RESUMEN
Puerarin was conjugated with bovine serum albumin(BSA) and ovalbumin(OVA) by periodate oxidation to serve as the immunogen and coating antigen, respectively. BALB/c mice were immunized with puerarin-BSA according to the routine immunization procedure, and the titer and specificity of serum were detected after three immunization. After booster immunization, mouse spleen lymphocytes were fused with mouse myeloma cells, and 24 hybridoma cell lines of the monoclonal antibodies against puerarin were screened by monoclonal antibody screening technique. Ascites was prepared and purified. The cross-reactivity of monoclonal antibody(mAb) M1 with 4'-methoxy puerarin, daidzin, puerarin-6â³-O-xyloside, daidzein, mirificin, 3'-methoxy puerarin, and 3'-hydroxy puerarin was 239.84%, 112.18%, 67.89%, 58.28%, 22.37%, 0.40%, and 0.20%, respectively, and those with other analogs such as baicalein and baicalin were all less than 0.10%. The IC_(50) and the working range of the indirect competitive enzyme-linked immunosorbent assay(icELISA) for puerarin were 44.80 ng·mL~(-1) and 8.20-292.30 ng·mL~(-1), respectively. The average recovery was 91.95%-98.20% with an RSD in the range of 0.70%-2.60%. The content of puerarin in different Puerariae Lobatae Radix samples was determined with icELISA and validated by UPLC-MS. The correlation between data obtained from icELISA and UPLC-MS was 0.999 0, indicating that icELISA is suitable for the rapid detection of puerarin in Puerariae Lobatae Radix samples.
Asunto(s)
Anticuerpos Monoclonales , Espectrometría de Masas en Tándem , Animales , Cromatografía Liquida , Ensayo de Inmunoadsorción Enzimática/métodos , Hibridomas/metabolismo , Isoflavonas , Ratones , Ratones Endogámicos BALB CRESUMEN
Antiribosomal P protein (anti-P) autoantibodies commonly develop in patients with systemic lupus erythematosus. We have previously established hybridoma clones producing anti-P mAbs. In this study, we explored the pathogenesis of behavioral disorders induced by anti-P Abs using these mAbs. New Zealand Black × New Zealand White F1, New Zealand White, C57BL/6, and BALB/c mice were treated with 1 mg of anti-P Abs once every 2 wk. The behavioral disorder was evaluated by the tail suspension test, forced swim test, and open field test. Following administration of anti-P Abs, New Zealand Black × New Zealand White F1 and C57BL/6 mice developed depressive behavior and showed increased anxiety with elevated serum TNF-α and IL-6 levels. Anti-P Abs were not deposited in the affected brain tissue; instead, this mood disorder was associated with lower serum and brain tryptophan concentrations. Tryptophan supplementation recovered serum tryptophan levels and prevented the behavioral disorder. TNF-α and IL-6 were essential for the decreased serum tryptophan and disease development, which were ameliorated by treatment with anti-TNF-α neutralizing Abs or dexamethasone. Peritoneal macrophages from C57BL/6 mice produced TNF-α, IL-6, and IDO-1 via interaction with anti-P Abs through activating FcγRs, which were required for disease development. IVIg, which has an immunosuppressive effect partly through the regulation of FcγR expression, also prevented the decrease in serum tryptophan and disease development. Furthermore, serum tryptophan concentrations were decreased in the sera of systemic lupus erythematosus patients with anti-P Abs, and lower tryptophan levels correlated with disease activity. Our study revealed some of the molecular mechanisms of mood disorder induced by anti-P Abs.
Asunto(s)
Complejo Antígeno-Anticuerpo/metabolismo , Encéfalo/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Macrófagos/inmunología , Trastornos del Humor/prevención & control , Suero/metabolismo , Triptófano/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Autoanticuerpos/metabolismo , Suplementos Dietéticos , Humanos , Hibridomas , Lupus Eritematoso Sistémico/complicaciones , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Trastornos del Humor/etiología , Fosfoproteínas/inmunología , Receptores de IgG/metabolismo , Proteínas Ribosómicas/inmunología , Triptófano/administración & dosificación , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We recently reported identification of sarcoplasmic/endoplasmic reticulum calcium-ATPase2a (SERCA2a) 971-990, which induces atrial myocarditis by generating autoreactive T cells in A/J mice. However, it was unknown how antigen-sensitized T cells could recognize SERCA2a 971-990, since SERCA2a-expression is confined to an intracellular compartment. In this report, we present evidence that antigen-presenting cells (APCs) from lymphoid and non-lymphoid organs in naïve animals present SERCA2a 971-990 and stimulate antigen-specific T cells. Using major histocompatibility complex (MHC) class II dextramers for SERCA2a 971-990, we created a panel of T cell hybridomas and demonstrated that splenocytes from naïve A/J mice stimulated the hybridoma cells without exogenous supplementation of SERCA2a 971-990. We then recapitulated this phenomenon by using SERCA2a 971-990 -specific primary T cells, verifying that the T cell responses were MHC-restricted. Furthermore, SERCA2a 971-990 -sensitzed T cells exposed to APCs from naïve mice were found to produce the inflammatory cytokines interferon-γ, granulocyte macrophage colony stimulating factor, and interleukin-17A, which are implicated in the induction of myocarditis. Finally, while T cells exposed to mononuclear cells (MNCs) obtained from heart and liver also responded similarly to splenocytes, endothelial cells (ECs) generated from the corresponding organs displayed opposing effects, in that the proliferative responses were suppressed with the heart ECs, but not with the liver ECs. Taken together, our data suggest that the surface expression of SERCA2a 971-990 by naïve APCs can potentially trigger pathogenic autoreactive T cell responses under conditions of autoimmunity, which may have implications in endothelial dysfunction.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Enfermedades Autoinmunes/inmunología , Autoinmunidad/inmunología , Epítopos/inmunología , Miocarditis/inmunología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/inmunología , Animales , Citocinas/inmunología , Células Endoteliales/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Hibridomas/inmunología , Inflamación/inmunología , Leucocitos Mononucleares/inmunología , Masculino , Ratones , Ratones Endogámicos , Linfocitos TRESUMEN
The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (<1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Productos Biológicos/inmunología , Animales , Especificidad de Anticuerpos , Bovinos , Línea Celular Tumoral , Reacciones Cruzadas/inmunología , Femenino , Flavanonas/inmunología , Ácido Glicirrínico/inmunología , Hibridomas/metabolismo , Ratones Endogámicos BALB C , Albúmina Sérica Bovina/inmunologíaRESUMEN
Peripheral blood lymphocytes from a patient allergic to Japanese cedar pollens were transformed by Epstein-Barr virus infection. Some transformed B-lymphoblastoid cells (BLCs) secreted IgM class antibodies to cedar pollen extracts and tomato fruit extracts. One stable human-mouse hybridoma clone Y-22-3-3 secreting IgM class monoclonal antibody to tomato fruit extracts was established by cell fusion of BLCs with mouse myeloma cells. Western blot analysis of tomato extracts showed Y-22-3-3 monoclonal antibody recognized a tomato protein with a molecular weight of 40 kDa. The CBB-stained 40 kDa protein from antibody-affinity chromatography was analyzed by MALDI-TOF/TOF, and identified as tomato endo-beta-mannanase, which was previously reported as one of the potential candidates for tomato allergens.
Asunto(s)
Alérgenos/inmunología , Anticuerpos Monoclonales/inmunología , Cedrus/inmunología , Cryptomeria/inmunología , Polen/inmunología , Solanum lycopersicum/inmunología , beta-Manosidasa/inmunología , Secuencia de Aminoácidos , Animales , Humanos , Hibridomas/inmunología , Inmunoglobulina M/inmunología , Ratones , Peso Molecular , Proteínas de Plantas/inmunología , Rinitis Alérgica Estacional/inmunologíaRESUMEN
Hybridoma screening is a key step for the successful generation of high-affinity analyte-specific monoclonal antibodies (MAbs). This work presents an innovative screening method, known as a bacterial macroarray, generated by contact printing of hybridoma cell supernatant samples on a nitrocellulose (NC) membrane initially coated with fluorescein isothiocyanate (FITC)-labeled bacteria. Given that bacterial fixation will be influenced by complex bacterial surface structures, we selected both gram-positive bacteria ( Staphylococcus aureus and Listeria monocytogenes) and gram-negative bacteria ( Escherichia coli O157:H7 and Cronobacter sakazakii) to optimize the fixation conditions for binding to the NC membrane, such as the aperture of the NC membrane, the concentration of bacteria, the dosage of glycerin in the spotting buffer, and the fixation time and temperature. As a result, we found that a better bacterial macroarray could be developed when the spotting buffer, containing 1011 CFU mL-1 of FITC-labeled bacteria and 15% (V/V) glycerol, was spotted onto a 0.45 µm NC membrane with an incubation of 2 h at 37 °C. Finally, we verified the stability and specificity of the prepared bacterial macroarray by detecting cell cultures with the addition of two MAbs ( Escherichia coli O157:H7 MAb E7 and Cronobacter sakazakii MAb 1E9) to simulate the screening experiments. Here, we describe a bacterial macroarray to efficiently screen the targeted antibody-secreted hybridomas.
Asunto(s)
Anticuerpos/metabolismo , Bacterias/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Hibridomas/metabolismo , Animales , Colodión , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Membranas Artificiales , Temperatura , Factores de TiempoRESUMEN
The tedious sample preparation for flow cytometry limits the throughput and thus its usage as a primary screening method despite its sensitivity and accuracy. With the growing focus on utilizing antibodies as a therapeutic modality in drug discovery, it is critical to develop a high-throughput flow cytometry (HTFC) workflow to cope with the increasing need to support antibody discovery programs. We have developed a seamless HTFC sample preparation and readout workflow using the HighRes modular robotic system and the IntelliCyt iQue Screener PLUS. To fully utilize the advantages offered by flow cytometry, we typically multiplex multiple cell lines of interest in one well to simultaneously quantitate on-target activity and nonspecific activity along with measurement of antibody concentration. The ability to measure multiple parameters coupled with speed and increased accuracy provides gains in productivity and helps speed up antibody lead discovery.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Descubrimiento de Drogas , Citometría de Flujo , Animales , Automatización , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos , Citometría de Flujo/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Hibridomas , Inmunoglobulina G/farmacología , Ratones , Flujo de TrabajoRESUMEN
The goal of high-throughput screening is to enable screening of compound libraries in an automated manner to identify quality starting points for optimization. This often involves screening a large diversity of compounds in an assay that preserves a connection to the disease pathology. Phenotypic screening is a powerful tool for drug identification, in that assays can be run without prior understanding of the target and with primary cells that closely mimic the therapeutic setting. Advanced automation and high-content imaging have enabled many complex assays, but these are still relatively slow and low throughput. To address this limitation, we have developed an automated workflow that is dedicated to processing complex phenotypic assays for flow cytometry. The system can achieve a throughput of 50,000 wells per day, resulting in a fully automated platform that enables robust phenotypic drug discovery. Over the past 5 years, this screening system has been used for a variety of drug discovery programs, across many disease areas, with many molecules advancing quickly into preclinical development and into the clinic. This report will highlight a diversity of approaches that automated flow cytometry has enabled for phenotypic drug discovery.
Asunto(s)
Descubrimiento de Drogas , Citometría de Flujo , Ensayos Analíticos de Alto Rendimiento , Automatización de Laboratorios , Plaquetas/efectos de los fármacos , Línea Celular , Biología Computacional/métodos , Análisis de Datos , Descubrimiento de Drogas/instrumentación , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos , Citometría de Flujo/instrumentación , Citometría de Flujo/métodos , Ensayos Analíticos de Alto Rendimiento/instrumentación , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Hibridomas , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Murine hybridoma cells can produce monoclonal antibody (MAb) and the production of these antibodies in culture and peritoneum can be affected by different factors, including stimulants, inhibitors and supplements. Among these factors, the impact of micronutrients on the production of MAbs by mouse hybridoma cells has not fully been explored. In this study the murine hybridoma cells, M3C5, were cultured and treated with different concentrations of ATRA and DHA, alone, in combinations, and at different time of exposure. Then, changes in the production of MAb in culture medium were evaluated using ELISA. The hybridoma cells after single and combined treatment with ATRA, DHA and vehicles were IP injected to Balb/c mice and the changes in production of MAb in ascites were determined by ELISA. The results showed that single and combined treatment of ATRA and DHA elevated the production of MAb by hybridoma cells in both in vivo and in vitro. The production of MAb following in vitro single treatment with 1⯵M of ATRA and 10⯵M of DHA for 2â¯days was significantly increased. The in vitro effects of ATRA on increase of MAb production was obtained more than DHA. The MAb productions in combined treatment with 0.5â¯µΜ of ATRA plus 5â¯µΜ of DHA were significantly increased in in vivo and in vitro. However, the effect of DHA was obtained more significant in in vivo conditions. The results of this study showed for the first time that in vitro and in vivo treatments of ATRA and DHA could increase the production of MAb in mouse M3C5 hybridoma cells.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/biosíntesis , Ácidos Docosahexaenoicos/farmacología , Inmunoglobulina G/biosíntesis , Tretinoina/farmacología , Animales , Líquido Ascítico/metabolismo , Gonadotropina Coriónica/inmunología , Hibridomas , Masculino , Ratones Endogámicos BALB CRESUMEN
Leucine aminopeptidase (LAP, EC: 3.4.11.1) is an important metalloexopeptidase that catalyze the hydrolysis of amino-terminal leucine residues from polypeptides and proteins. In this study, a full length of cDNA encoding leucine aminopeptidase of Taenia pisiformis (TpLAP) was cloned by rapid amplification of cDNA-ends using the polymerase chain reaction (RACE-PCR) method. The full-length cDNA of the TpLAP gene is 1823 bp and contains a 1569 bp ORF encoding 533 amino acids with a putative mass of 56.4â¯kDa. TpLAP contains two characteristic motifs of the M17LAP family in the C-terminal sequence: the metal binding site 265-[VGKG]-271 and the catalytic domain motif 351-[NTDAEGRL]-357. The soluble GST-TpLAP protein was expressed in Escherichia coli Transetta (DE3) and four specific anti-TpLAP monoclonal antibodies (mAbs) were prepared. In enzymatic assays, the optimal activity was observed at pH 9.5â¯at 45⯰C. GST-TpLAP displayed a hydrolyzing activity for the Leu-pNA substrate with a maximum activity of 46 U/ml. The enzymatic activity was significantly enhanced by Mn2+ and completely inhibited by 20â¯nM bestatin and 0.15â¯mM EDTA. The native TpLAP was detected specifically in ES components of adult T. pisiformis by western blotting using anti-TpLAP mAb as a probe. Quantitative real-time PCR revealed that the TpLAP gene was expressed at a high level in adult worm tissues, especially in the gravid proglottids (50.71-fold). Immunolocalization analysis showed that TpLAP was located primarily in the subtegumental parenchyma zone and the uterine wall of adult worms. Our results indicate that TpLAP is a new member of the M17LAP family and can be considered as a stage-differentially expressed protein. These findings might provide new insights into the study of the mechanisms of growth, development and survival of T. pisiformis in the final host and have potential value as an attractive target for drug therapy or vaccine intervention.
Asunto(s)
Leucil Aminopeptidasa/genética , Taenia/enzimología , Taenia/genética , Secuencia de Aminoácidos , Compuestos de Anilina/metabolismo , Animales , Anticuerpos Monoclonales/biosíntesis , Western Blotting , Clonación Molecular , ADN Complementario/aislamiento & purificación , ADN Complementario/metabolismo , ADN de Helmintos/aislamiento & purificación , ADN de Helmintos/metabolismo , Hibridomas , Concentración de Iones de Hidrógeno , Inmunohistoquímica , Leucil Aminopeptidasa/química , Leucil Aminopeptidasa/inmunología , Leucil Aminopeptidasa/metabolismo , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Alineación de Secuencia , Análisis de Secuencia , Taenia/inmunología , TemperaturaRESUMEN
Potato virus S (PVS) often causes significant losses in potato production in potato-growing countries. In this study, the ordinary strain of PVS (PVSO) was purified from PVS-infected potato plants and used as the immunogen to produce hybridomas secreting monoclonal antibodies (MAbs). Five highly specific and sensitive murine MAbs (1A3, 16C10, 18A9, 20B12, and 22H4) against PVS were prepared using conventional hybridoma technology. Using these MAbs, tissue print-enzyme-linked immunosorbent assay (ELISA), dot-ELISA, and double-antibody sandwich (DAS)-ELISA were developed for sensitive and specific detection of PVS infection in potato plants. The results of sensitivity assays revealed that PVS could be reliably detected in PVS-infected leaf crude extracts diluted at 1:10 240 and 1:163 840 (w/v, g/ml) in phosphate buffer saline (PBS) by dot-ELISA and DAS-ELISA, respectively. Twenty-two samples collected from potato fields in Yunnan Province, China were tested for PVS infection using the serological assays we had developed, and 14 of them were found to be positive. This indicates that PVS is now prevalent in potato fields in Yunnan Province.
Asunto(s)
Anticuerpos Monoclonales/química , Carlavirus/aislamiento & purificación , Solanum tuberosum/virología , Animales , Bioensayo , China , Ensayo de Inmunoadsorción Enzimática , Femenino , Hibridomas , Ratones , Ratones Endogámicos BALB C , Enfermedades de las Plantas/virología , Hojas de la Planta/virología , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
A monoclonal antibody (Mab) against lycopene was developed from hybridoma clones obtained from BALB/c mice immunized with trans-isomer of lycopene (t-lycopene, t-LC) conjugated with colloidal gold particles. An alternating immunization schedule which included injection of both formulations of immunogen (without and with Freund's adjuvant) was most effective in the elucidation of a measurable immune response to the t-Lycopene conjugate. Selected hybridoma clones were able to produce an Mab positive in competition assay. In particular, preincubation of 6B9 Mabs with t-LC abolished the ability of 6B9 Mabs to bind LC in the competition assay. Mabs produced by other clones (4F10, 4A3, and 3B12) worked similarly. Analysis of antigen specificity showed that 6B9 Mab raised against t-LC did not recognize other carotenoids such as lutein and carotene. Mab 6B9 was shown to recognize lycopene on a glass surface and in the settings of indirect immunofluorescence experiments performed in cultured hepatocytes and alveolar macrophages incubated with and without lycopene, as well as in sebum and corneocyte specimens from the skin of volunteers supplemented with nutraceutical formulation of lycopene. Newly generated Mabs against lycopene may provide a valuable tool for different analytical assays of lycopene content in various biological, agricultural, and food products.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Antígenos/inmunología , Carotenoides/inmunología , Inmunización Secundaria/métodos , Inmunoconjugados/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/química , Especificidad de Anticuerpos , Antígenos/administración & dosificación , Antígenos/química , Western Blotting , Carotenoides/administración & dosificación , Carotenoides/química , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Adyuvante de Freund/administración & dosificación , Oro Coloide/administración & dosificación , Oro Coloide/química , Hepatocitos/química , Hepatocitos/ultraestructura , Humanos , Hibridomas/inmunología , Inmunoconjugados/administración & dosificación , Inmunoconjugados/química , Luteína , Licopeno , Macrófagos Alveolares/química , Macrófagos Alveolares/ultraestructura , Ratones , Ratones Endogámicos BALB C , Bazo/citología , Bazo/inmunologíaRESUMEN
Japanese cedar pollen allergen Cry j2 is a causal allergen of seasonal pollinosis in Japan. To analyze B cell epitopes of Cry j2, we established two human-mouse hybridomas secreting IgM class human monoclonal antibodies to Cry j2. A pin-peptide enzyme-linked immunosorbent assay with synthesized icosa peptides showed that 404-117 monoclonal antibody bound to peptides #11-13 with cry j2 amino acid sequence of 101F-L140. Detailed analysis with octa peptides and alanine substituted peptides indicated that an amino acid sequence of 118FKVD121 was an essential for antibody binding. When K119 (Asn) was substituted with alanine, 404-117 monoclonal antibody did not bind to the alanine substituted peptide. We concluded that the 118FKVD121 sequence might have a very important role in early recognition by Cry j2-specific B cells, which could act as antigen presenting cells.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Epítopos/inmunología , Inmunoglobulina M/biosíntesis , Proteínas de Plantas/inmunología , Polen/inmunología , Rinitis Alérgica Estacional/inmunología , Alérgenos/química , Alérgenos/inmunología , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/patología , Antígenos de Plantas/química , Antígenos de Plantas/inmunología , Linfocitos B/inmunología , Linfocitos B/patología , Sitios de Unión , Cryptomeria/química , Cryptomeria/inmunología , Epítopos/química , Humanos , Hibridomas/inmunología , Hibridomas/metabolismo , Japón , Ratones , Péptidos/química , Péptidos/inmunología , Proteínas de Plantas/química , Polen/química , Unión Proteica , Rinitis Alérgica Estacional/inducido químicamente , Rinitis Alérgica Estacional/patologíaRESUMEN
In the present study, a novel monoclonal antibody (MAb) specific for icariin (ICA) was prepared and characterized. A hybridomasecreting MAb against icariin was produced by fusing splenocytes immunized with an ICAbovine serum albumin conjugate with a hypoxanthineaminopterinthymidinesensitive mouse myeloma SP2/0 cell line. The antibody showed high specificity for ICA with almost no crossreactivity against the majority of structurallyrelated chemicals. Subsequently, an indirect competitive enzymelinked immunosorbent assay (ELISA) for ICA was established and characterized. In this assay, an effective measuring range of 101,000 ng/ml of ICA (R2=0.9828) was detected. Intra and interassay repeatability and precision were achieved with a relative standard deviation (RSD) of <10%. A mean recovery of 95115% was obtained, with an RSD of <10%. In addition, the levels of ICA in traditional Chinese herbal prescriptions were determined, and correlation between the ELISA and highperformance liquid chromatography analyses of total ICA was obtained. These results demonstrated that a reliable ELISA method had been successfully developed to determine ICA in traditional Chinese herbs and may contribute to further clinical investigations.
Asunto(s)
Medicamentos Herbarios Chinos/química , Ensayo de Inmunoadsorción Enzimática/métodos , Epimedium/química , Flavonoides/análisis , Animales , Anticuerpos Monoclonales/inmunología , Reacciones Cruzadas , Femenino , Flavonoides/inmunología , Hibridomas/inmunología , Inmunización , Límite de Detección , Ratones , Ratones Endogámicos BALB CRESUMEN
In this work we characterize the interaction of pomegranate hydrolyzable tannins (HT) with hen egg-white lysozyme (HEL) and determine the effects of non-covalent tannin-protein complexes on macrophage endocytosis, processing and presentation of antigen. We isolated HT from pomegranate and complex to HEL, the resulting non-covalent tannin-protein complex was characterized by gel electrophoresis and MALDI-TOF MS. Finally, cell culture studies and confocal microscopy imaging were conducted on the non-covalent pomegranate HT-HEL protein complexes to evaluate its effect on macrophage antigen uptake, processing and presentation to T-cell hybridomas. Our results indicate that non-covalent pomegranate HT-HEL protein complexes modulate uptake, processing and antigen presentation by mouse peritoneal macrophages. After 4 h of pre-incubation, only trace amounts of IL-2 were detected in the co-cultures treated with HEL alone, whereas a non-covalent pomegranate HT-HEL complex had already reached maximum IL-2 expression. Pomegranate HT may increase rate of endocytose of HEL and subsequent expression of IL-2 by the T-cell hybridomas.
Asunto(s)
Taninos Hidrolizables/química , Taninos Hidrolizables/inmunología , Lythraceae/química , Lythraceae/inmunología , Muramidasa/química , Muramidasa/inmunología , Animales , Presentación de Antígeno , Técnicas de Cocultivo , Suplementos Dietéticos/análisis , Proteínas del Huevo/química , Proteínas del Huevo/inmunología , Alimentos Funcionales/análisis , Humanos , Hibridomas/inmunología , Macrófagos Peritoneales/inmunología , Ratones , Complejos Multiproteicos/química , Complejos Multiproteicos/inmunología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Linfocitos T/inmunologíaRESUMEN
Statistically designed experiments were used in developing a low-serum medium for the production of a diagnostic monoclonal antibody against congenital adrenal hyperplasia using hybridoma 192. A two-level half-fractional factorial design was used for screening six components (Minimum Essential Medium Eagle amino acids, 2-mercaptoethanol, ethanolamine, ferric citrate, zinc sulfate, and sodium selenite). The experimental design was then augmented to central composite design. The basal Dulbecco's modified Eagle's medium (DMEM; containing 4 mM L-glutamine, 1% antibiotic-antimycotic agent) supplemented with 0.4% by volume fetal bovine serum (FBS), 311.8 mM ferric citrate, 17.3 nM sodium selenite, and 4.5 mM zinc sulfate (LSD) was found to support the growth of the hybridoma. Specific cell growth rate in the LSD (0.033 ± 0.001/h) was slightly lower than in the control medium (i.e., basal DMEM supplemented with 2% FBS; 0.0045 ± 0.003/h). Nevertheless, the specific MAb production rate for LSD was higher (0.057 ± 0.015 pg/cell · h versus 0.004 ± 0.002 pg/cell · h in LSD and control, respectively). The antibody produced in the LSD showed high specificity and no cross-reactivity with the other structural resemblance's steroid hormones, revealing no structural changes owing to the new medium formulation developed. The new medium formulation effectively reduced the medium cost by up to 64.6%.
Asunto(s)
Hiperplasia Suprarrenal Congénita/inmunología , Anticuerpos Monoclonales/biosíntesis , Hibridomas/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Sangre , Bovinos , Línea Celular , Reacciones Cruzadas , Medios de CultivoRESUMEN
Fermentanomics is an emerging field of research and involves understanding the underlying controlled process variables and their effect on process yield and product quality. Although major advancements have occurred in process analytics over the past two decades, accurate real-time measurement of significant quality attributes for a biotech product during production culture is still not feasible. Researchers have used an amalgam of process models and analytical measurements for monitoring and process control during production. This article focuses on using multivariate data analysis as a tool for monitoring the internal bioreactor dynamics, the metabolic state of the cell, and interactions among them during culture. Quality attributes of the monoclonal antibody product that were monitored include glycosylation profile of the final product along with process attributes, such as viable cell density and level of antibody expression. These were related to process variables, raw materials components of the chemically defined hybridoma media, concentration of metabolites formed during the course of the culture, aeration-related parameters, and supplemented raw materials such as glucose, methionine, threonine, tryptophan, and tyrosine. This article demonstrates the utility of multivariate data analysis for correlating the product quality attributes (especially glycosylation) to process variables and raw materials (especially amino acid supplements in cell culture media). The proposed approach can be applied for process optimization to increase product expression, improve consistency of product quality, and target the desired quality attribute profile.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Técnicas de Cultivo de Célula/métodos , Biología Computacional/métodos , Medios de Cultivo/metabolismo , Fermentación/fisiología , Aminoácidos/análisis , Aminoácidos/química , Aminoácidos/metabolismo , Animales , Anticuerpos Monoclonales/análisis , Medios de Cultivo/análisis , Medios de Cultivo/química , Fermentación/genética , Glicosilación , Hibridomas/metabolismo , Ratones , Análisis Multivariante , Proyectos de InvestigaciónRESUMEN
The aim of this study was to investigate the use of tomatine adjuvant to deliver soluble antigen for crosspresentation by bone marrow-derived dendritic cells (BMDCs). BMDCs were incubated with tomatine adjuvantovalbumin (OVA) complex and analyzed for antigen uptake by flow cytometry. Adjuvant-induced cell death was examined in situ by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. To elucidate the effect of antigen internalization on tomatine adjuvant-mediated antigen presentation, BMDCs were treated with several endocytosis inhibitors, and antigen presentation was analyzed by B3Z activity assay. Our data indicated that tomatine adjuvant enhanced antigen internalization by antigen presenting cells (APCs) and induced significant cell death and leukocyte infiltration at the injection sites. In vitro tomatine adjuvant treatment of BMDCs activated Ova/K(b) restricted B3Z T cell hybridomas, whereas this activation was impaired by pretreatment with brefeldin A, cytochalasin B, wortmannin, or ZnCl2. Our results demonstrated the role of tomatine adjuvant in antigen delivery to antigen presenting cells (APCs) and suggested the involvement of phagocytosis and PI3K signaling during the delivery of soluble antigens in the context of MHC class I.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Presentación de Antígeno/inmunología , Tomatina/administración & dosificación , Adyuvantes Inmunológicos/farmacología , Animales , Células de la Médula Ósea/inmunología , Células Dendríticas/inmunología , Endocitosis , Citometría de Flujo , Hibridomas , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/inmunología , Fagocitosis/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Linfocitos T/inmunología , Tomatina/farmacologíaRESUMEN
Expression of matrix metalloproteinase 9 (MMP9) is elevated in a variety of inflammatory and oncology indications, including ulcerative colitis and colorectal cancer. MMP9 is a downstream effector and an upstream mediator of pathways involved in growth and inflammation, and has long been viewed as a promising therapeutic target. However, previous efforts to target matrix metalloproteinases (MMPs), including MMP9, have utilized broad-spectrum or semi-selective inhibitors. While some of these drugs showed signs of efficacy in patients, all MMP-targeted inhibitors have been hampered by dose-limiting toxicity or insufficient clinical benefit, likely due to their lack of specificity. Here, we show that selective inhibition of MMP9 did not induce musculoskeletal syndrome (a characteristic toxicity of pan-MMP inhibitors) in a rat model, but did reduce disease severity in a dextran sodium sulfate-induced mouse model of ulcerative colitis. We also found that MMP9 inhibition decreased tumor growth and metastases incidence in a surgical orthotopic xenograft model of colorectal carcinoma, and that inhibition of either tumor- or stroma-derived MMP9 was sufficient to reduce primary tumor growth. Collectively, these data suggest that selective MMP9 inhibition is a promising therapeutic strategy for treatment of inflammatory and oncology indications in which MMP9 is upregulated and is associated with disease pathology, such as ulcerative colitis and colorectal cancer. In addition, we report the development of a potent and highly selective allosteric MMP9 inhibitor, the humanized monoclonal antibody GS-5745, which can be used to evaluate the therapeutic potential of MMP9 inhibition in patients.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/farmacología , Colitis Ulcerosa/tratamiento farmacológico , Neoplasias Colorrectales/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica , Metaloproteinasa 9 de la Matriz/genética , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Regulación Alostérica , Animales , Anticuerpos Monoclonales Humanizados/biosíntesis , Anticuerpos Monoclonales Humanizados/aislamiento & purificación , Antineoplásicos/aislamiento & purificación , Antineoplásicos/metabolismo , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/enzimología , Colitis Ulcerosa/genética , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Mapeo Epitopo , Femenino , Humanos , Hibridomas/inmunología , Masculino , Metaloproteinasa 9 de la Matriz/administración & dosificación , Metaloproteinasa 9 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz/aislamiento & purificación , Inhibidores de la Metaloproteinasa de la Matriz/metabolismo , Ratones , Ratones Desnudos , Ratas , Ratas Endogámicas Lew , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Repeated exposure to an allergen induces allergic symptoms by activating mast cells that express anti-allergen IgE, which results in further sensitization to an allergen. Considering that additional sensitization elicits more severe allergic reactions upon the next allergen challenge, suppression of the boosting phase represents an efficacious way to prevent and ameliorate allergic diseases. In this study, we investigated the therapeutic potential of allergen-specific monoclonal IgA on allergic diseases. This antibody acts by decreasing immune responses upon exposure to allergens in mice previously sensitized by a monoclonal IgE that recognizes the allergen. The lack of inhibitory effects of anti-ovalbumin monoclonal IgA (OA-4) on either the binding of anti-ovalbumin monoclonal IgE (OE-1) to ovalbumin by ELISA or on ovalbumin-induced degranulation of rat basophilic leukemia RBL2H3 cells sensitized with OE-1 indicated that OA-4 and OE-1 recognized different epitopes on ovalbumin. Immune responses (anti-ovalbumin IgG1 production and cytokine release from splenocytes) induced by intravenous ovalbumin challenge in DBA/1J mice passively sensitized with OE-1 were inhibited by intravenous injection of OA-4 15 min before challenge without affecting anaphylaxis. Moreover, OA-4 injection 1 h after ovalbumin challenge also effectively suppressed immune responses. The achievement of immunosuppression by IgA injection occurred even after allergen challenge in mice in an epitope-independent fashion. These findings suggest that monoclonal IgA administered at the time of hospitalization of a patient with allergic symptoms, who was already exposed to the allergen in the presence of IgE recognizing an undefined epitope(s) on the allergen, should effectively relieve allergic disease through its immunosuppressive effects.