Immunoadsorbents for the isolation of high-affinity anti-hapten antibodies in high yield.
Anti-hapten antibodies with affinities close to 10(8)/M can be isolated in high yield by elution under mild conditions from immunoadsorbents prepared by coupling haptens directly to an aminoethylamino (AEA) derivative of beaded agarose. In experiments with rabbit anti-2,4-dinitrophenyl (DNP) antibody with an average association constant (Ko) of 1.2 X 10(9)/M, elution of antibody bound to a Sepharose-AEA-DNP column produced a yield of 57%. With rabbit antibody to the 5-dimethylaminonaphthalene-1-sulfonyl (DANS) hapten with Ko=2.7 X 10(8)/M and a Sips heterogeneity index a=0.52, a 20% yield of the antibody found in the starting material by quantitative precipitin analysis was obtained by specific precipitation and dissolution of the precipitate with 0.18 M Na DANSulfonate; this antibody had Ko=6.6 X 10(8)/M and a=0.66. Purification of another aliquot of the same antibody preparation by immunoadsorption on DANS-AEA-Sepharose and elution with the same hapten concentration produced a yield of 75% of antibody with Ko=5.7 X 10(7)/M and a=0.29. In 3 production runs with the new method, apparent yields of 181, 101 and 94% (based on quantitative precipitin analysis of the starting material) of anti-DANS antibody were obtained. In 2 of these runs, the immunoadsorbent was further eluted with 0.1 M acetic acid and then with 0.1 M acetic acid in 10% dioxane; additonal yields of 9 and 4% were obtained with acetic acid and further yields of 15 and 11% with acetic acid/dioxane; the antibody eluted with acetic acid was damaged as shown by its failure to mediate passive immune lysis of DANS-coupled sheep erythrocytes, but the antibody eluted with acetic acid/dioxane was recovered intact.
D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000302:isolation & purification*; D000918:Antibody Specificity; D000936:Antigen-Antibody Complex; D001666:Binding Sites, Antibody; D011232:Chemical Precipitation; D003619:Dansyl Compounds / Q000276:immunology; D004136:Dinitrobenzenes / Q000276:immunology; D006241:Haptens; D007074:Immunoglobulin G / Q000378:metabolism; D007164:Immunosorbents; D011817:Rabbits
D000906:Antibodies; D000936:Antigen-Antibody Complex; D003619:Dansyl Compounds; D004136:Dinitrobenzenes; D006241:Haptens; D007074:Immunoglobulin G; D007164:Immunosorbents