Immunoaffinity purification of recombinant hepatitis B surface antigen from yeast using a monoclonal antibody

Agraz, Alberto; Duarte, Carlos A.; Costa, Lourdes; Pérez, Lincidio; Páez, Rolando; Pujol, Vivian; Fontirrochi, Giuvel

doi:10.1016/0021-9673(94)80591-1

Cited by 44 publications

(14 citation statements)

References 14 publications

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“…The high purity of the purified antibodies was demonstrated with sodium dodecyl sulphate-polyacrylamide gel electrophoresis. ability to dissociate complexes formed between digoxin and a rabbit anti-digoxin Ab [11][12][13][14][15]. We also compared the effectiveness of these eluents.…”

Section: Introductionmentioning

confidence: 99%

Application of an ELISA-elution Assay to Dissociate Digoxin-antibody Complexes in Immunoaffinity Chromatography

Lin

Wang

et al. 2010

Scandinavian Journal of Immunology

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In this study, we used a modified enzyme‐linked immunosorbent assay (ELISA)‐elution technique as a screening tool to select specific elution conditions. We examined 12 different elution conditions for the removal of antibodies from a complex on an ELISA plate; 0.2 mol/l glycine‐HCl (pH 2.5), 1.0 mol/l acetic acid (pH 2.5), 25% methanol (pH 2.5) and 3 mol/l NaSCN showed a higher elution efficiency. We conducted affinity chromatography with these four conditions for the purification of anti‐digoxin antibodies from hyperimmune sera with a digoxin‐specific column using ω‐aminoalkyl derivatives of Sepharose 4B, whose elution efficiency was similar to that of ELISA. We also monitored the relative specific activities during elution from the digoxin‐specific column. The optimum, general‐purpose dissociation reagent for this immunoaffinity system was identified as 25% methanol (pH 2.5) with an elution efficiency and relative specific activity of 88.40% and 62.25%, respectively. The high purity of the purified antibodies was demonstrated with sodium dodecyl sulphate‐polyacrylamide gel electrophoresis.

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Section: Introductionmentioning

confidence: 99%

Application of an ELISA-elution Assay to Dissociate Digoxin-antibody Complexes in Immunoaffinity Chromatography

Lin

Wang

et al. 2010

Scandinavian Journal of Immunology

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“…The aforementioned motivated us to evaluate the feasibility of producing an anti‐HBsAg [anti‐(hepatitis B surface antigen)] mouse mAb (monoclonal antibody) in transgenic tobacco. mAb CB‐Hep.1 is currently used for the immunoaffinity‐chromatography purification of a recombinant HBsAg and the production of a commercially successful vaccine [21].…”

Section: Introductionmentioning

confidence: 99%

Expression and characterization of an anti‐(hepatitis B surface antigen) glycosylated mouse antibody in transgenic tobacco (Nicotiana tabacum) plants and its use in the immunopurification of its target antigen

Ramírez

Rodríguez

Ayala

et al. 2003

Biotech and App Biochem

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Transgenic plants expressing recombinant immunoglobulins have arisen as an alternative technology for the large-scale production of antibodies useful in therapeutics and in industrial processes. In the present paper we report the expression in transgenic tobacco ( Nicotiana tabacum ) of an anti-HBsAg [anti-(hepatitis B virus surface antigen)] mouse IgG1 mAb (monoclonal antibody), currently used for the industrial purification of the recombinant vaccine antigen. Using the sweet potato sporamin signal peptide, a KDEL (Lys-Asp-Glu-Leu) ER (endoplasmic reticulum) anchorage domain, and a heavy- and light-chain gene tandem construction, we generated F1 plants in which the expression of the antibody accounted for 0.5% of the total soluble proteins. The 'plantibody' (functional IgG antibody produced in plants) was easily purified by Protein A-Sepharose chromatography with a yield of approximately 35 microg/g of fresh leaf material, and its glycosylation indicated that, irrespective of the KDEL signal, the molecule is modified in both the ER and Golgi. Finally, a successful comparison of the plantibody with the ascites-derived mAb in the immunoaffinity purification of the vaccine recombinant HBsAg was performed. Taken as a whole, our results show that the large-scale production of this antibody of industrial relevance in transgenic tobacco is feasible.

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“…Several factors can cause a loss of column capacity during a repeated operation. One of the most important is the irreversible denaturalization of the antibody, usually caused by harsh elution conditions [24]. The plantibody HB-01 columns (ligand density: 3.41, 4.45, and 5.31 mg/mL) showed rapid elution capacity decrease similar to that of the mAb column.…”

Section: Resultsmentioning

confidence: 98%

Comparison of different ligand densities in immunoaffinity chromatography of the plantibody HB-01 coupled to Sepharose CL-4B to purify the rHBsAg

Valdés¹,

Medina²,

Ferro³

et al. 2007

Journal of Chromatography B

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Immunoaffinity purification of recombinant hepatitis B surface antigen from yeast using a monoclonal antibody

Cited by 44 publications

References 14 publications

Application of an ELISA-elution Assay to Dissociate Digoxin-antibody Complexes in Immunoaffinity Chromatography

Application of an ELISA-elution Assay to Dissociate Digoxin-antibody Complexes in Immunoaffinity Chromatography

Expression and characterization of an anti‐(hepatitis B surface antigen) glycosylated mouse antibody in transgenic tobacco (Nicotiana tabacum) plants and its use in the immunopurification of its target antigen

Comparison of different ligand densities in immunoaffinity chromatography of the plantibody HB-01 coupled to Sepharose CL-4B to purify the rHBsAg

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