RESUMEN
Mice immunized with a synthetic peptide located on an intracellular segment of the polytopic Kx protein (37 kDa) from human red blood cells (RBCs) produced a monoclonal antibody called C7B8. As expected, this antibody did not agglutinate common RBCs but reacted with permeabilized cells in flow cytometry. C7B8 recognizes the Kx protein on Western blots. Cross-reactivity of C7B8 with human calpain of human muscle extracts was demonstrated by Western blot analysis. This cross-reactivity precludes the use of C7B8 for Kx tissue distribution studies, but immobilized C7B8 was a convenient tool for purification of the Kell-Kx complex from RBC membrane extract by immunochromatography.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/inmunología , Anticuerpos Monoclonales/inmunología , Sistemas de Transporte de Aminoácidos Neutros/aislamiento & purificación , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/aislamiento & purificación , Reacciones Antígeno-Anticuerpo , Western Blotting , Calpaína/inmunología , Cromatografía de Afinidad , Reacciones Cruzadas , Citometría de Flujo , Humanos , Sistema del Grupo Sanguíneo de KellRESUMEN
The epitope recognized by a new anti-Fy6 monoclonal antibody (MoAb) (clone name: NaM185-2C3) was characterized using peptides synthesized on pins (Epitope scanning kit). The clone was obtained from splenocytes of mice immunized with CHO cells expressing the recombinant Duffy glycoprotein. NaM185-2C3 recognized a linear epitope, the essential portion of which was pentapeptide Phe-Glu-Asp-Val-Trp comprising amino acid residues 22-26 of the main (336aa) isoform of the Duffy antigen. All the amino acid residues of the epitope, except Asp, were essential for the antibody-binding, because they could not be replaced by any or most other amino acid residues. The Asp residue could be replaced by most other amino acid residues and its replacement by some amino acid residues gave a distinct increase in the antibody-binding. The MoAb NaM185-2C3, similarly as other anti-Fy6 antibodies, inhibits interleukin (IL)-8-binding to the Duffy antigen. A part of the results was presented at ISBT meeting (Blanchard et al., 1998, Vox Sanguinis, 74, S1, Abstract no. 71).
Asunto(s)
Anticuerpos Monoclonales/inmunología , Sistema del Grupo Sanguíneo Duffy/inmunología , Epítopos/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Especificidad de Anticuerpos , Mapeo Epitopo , Epítopos/inmunología , Humanos , RatonesRESUMEN
Kx is a polytopic membrane protein of human erythrocytes carrying the Kx blood group antigen, which is deficient in rare patients with McLeod syndrome. Kx is disulphide bond linked to the Kell glycoprotein, which is a bitopic type II membrane protein carrying the Kell blood group antigen. Mice immunized with a synthetic peptide predicted to be located on the second external loop of Kx produced a monoclonal antibody called 3E12 which does not recognize red cells with common Kell phenotype by agglutination and flow cytometry. 3E12 recognizes the Kx protein and the spectrin beta-chain on western blots, the affinity for these two proteins being lowered with increasing ionic strength. Linear epitopes recognized by 3E12 are E116EIEKE121 and L484AQELEKE491 on the Kx protein and spectrin beta-chain, respectively. To quantify the relative amount of Kx in Empigen BB extracts of red cell membranes, an ELISA for Kx was set up which showed conclusively that (i) there is less Kx in membranes of K0 individuals (lacking the Kell glycoprotein) than in membranes of common individuals, and (ii) that all common individuals, typed as K+k-, K-k+ and K+k+, have the same amount of Kx on their red cell membranes. When an erythrocyte membrane detergent extract from one K0 individual was chromatographed on an immobilized 3E12 column, a minute amount of authentic Kell glycoprotein was recovered in acid eluted fractions, indicating that at least the K0 individual under study may still produce some Kell protein.