title: Purification and partial characterization of a highly immunogenic human leukocyte protein, the L1 antigen.
abstract: L1 is a major and highly immunogenic protein component of human granulocytes. It was purified from random samples of blood-donor leukocytes. An antiserum to L1 was initially raised in rabbits by immunization with protein fractions from preparative agarose gel electrophoresis. The main problem in purifying L1 was its poor stability when carried through multiple steps. Preparative isoelectric focussing was therefore adopted as an efficient one-step method. The isoelectric focussing pattern of L1 was greatly influence by the presence of EDTA or calcium ions in the sample buffer. With low EDTA concentrations or calcium excess, L1 focussed as seven protein bands in two regions, pH 6.1-6.5 and pH 7.6-8.4. Conversely, L1 was found only in the pH-6.1-6.5 region when excess EDTA was added to the sample. Irrespective of conditions, the bulk of L1 focussed at pH 6.3 and pH 6.5. Also the electrophoretic mobility of L1 was strongly influenced by calcium ions, suggesting uptake of calcium by the protein. In the presence of calcium, L1 adhered to glass surfaces and filters used for concentration of protein solutions. The latter problem could be prevented by addition of EDTA. The molecular mass of L1 was determined to be about 36.5 kDa. The molecule was shown to consist of three non-covalently linked 12.5-kDa subunits.
mesh_terms: D000818:Animals; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007106:Immune Sera / Q000032:analysis; D007120:Immunochemistry; D007525:Isoelectric Focusing; D007962:Leukocytes / Q000276:immunology*; D008970:Molecular Weight; D011817:Rabbits
chemical_list: D000941:Antigens; D007106:Immune Sera