Additionally, according to a Reverse Phase-HPLC used to measure the level of adventitiously introduced contaminants, it can be concluded that aflatoxins B1, B2, G1 and G2 were undetected in the purified Plantibody HB-01 samples.

Expression and characterization of an anti-(hepatitis B surface antigen) glycosylated mouse antibody in transgenic tobacco (Nicotiana tabacum) plants and its use in the immunopurification of its target antigen.

Transgenic plants expressing recombinant immunoglobulins have arisen as an alternative technology for the large-scale production of antibodies useful in therapeutics and in industrial processes. In the present paper we report the expression in transgenic tobacco ( Nicotiana tabacum ) of an anti-HBsAg [anti-(hepatitis B virus surface antigen)] mouse IgG1 mAb (monoclonal antibody), currently used for the industrial purification of the recombinant vaccine antigen. Using the sweet potato sporamin signal peptide, a KDEL (Lys-Asp-Glu-Leu) ER (endoplasmic reticulum) anchorage domain, and a heavy- and light-chain gene tandem construction, we generated F1 plants in which the expression of the antibody accounted for 0.5% of the total soluble proteins. The 'plantibody' (functional IgG antibody produced in plants) was easily purified by Protein A-Sepharose chromatography with a yield of approximately 35 microg/g of fresh leaf material, and its glycosylation indicated that, irrespective of the KDEL signal, the molecule is modified in both the ER and Golgi. Finally, a successful comparison of the plantibody with the ascites-derived mAb in the immunoaffinity purification of the vaccine recombinant HBsAg was performed. Taken as a whole, our results show that the large-scale production of this antibody of industrial relevance in transgenic tobacco is feasible.

Seroprevalence of hepatitis B virus infection in individuals with clinical evidence of hepatitis in Goiânia, Goiás. Detection of viral DNA and determination of subtypes.

The presence of serological markers for hepatitis B virus (HBsAg, anti-HBc IgM and Anti-HBc total) was investigated in the serum of 1,396 individuals who had clinical suspect of hepatitis. It was observed that 50.7% of the individuals were positive and, from the total of the studied individuals, 14.5% were positive for HBsAg. From these, 8.5% were also positive for anti-HBc IgM. The analysis in relation to gender showed a higher seroprevalence index among male individuals (p < 0.0001). It was observed the occurrence of subtypes adw2 (62.7%), ayw3 (23.5%), ayw2 (9.8%) and adw4 (3.9%). The viral DNA was detected in 61 (33.9%) HBsAg positive samples and in one sample positive only for anti-HBc total. These results indicate an important incidence of the HBV infection in this population, and reinforce previous studies regarding this virus in the central west region of Brazil.

[Prevalence of serologic markers of hepatitis B virus in hospital personnel]. / Prevalência de marcadores sorológicos do vírus da hepatite B em trabalhadores do serviço hospitalar.

OBJECTIVE: To verify the prevalence of the anti-HBc, anti-HBs and HBsAg markers of hepatitis B virus, and to identify the risk factors determining occupational infection with this virus among hospital personnel. METHODS: Samples of serum from 210 persons both male and female who work in different occupations at a hospital university, were analysed. The technique employed was the immunoenzymatic assay using commercial kits. RESULTS: As a control group, samples of serum from 45 volunteer blood donors were utilized. It was verified that 20.5% of the hospital personnel presented a positive reaction to at least one of the markers songht, as against 6.6% of the control group. The prevalence of each marker separately was: anti-HBc 8.1%, anti-HBs 5.2%, and HBsAg 2.9% in the hospital personnel; and 4.4%, 2.2% and 0.0% in the control group. The simultaneous presence of the anti-HBc and anti-HBs markers was detected in 4.3% of the workers. In the control group, the presence of the anti-HBc and anti-HBs markers was detected, isolately, with respective prevalences of 4.4% and 2.2%. Those who presented the highest rates of positivite reaction were: laboratory technicians 24.0%, nurses 23.6%, physicians 20.8%, and cleaning personnel 18.2%. CONCLUSIONS: The findings suggest that direct contact with patients and handling of blood and other body fluids are risk factors related to occupational infection with HBV. Therefore, it is recommended that hospital personnel be vaccinated against hepatitis B.

[HCV and HBV prevalence in hemodialyzed pediatric patients. Multicenter study]. / Prevalencia de HCV y HBV en pacientes pediátricos hemodializados. Estudio multicéntrico.

Hemodialized pediatric patients are a risk population for the hepatitis B and C virus infection. The aim of this paper was to study the serum prevalence of HBV and HCV infection in hemodialized children. We study 61 pediatric patients at hemodialisis, 12 on renal transplant, range between 2 and 20 years old (mean: 12.9 years), 23 male and 38 female. The specific anti-HCV IgC were measured by enzyme immunoassay (ELISA Abbott) and confirmed by LIA-TEK (Organon). The anti-HBV were measured by ELISA Abbott and transaminases by cinetic method (ASAT: 29 UI/L and ALT: 33 UI/L). The 19.7% of studied children were HCV (+) and 29.5% were HBV (+), 38.9% of them were HbsAg (+) and 50% anti-HBs (+). The HCV and HBV infection was more elevated in relation to the transfusion number and the hemodilisis time. The elevation of ALT/ASAT activity isn't a right infection index for HCV and HBV in this children.

Prevalencia de HCV y HBV en pacientes pediatricos hemodializados: estudio multicentrico / HCV and HBV prevalence in hemodialyzed pediatric patients: multicenter study

Los pacientes Hd constituyen una población de riesgo para la infección por los virus de las hepatitis B y C. El objetivo de este trabajo fue evaluar la prevalencia de la infección por HCV y HBV en un corte de una población de niños hemodializados. Se estudiaron 61 pacientes pediátricos en hemodiálisis, 12 de los cuales eran Tx, con edades comprendidas entre 2 y 20 años (media: 12,9), 23 varones y 38 mujeres. Se determinaron los anticuerps anti-HCV IgG por enzimoinmunoensayo (EIE) (Abbott 2 generación) y como método confirmatorio se utilizó inmunoblotting de péptidos sintéticos linearizados (LIA-TEK) (Organon). La detección de los marcadores para HBV fue por EIE y el nivel de actividad de las transaminasas se determinó por método cintético. El 19,7 por ciento de los niños estudiados fueron HCV positivos y el 29,5 por ciento fueron HBV positivos, de los cuales el 38,9 por ciento fueron HBsAg positivos el 50 por ciento Anti-HBs positivos. La infección por HCV y HBV se vió incrementada en relación al número de transfusiones y al tiempo de inclusión en Hd. La elevación de nivel de actividad de las transaminasas no fue un buen índice de infección para HBV y HCV en estos pacientes. (AU)

Prevalencia de HCV y HBV en pacientes pediatricos hemodializados: estudio multicentrico / HCV and HBV prevalence in hemodialyzed pediatric patients: multicenter study

Los pacientes Hd constituyen una población de riesgo para la infección por los virus de las hepatitis B y C. El objetivo de este trabajo fue evaluar la prevalencia de la infección por HCV y HBV en un corte de una población de niños hemodializados. Se estudiaron 61 pacientes pediátricos en hemodiálisis, 12 de los cuales eran Tx, con edades comprendidas entre 2 y 20 años (media: 12,9), 23 varones y 38 mujeres. Se determinaron los anticuerps anti-HCV IgG por enzimoinmunoensayo (EIE) (Abbott 2 generación) y como método confirmatorio se utilizó inmunoblotting de péptidos sintéticos linearizados (LIA-TEK) (Organon). La detección de los marcadores para HBV fue por EIE y el nivel de actividad de las transaminasas se determinó por método cintético. El 19,7 por ciento de los niños estudiados fueron HCV positivos y el 29,5 por ciento fueron HBV positivos, de los cuales el 38,9 por ciento fueron HBsAg positivos el 50 por ciento Anti-HBs positivos. La infección por HCV y HBV se vió incrementada en relación al número de transfusiones y al tiempo de inclusión en Hd. La elevación de nivel de actividad de las transaminasas no fue un buen índice de infección para HBV y HCV en estos pacientes.

Antigen recognition characteristics and comparative performance in immunoaffinity purification of two monoclonal antibodies specific for the hepatitis B virus surface antigen.

In this paper we describe the antigen recognition characteristics, variable region base and amino acid sequence, and performance as immunoaffinity chromatography ligands of two MAb specific to the alpha determinant of the HBsAg, derived from the same fusion. We show that the epitope recognized by CB-Hep.0 (IgM) is probably associated to an intrachain disulfide bond in the antigen. On the other hand, CB-Hep.1 (IgG2b) recognizes a heat-resistant non-conformation dependent antigenic determinant on HBsAg. PCR-cloning and sequencing of the variable regions of these two MAb indicated that both heavy chain variable regions were originated from the usage of the same germinal V and J genes. However, the outstanding differences in the size of the VH CDR3, and the absolute difference in the light chain sequences, suggest that the hybridomas were originated from different precursor B lymphocytes. With respect to their use as immunoaffinity chromatography ligands for the purification of a recombinant HBsAg, we found that the IgM immunogel exhibited increased performance with respect to amount of eluted antigen, and final recovery. This difference in overall performance could be attributed to a series of factors: the higher valence number of IgM, a dissimilar distribution of IgM and IgG in the activated gel particles, and differences in antigen recognition between both MAb. Our results suggest that IgM antibodies may be useful in immunopurification, particularly if the antigen is structurally complex and has a high density of repeating epitopes.

Infección con los virus de la hepatitis B y C en niños con trastornos congénitos de la coagulación / Infection with B and C hepatitis virus in children with congenital coagulation disorders

The prevalence of hepatitis B and C virus infections, transmitted by blood transfusions, was studied in 79 children with congenital coagulation disorders. Twenty nine percent had evidences of hepatitis B virus infection and 52 per cent evidences of hepatitis C virus infection. Older children and those with the higher number of transfussions had the highest rates of infections. It is concluded that children with congenital coagulation disorders constitute a high risk group for hepatitis B and C virus infections

Generation of murine triomas secreting bi-specific monoclonal antibodies that recognize HBsAG ad and ay subtypes.

We report the generation of murine triomas by fusing splenocytes from mice previously immunized with HBsAg ay-subtype and a hybridoma, secreting anti-HBsAg ad-subtype monoclonal antibody, which was rendered HGPRT- by induced mutagenesis with N-methyl-N'nitro-N-nitrosoguanidine. The fusion yielded a 83.8% of hybrids showing the antigen specificity of the parental hybridoma and a 16.1% of bi-specific monoclonal antibodies. One of them, coded as 1C8A5, showing a heavy chain isotype (IgG1/IgG2b) was used as capture reagent in an ultramicro-ELISA. As little as 0.78 I.U. of both HBsAg ad- and ay-subtypes could be realiably detected.

Prevalencia de los marcadores del virus de la hepatitis B en personal de alto riesgo de un hospital general / Viral B hepatitis markers: it`s prevalence among hig-risk personal in a general hospital

Se estudia a todo el personal de los departamentos y servicios de un hospital general considerados de alto riesgo para la contaminación con el virus de la hepatitis B (Laboratorio Clínico, Banco de Sangre, Clínica Estomatológica, Anatomía Patológica, Gastroenterología, Nefrología, Terapia Intensiva y Unidad Quirúrgica), realizándose determinaciones del antígeno de superficie de la hepatitis B y su anticuerpo (HBsAg y anti HBs) y una encuesta epidemiológica. La prevalencia de estos marcadores en el personal estudiado fue del 21,1 %. El Servicio de Nefrología fue el de mayor prevalencia con 50 %. Los técnicos de la salud y enfermeras fueron las categorías ocupacionales con mayor prevalencia. El tiempo de trabajo dentro del área de alto riesgo, el contacto frecuente con la sangre y con el paciente resultaron ser factores de importancia en la prevalencia de los marcadores virales

Prevalencia de marcadores sericos del virus de la hepatitis B en el personal femenino de un hospital general / Prevalence of serum markers of hepatitis B virus in female personnel of a general hospital

Hemos determinado los marcadores del virus de la hepatitis B a 209 mujeres del personal de un hospital general: 12 Médicos, 90 ATS, 70 Auxiliares de clínica y de laboratorio y 37 personas de Personal de limpieza y office. La prevalencia fue del 15,31% (32 personas). Cuatro personas (1,91%) presentaron HBs-Ag. La prevalencia fue más baja que las encontradas por otros autores de nuestro país. Las prevalencias más altas se encontraron en el personal de limpieza y office, seguido de los ATS. Por Servicios, las prevalências más altas se encontraron en el personal en contacto con sangre y hemoderivados. No se encontraron marcadores en el personal de la UVI. La prevalencia aumentó y generalmente con la edad y la antigüedad. Asociamos los bajos índices de prevalencia encontrados por nosotros con los elevados porcentajes de personal joven (33,49%) tienen menos de 30 años y con poca antigüedad en el hospital (26,31% con menos de 4 años

Prevalencia de marcadores sericos del virus de la hepatitis B en el personal femenino de un hospital general / Prevalence of serum markers of hepatitis B virus in female personnel of a general hospital

Hemos determinado los marcadores del virus de la hepatitis B a 209 mujeres del personal de un hospital general: 12 Médicos, 90 ATS, 70 Auxiliares de clínica y de laboratorio y 37 personas de Personal de limpieza y office. La prevalencia fue del 15,31% (32 personas). Cuatro personas (1,91%) presentaron HBs-Ag. La prevalencia fue más baja que las encontradas por otros autores de nuestro país. Las prevalencias más altas se encontraron en el personal de limpieza y office, seguido de los ATS. Por Servicios, las prevalÛncias más altas se encontraron en el personal en contacto con sangre y hemoderivados. No se encontraron marcadores en el personal de la UVI. La prevalencia aumentó y generalmente con la edad y la antig³edad. Asociamos los bajos índices de prevalencia encontrados por nosotros con los elevados porcentajes de personal joven (33,49%) tienen menos de 30 años y con poca antig³edad en el hospital (26,31% con menos de 4 años (AU)

[Prevalence of serum markers of hepatitis B virus in the female personnel at a general hospital]. / Prevalencia de marcadores séricos del virus de la hepatitis B en el personal femenino de un hospital general.

We have determined hepatitis B virus markers to 208 female personnel of a general hospital: 12 physicians 90 nursing personnel, 70 assistants of clinic and laboratory and 37 cleanliness, and office personnel. The prevalence was 15.31% (31 persons). Four persons (1.91%) presented HBs-Ag. The prevalence was more low that the prevalences found by other authors of our country. The highest prevalences were found in the cleanliness and office personnel, pursued of nursing personnel. Among different services, the highest prevalences were found in the personnel in contact with blood and its derivates. Not were found markers in the intensive care unit personnel. The prevalence increased generally with age and with years in occupation. We associated the low young personnel (33.49% are more low of 30 years age) and with short years occupation in the hospital (26.31% are more low of 4 years).

Dependencia de anticuerpos contra el Virus B de la hepatitis (AC-Ags VBH) en Caldas estudio en personal de salud / Prevalence of hepatitis B Virus antibodies in Caldas: study in health personnel

En el presente estudio realizado en el Departamento de Caldas, se analiza por primera vez la prevalencia de anticuerpos al antigeno de superficie del virus B de la Hepatitis (Ac-Ags VBh) mediante la tecnica de analisis inmunoenzimatico. Se procesaron 2226 sueros procedentes de personal vinculado al area de la salud y se incluyeron en dicho estudio 70 sueros controles provenientes de donadores de sangre del Hospital Universitario de Caldas. De los 2152 sueros del personal de salud, 81 fueron positivos (3.8 X 100), del grupo control, 4 fueron positivos (5.4 X 100). Las bacteriologas presentaron una positividad de 6.6 X 100 y los odontologos estudiados no tenian anticuerpos. En el estudio, se analizan los factores de sexo, edad, profesion, tiempo de vinculacion al area de trabajo, antecedentes clinicos, personales y familiares de Hepatitis e importancia de la prueba empleada

[Anti-HBs antibodies from rabbits and their use as reagents for serological tests in the diagnosis of hepatitis B]. / Anticorpos anti-HBs obtidos em coelhos e sua utilizacão como reagentes para testes sorológicos no diagnóstico da hepatite B.

By the method of affinity chromatography a partially purified antigen was obtained after passing the plasma of an asymptomatic carrier of HBsAg through a column of Sepharose 4B linked to angi-HBs. This antigen was inoculated in rabbits using a schedule of 1,0 mg in the first dose and 4 other doses of 0,5 mg with intervals of approximately 15 days. Observing that blood samples collected after the 5th inoculation showed no change in antibody levels, the animals were bled on the 62th day and these immune sera were standardized with the following tests for the detection of HBsAg: Reverse passive hemagglutination (R-PHA) - using specific gamma globulin that was obtained from rabbit sera by affinity chromatography and reaching an optimal concentration of 10 micrograms/ml to sensitise SRBC at 5% fixed in glutaraldehyde. Counter immuno electrophoresis (CIEP) - using the rabbit immune sera diluted to 1/20 as a reagent for the detection of HBsAg. The immune sera was also used to conjugate new Sepharose 4B for affinity chromatography and was found having a linking capacity of approximately 0,5 to 1,0 mg of HBsAg per ml of Sepharose after complete saturation.

Membranous glomerulonephritis and hepatitis B surface antigen in children.

Of 33 children with membranous nephropathy screened for HBs Ag, 14 were found to be HBs Ag carriers, whereas HBs Ag was detected in 3 of 170 and 4 of 100 children with glomerular and nonglomerular kidney diseases, respectively. HBs Ag was often associated with acute hepatitis at onset (five patients) or with elevated transminases values. This high incidence and the prevalence of an unusual subtype (ayw2) suggest a relationship between HBs Ag and the glomerular lesions. Using immunofluorescence, however, HBs Ag could not be detected within the deposits, so that the nature of the relationship cannot be considered as established. The clinical outcome (50% remission), the plasma complement component disturbances, and findings by immunofluorescence did not differ from those observed in children with MGN without detectable HBs Ag.