W e h a v e d e m o n s t r a t e d t h a t r a t b a s o p h i l i c l e u k e m i a cells ( R B L -1 cells) 1 b i n d rat I g E (1). T h o s e s t u d i e s a n d o t h e r s (2) s h o w e d t h a t t h e I g E is b o u n d to t h e cell s u r f a c e w i t h a s p e c i f i c i t y s i m i l a r to t h a t o b s e r v e d w i t h n o r m a l rat m a s t cells.In t h i s p a p e r we p r e s e n t a q u a n t i t a t i v e a n a l y s i s of t h e b i n d i n g . F o r s o m e of t h e e x p e r i m e n t s larger a m o u n t s of I g E were n e c e s s a r y t h a n c o u l d be o b t a i n e d easily f r o m r e a g i n i c s e r u m . F o r t h i s reason an I g E m y e l o m a p r o t e i n (3) was u s e d a f t e r d e m o n s t r a t i n g t h a t its b i n d i n g p r o p e r t i e s were i n d i s t i n g u i s h a b l e f r o m those o b s e r v e d for t h e r e a g i n i c a n t i b o d i e s u s e d p r e v i o u s l y . Materials and MethodsIgE Preparations. IgE was purified from rat anti-Nippostrongylus brasiliensis serum by the method described previously (4). A rat IgE myeloma protein was purified from serum of rats bearing tumor IR 162 (3) in an analogous fashion: 12 ml serum were dialyzed vs. 0.2 M borate-buffered saline pH 8.0 (BBS) and the protein precipitating between 38 and 48% saturated (NH4)2SO4 at 4°C collected. This fraction was chromatographed on a 4.1 × 105 cm column of Sepharose 6B and the major component in the effluent concentrated by ultrafiltration (Amicon Corp., Lexington, Mass.) and dialyzed against a solution which was 33 mM in Tris base, 25 mM in phosphate, having a conductivity of 2.3 mmho and a pH of 8.0 at room temperature. The protein (~ 100 absorbancy units at 280 nm) was next chromatographed on a 2.6 × 7.2 cm column of "DE-52" diethylaminoethyl cellulose (Whatman Biochemicals Ltd., Maidstone, England). The excluded component was dialyzed against BBS and applied successively to two Sepharose 2B immunoadsorbents containing rabbit antibodies to the normal rat serum fraction precipitating at 50% saturated (NH4)2S04 and rabbit antibodies to normal rat proteins excluded from DE-52 respectively (4). Both antibody preparations had been absorbed with a Sepharose 2B immunoadsorbent containing Fab fragments of rat IgG. The purified IgE preparations were stored at 4°C in BBS and radiolabeled with ~25I {New England Nuclear, Boston, Mass.) by the chloramine-T method (4, 5) as required.RBL-1 Cells. Cells were cultured in Falcon flasks (Falcon Plastics, Div. of BioQuest, Oxnard, Calif.) as described previously (1) or in spinner cultures (footnote 2). In the latter case the medium
A rat basophilic leukemia cell line originally described by Eccleston et al. (3) has been successfully transplanted into eight Wistar strains and adapted to suspension cell culture without noticeable morphological changes. The cells deplete the reaginic activity of mouse and rat immune sera to an extent roughly equivalent to that reported for normal rat mast cells. Studies utilizing radioiodinated antilight-chain antibodies and radioiodinated partially purified rat IgE indicate that the cells bind IgE to their surface membrane with high specificity. Heating or mildly reducing the rat IgE destroyed its binding activity. The binding is unaffected by the presence or absence of Ca++ and Mg++, and is markedly inhibited by reaginic but not by normal rat or mouse serums. The affinity of these cells for human IgE, if present at all, is substantially lower than for rat IgE.
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