Purification of human immunoglobulin M by affinity chromatography on protamine-sepharose

Wichman, Anders; Borg, Håkan

doi:10.1016/0005-2795(77)90011-3

Cited by 23 publications

(8 citation statements)

References 20 publications

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“…Similar results were obtained when D23 TBS was subjected to aUinity chromatography on protamine Sepharose [20]. followed by column chromatography of the protaminebound material on Sepharose CL-4B (Fig.…”

Section: Resultssupporting

confidence: 80%

“…The binding of immune complexes to Con A seemed to depend on the glycoprotein nature of the antigen involved rather ihan on binding of the IgG molecules making up these complexes. Protamine-conjugaled Scpharose has been described as having strong allinity for human IgM [20]. The interaction <^t' protamine with human IgM and othci suruin proteins was assumed to be of electrostatic nature and to depend on multiple-point binding between tbe molecules involved.…”

Section: Discussionmentioning

confidence: 99%

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Determination of the Immunoglobulin Class of Complement‐dependent Cytotoxic Antibodies in Serum of D23 Hepatoma‐bearing Rats

et al. 1980

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The immunoglobulin class of the complement-dependent cytotoxic antibodies in serum from D23 hepatoma-bearing rats (D23 TBS) for D23 hepatoma cells was analysed. When studied by affinity chromatography with concanavalin A, protamine, or staphylococcal protein A conjugated to Sepharose, the cytotoxic activity bound to the former two but not protein A. The binding fractions were further characterized by column chromatography on Sepharose CL-4B. The cytotoxic activity was recovered exclusively in the high molecular weight fractions corresponding to human IgM. Monitoring with IgG- or IgM-specific rabbit antibodies indicated that these high molecular weight cytotoxic fractions contained both IgG and IgM. However, fractionation of D23 TBS at low pH suggested that cytotoxicity was due to IgM antibodies rather than to immune-complexed IgG antibodies. This was supported by the findings that rabbit antirat IgM antibodies inhibited the cytotoxicity of TBS completely when added at high dilutions.

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Section: Resultssupporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

Determination of the Immunoglobulin Class of Complement‐dependent Cytotoxic Antibodies in Serum of D23 Hepatoma‐bearing Rats

et al. 1980

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“…Purification of IgM containing IgM–IgG IC from plasma was achieved by modifying an existing fast protein liquid chromatography (FPLC)‐based protocol (22). FPLC was performed using the ÄKTA TM explorer chromatography system and the UNICORN TM analysis software (Amersham Biosciences/Pharmacia Biotech, Uppsala, Sweden).…”

Section: Methodsmentioning

confidence: 99%

IgG2 containing IgM–IgG immune complexes predominate in normal human plasma, but not in plasma of patients with warm autoimmune haemolytic anaemia

Stahl

Sibrowski

2006

European J of Haematology

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The different physicochemical and sterical properties of IgG subclasses may favour a selective enrichment of defined IgG subclasses in IgM-IgG immune complexes (IC) of human plasma under physiological conditions. Such enrichment of IgG subclasses in IgM-IgG IC of plasma may differ from the normal IgG subclass distribution in plasma itself, and contribute to the physiological functions of IgM-IgG IC. Systematic studies on the IgG subclass distribution in IgM-IgG IC in humans are lacking. Using specific analytical techniques to characterise IgM-IgG IC in human plasma (i.e. fast protein liquid chromatography, enzyme-linked immunosorbent assay, affinity biosensor technology), and taking warm autoimmune haemolytic anaemia (WAIHA) of humans as a disease model, we here demonstrate that: (i) IgG2 is the predominant IgG subclass in IgM-IgG IC under physiological conditions, (ii) the predominance of IgG2 within IgM-IgG IC may get lost in polyclonal IgG-mediated autoimmune disease and (iii) the IgG subclass distribution in IgM-IgG IC influences the interaction between IC and blood cells involved in antigen presentation. The data presented here therefore extend the physiological function of IgG2, which is the protective immune response towards carbohydrate antigens in bacterial infections, and suggest IgG2-dependent regulation of immune responses to self-immunoglobulin in humans. The disturbed IgG subclass distribution in IgM-IgG IC of patients with WAIHA might influence activity of self-reactive B cells involved in the pathophysiology of the disease.

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“…The resulting IgM fraction was adjusted to pH 7.4 using a Tris buffer, pH 8.0, and was then subjected to size exclusion chromatography (SEC) (HiPrep Ò Sephacryl S-200 High Resolution, Pharmacia Biotech). This approach results in a highly pure IgM fraction exhibiting the same behavior as IgM in plasma in immunoelectrophoresis and isoelectric focusing [24]. It avoids using acids in the purification process, which might destroy IgMeIgG immune complexes, and it minimizes the risk of formation of IgM aggregates by adding 0.01 M glycine to the Tris buffer.…”

Section: Purification Of Igm From Plasmamentioning

confidence: 96%

Warm autoimmune hemolytic anemia is an IgM–IgG immune complex disease

Stahl

Sibrowski

2005

Journal of Autoimmunity

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Purification of human immunoglobulin M by affinity chromatography on protamine-sepharose

Cited by 23 publications

References 20 publications

Determination of the Immunoglobulin Class of Complement‐dependent Cytotoxic Antibodies in Serum of D23 Hepatoma‐bearing Rats

Determination of the Immunoglobulin Class of Complement‐dependent Cytotoxic Antibodies in Serum of D23 Hepatoma‐bearing Rats

IgG2 containing IgM–IgG immune complexes predominate in normal human plasma, but not in plasma of patients with warm autoimmune haemolytic anaemia

Warm autoimmune hemolytic anemia is an IgM–IgG immune complex disease

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