Purification of human IgG4 subclass with allergen-specific blocking activity.
Blocking antibodies (bAb) induced by allergen immunotherapy are restricted to the IgG1 and IgG4 subclasses, with IgG1 predominating early and IgG4 coming later. Study of IgG4 bAb has been limited, in part, by the absence of a method to purify IgG4. We describe a rapid immunoaffinity chromatographic method for the purification of that subclass from whole serum. Starting serum (TR) contained 90 micrograms/ml Dactylis glomerata (orchard grass) pollen (DGP)-specific IgG4, measured by indirect ELISA. The blocking activity of TR was assayed in vitro on IgE-sensitized human basophils. Immunoadsorption on a strong-binding anti-IgG4 monoclonal antibody (mAb) removed about 90% of the total and allergen-specific IgG4 and nearly all of the blocking activity from TR. An IgG4-rich fraction was then obtained by absorption of several small volumes of TR on a weak-binding anti-IgG4 mAb column at neutral pH followed by elution with glycine-HCl buffer. The pooled eluates contained 82% IgG4, amounting to a 65-fold purification of the serum IgG4; the yield was approximately 30%. Nearly all the DGP-specific antibody was in the IgG4 component of the eluate. The blocking activity of the eluate was approximately equal to that of TR. Immunoblot patterns with the eluate and with TR on SDS-PAGE of DGP were nearly identical. This method thus provides a fully active, relatively pure IgG4 blocking antibody. Moreover, the results reinforce the importance of using a well-chosen mAb when purifying proteins by immunoaffinity chromatography.
Lambin
P
P
Institut National de Transfusion Sanguine, Paris, France.
Bouzoumou
A
A
Murrieta
M
M
Debbia
M
M
Rouger
P
P
Leynadier
F
F
Levy
D A
DA
D000328:Adult; D000485:Allergens / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D015549:Basophil Degranulation Test; D001491:Basophils / Q000276:immunology; D002846:Chromatography, Affinity; D003888:Desensitization, Immunologic; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology; D007073:Immunoglobulin E / Q000276:immunology; D007074:Immunoglobulin G / Q000145:classification / Q000302:isolation & purification*; D008297:Male; D011058:Pollen / Q000276:immunology*
D000485:Allergens; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007073:Immunoglobulin E