RESUMEN
Abalone species are different from most mollusks utilized in aquaculture as they are known to hybridize in laboratory-induced matings. Allotriploidization of hybrid abalone has not yet been studied, and methodology useful in verifying the genotypic condition of such allotriploids do not exist. Genotypic verification of hybridization and allotriploidization in a cross of Haliotis fulgens and Haliotis rufescens was performed utilizing 6 crossamplifying microsatellite loci. Five H. rufescens spawns were used in this experiment, dividing each spawn into control and experimental hybrid groups and further into diploids and triploids. Two microsatellite loci developed for H. fulgens and H. rufescens allowed for the genotypic identification of hybrids within diploid and triploids. To further verify the percentage of allotriploids within the genotypic hybrids in the triploid hybrid groups, microsatellite loci originally developed in Haliotis corrugata and Haliotis kamtschatkana were tested for crossamplification in H. fulgens and H. rufescens. Of 21 loci, 4 were chosen for this study based on their crossamplification, heterozygosity in the females, and centromere recombination frequencies. Allotriploids in triploid-hybrid larvae were then detected by identifying larvae with recombinant genotypes at any of those loci. One family had low success verification associated with reduced recombination frequencies for all loci in that family. These results demonstrate that allotriploid verification at larval stages is feasible but depends on the number of loci available, their crossamplification in the species, and their recombination frequencies.
Asunto(s)
Repeticiones de Microsatélite/genética , Moluscos/genética , Poliploidía , Animales , Femenino , Variación Genética , Genotipo , Heterocigoto , Larva/genética , Moluscos/crecimiento & desarrollo , Recombinación GenéticaRESUMEN
A comparison between expanded bed adsorption and conventional packed bed Protein A Fast Flow to purify the anti-rHBsAg mAbs from feedstock is presented in this work. Direct capture by STREAMLINE expanded bed adsorption chromatography resulted in 92% product recovery and sevenfold more concentrated product with similar purity levels compared to that obtained by the standard packed method. The process time and buffer consumption were reduced in the expanded bed adsorption method not only with the binding-elution conditions but also with the use of NaOH during the cleaning-in-place step. The latter is the most widely accepted agent in downstream processing, being a cost effective technique that provides not only efficient cleaning but also sanitizes complete column systems and destroys pirogens.
Asunto(s)
Cromatografía de Afinidad/métodos , Adsorción , Animales , Técnicas de Cultivo de Célula , Cromatografía en Gel/métodosRESUMEN
Different ligand densities of monoclonal antibody (Mab) CB.Hep-1 were studied during covalent coupling on Sepharose CL-4B for recombinant hepatitis B surface antigen (rHBsAg) immunoaffinity purification. Ligand densities of 2.2, 3.2, 4.2 and 5.2 mg Mab/ml immunosorbents, respectively, were assayed during five cycles of immunoaffinity chromatography (IAC). Adsorption capacities averaged either 3.2 mg/ml (0.57 mg rHBsAg/ml immunosorbent/5.42 mg of total purified protein) or 5.2 mg/ml (0.56 mg rHBsAg/ml immunosorbent/5.05 mg total purified protein). Immunosorbents showed ligand leakage levels below 3 ng Mab/microg rHBsAg. Antigen purity was higher than 95% in all cases. The results suggest that a ligand density (LD) of 3.2 mg Mab/ml immunosorbent should be used for immunoaffinity chromatography because no significant differences were found in the ligand densities studied (P-value=0.012), which saves 40% of CB.Hep-1 immunosorbent manufacturing cost in comparison with 5 mg Mab/ml immunosorbent, which is currently used in large-scale production.
Asunto(s)
Anticuerpos Monoclonales/química , Cromatografía de Afinidad/métodos , Antígenos de Superficie de la Hepatitis B/química , Antígenos de Superficie de la Hepatitis B/aislamiento & purificación , Técnicas de Inmunoadsorción , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Ligandos , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
The use of cholinesterase activity as a biochemical method for monitoring organophosphate pesticide exposure in cattle is described herein. Determination of cholinesterase activity of whole blood, erythrocyte, and plasma was carried out according to the Ellman modified kinetic method. The mean baseline acetylcholinesterase activities of 9.549 +/- 3.619 IU/mL in whole blood, 9.444 +/- 3.006 IU/mL in erythrocytes, and 0.149 +/- 0.063 IU/mL in plasma were estimated for steers from the control group. Results of multivariate analysis showed that the general responses between the control and experimental groups (in vivo, monitoring and case studies) treated with Coumaphos and Fenthion were statistically different, and the general responses of these experimental groups were statistically different over time as well. Among the fractions that were analyzed, the erythrocyte acetylcholinesterase activity could be adequate for the diagnosis of exposure or acute poisoning in cattle as it showed a good within-run and between-run precision with CVs <10% better than those in plasma.
Asunto(s)
Acetilcolinesterasa/sangre , Insecticidas/farmacocinética , Compuestos Organofosforados , Animales , Butirilcolinesterasa/sangre , Bovinos , Colinesterasas/sangre , Exposición a Riesgos Ambientales , Monitoreo del Ambiente , Insecticidas/farmacología , México , Análisis Multivariante , Valores de Referencia , Clima TropicalRESUMEN
Four mouse monoclonal antibodies (MAbs) that react with filamentous M13KO7 and R408 phage were obtained. Three of these MAbs (two IgG2a and one IgG3) recognize linear sequences of the p8 main structural coat protein, and one (IgG2a) identifies a putatively conformational epitope, as suggested by Western blot. These MAbs also react with recombinant phage expressing peptide antigens fused to p8, and are though useful reagents for peptide/protein phage display screening based methods. The latter was shown in an enzyme-linked immunoadsorbent assay (ELISA) and a visual immunoassay where one of the anti-p8 MAbs was used to capture recombinant phages displaying a peptide characteristic of the Hepatitis B virus surface antigen or a Dengue virus-related peptide antigen.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Bacteriófago M13/inmunología , Cápside/inmunología , Inovirus/inmunología , Biblioteca de Péptidos , Animales , Anticuerpos Antivirales/metabolismo , Bacteriófago M13/metabolismo , Sitios de Unión de Anticuerpos , Unión Competitiva/inmunología , Línea Celular , Femenino , Humanos , Inmunoglobulina G/metabolismo , Ratones , Ratones Endogámicos BALB C , Células Tumorales CultivadasRESUMEN
An immunoaffinity chromatographic method was developed using a mAb immunosorbent to purify recombinant hepatitis B surface antigen (r-HBsAg) from yeast. Elution conditions using a mAb-coated ELISA were improved to select the best conditions to purify r-HBsAg. The optimum results in terms of total quantitative recovery were obtained using 20 mM Tris pH 11.6. An increase in the CB.Hep-1 mAb (anti-HBsAg) useful immunosorbents half-life and in its yield per cycle was obtained when alkaline elution conditions were used. Moreover, the basic conditions do not affect either the antigenic characteristics or the purity or the molecular integrity of r-HBsAg.