A method for preparing IgG F(ab′)2 fragments using small amounts of serum

Madsen, L.H.; Rodkey, L. Scott

doi:10.1016/0022-1759(76)90210-6

Cited by 55 publications

(15 citation statements)

References 16 publications

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“…Anti-MCMV IgG was precipitated three times by one-third saturated (NH~)2SO~, digested with pepsin (Sigma, 1:60000), and purified F(ab')z was obtained with a Sephadex G-200 column (Madsen & Rodkey, 1976). Specificity of purified F(ab') 2 was confirmed by Ouchterlony double immunodiffusion test using rabbit anti-mouse IgG Fc fragment antibody (Nordic Immunological Laboratories, Maidenhead, U.K.).…”

confidence: 99%

Enhancing Antibodies, Macrophages and Virulence in Mouse Cytomegalovirus Infection

Inada

Chong

Mims

1985

Journal of General Virology

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SUMMARYThe infectivity of tissue culture-passed mouse cytomegalovirus (MCMV) for resident mouse peritoneal macrophages in the presence of serial dilutions of antiviral antibody was studied by fluorescent antibody staining and virus yields. Although MCMV was neutralized by high concentrations of antiserum, there was a twofold enhancement of infectivity by subneutralizing antibody concentrations. On further dilution of antiserum, significant neutralization appeared again. When F(ab')_, fragments of anti-MCMV IgG were used or when macrophages were pretreated with monoclonal antibody to Fc receptor, there was no enhancement, and no neutralization at high dilutions of antiserum. This suggests that both enhancement and high dilution neutralization are mediated via the Fc portion of IgG and Fc receptors of macrophages. Tissue culture-passed virus whose infectivity for macrophages was reduced by high dilutions of antibody was converted to a more infectious state by addition of anti-mouse immunoglobulin. Similar results were obtained with salivary gland virus, which is less infectious for macrophages and is coated with nonneutralizing antibody. Tissue culture-passed virus is known to be less virulent for suckling mice and more infectious for macrophages than salivary gland-passed virus. When tissue culture-passed virus was coated with appropriately diluted antiviral antibody, not only was its infectivity for macrophages reduced, but it also became more virulent than control virus treated with normal mouse serum. These results are interpreted in terms of the optimal density of Fc on the virus-immunoglobulin complex in relation to the density of Fc receptors on macrophages.

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confidence: 99%

Enhancing Antibodies, Macrophages and Virulence in Mouse Cytomegalovirus Infection

Inada

Chong

Mims

1985

Journal of General Virology

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“…This peak was pooled, concentrated, and applied to a 500-ml Biogel AO.5m column (Bio-Rad Laboratories, Richmond, CA). The antibody peak was again pooled and concentrated and then digested for 24 h with pepsin as described (8). The pepsin digest was reapplied to the Biogel AO.5m column, and the column fractions were assayed for the presence of F(ab')2 and intact IgG by SDS-PAGE (9).…”

Section: Monoclonal Antibodiesmentioning

confidence: 99%

Mechanism of inhibition of immunoglobulin G-mediated phagocytosis by monoclonal antibodies that recognize the Mac-1 antigen.

Brown¹,

Bohnsack²,

Gresham³

1988

J. Clin. Invest.

116

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“…Fractions after isoelectric focusing were adjusted to pH 7, dialyzed against 0.14 M NaCl/0.01 M Tris-HCI at pH 7 (17) or papain (18), respectively. The F(ab')2 fragments were isolated by chromatography on Sephadex G-150 (17). The Fab fragments were separated from Fc fragments by affinity chromatography on anti-Fab columns.…”

Section: Methodsmentioning

confidence: 99%

Host specificity of a serum marker for hepatitis B: evidence that "e antigen" has the properties of an immunoglobulin.

Neurath¹,

Strick²

1977

Proc. Natl. Acad. Sci. U.S.A.

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A family of antigens, referred to collectively as e antigen (eAg), has been detected in sera of some individuals with liver disease who test positive for hepatitis B surface antigen. Studies on eAg partially purified by affinity chromatography on insolubilized antiboies to eAg revealed the following: (i) (eAg). Though distinct from HBsAg in both immunologic and physicochemical properties (8), eAg is also specifically associated with HBV infections and appears to be a marker for the infectivity of HBsAg-positive sera (7, 9, 10); its persistence seems to be associated with chronic liver disease (11). Our attempts to characterize eAg, described here, led to the surprising finding that eAg has the properties of an immunoglobulin. MATERIALS AND METHODSeAg was partially purified by precipitation with polyethylene glycol 6000, followed either by affinity chromatography on columns of insolubilized antibodies to eAg (eAbl (12) or by chromatography on DEAE-Bio-Gel (13). eAb was prepared from human sera by sequential precipitation with Rivanol and Na2SO4 (14). Antibodies to individual human plasma proteins and to IgG subclasses were each linked to Sepharose 4B as described (15). Normal human serum or IgG was insolubilized by the same procedure.Partially purified eAg (total amount of protein: 90-250 mg in 30 mM sodium citrate, pH 7.4) was submitted to isoelectric focusing (16). Fractions after isoelectric focusing were adjusted to pH 7, dialyzed against 0.14 M NaCl/0.01 M Tris-HCI at pH 7.2 (Tris/saline), concentrated about 10-fold, and tested for eAg. Identical results were obtained with either the cathode or the anode positioned in the bottom of the electrofocusing column.To compare the sedimentation rates of eAg and an IgG (18), respectively. The F(ab')2 fragments were isolated by chromatography on Sephadex G-150 (17). The Fab fragments were separated from Fc fragments by affinity chromatography on anti-Fab columns. F(ab")2 fragments from samples containing eAg were prepared by cleavage with CNBr (19), followed by affinity chromatography on anti-Fab columns.eAg purified by affinity chromatography or proteins eluted with 50 mM Na2B407 (pH 10.9) from extensively washed eAg-eAb precipitin lines excised from the agarose gel of rheophoresis plates were each labeled with 1251 (20) or with [125I]-iodinated p-hydroxyphenylpropionic acid N-hydroxysuccinimide ester (Bolton-Hunter reagent; New England Nuclear, Boston, MA). Similar results were obtained by either of the two methods. Labeled eAg was further purified by isoelectric focusing. Fractions collected in the pH range of 4.9-5.7 were pooled, dialyzed against Tris/saline, mixed with 2 ml of lamb serum and with normal human serum proteins, precipitated from 10 ml of serum with polyethylene glycol 6000 (600 mg), and resuspended in 2 ml of Tris/saline. Labeled eAg from this mixture was repurified by affinity chromatography on a 15-ml column of insolubilized eAb.The method for polyacrylamide gel electrophoresis described earlier (16) was used. Samples were dissociated...

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A method for preparing IgG F(ab′)2 fragments using small amounts of serum

Cited by 55 publications

References 16 publications

Enhancing Antibodies, Macrophages and Virulence in Mouse Cytomegalovirus Infection

Enhancing Antibodies, Macrophages and Virulence in Mouse Cytomegalovirus Infection

Mechanism of inhibition of immunoglobulin G-mediated phagocytosis by monoclonal antibodies that recognize the Mac-1 antigen.

Host specificity of a serum marker for hepatitis B: evidence that "e antigen" has the properties of an immunoglobulin.

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