MOLECULAR COMPOSITION AND SEDIMENTATION CHARACTERISTICS OF SOLUBLE ANTIGEN-ANTIBODY COMPLEXES
MOLECULAR COMPOSITION AND SEDIMENTATION CHARACTERISTICS OF SOLUBLE ANTIGEN-ANTIBODY COMPLEXES
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DOI：
10.1021/bi00772a008
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发表时间：
1972-01-01
期刊：
影响因子：
2.9
通讯作者：
TELLER, DC
中科院分区：
文献类型：
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作者：
AREND, WP;MANNIK, M;TELLER, DC
材料和方法抗原和抗体的制备。准备程序的详细信息已经介绍过（Mannik 等人，1971），因此，这里仅包括摘要。使用弗氏完全佐剂中的 HSA 对新西兰兔进行超免疫。此后，每周对它们进行一次放血。对于一些研究，用10mg HSA的生理盐水对兔子进行静脉内免疫，并在第10天腹膜内注射20mg HSA的盐水进行加强免疫。这些兔子在初次静脉免疫后第15天和第17天放血，并在第20天放血。约 5 公斤重的恒河猴（Macaca mulatto）使用弗氏完全佐剂中的 BSA 进行超免疫，免疫和放血时间表与兔子所用的类似。所有抗血清均储存于—20℃，直至进行抗体的进一步纯化。利用抗原琼脂糖柱通过固相免疫吸附剂分离抗体（Mannik 和 Stage，1971）。从抗血清中吸附特异性抗体，并用硼酸钠缓冲液（0.2 m 硼酸盐-0.16 m NaCl，pH 8.0）彻底洗涤柱。使用 0.01 m HC1-0.15 m NaCl (pH 2.0) 或未缓冲（中性 pH）2.5 m KI 从免疫吸附柱上洗脱抗体，然后用大量硼酸盐缓冲液进行透析。通过一氯化碘法（Helmkamp 等，1960）分别用 125I 或 31I 痕量标记抗体和抗原，每摩尔蛋白质产生 1-2 摩尔碘。通过对硼酸盐缓冲液进行彻底透析去除游离同位素。标记后，所有抗原和抗体制剂均通过 Sephadex G-200 凝胶过滤去除聚集体，单体 7G 或白蛋白溶液在几周内用于研究。通过蔗糖密度梯度超速离心测定，在所有制剂中，超过 98% 的蛋白质呈单体形式，并且超过 99% 的放射性与蛋白质结合。免疫复合物的制备。为每个抗原-抗体系统构建沉淀素曲线，并确定等价点。等价时，85-90%的兔抗HSA抗体沉淀，45%的恒河猴抗BSA抗体沉淀。分离抗体沉淀不完全的原因尚未阐明，但存在三种可能性：一些抗体可能因洗脱程序而变性，一些抗体可能不沉淀，或者存在非特异性粘附在免疫吸附柱上的正常7G。为了制备可溶性免疫复合物，在混合时将抗体以所需的抗原过量程度（按重量计）添加到抗原中。让复合物在室温下稳定1小时，然后在蔗糖密度梯度超速离心之前在4℃稳定2小时，或在分析超速离心之前在4℃稳定过夜。在对重新溶解的复合物进行的研究中，复合物被允许在当量沉淀并在 4°C 下稳定过夜。然后将沉淀洗涤四次以除去可溶性蛋白质。此后，将抗原添加到沉淀物中以产生抗原过量五倍的终浓度。将混合物在37℃下孵育过夜，并在4℃下孵育一周，并重复混合。将溶液离心并对含有可溶性复合物的上清液进行进一步研究。在这些条件下，沉淀物中只有 25% 的原始抗体被溶解。蔗糖密度梯度超速离心。线性蔗糖密度梯度（10-30% 或 10-40…
Materials and MethodsPreparation of Antigens and Antibodies. The details of preparative procedures have been presented (Mannik et al., 1971), therefore, only a summary is included here. New Zealand rabbits were hyperimmunized with HSA in complete Freund’s adjuvant. Thereafter they were bled at weekly inter-vals. For some studies, rabbits were immunized intravenously with 10 mg of HSA in normal saline, and boosted at10 days with 20 mg of HSA in saline injectedintraperitoneally. These rabbits were bled at 15 and 17 days and exsanguinated at 20 days after the initial intravenous immunization. Approxi-mately 5-kg rhesus monkeys (Macaca mulatto) were hyperimmunized with BSA in complete Freund’s adjuvant, with an immunization and bleeding schedule similar to that utilized for the rabbits. All antisera were stored at—20 until further purification of antibodies was carried out. Antibodies were isolated by solid-phase immunoadsorbents, utilizing antigen-agarose columns (Mannik and Stage, 1971). Specific antibodies were adsorbed from the antisera, and the columns were exhaustively washed with sodium borate buffer (0.2 m borate-0.16 m NaCl, pH 8.0). The antibodies were eluted from the immunoadsorbent columns with 0.01 m HC1-0.15 m NaCl (pH 2.0) or with unbuffered (neutral pH) 2.5 m KI, and then dialyzed against large volumes of borate buffer. Anti-bodies and antigens were trace labeled with 125I or’31I, respectively, by the iodine monochloride method (Helmkamp et al., 1960), yielding 1-2 moles of iodine per mole of protein. Free isotope was removed by exhaustive dialysis against borate buffer. All antigen and antibody preparations were made aggregate free by gel filtration on Sephadex G-200 after labeling, and the monomeric 7G or albumin solutions were utilized for studies within a few weeks. In all preparations more than 98% of the proteins were in monomeric form, as determined by sucrose density gradient ultracentrifugation, and greater than 99% of the radioactivity was protein bound. Preparation of Immune Complexes. Precipitin curves were constructed for each antigen-antibody system, and the point of equivalence was determined. At equivalence, 85-90% of the rabbit antibodiesagainst HSA precipitated, and 45% of the rhesus antibodies against BSA were precipitated. The reasons for incomplete precipitation of isolated antibodies were not elucidated, but three possibilities existed: some antibodies may have been denatured by elution procedures, possibly some antibodies were nonprecipitating, or normal 7G was present which had nonspecifically adhered to the immuno-adsorbent column. To prepare soluble immune complexes, antibodies were added to antigens while mixing, at the desired degree of antigen excess (by weight). Complexes were allowed to stabilize at room temperature for 1 hr, then at 4 for 2 hr prior to sucrose density gradient ultracentrifugation, or at 4 overnight prior to analytical ultracentrifugation. In the studies performed on redissolved complexes, the complexes were allowed to precipitate at equivalence and stabilize overnight at 4C. Then the precipitate was washed four times to remove soluble proteins. Thereafter, antigen was added to the precipitate to yield a final concentration of five times antigen excess. The mixture was incubated overnight at 37 and for a week at 4 with repeated mixing. The solution was centrifuged and further studies carried out on the super-natant containing the soluble complexes. Only 25% of the original antibodies in the precipitate was solubilized under these conditions.Sucrose Density Gradient Ultracentrifugation. Linear sucrose density gradients (10-30% or 10-40 …