Fractionation of polyclonal antibody by isoelectric focusing--differences in cross-reactivity and affinity of rabbit clonotype anti-human thyrotropin antibody.
Immunoglobulin G (IgG) fractions prepared from three different batches of rabbit antihuman thyrotropin (hTSH) antisera were fractionated by agarose isoelectric focusing (IEF) in the pH ranges 3 to 10 and 5 to 8. Staining of protein in agarose gel after IEF showed that polyclonal IgG separated into more than 20 protein bands with isoelectric points (pIs) ranging from 6 to 9. The clonotype antibodies to hTSH were recovered from the fractions and subjected to radioimmunoassay for determination of the binding-affinity for hTSH and the cross-reactivity with human chorionic gonadotropin (hCG). The affinity constants of the antibodies recovered ranged from 6.4 X 10(9) M-1 to 3.1 X 10(10) M-1, and the cross-reactivities of the clonotype antibodies differed greatly. A good correlation was observed between the pIs of antibody molecules and their cross-reactivities: antibodies with higher pIs bound hCG more strongly than those with lower pIs. The correlation coefficients between the pIs and cross-reactivities were 0.83, 0.84, and 0.87 in three batches of antibody.
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D000915:Antibody Affinity; D000918:Antibody Specificity; D003429:Cross Reactions; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000302:isolation & purification; D007525:Isoelectric Focusing; D011817:Rabbits; D013972:Thyrotropin / Q000276:immunology*
D000906:Antibodies; D007074:Immunoglobulin G; D013972:Thyrotropin