RESUMEN
The antigenic P64k protein from the pathogenic bacterium Neisseria meningitidis has been used as an immunological carrier in several conjugated vaccines. The aim of this report was to develop and validate a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of recombinant p64k protein, to perform both manufacturing process and identification in different vaccine preparations. Validation studies were performed according to the guidelines of the International Conference of Harmonization (ICH). The reference curve showed to be precise and accurate over the entire linear range of 1.25 and 20ng/mL with a limit of quantification validated to 1.25ng/mL. The intra- and inter-assay coefficient of variation ranged from 0.35 to 6.65% and 4.70 to 10.63%, respectively. The ANOVA test used in the specificity/interference study revealed parallelism among curves (p>0.1), which indicates the lack of interference in the working range. Recovery obtained from the accuracy test, using three concentration levels, varied between 94 and 111%, confirming the assay's reliability. The short-term study shown the P64k is stable to -20°C up to 1-week. This ELISA was fully used to assess its manufacturing process and molecular interaction issues in several vaccine preparations. Thus, this immunoassay could be an excellent analytical choice to characterize the quality of that recombinant protein in several contexts as manufacturing process and molecular conjugates.
Asunto(s)
Antígenos Bacterianos/análisis , Proteínas de la Membrana Bacteriana Externa/análisis , Vacunas Bacterianas , Ensayo de Inmunoadsorción Enzimática/métodos , Neisseria meningitidis/inmunología , Proteínas Recombinantes/análisis , Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/genética , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Límite de Detección , Estabilidad Proteica , Proteínas Recombinantes/genética , Reproducibilidad de los ResultadosRESUMEN
Antibodies have been one of the proteins widely expressed in tobacco plants for pharmaceutical purposes, which demand contaminant free preparations. Rubisco constitutes 40-60% of tobacco leaf soluble proteins; therefore it is the major potential protein contaminant of plantibodies, while mycotoxins are toxic compounds that could be introduced during the biomass production and post-harvest stages with important consequences to human health. The objective of this paper was to investigate whether Rubisco and mycotoxins are present in Plantibody HB-01 preparations used in the immunopurification of the hepatitis B surface antigen. Rubisco was purified from Nicotiana tabacum yielding 154 microg of protein per gram of leaves and purity over 95%. Among mouse monoclonal antibodies generated against this enzyme, the CBSS.Rub-2 was selected for its immunodetection. It recognizes a conserved sequential epitope of Rubisco large subunit with an affinity constant of 0.13 x 10(8)M(-1). Rubisco quantification limit was 1 microg spreading to the measurement of this contaminant less than 4% of plantibodies samples. Additionally, according to a Reverse Phase-HPLC used to measure the level of adventitiously introduced contaminants, it can be concluded that aflatoxins B1, B2, G1 and G2 were undetected in the purified Plantibody HB-01 samples.
Asunto(s)
Aflatoxinas/análisis , Anticuerpos contra la Hepatitis B/aislamiento & purificación , Planticuerpos/aislamiento & purificación , Ribulosa-Bifosfato Carboxilasa/análisis , Aflatoxinas/química , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/aislamiento & purificación , Western Blotting , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Líquida de Alta Presión/estadística & datos numéricos , Contaminación de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Anticuerpos contra la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/inmunología , Humanos , Ratones , Planticuerpos/genética , Plantas Modificadas Genéticamente , Nicotiana/genética , Nicotiana/inmunologíaRESUMEN
BACKGROUND: Prostate specific antigen (PSA) is a relevant antigen in diagnosis; follow-up, and therapeutic approaches for fighting the prostate cancer. Several methods have been published previously to obtain a high purity preparation of PSA. In general, these methods are expensive, time-consuming, laborious, and in some cases produce low yields. METHODS: Based on a panel of 7 anti-PSA Mab's we carried on binding and elution experiments of PSA antigen in 96-well plates. The selected Mab were immobilized in a Sepharose CL-4B activated matrix with the purpose of purify PSA from human seminal fluid. In order to optimize the purification procedure, we test several washing and elution conditions (chaotropic agents, high ionic strength solution, and extreme pH). RESULTS: We selected a high ionic strength solution (2 M MgCl2) as elution condition, and a previous washing step with a mix of two ionic solutions (2.5 M NaCl pH 8/1 M MgCl2 pH 5.5) in order to purify PSA. Using such conditions we obtained a PSA preparation with 90% of purity and 50% of recovery. CONCLUSION: In this article, we report a simple, quickly, and non-expensive procedure to obtain free-PSA from human seminal plasma at high purity levels.