Application of dye-ligands affinity adsorbent in capturing of rabbit immunoglobulin G

The applicability of dye-ligands attached to an expanded bed chromatography quartz base matrix (Streamline™) for the affinity bioseparation of rabbit immunoglobulin G (IgG) was investigated. Reactive Green 5 (RG-5) immobilized onto adsorbent was selected for capturing of rabbit-IgG due to its higher binding capacity compared to other dye-ligands possessing similar ligand density. Adsorption parameters such as pH, temperature, ionic strength and initial rabbit-IgG concentration were optimized for the adsorption of rabbit-IgG on the RG-5-immobilized adsorbent. The highest rabbit-IgG adsorption was recorded in pH 7.0, while the maximum binding capacity for BSA was achieved at pH 4.0. The adsorption of rabbit-IgG on RG-5-immobilized adsorbent was declined as the increase of ionic strength. There is no significant influence of temperature against adsorption efficiency of RG-5-immobilized adsorbent for rabbit-IgG. The adsorption phenomenon of rabbit-IgG on RG-5-immobilized adsorbent appeared to follow the Langmuir–Freundlich adsorption isotherm model. The theoretically maximum binding capacity (qm) of RG-5-immobilized adsorbent estimated from this isotherm was 49.3 mg ml−1, which is very close to that obtained experimentally (49.0 mg ml−1). About 50% of bound BSA on RG-5-immobilized adsorbent in binary adsorption system was removed with washing buffer containing 1 M NaCl

Purification of rabbit polyclonal immunoglobulin G using anion exchangers.

Negative chromatography antibody purification (N-CAP) using the weak anion exchanger STREAMLINE™ DEAE to extract impurities while retaining the target antibody is proposed as an effective method for the recovery of antibody from rabbit serum. The effects of pH and initial protein concentration on the removal of albumin were investigated. The optimal pH and initial protein concentration for the efficient removal of albumin from rabbit serum were pH 8.0 and 0.5 mg/ml, respectively. Under optimal binding conditions, DEAE successfully removed more than 90% of the albumin from rabbit serum with less than 20% IgG loss. This process offered good polyclonal IgG yield of 80% with a purity of 83% and a purification factor of 5.5. The use of a strong anion exchanger like STREAMLINE™ Q XL for albumin removal was also explored. Under similar optimized conditions, albumin removal by Q XL was as high as 90%. However, IgG recovery and purity were reduced to about 70% and 62%, respectively. Thus, N-CAP using the anion exchanger DEAE removes albumin from rabbit serum and thereby offers an efficient means of purifying polyclonal antibodies

Polyclonal antibody production anti Pc_312-324 peptide: Its potential use in electrochemical immunosensors for transgenic soybean detection

A new polyclonal antibody that recognizes the CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS), which provides resistance to glyphosate in soybean (Roundup Ready®, RR soybean), was produced. New Zealand rabbits were injected with a synthetic peptide (Pc_312-324, (PEP)) present in the soybean CP4-EPSPS protein. The anti-PEP antibodies production was evaluated by electrophoresis (SDS-PAGE) and an enzyme-linked immunosorbent assay (ELISA) was developed in order to study their specificity. The ELISA showed that the polyclonal antibody was specific to PEP. In addition, the anti- PEP was immobilized onto a gold disk electrode and the antigen-antibody interaction was evaluated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Moreover, the EIS showed that the electron transfer resistance of the modified electrode increased after incubation with solutions containing CP4-EPSPS protein from RR transgenic soybean, while no changes were detected after incubation with no-RR soybean proteins. These results suggest that the CP4-EPSPS was immobilized onto the electrode, due to the specific interaction with the anti-PEP. These results show that this antigen-antibody interaction can be detected by electrochemical techniques, suggesting that the anti-PEP produced can be used in electrochemical immunosensors development to quantify transgenic soybean.Fil: Farias, Marcos Ezequiel. Universidad Nacional de Río Cuarto. Instituto para el Desarrollo Agroindustrial y de la Salud. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto para el Desarrollo Agroindustrial y de la Salud; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Marani, Mariela Mirta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto Patagónico para el Estudio de los Ecosistemas Continentales; ArgentinaFil: Ramirez, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: Niebylski, Ana Maria. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Biotecnología Ambiental y Salud - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Biotecnología Ambiental y Salud; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Correa, Nestor Mariano. Universidad Nacional de Río Cuarto. Instituto para el Desarrollo Agroindustrial y de la Salud. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto para el Desarrollo Agroindustrial y de la Salud; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; ArgentinaFil: Molina, Patricia Gabriela. Universidad Nacional de Río Cuarto. Instituto para el Desarrollo Agroindustrial y de la Salud. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto para el Desarrollo Agroindustrial y de la Salud; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentin

Development of dye affinity adsorbents for recovery of polyclonal anti-hepatitis B core antigen immunoglobulin G

Antibodies such as immunoglobulin G (IgG) have been used extensively for therapeutic and diagnostic purposes. Protein A affinity chromatography which is highly specific towards IgG is a standard method to purify it. However, using expensive and unstable protein A in large-scale production has increased the antibody production cost accordingly. Affinity dye-ligands which are widely used for protein purification has demonstrated their high binding capacity as 40 mg/mL comparable to protein A. Moreover, their widespread availability, ease and speed of preparation, chemical stability, and ease of storage, render them an attractive alternative choice. Especially, their economy is also a major consideration in replacement of expensive protein A. Thus, the development of selective recovery of polyclonal anti-hepatitis B core antigen immunoglobulin G (anti-HBcAg IgG) from rabbit sera has been investigated. Four different reactive dye-ligands; Cibacron Blue 3GA (CB), Reactive Brown 10 (RB 10), Reactive Red 120 (RR 120) and Reactive Green 5 (RG 5) were covalently attached on the Streamline quartz base matrix via triazine linkage under alkali condition. Essentially at start, IgG antibody’s binding capacity screening of these immobilized dyes was required. Similar amount of dye-ligands attached on the bare matrix, determined by mass balance method was attributed relatively in comparison of adsorption capacities for different dye-ligands. From the simulating adsorption study in single protein system, the immobilized RG 5 was chosen as its capacity for fewer albumins and more IgG adsorbed at pH 7.0, compared to other immobilized dye-ligands possessing similar ligand density. The content of RG 5 immobilized on the matrix was 17.4 µmol/mL adsorbent. About 64% of rabbit IgG was bound on the immobilized RG 5 at pH 7.0 in binary protein binding system with similar ratio of both albumin and rabbit IgG. The maximum adsorption capacity (qm) of RG-5 immobilized adsorbent for rabbit IgG was 49.0 mg/mL adsorbent and the dissociation constant (Kd) value was found to be 3.33×10–6 M. The phenomenon of reversible IgG adsorption on the adsorbent appeared to follow the Langmuir-Freundlich isotherm model. Serum from the immunized rabbits against hepatitis B core antigen (HBcAg) was used as a feedstock containing polyclonal anti-HBcAg IgG solely for batch antibody purification study. Highly abundant albumin and other serum proteins which constitute about 80% of total serum protein are a major interference in dye-ligand affinity chromatographic studies. This leads to the strategy of removing contaminant proteins before subjecting to dye-ligand immobilized system. Anion exchange adsorbents like the Streamline DEAE and Streamline Q XL were introduced as their high capacity available for albumin. Although both anion exchangers were capable of removing most of albumin and other contaminants greater than 90%, the loss of IgG was higher in the presence of Q XL. As a result, the removal of albumin was accomplished in high efficiency via a strong adsorption on DEAE under optimized conditions as followed: 0.5 mg/mL initial protein concentration, pH 8.0; 0.25 mL settled bed volume of Streamline DEAE. Consequently, 80% of polyclonal anti-HBcAg IgG was recovered. A two step procedure using Streamline DEAE anion exchanger and RG-5 immobilized adsorbent was performed for removing albumin and capturing IgG, respectively, under the optimized conditions. After antibody adsorption, bound IgG was eluted in elution medium, pH 8.0 containing 1.0 M NaCl, resulting about 53% IgG recovered with 86% purity and a purification factor of 6. As exhibited in the current study, DEAE anion exchanger is credited for high efficacy to remove most contaminant proteins from rabbit serum. The purified antibodies can be a useful reagent in diagnosis of chronically infected hepatitis B carriers. Moreover, synthetic dye-ligands can be a potential alternative possessing a tendency of binding to biomolecules for several biological purposes

Analysis of Lead and Cadmium Contents in Local Vegetables in Surat Thani, Thailand

Two toxic heavy metals, cadmium (Cd(II)) and lead (Pb(II)), in samples of local vegetables were analyzed by graphite furnace atomic absorption spectroscopy (GFAAS). Pak-Leang (Gnetum gnemon Linn.), Pak-Waen (Marsilea crenata Presl.), Mun-Poo (Glochidion littorale Blume Baill.), and Chamuang (Garcinia cowa Roxb.) were from fresh markets in 4 districts namely Muang, Phunphin, Kanchanadit and Ban Na Doem, Surat Thani province. The preparation of samples was carried out by mixed acid digestion procedure in order to extract the heavy metals. From the GFAAS analysis of sample solutions, the average lead contents were as follows: 0.10 ± 0.11 mg kg-1 in Pak-Leang, 0.04 ± 0.07 mg kg-1 in Pak-Waen, 0.14 ± 0.17 mg kg-1 in Mun-Poo and 0.02 ± 0.05 mg kg-1 in Chamuang. The results indicated that the concentrations of lead within these local vegetables were under the maximum allowable level according to the standard of the Ministry of Public Health, Thailand. On the other hand, analysis of cadmium found that 3 certain vegetables including Pak-Waen (0.48 ± 0.27 mg kg-1), Mun-Poo (0.78 ± 0.72 mg kg-1) and Chamuang (0.34 ± 0.27 mg kg-1), were contaminated with cadmium higher than the maximum allowable levels in the average for the standards of Australia-New Zealand, Codex, China and the European Union. The assessment of heavy metal indicated that these accumulation quantities in edible plants could be valuably evident for public concerns and research-based food safety. doi:10.14456/WJST.2014.