Development of monoclonal antibodies against prM of Dengue virus 4 / Desenvolvimento de anticorpos monoclonais para prM de Dengue vírus 4

Introduction: dengue is a most common mosquito-borne viral disease in the Americas and tropical countries. Objective: in this work, mice were hyperimmunized with DENV 4 antigen to produce monoclonal antibodies (mAbs). Methodology: DENV 4 (GenBank KC806069) was inoculated in C6/36 cell monolayers cultivated in Leibovitz's 15 medium supplemented with 5% fetal bovine serum and incubated at 28 oC. The virus stock was submitted to concentration and ultracentrifugation and stored at -80 oC until use (VC DENV 4). Balb/c mice were injected intraperitoneally with 50µg of DENV-4 and successive intraperitoneal injections of 25 µg of VCDENV 4 with Freund's incomplete adjuvant were performed. The spleen cells were fused to SP2/0 myeloma cells with PEG 1540 and distributed in 96-well microplates with Iscove's modified medium with Hipoxantina­Aminopterina­Timidina. Hybridoma screening by indirect ELISA showed positive results for six mAbs, and their characterization was performed by Western blotting and Indirect Immunofluorescence (IFI) techniques. Results: the six mAbs showed strong recognition of prM (24/29 kDa), and minor reaction to E protein (66 kDa), E/E protein dimer (105 kDa), and NS1 (49 kDa) protein in two mAbs. The use of mAbs anti-prM as a diagnostic tool using IFI has been demonstrated to detect DENV-4 antigen in infected cells or tissues. Conclusion: DENV 4 generate mAbs with strong reactivity to prM with potential use to confirm the presence of DENV 4 antigen in tissues or infected cells.

Introdução: a dengue é uma doença viral transmitida por mosquitos comumente das Américas e países tropicais. Objetivo: neste trabalho, camundongos foram hiperimunizados com antígeno DENV 4 para produzir anticorpos monoclonais (mAbs). Metodologia: DENV 4 (GenBank KC806069) foi inoculado em monocamadas de células C6 / 36 cultivadas em meio Leibovitz 15 suplementado com 5% de soro fetal bovino e incubadas a 28oC. O estoque viral foi submetido à concentração, ultracentrifugação e armazenado a -80 oC (VC DENV 4). Camundongos Balb / c foram injetados intraperitonealmente com 50 µg de VC DENV-4 e injeções intraperitoneais sucessivas de 25 µg de antigeno com adjuvante incompleto de Freund. As células do baço foram misturadas a células SP2/0 com PEG 1540 e distribuídas em microplacas de 96 poços com meio Iscove Modificado em presença de Hipoxantina ­ Aminopterina ­ Timidina. A triagem de hibridomas por ELISA indireto apresentou resultados positivos para seis mAbs, e sua caracterização foi realizada por técnicas de Western blotting e Imunofluorescência Indireta (IFI). Resultados: os seis mAbs mostraram forte reconhecimento de prM (24/29 kDa) e reação menor à proteína E (66 kDa), dímero de proteína E / E (105 kDa) e proteína NS1 (49 kDa) em dois mAbs. O uso de mAbs anti-prM como uma ferramenta de diagnóstico utilizando IFI demonstrou eficacia em detectar o antígeno DENV-4 em células ou tecidos infectados. Conclusão: o mAbs produzidos para DENV 4 demonstraram uma forte reatividade contra prM, e poderiam ser uma ferramenta de uso potencial no diagnóstico de DENV 4 .

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.

Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1

Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.

Development, characterization and clinical validation of new sensitive immunofluorometric assay for the measurement of serum thyroglobulin / Desenvolvimento, caracterização e validação clínica de um novo ensaio sensível para a dosagem da tiroglobulina sérica

OBJECTIVE: In the last decade, data published stressed the role of highly-sensitive thyroglobulin (Tg) assays in the follow-up of differentiated thyroid carcinoma (DTC) patients. The present study describes a new, highly-sensitive Tg assay, compares it with an available commercial assay, and validates it in the follow-up of DTC patients. SUBJECTS AND METHODS: The immunofluorometric high-sensitivity Tg assay is based on monoclonal and polyclonal antibodies produced at our laboratories. It was validated in 100 samples of 87 patients with DTC submitted to total thyroidectomy, 87% of whom also received radioiodine. For correlation, all samples were also tested using a commercial Tg assay (Beckman Access) with functional sensitivity (FS) of 0.1 ng/mL. RESULTS: The new method showed FS of 0.3 ng/mL. The correlation between the two methods was good (r = 0.74; p < 0.0001). The diagnostic sensitivity was 88.9%, and it was increased to 100% when combined with neck US. CONCLUSION: This new, high-sensitivity Tg assay presented a good correlation with Beckman Access assay and with the clinical outcome of the patients. The continuous availability of a validated assay is an additional advantage for long term follow-up of DTC patients. Arq Bras Endocrinol Metab. 2012;56(9):658-65.

OBJETIVO: Na última década, estudos mostraram a importância dos ensaios de tiroglobulina (Tg) com melhor sensibilidade funcional no seguimento dos pacientes com carcinoma diferenciado de tiroide (CDT). Neste estudo, descrevemos o desenvolvimento de um novo ensaio de Tg de alta sensibilidade, que foi validado no seguimento de pacientes com CDT e correlacionado com um ensaio comercialmente disponível. SUJEITOS E MÉTODOS: O ensaio imunofluorométrico de Tg baseia-se em anticorpos, um monoclonal e um policlonal desenvolvidos em nosso laboratório. Avaliamos 100 amostras de soro de 87 pacientes com CDT submetidos à tiroidectomia total, sendo que 87% deles também receberam 131I. A Tg foi dosada também em ensaio comercial (Beckman Access). RESULTADOS: A correlação entre os dois métodos foi de 0,74 (p < 0,0001). O novo ensaio mostrou uma sensibilidade funcional de 0,3 ng/mL. A sensibilidade diagnóstica foi de 88,9%, que aumentou para 100% quando associada ao ultrassom cervical (US). CONCLUSÃO: O novo método de dosagem de Tg mostra boa correlação com o ensaio comercial Beckman Access e com a evolução clínica dos pacientes. O novo ensaio será fundamental no seguimento dos nossos pacientes com CDT. Arq Bras Endocrinol Metab. 2012;56(9):658-65.

Development and characterization of a potential diagnostic monoclonal antibody against capsid protein VP1 of the chicken anemia virus

Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. In this study, a potential diagnostic monoclonal antibody against the CAV VP1 protein was developed which can precisely recognize the CAV antigen for diagnostic and virus recovery purposes. The VP1 gene of CAV encoding the N-terminus-deleted VP1 protein, VP1Nd129, was cloned into an Escherichia (E.) coli expression vector. After isopropyl-beta-D-thiogalactopyronoside induction, VP1Nd129 protein was shown to be successfully expressed in the E. coli. By performing an enzyme-linked immunoabsorbent assay using two coating antigens, purified VP1Nd129 and CAV-infected liver tissue lysate, E3 monoclonal antibody (mAb) was found to have higher reactivity against VP1 protein than the other positive clones according to the result of limiting dilution method from 64 clones. Using immunohistochemistry, the presence of the VP1-specific mAb, E3, was confirmed using CAV-infected liver and thymus tissues as positive-infected samples. Additionally, CAV particle purification was also performed using an immunoaffinity column containing E3 mAb. The monoclonal E3 mAb developed in this study will not only be very useful for detecting CAV infection and performing histopathology studies of infected chickens, but may also be used to purify CAV particles in the future.

Tissue expression and subcellular localization of Mipu1, a novel myocardial ischemia-related gene

Myocardial ischemic preconditioning up-regulated protein 1 (Mipu1), a novel zinc finger protein, was originally cloned using bioinformatic analysis and 5' RACE technology of rat heart after a transient myocardial ischemia/reperfusion procedure in our laboratory. In order to investigate the functions of Mipu1, the recombinant prokaryotic expression vector pQE31-Mipu1 was constructed and transformed into Escherichia coli M15(pREP4), and Mipu1-6His fusion protein was expressed and purified. The identity of the purified protein was confirmed by mass spectrometry. The molecular mass of the Mipu1 protein was 70.03779 kDa. The fusion protein was intracutaneously injected to immunize New Zealand rabbits to produce a polyclonal antibody. The antibody titer was approximately 1:16,000. The antibody was tested by Western blotting for specificity and sensitivity. Using the antibody, it was found that Mipu1 was highly expressed in the heart and brain of rats and was localized in the nucleus of H9c2 myogenic cells. The present study lays the foundation for further study of the biological functions of Mipu1.

Production and Characterization of Anti-Staphylococcal Toxic Shock Syndrome Toxin-1 Monoclonal Antibody / 대한진단검사의학회지

BACKGROUND: Recently the association between the virulence factors of Staphylococcus aureus and the outcome of the patients infected with the organism appears to be the subject of active investigation. Toxic shock syndrome toxin-1 (TSST-1) is thought to be a clinically more significant virulence factor than other staphylococcal toxins. We attempted to produce and characterize monoclonal antibodies to staphylococcal TSST-1. METHODS: An important epitope of TSST-1, amino acids 1-15 region, was synthesized into a peptide antigen, and Balb/c mice were immunized by intraperitoneal injection of the synthetic antigen. Hybridomas were produced by fusing immunized murine splenocytes with immortal myeloma cells. Hybridomas were cloned through a limiting dilution method. Stable cultured hybridoma was injected into the peritoneal cavity of Balb/c mice, and peritoneal fluid containing the monoclonal antibody was produced. RESULTS: One IgG2b type monoclonal antibody and two IgM type monoclonal antibodies were obtained. The IgG2b type monoclonal antibody was able to detect 5 microgram of TSST-1 with Western blot analysis and showed a strong reactivity to TSST-1 with ELISA. CONCLUSIONS: Highly immunoreactive anti-TSST-1 monoclonal antibody was produced by the use of synthesized peptide antigen. Diagnostic and protective capacity of this monoclonal antibody should be evaluated in the future.

Human Monoclonal Antibody Inhibiting Reverse Transcriptase Activity of Hepatitis B Virus Polymerase Protein / 대한소화기학회지

BACKGROUND/AIMS: To develop a novel treatment method for hepatitis B virus (HBV) infection, we aimed to make a human monoclonal antibody inhibiting reverse transcriptase (RT) activity of P protein which was important in HBV replication by using phage display technique. Therefore, we analysed the usability of human monoclonal antibody as a protein based gene therapy. METHODS: Reverse transcriptase/polymerase (RT/POL) functional motif of P protein of HBV was cloned in pMAL-c vector and expressed as maltose binding fusion protein form. The RT/POL recombinant protein (pMRT/POL) was purified by amylose resin column. Using human single chain Fv phage antibody library with 1.1x10(10) size, human antibody against pMRT/POL was selected with BIAcore panning. Selected antibody fragments were analyzed for the activity of RT inhibition. Finally, they were analyzed for the affinity with BIAcore and the complementarity determining regions with nucleotide sequencing. RESULTS: pMRT/POL recombinant protein expressed in E. coli showed RT activity, 1microgram of recombinant protein had an activity equivalent to 5 unit of MMLV RT. By BIAcore panning, we could select 3 clones; POL-A5, POL-B8 and POL-B12. Each clone's RT inhibiting activity were 52-82%, affinity against antigen were 8.15x10(-8) M to 1.75x10(-6) M. CONCLUSIONS: Human monoclonal antibodies produced in this study showed low affinity, but efficiently inhibited the activity of RT in vitro. If POL-A5, POL-B8, and POL-B12 can be converted to intracellular antibody form, it can be used for protein-based gene therapy by inhibiting the replication through the neutralization of polymerase protein of HBV.

Production of monoclonal antibodies against Streptococcus mutans antigens

Several studies have been conducted in the last decades aiming to obtain an anti-caries vaccine, however some studies have demonstrated cross reactivity between Streptococcus mutans surface antigens and the human cardiac tissue. In this work, the reactivity of five anti-Streptococcus mutans monoclonal antibodies (MoAb) (24A, 56G, C8, E8 and F6) was tested against oral streptococci, cardiac antigens and skeletal and cardiac myosins, aiming to evaluate the specificity of these MoAb. The hybrid producers of immunoglobulins of the IgG2b class were cloned by limit dilution and expanded in vivo. MoAb were tested by ELISA. The hybrid 24A reacted with S. mutans CCT 1910, S. salivarius CCT 0365 and S. pyogenes T23. No reactivity difference was observed among the tested species. Cross reactivity with heart and cardiac myosin was not confirmed and only reaction with myosin of skeletal muscle was observed (p = 0.0381). The hybrid 56G reacted with all the tested microorganisms and there was statistically significant difference between S. mutans and S. pyogenes T23 (p < 0.001). This hybrid also reacted with myosin of skeletal muscle (p = 0.0095). C8, E8 and F6 presented low reactivity against oral streptococci strains and no reactivity against cardiac antigens. The data of this study showed that the 24A and 56G anti-S. mutans MoAb presented reactivity with S. pyogenes and S. salivarius, reinforcing the occurrence of common antigens between these species. The tested MoAb presented low cross-reactivity with myosin of skeletal muscle, but anti-heart activity could not be confirmed.

Diversos estudos foram realizados nas últimas décadas com o intuito de se obter uma vacina anticárie dentária, mas alguns trabalhos têm demonstrado reatividade cruzada entre antígenos de superfície de Streptococcus mutans e tecido cardíaco humano. Neste trabalho, foi testada a reatividade de cinco anticorpos monoclonais (AcMo) anti-Streptococcus mutans (24A, 56G, C8, E8 e F6) contra estreptococos orais, antígenos cardíacos e miosinas esquelética e cardíaca, no intuito de avaliar a especificidade desses AcMo. Os híbridos produtores de imunoglobulinas da classe IgG2b foram clonados por diluição limite e expandidos in vivo. Os AcMo foram testados por ELISA. O híbrido 24A reagiu com S. mutans CCT 1910, S. salivarius CCT 0365 e S. pyogenes T23. Nenhuma diferença de reatividade foi detectada entre as espécies analisadas. Reatividade cruzada com coração e miosina cardíaca não foi confirmada, existindo somente reação com miosina de músculo esquelético (p = 0,0381). O híbrido 56G reagiu com todos os microrganismos testados e houve diferença estatisticamente significante entre S. mutans e S. pyogenes T23 (p < 0,001). Este híbrido também reagiu com miosina de músculo esquelético (p = 0,0095). C8, E8 e F6 apresentaram baixa reatividade contra cepas de estreptococos orais e nenhuma reatividade com antígenos cardíacos. Os dados deste trabalho demonstraram que os AcMo 24A e 56G anti-S. mutans reagiram com S. pyogenes e S. salivarius, confirmando a existência de antígenos comuns entre essas espécies. Esses AcMo avaliados apresentaram baixa reatividade cruzada com miosina de músculo esquelético, porém a atividade anticoração não foi confirmada.

Production, characterization, and application of antibodies against heat-labile type-I toxin for detection of enterotoxigenic Escherichia coli

Strains of enterotoxigenic Escherichia coli (ETEC) are responsible for significant rates of morbidity and mortality among children, particularly in developing countries. The majority of clinical and public health laboratories are capable of isolating and identifying Salmonella, Shigella, Campylobacter, and Escherichia coli O157:H7 from stool samples, but ETEC cannot be identified by routine methods. The method most often used to identify ETEC is polymerase chain reaction for heat-stable and heat-labile enterotoxin genes, and subsequent serotyping, but most clinical and public health laboratories do not have the capacity or resources to perform these tests. In this study, polyclonal rabbit and monoclonal mouse IgG2b antibodies against ETEC heat-labile toxin-I (LT) were characterized and the potential applicability of a capture assay was analyzed. IgG-enriched fractions from rabbit polyclonal and the IgG2b monoclonal antibodies recognized LT in a conformational shape and they were excellent tools for detection of LT-producing strains. These findings indicate that the capture immunoassay could be used as a diagnostic assay of ETEC LT-producing strains in routine diagnosis and in epidemiological studies of diarrhea in developing countries as enzyme linked immunosorbent assay techniques remain as effective and economical choice for the detection of specific pathogen antigens in cultures.

Evolution of PTH assays / Evolução dos ensaios para paratormônio

PTH metabolism is complex and the circulating forms include the intact 1-84 molecule as well as several carboxyl-terminal fragments. The first generation of PTH assays included several types of competitive assays, with specificities that spanned carboxyl, mid-region and amino-terminal portions of the molecule. The limitations of these assays and the methodological evolution led to the description of 2nd generation non-competitive immunometric assays for PTH in the late 80's, based on the recognition of the PTH molecule by two different antibodies, one directed against de amino-terminal and other against the carboxyl-terminal segments. The observation that in some circumstances "long" carboxyl-terminal segments were also measured by 2nd generation assays led to the development of 3rd generation assays based on amino-terminal specific antibodies that are specific for the first amino acids, measuring only the molecular forms that activate PTH1R. The practical and cost-benefit advantages of these assays are still debatable. The recent observation that carboxyl-terminal fragments of PTH have biological activity via a distinct receptor than PTH1R, points to the future need of more than one assay in order to evaluate parathyroid hormone function.

O metabolismo do PTH e complexo e as formas circulantes incluem o PTH 1-84, assim como fragmentos C-terminal. A primeira geração de ensaios para o PTH incluía vários ensaios competitivos com especificidades para as regiões carboxi, meio da molécula e amino-terminal. A limitação destes ensaios e a evolução metodológica, levaram ao desenvolvimento dos ensaios não competitivos de 2ª. geração no final dos anos 80, baseados no reconhecimento por dois anticorpos diferentes, contra a porção amino e carboxi-terminal respectivamente. A observação que em algumas circunstâncias segmentos carboxiterminais longos também eram detectados, levou ao desenvolvimento dos ensaios de 3ª. geração, baseados em anticorpos específicos para a porção aminoterminal com maior especificidade para os primeiros aminoácidos, e assim mensurando apenas a forma molecular que ativa o PTH1R. As vantagens práticas e o custo-benefício deste ensaio ainda e motivo de debate. A observação recente de que fragmentos carboxiterminais têm atividade biológica via receptor distinto, aponta para a necessidade futura de mais de um ensaio para avaliar a função do paratormônio.

The anti-IRBP IgG1 and IgG2a response does not correlate with susceptibility to experimental autoimmune uveitis

Susceptibility to experimental autoimmune uveitis (EAU) in inbred mice has been associated with a dominant Th1 response. Elevated anti-inter-photoreceptor retinoid-binding protein (anti-IRBP) IgG2a/IgG1 antibody ratios have been implicated as candidate markers to predict disease severity. In the present study, both the anti-IRBP antibody isotype and severity of EAU phenotypes were examined in 4 non-isogenic genetically selected mouse lines to determine if they can be used as general markers of disease. Mice between 8 and 12 weeks old selected for high (H III) or low (L III) antibody response and for maximum (AIR MAX) or minimum (AIR MIN) acute inflammatory reaction (AIR) were immunized with IRBP. Each experiment was performed with at least 5 mice per group. EAU was evaluated by histopathology 21 days after immunization and the minimal criterion was inflammatory cell infiltration of the ciliary body, choroid and retina. Serum IgG1- and IgG2a-specific antibodies were determined by ELISA. EAU was graded by histological examination of the enucleated eyes. The incidence of EAU was lower in AIR MIN mice whereas in the other strains approximately 40 percent of the animals developed the disease. Low responder animals did not produce anti-IRBP IgG2a antibodies or interferon-gamma. No correlation was observed between susceptibility to EAU and anti-IRBP isotype profiles. Susceptibility to EAU is related to the intrinsic capacity to mount higher inflammatory reactions and increased production of anti-IRBP IgG2a isotype is not necessarily a marker of this immunologic profile.

Generation and characterisation of monoclonal antibodies specific to Plasmodium falciparum enolase.

BACKGROUND AND OBJECTIVES: Glycolysis is the sole source of energy for the intraerythrocytic stages of Plasmodium falciparum, making glycolytic enzymes putative therapeutic targets. Enolase, a single copy gene in P. falciparum is one such enzyme whose activity is elevated approximately 10-15 fold in infected RBC's. It holds the possibility of having multiple biological functions in the parasite and hence can be a suitable candidate for diagnostic and chemotherapeutic purposes. METHODS: We have aimed at generating parasite-specific reagents in the form of monoclonal antibodies. We have raised monoclonal antibodies against the recombinant P. falciparum enolase. RESULTS: Two IgG monoclonals were obtained with 1:1000 titre and specific for P. falciparum enolase. Apicomplexan parasites including P. falciparum enolase has a plant like pentapeptide sequence (104EWGWS108) which is uniquely different from the host counterpart. A peptide spanning this pentapeptide region (ELDGSKNEWGWSKSK) coupled to BSA was used to raise parasite-specific antibody. Four monoclonals were obtained with 1:1000 titre and of IgM isotype. INTERPRETATION AND CONCLUSION: All the monoclonals are specific for P. falciparum enolase and one of them display reactivity against native P. falciparum enolase signifying this pentapeptide to be surface exposed and immunogenic.

Production of murine monoclonal anti-B.

BACKGROUND & OBJECTIVE: Monoclonal antibodies against red blood cell antigens used in research and as diagnostics in India are commercially procured from western countries. Indigenously generated potent clones are not available in India. Hence, the objective of the present study was to raise potent murine monoclonal antibodies against A, B and H blood group antigens indigenously and establish a stable clone of anti-B secreting cells. METHODS: Spleen cells of female BALB/c mice immunized with B group red blood cells were fused in presence of polyethylene glycol (PEG) 1500 with a mouse myeloma cell line Sp 2/0 Ag. 14 in hypoxanthine aminopterine thymidine (HAT) selective medium and incubated at 37 degrees C, 5 per cent CO(2) and 95 per cent humidity for a week. RESULTS: The culture supernatant of the wells showing anti-B activity, were further subcloned and a clone 2C4D5F10 was generated which showed a good potency, avidity and specificity. INTERPRETATION & CONCLUSION: The anti-B clones thus produced indigenously provided a useful reagent in blood group typing. The unlimited availability unlike polyclonal antisera makes this reagent more cost-effective. It also ensures a regular supply with the similar specificity.

Production of monoclonal antibodies against serum immunoglobulins of black rockfish (Sebastes schlegeli Higendorf)

The present study was undertaken to produce monoclonal antibodies (MAbs) against immunoglobulin (Ig) purified from black rockfish (Sebastes schlegeli Higendorf) serum using protein A, mannan binding protein, and goat IgG affinity columns. These three different ligands were found to possess high affinity for black rockfish serum Ig. All of the Igs purified eluted at only 0.46 M NaCl concentration in anion exchange column chromatography and consisted of two bands at 70 kDa and 25 kDa in SDS-PAGE; they also had similar antigenicity for MAbs to Ig heavy chain in immunoblot assays. Therefore, black rockfish Ig is believed to exist as a single isotype within serum. The MAbs produced against Ig heavy chain reacted specifically with spots distributed over the pI range from 4.8 to 5.6 with a molecular weight of 70 kDa on two dimensional gel electrophoresis immunoblot profiles.

Overexpression of nicotinamide N-methyltransferase in gastric cancer tissues and its potential post-translational modification

Gastric cancer is one of the most common cancers worldwide. The purpose of this study was to find out potential markers for gastric cancer. Tumor and normal tissues from 152 gastric cancer cases were analyzed by two-dimensional gel electrophoresis (2-DE). The images of silver stained gels were analyzed and statistical analysis of spot intensities revealed that spot 4262 showed higher expression (5.7-fold increase) in cancer tissues than in normal tissues (P< 0.001). It was identified by peptide mass fingerprinting as nicotinamide N-methyltransferase (NNMT). A monoclonal antibody with a detection limit down to 10 ng was produced against NNMT in mouse. Using the prepared monoclonal antibody, western blot analysis of NNMT was performed for gastric tissues from 15 gastric cancer patients and two gastric ulcer patients. The results corroborated those of 2-DE experiments. A single spot was detected in gastric ulcer tissues while four to five spots were detected in gastric cancer tissues. In cancer tissues, two additional spots of acidic and basic form were mainly detected on 2-DE gels. This suggests that NNMT receives a post-translational modification in cancer- specific manner.

Anticuerpos monoclonales (AcMo) y recombinantes en medicina transfusional: desarrollos y desafíos / Monoclonal antibodies and recombinant in transfusion medicine: developments and challenges

Desde el advenimiento de los AcMo logrados a través de tecnologías de biología celular, el campo del inmunodiagnóstico, se ha visto altamente beneficiado al cambiar los reactivos policlonales, obtenidos de animales o humanos sensibilizados, por reactivos monoclonales, altamente específicos y muy potentes producidos en laboratorios. En un comienzo se utilizaron técnicas de fusión celular (las cuales se continúan utilizando para cierto tipo de desarrollos tecnológicos). Posteriormente, la biología molecular y la biología celular se unieron para producir anticuerpos quiméricos y humanizados, y la tecnología recombinante generó la producción de los repertorios o muestrarios de fagos (phage display). Estos anticuerpos han sido utilizados para múltiples propósitos: investigación básica y aplicada, para producción de reactivos y en el campo terapéutico, por lo que su impacto en el campo de la medicina tranfusional ha sido innegable. Todos estos avances nos permitirán en el futuro producir en forma rápida y barata, anticuerpos genéticamente diseñados según el objetivo que se persiga.

Production of a monoclonal antibody against a yeast secreted antigen of Penicillium marneffei.

Monoclonal antibody against P. mameffei yeast secreted antigen was produced in order to develop a serological test for penicilliosis marneffei. The yeast form of P. marneffei was cultured in brain heart infusion broth at 37 degrees C for 7 days. A secreted antigen was prepared, partially purified from culture supernatant and subsequently immunized in a BALB/c mouse. Mouse monoclonal antibody was produced from immune spleen cells by a standard hybridoma technique. Specificity of the obtained monoclonal antibody was assessed with yeast secreted antigens for P. mameffei, C. alblicans, C. neoformans, and H. capsulatum by an indirect ELISA. Three of 46 hybrid clones (1 F1, 2G5, and 3G4) reacted positively with P mameffei secreted antigen. 1 F1 and 3G4 were cloned by two rounds of limiting dilution. Partially purified monoclonal antibody and rabbit polyclonal antibody against P. marneffei yeast secreted antigen were used to develop a double antibody sandwich ELISA to detect P. marneffei antigen in plasma or serum samples of 7 patients with penicilliosis marneffei and 5 healthy controls. The sandwich ELISA developed using monoclonal antibody as a capture antibody and rabbit polyclonal antibody as a detector was able to detect P. marneffei antigen in all the plasma and serum samples of penicilliosis marneffei patients, while negative in all the healthy controls. Thus, the monoclonal antibody produced in the present study appeared to be highly specific for P. marneffei and the double antibody sandwich ELISA developed using monoclonal and polyclonal antibodies against the yeast secreted antigen of P. marneffei showed a strong potential for the diagnosis of penicilliosis marneffei.

Production of monoclonal antibody to subtype 9 of Neisseria meningitidis and the distribution of this subtype in Brazil

A new monoclonal antibody (5F81A4P1.9), which is specific for subtype 9 antigen of meningococci, was studied. The antibodies were raised against a previously non-typable (NT) serogroup B strain from Brazilian patients and were found to react with the subtype antigen of prototype reference strains for subtype 9 (M982), as well as with those of homologous strains. The subtype 9 epitope was found in 6.8 percent of serogroup B strains among 602 strains of Neisseria meningitidis case isolates, including representative isolates from Brazilian states. Subtype P1.9 was predominantly related to serogroup B in Brazil among the isolates collected during the N. meningitidis epidemic in 1992. No significant differences were observed in the occurrence of subtype P1.9 among strains isolated from several Brazilian states. Fluorescence-activated cell-sorter analysis showed that 5F81A4 MAb recognized a 46 kDa protein on the surface of a homologous strain of N. meningitidis (B:4:P1.9). These results, in association with a bactericidal activity assay for 5F81A4, and with experimental passive protection in mice, demonstrated the importance of subtype 9 class 1 proteins of N meningitidis in Brazil. Serotyping is essential for the development of vaccination strategies.

An antigen detection assay for diagnosing filariasis.

In this study we examined the diagnostic potential of monoclonal antibodies (MAb) reactive to antigens of adult Brugia malayi, their microfilariae and antigen of Dirofilaria immitis. The MAb of clone 17E10, which were of IgM isotype, reacted to the inner cuticles and internal content of both male and female worms and also to the sheath and internal content of microfilariae in utero. However, these MAb did not react to the sheath of blood circulating microfilariae. The MAb 17E10 produced a smear pattern between 37 to > 200 kDa in the Western blot analysis against a SDS-PAGE separated extract of B. malayi. The epitopes were non-protein in nature as indicated by their resistance to proteinase-K treatment. The MAb 17E10 were applied in a sandwich ELISA to detect filarial antigen in the buffy coat and plasma of patients. We tested patients with different clinical manifestations of brugian filariasis, i.e. microfilaremia (M), lymphangitis (L) and elephantiasis (E), as well as non-symptomatic inhabitants of a filariasis endemic area (NE), and compared them to samples from non-symptomatic inhabitants of disease non-endemic areas (NNE). It was found that 22 of 31 (70.9%) of M, 7 of 13 (53.8%) of L, 2 of 14 (14.2%) of E, 10 of 100 (10.0%) of NE and none (0%) of the NNE were positive for antigenaemia. The assay was also positive in 14 of 15 (93.3%) blood samples from B. malayi microfilaremic cats and in 7 of 7 (100%) blood samples of Dirofilaria immitis microfilaremic dogs. The so-developed test has a high potential for routine diagnosis of active filariasis, for epidemiological studies in both humans and reservoir animals and for monitoring treatment efficacy.