Fractionation of polyclonal antibody by isoelectric focusing and chromatofocusing: separation of high-affinity rabbit clonotype anti-thyroxine antibody.
Immunoglobulin G fractions prepared from conventional rabbit anti-thyroxine (T4) antisera were fractionated by agarose gel isoelectric focusing (IEF) in the range of pH 3 to 10, and by chromatofocusing using a Fast Protein Liquid Chromatography (FPLC) system. The clonotype antibodies were recovered from the fractions and subjected to Scatchard plot analysis. The highest affinity constants of the initial antibody (shown in parentheses) and those of the antibodies recovered were IEF, 1.8 X 10(9) to 8.3 X 10(9) M-1 (2.2 X 10(9) M-1); FPLC, 2.4 X 10(9) to 6.0 X 10(9) M-1 (2.5 X 10(9) M-1). A sensitive radioimmunoassay of T4 was achieved with the isolated high-affinity anti-T4 antibody. The minimum detectable concentration of T4 was 6.3 X 10(-15) to 1.5 X 10(-14) mol/tube, which was three to five times lower than detectable with the initial antibodies.
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D000915:Antibody Affinity; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid; D007074:Immunoglobulin G / Q000302:isolation & purification; D007525:Isoelectric Focusing; D011817:Rabbits; D013974:Thyroxine / Q000276:immunology*
D000906:Antibodies; D007074:Immunoglobulin G; D013974:Thyroxine