A one-step purification method for monoclonal antibodies based on salt-promoted adsorption chromatography on a ‘thiophilic’ adsorbent

Belew, Makonnen; Juntti, N.; Larsson, Anders; Porath, Jerker

doi:10.1016/0022-1759(87)90074-3

Cited by 105 publications

(27 citation statements)

References 9 publications

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“…IgG supernatants were isolated by affinity chromatography using Sepharose-coupled protein G (Pharmacia, Piscataway, NJ). IgA and IgM were isolated from supernatants using Affi-T columns (BIOZyme, Landgraaf, The Netherlands) (25). Purified Abs were submitted to electrophoresis on 8 -18% SDS gradient polyacrylamide gels (Pharmacia) and stained with Coomassie blue.…”

Section: Ig Purificationmentioning

confidence: 99%

Activity of Human IgG and IgA Subclasses in Immune Defense Against Neisseria meningitidis Serogroup B

Vidarsson¹,

Pol²,

Elsen

et al. 2001

The Journal of Immunology

122

125

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Both IgG and IgA Abs have been implicated in host defense against bacterial infections, although their relative contributions remain unclear. We generated a unique panel of human chimeric Abs of all human IgG and IgA subclasses with identical V genes against porin A, a major subcapsular protein Ag of Neisseria meningitidis and a vaccine candidate. Chimeric Abs were produced in baby hamster kidney cells, and IgA-producing clones were cotransfected with human J chain and/or human secretory component. Although IgG (isotypes IgG1–3) mediated efficient complement-dependent lysis, IgA was unable to. However, IgA proved equally active to IgG in stimulating polymorphonuclear leukocyte respiratory burst. Remarkably, although porin-specific monomeric, dimeric, and polymeric IgA triggered efficient phagocytosis, secretory IgA did not. These studies reveal unique and nonoverlapping roles for IgG and IgA Abs in defense against meningococcal infections.

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Section: Ig Purificationmentioning

confidence: 99%

Activity of Human IgG and IgA Subclasses in Immune Defense Against Neisseria meningitidis Serogroup B

Vidarsson¹,

Pol²,

Elsen

et al. 2001

The Journal of Immunology

122

125

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“…However, MAB of the IgM class have been considered unstable, hard to purify and difficult to work with for large-scale purification (Parkhouse, 1984;Knutson et al, 1991). The approaches used have included ammonium sulphate purification (Reik et al, 1987), euglobulin purification (Garcia-Gonzalez et al, 1988), gel filtration, affinity chromatography using immobilized lectins or thiophilic absorbents, or a combination of these procedures (Belew et al, 1987;Danielsson et al, 1988;Juronen et al, 1991). These techniques have proved to be time consuming and cumbersome, especially when large amounts of pure antibody are required.…”

Section: Introductionmentioning

confidence: 98%

Purification of two murine monoclonal antibodies of the IgM class by hydroxylapatite chromatography and gel filtration

Henniker

Bradstock

1993

Biomedical Chromatography

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A two-step method using hydroxylapatite chromatography and gel filtration is described for the purification of two murine monoclonal antibodies of the IgM class. Ascites fluid from each hybridoma was diluted in sodium phosphate buffer (0.01 M, pH 6.8), loaded onto a hydroxylapatite column and eluted with a stepwise sodium phosphate gradient. The immunoreactive protein peaks were concentrated and subjected to gel filtration using either Sephadex G-200 or Sephacryl S-200HR. The biological activity of the end-products was confirmed by complement lysis assay and by indirect immunofluorescence and flow cytometry. The purity of the end-products as assessed by SDS polyacrylamide gel electrophoresis and densitometry was at least 90%. The methods described produced immunoreactive material with a high level of purity. The procedure for each antibody was reproducible and provides a reliable method for purification of monoclonal antibodies of the IgM class.

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“…Although several methods are available for TgM purification, the purified material is usually low in immunoreactivity, probably as a result of steric dissociation occurring during the truculent adsorptiondesorption process (Belew et al, 1987). Furthermore, most of these methods are only applicable for ascites fluid or serum which contain high concentrations of IgM .…”

Section: Introductionmentioning

confidence: 99%

Purification of murine immunoglobulin M from spent tissue culture supernatant by one‐step gel filtration chromatography procedure

Novales-Li

1995

Biomedical Chromatography

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Optimal purification of immunoglobulin M (IgM) grown as spent tissue culture supernatant can be achieved by a straightforward size-exclusion chromatography procedure. Preparative isolation of IgM was achieved by precipitation with saturated ammonium sulphate to achieve a 45% solution. IgM was then purified by gel filtration chromatography with a solution of 100 mM Tris + 150 mM NaCl at pH 8. Denaturing electrophoresis and Western immunoblotting revealed two prominent bands at 80 and 25 kDa, indicative of the heavy and light chains of IgM respectively. The specific immunoreactivity of this IgM was assessed by a modified enzyme antigen capture assay. In contrast to affinity and ion-exchange chromatographies, gel filtration allows retention of IgM immunoreactivity.

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A one-step purification method for monoclonal antibodies based on salt-promoted adsorption chromatography on a ‘thiophilic’ adsorbent

Cited by 105 publications

References 9 publications

Activity of Human IgG and IgA Subclasses in Immune Defense Against Neisseria meningitidis Serogroup B

Activity of Human IgG and IgA Subclasses in Immune Defense Against Neisseria meningitidis Serogroup B

Purification of two murine monoclonal antibodies of the IgM class by hydroxylapatite chromatography and gel filtration

Purification of murine immunoglobulin M from spent tissue culture supernatant by one‐step gel filtration chromatography procedure

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