Cyanogen bromide‐activated nitrocellulose membranes: A new tool for immunoprint techniques

Abstract

C yanogen bromide-activated nitrocellulose membranes: A new tool for immunoprint techniquesNitrocellulose membranes were activated by cyanogen bromide in order to improve their binding capacities. Crude extracts of a grass pollen, Dactylis glomerata, and a mite, Dermatophagoides farinae, were separated by isoelectric focusing in agarose and blotted by capillary transfer onto nitrocellulose or activated nitrocellulose. The Dermatophagoides farinae components were visualized by Coomassie Brilliant Blue, Indiaink or Aurodye staining. The Dactylis glomerata and Dermatophagoides farinae allergens were revealed by radioimmunodetection using sera from allergic patients. A great increase of allergen binding was observed visually and by radioactive counting on activated nitrocellulose as compared with untreated nitrocellulose. New allergens which were not detectable on nitrocellulose appeared on activated nitrocellulose.Characterization of allergen molecules is of great interest in the standardization of allergens and elucidation of the mechanisms underlying the allergic manifestations [ 1-31. Immobilization by electrophoretic transfer [4] or capillary transfer 1. 51 onto membranes of nitrocellulose (NC) after electrophoretic separation of a complex crude extract is useful for characterization of antigens [6, 71. Cellulose acetate and cellulose membranes activated by cyanogen bromide (CNBr) had already been used for transfer [S, 91 but NC membranes were usually preferred owing to their facilitated handling and good binding capacity. In the present report, NC membranes were activated with CNBr to improve their binding capacity. Crude extracts of Dactylis glomerata (Dg) pollen and of Dermatophagoides farinae (Df) were separated by isoelectric focusing (IEF) followed by blotting of the separated constituents onto NC or CNBr-activated NC (a-NC) membranes and visualization by carbon or gold colloids. The aller-.gen binding was revealed by radioimmunodetection using sera from Dg or Df allergic patients.The Dg-soluble extract was obtained by stirring 60 mg Dg pollen from our own production, suspended in 500 pL distilled water for 1 h at room temperature. The Df-soluble extract was obtained from a whole mite culture as previously described {lo], lyophilized and dissolved in distilled water to a protein content of 50 mg/mL. The Dg and Df extracts were centrifugedfor 5 min at 9000 gjust beforeuse. Rabbit 1251-labelled antihuman IgE antibodies were obtained from Pharmacia (Uppsala, Sweden). Anti-Dg sera were obtained from Dg-pollensensitive patients whose radioallergo sorbent tests (RAST, Pharmacia) were of class IV, with total IgE levels less than 200 IU/mL. Anti-Df sera were obtained from Df-sensitive patients with different RAST class values (Fig. 3) and total IgE levels less than 400 IU/mL.A previously described technique [ 111 was modified to activate NC. Ten g CNBr (Merck, Darmstadt, FRG) were dis-~ ,.Abbreviations: a-NC, cyanogen bromide-activated nitrocellulose; CNBr, cyanogen bromide; Df, Dermatophagoides farinae; Dg, Dact...

Differential IgE sensitization to cypress pollen associated to a basic allergen of 14 kDa

“…After IEF separation, 5-mm strips of the gel were cut, incubated in the equilibration buffer [114 mmolAEL )1 Tris pH 6.8 containing 12% (w ⁄ v) SDS] for 30 min and submitted to the second dimension SDS ⁄ PAGE in the same conditions as described above. One-DE and 2-DE gels were either silver stained according to Blum et al [39], Coomassie Blue stained or blotted onto a cyanogen bromide activated nitrocellulose (NCa) sheet [40] (Optitran Ò BA-S 83; Schleicher and Schuell, Dassel, Germany).…”

Isotypic Analysis of Grass Pollen-Specific Immunoglobulins in Human Plasma

“…This was ac complished using NC filter paper (BA 83, Schleicher and Schuell. Dassel, FRG) that had been previously activated with cyanogen bro mide according to the method of Dcmeulemestcr et al [23], Deion ized water (5 ml/gel) was first layered on the horizontally positioned agarose gel followed by the gentle layering of a wet NC filter over the gel surface. After the excess water was drained, the NC filter was cov ered with a single sheet of dry Whatman No.…”

Identification of <i>Psilocybe cubensi</i><i>s</i> Spore Allergens by Immunoprinting

“…Before staining, portions of gels were fixed in a solution of 3.5% sulfosalicylic acid and 11.5% trichloroacetic acid for 1 h. After equilibration in de staging solution for 20 min, the fixed gels were stained for 20 min in 0.1% Coomassie brilliant blue R 250 preheated to60°C. Focused pro teins were blotted by passive transfer to a nitrocellulose membrane (0.2 pm pore size) previously activated with CNBr [22].…”