Human granulocyte colony stimulating factor (hG-CSF): cloning, overexpression, purification and characterization

Background: Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli ( Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells.Results: Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-beta-D-thiogalactopyranoside ( IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture.Conclusion: The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community.

High-fat diet associated with obesity induces impairment of mouse corpus cavernosum responses

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Imunossensor amperométrico

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Microscopia de força atômica aplicada em imunoensaios

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Anti-inflammatory activity of Alchornea triplinervia ethyl acetate fraction: Inhibition of H2O2, NO and TNF-alpha

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Caracterização eletroforética e análise de subgrupo de rotavírus em rebanhos bovinos leiteiros do Estado de São Paulo

Foi realizado um estudo para determinar a ocorrência de infecção por rotavírus em rebanhos bovinos leiteiros. Foram analisadas 375 amostras de fezes de bezerros, na faixa etária 1 a 45 dias, provenientes de animais pertencentes a nove propriedades rurais, situadas em seis municípios da região nordeste do Estado de São Paulo. Destas, 193 pertenciam a animais com diarréia e 182 foram obtidas de animais clinicamente sadios. As técnicas utilizadas para a detecção de rotavírus foram o ensaio imunoenzimático (EIE) e a eletroforese em gel de poliacrilamida (EGPA). Por meio do EIE foram detectadas 11,2% (42/375) de amostras positivas, 15% delas (29/193) obtidas de animais com diarréia e 7,1% (13/182) colhidas de animais sem diarréia. A análise do perfil do genoma indicou a presença de seis eletroferótipos distintos, característicos de rotavírus do grupo A. Um único eletroferótipo foi detectado em três rebanhos, o qual permaneceu constante durante o período de amostragem. em dois rebanhos diferentes eletroferótipos foram detectados, embora com maior prevalência de um dado perfil. A caracterização das amostras positivas em subgrupos foi realizada por meio do EIE com duplo sanduíche, utilizando-se anticorpos monoclonais (MAb) específicos para antígenos de subgrupo (I e II). Foram caracterizadas como subgrupo I 52,4% (22/42) das amostras testadas, nenhuma reagiu com MAb de subgrupo II, enquanto as demais, 47,6% (20/42), não reagiram com nenhum dos dois subgrupos.

Atrazine interaction with tropical humic substances by Enzyme Linked Immunosorbent Assay

Enzyme-Linked Immunosorbent Assay (ELISA) has been evaluated by analyzing rich-humic water samples from tropical rivers. The samples were spiked with atrazine at ppb level Different pHs (4 to 9) and humic concentrations (2.5 to 40 mg L-1) were investigated. The assay performance showed a strong dependence on the pH values and amount of humic matter at low atrazine concentration. From all the conditions studied the low pH (pH 4) and high humic substances concentrations (40 mg L-1) showed the greatest influence. The IC50 value to control sample (no humic) diminished from 0.28 nmol L-1 to 0.64 nmol L-1 to humic acid solution. This effect is specially noted for the humic acid fractions, since fulvic acid fractions showed no significant change on the immunoassay results. Additionally, it has been demonstrated that at basic pH the matrix effect produced by the natural Brazilian water sample containing humic substances even at 40 mg L-1 disappears. Therefore, the ELISA method used to determine atrazine, can be employed to determine this pesticide in water samples containing humic substances without prior preparation.

Evaluation of a novel kit (TF-test) for the diagnosis of intestinal parasitic infections

Intestinal parasitic infections are currently a source of concern for Public Health agencies in developing and developed countries. Since three ovum-and-parasite stool examinations have been demonstrated to provide sensitive results, we designed a practical and economical kit (TF-Test) that is now commercially available (Immunoassay Com. Ind. Ltda., S (a) over tildeo Paulo, Brazil). This kit allows the separate collection of three fecal specimens into a preservative solution. The specimens are then pooled, double-filtered, and concentrated by a single rapid centrifugation process. The TF-Test was evaluated in four different laboratories in a study using 1,102 outpatients and individuals living in an endemic area for enteroparasitosis. The overall sensitivity found using the TF-Test (86.2-97.8%) was significantly higher (P<0.01) than the sensitivity of conventional techniques such as the Coprotest (NIL Comercio Exterior Ltda, São Paulo, Brazil) and the combination of Lutz/Hoffman, Faust, and Rugai techniques (De Carli, Diagnostico Laboratorial das Parasitoses Humanas. Metodos e Tecnicas, 1994), which ranged from 48.3% to 75.9%. When the above combined three specimen technique was repeated with three specimens collected on different days, its sensitivity became similar (P > 0.01) to that of the TF-Test. The kappa index values of agreement for the TF-Test were consistent (P < 0.01), being higher and ranking in a better position than conventional techniques. The high sensitivity, cost/benefit ratio, and practical aspects demonstrate that the TF-Test is suitable for individual diagnosis, epidemiological inquiries, or evaluation of chemotherapy in treated communities. (C) 2004 Wiley-Liss, Inc.

Differences in reactivity of paracoccidioidomycosis sera with gp43 isoforms

The glycoprotein gp43 from Paracoccidioides brasiliensis is the main antigenic component in paracoccidioidomycosis (PCM) because it is recognized by 100% of PCM patients. It has also been shown that different fungal strains produce gp43 with at least four isoform profiles. In this study, different isoform profiles from gp43, with pIs ranging from 5.8 to 8.5, were affinity purified from various P. brasiliensis (B-339, S.S., 1925 and I-9) exoantigens. Because of the isoform heterogeneity, we questioned whether those isoform profiles could be similarly recognized by acute or chronic PCM patients. By using a specific and sensitive method for detection of human IgG anti-gp43 antibodies, the monoclonal antibody capture immunoassay, we report that not all gp43 isoform profiles are equally recognized in PCM sera when anti-gp43 MAb 17c was employed as capturing antibody. Our result showed that recognition of pI 8.5 gp43 isoform was significantly lower for both acute (56%) and chronic patients (71%), compared with gp43 isoforms from the standard strain B-339. on the other hand, when anti-gp43 MAb 8a, which recognizes a different antigenic epitope was used to capture the different gp43 isoform profiles, all patient's sera reacted similarly. The results described suggest that not all the antigenic epitopes expressed by gp43 are equally present in all P. brasiliensis strains.

Immunoassays for pesticide analysis in environmental and food matrices

Immunochemical methods have increased considerably in the past years, and many examples of small and large scale studies have demonstrated the reliability of the immunotechniques for control and monitoring gf contaminant residues in different kinds of samples. Application of the immunoassay (IA) methods in pesticide residue control is an area with enormous potential for growth. The most extensively studied IA is the enzyme-linked absorbent assay (ELISA), but several other approaches, that include radioimmunoassay and immunoaffinity chromatography, have been also developed recently. In comparison with classical analytical methods, IA methods offer the possibility of highly sensitive, relatively vapid, and cost-effective measurements. This paper introduces the general IAs used until now, focusing on their use in pesticide analysis, and discussing briefly the effects of interferences from solvent residues or matrix components on the IA performance. Numerous immunochemical methods commonly used for pesticide determination in different samples such as food, crop and environmental samples are presented.

Analysis of pesticides in food and environmental samples by enzyme-linked immunosorbent assays

Enzyme-linked immunosorbent assays (ELISAs) are the most extensively studied types of immunoassay and their application in pesticide residue monitoring is an area with enormous potential for growth. In comparison with classical analytical methods, ELISA methods offer the possibility of highly sensitive, relatively rapid, and cost-effective measurements. This review introduces the general ELISA formats used, focusing on their use in pesticide analysis. Identifying and studying the effects of interferences in immunoassays is an active area of research and we discuss the matrix effects observed in several studies involving e.g. food, crop and environmental samples. The procedures to eliminate the matrix interferences are briefly discussed. (C) 1998 Elsevier B.V. B.V.

Serologic profile in hepatitis B virus and hepatitis C virus in a Brazilian liver transplant waiting list

Chronic viral hepatitis is currently the most common indication for liver transplantation (OLT). Knowing the serological profile of patients on the liver transplant waiting list (LTWL) is essential to manage prophylactic and therapeutic strategies pre- and post-OLT. The aim of this study was to determine the hepatitis B virus (HBV) and hepatitis C virus (HCV) serological profile on the LTWL.Methods. Serological data were collected from 44 candidates included on, the LTWL from May 2003 to November 2004. HBV and HCV serological profiles were performed by microenzyme immunoassay.Results. Twenty-eight patients (66.7%) lacked H13V serological markers. Anti-HBs was detected in 9.5% and was positive for HBsAg, anti-HBc, IgM anti-HBc, or HbeAg in 4.8% of patients, probably related to reactivation of chronic infection. In 7.1% of patients, the markers demonstrated serological cure of infection. In HCV patients, 41.5% were positive. There was H13V and HCV co-infection in 12.2% of patients.Conclusion. HBV infection in 21.4% of the patients corroborates the need to use more efficient protocols for prophylactic and therapeutic management pre- and post-OLT. The high prevalence of HCV infection reinforces the need to follow adequate protocols to avoid related complications and guarantee rational and universal use of more efficient drugs.

Magneto Immunoassays for Plasmodium falciparum Histidine-Rich Protein 2 Related to Malaria based on Magnetic Nanoparticles

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

A disposable chitosan-modified carbon fiber electrode for dengue virus envelope protein detection

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Utilidade dos métodos imuno-histoquímicos para o diagnóstico anátomo-patológico.

In order to determine the value of immunohistochemical staining methods for the morphologic diagnosis, we studied 949 histologic specimens sent for consultation to the Immunohistochemistry Laboratory of Department of Pathology of the Medical School of Botucatu in the period 1984-1989. All case were submitted to the immunoperoxidase staining with the methods PAP or ABC. Immunohistochemical stains confirmed the original morphologic diagnosis in 468 cases (49.3%); made the definitive diagnosis from a list of differential diagnostic possibilities in 244 cases (25.7%); provided contributory information in 74 cases (7.8%); were non-contributory in 114 cases (12%) and rendered an unsuspected diagnosis in 49 cases (5.2%). In some cases with non-contributory information the differences in methods of fixation might have led to suboptimal preservation of tissue antigens. The immunohistochemical staining may provide important and sometimes essential informations for definitive diagnosis. This technique was particularly useful for differential diagnosis between carcinoma, lymphoma and melanoma.