Hoffmann L G, Kuo C Y
J Immunol Methods. 1977;15(2):101-19. doi: 10.1016/0022-1759(77)90021-7.
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.
通过在温和条件下从直接将半抗原偶联到珠状琼脂糖的氨乙基氨基（AEA）衍生物制备的免疫吸附剂上洗脱，可以高收率分离出亲和力接近10⁸/M的抗半抗原抗体。在对平均缔合常数（Ko）为1.2×10⁹/M的兔抗2,4-二硝基苯基（DNP）抗体进行的实验中，从琼脂糖-AEA-DNP柱上洗脱结合的抗体，收率为57%。对于对5-二甲基氨基萘-1-磺酰基（DANS）半抗原的兔抗体，Ko = 2.7×10⁸/M，Sips非均一性指数a = 0.52，通过定量沉淀分析在起始材料中发现的抗体，通过特异性沉淀并用0.18 M Na DANSulfonate溶解沉淀物，获得了20%的收率；该抗体的Ko = 6.6×10⁸/M，a = 0.66。通过在DANS-AEA-琼脂糖上进行免疫吸附并用相同的半抗原浓度洗脱，对同一抗体制备物的另一等份进行纯化，得到了收率为75%的抗体，其Ko = 5.7×10⁷/M，a = 0.29。在用新方法进行的3次生产运行中，抗DANS抗体的表观收率分别为181%、101%和94%（基于对起始材料的定量沉淀分析）。在其中2次运行中，用0.1 M乙酸进一步洗脱免疫吸附剂，然后用10%二氧六环中的0.1 M乙酸洗脱；用乙酸额外获得了9%和4%的收率，用乙酸/二氧六环进一步获得了15%和11%的收率；用乙酸洗脱的抗体受损，表现为它不能介导DANS偶联的绵羊红细胞的被动免疫裂解，但用乙酸/二氧六环洗脱的抗体完好无损地回收。
