IMMUNOLOGIC ADSORBENTS .1. ISOLATION OF ANTIBODY BY MEANS OF A CELLULOSE-PROTEIN ANTIGEN
IMMUNOLOGIC ADSORBENTS .1. ISOLATION OF ANTIBODY BY MEANS OF A CELLULOSE-PROTEIN ANTIGEN
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DOI：
10.1073/pnas.37.9.575
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发表时间：
1951-01-01
影响因子：
11.1
通讯作者：
LERMAN, LS
中科院分区：
文献类型：
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作者：
CAMPBELL, DH;LUESCHER, E;LERMAN, LS
在过去的几年里，我们一直有兴趣设计的方法，将提供实用的程序，从免疫血清中分离和纯化抗体，特别是从动物血清中分离非沉淀抗体和从人血清中分离过敏性抗体（re-agins）。1.一般的方法是试图生产一种不溶性蛋白质抗原，它将与抗体特异性结合联合收割机，得到一种复合物，该复合物可以解离成可溶性抗体和不溶性抗原，然后可以通过离心分离。尝试赋予蛋白质抗原如卵清蛋白和结晶牛血清白蛋白不溶性而不破坏它们的抗原性。产物通过使用通常的变性剂、单宁酸和双官能偶联试剂（例如，四唑联苯胺）以及通过偶联到不溶性底物（例如酚醛树脂或不溶性蛋白质（例如纤维蛋白））而获得。然而，在每种情况下，所得产物要么失去了太多的天然抗原性（即，吸附的血清总是含有与天然抗原反应的抗体），要么太可溶，要么对非特异性蛋白质具有太大的吸附能力。现在，通过将蛋白质偶联到粉末状纤维素（Solka-floc）上，可以制备出最有前途的抗原。合成纤维素-蛋白质化合物的一般方法包括将纤维素转化为对硝基苄基衍生物，还原硝基，随后以通常的方式重氮化和偶联至蛋白质。对硝基苄基纤维素用稀酸、稀碱和水彻底洗涤粉末状纤维素（Solka-floc），然后干燥。然后将4克这种材料与12克混合。的对硝基苄基氯和30 ml. 40%氢氧化钠，并在95 ℃剧烈搅拌。(The在放热反应的第一部分期间冷却反应混合物）。4小时后。将混合物倒入大量过量的冷水中，过滤，在索格利特萃取器中用水、乙醇、最后用丙酮洗涤残余物。产物中每单位葡萄糖含有约1.5个苄基，以所用纤维素计，产率接近100%。对氨基苄基纤维素将5克硝基化合物悬浮于50 ml中。将乙醇加热至接近沸腾。然后将混合物剧烈搅拌，并将5g.连二亚硫酸钠（Na 2S 2 O 4 2 H2O）的溶解度为
For the past several years we have been interested in devising methods that would afford practical procedures for the isolation and purification of antibodies from immune serums and in particular the isolation of non-precipitating antibodies from animal serums and allergic antibodies (re-agins) from human serums. 1 The general approach has been to attempt to produce an insoluble protein antigen which would combine specifically with antibody to give a complex that could be dissociated into soluble antibody and insoluble antigen, which could then be separated by centrifugation. Attempts were made to confer insolubility on protein antigens such as oval-bumin and crystalline bovine serum albumin without destroying their anti-genicity. Products were obtained by use of the usual denaturing agents, tannic acid, and bifunctional coupling reagents (eg, tetrazotized benzi-dine), and also by coupling to an insoluble substrate such as phenolic resins or insoluble proteins (such as fibrin). However, in every case the resulting product either had lost too much native antigenicity (ie, adsorbed serum always contained antibody which reacted with native antigen), was too soluble, or had too much power of adsorption for non-specific protein. A most promising antigen has now been prepared by coupling protein to a powdered cellulose (Solka-floc). The general method of synthesizing the cellulose-protein compound consists in conversion of the cellulose to a p-nitrobenzyl derivative, reduction of the nitro group, and subsequent diazotization and coupling to protein in the usual manner. p-Nitrobenzylcellulose.-Powdered cellulose (Solka-floc) was thoroughly washed with dilute acid, dilute alkali, and water, and then dried. Four grams of this material was then mixed with 12 g. of p-nitrobenzylchloride and 30 ml. of 40% sodium hydroxide and stirred vigorously at 95 C.(The reacting mixture was cooled during the first part of the exothermic reac-tion.) After 4 hrs. the mixture was poured into a large excess of cold water and filtered, and the residue was washed with water, with ethanol, and finally with acetone in a Soxhlet extractor. The product contained about 1.5 benzyl groups per glucose unit, and the yield, based on the cellu-lose used, was close to 100%. p-Aminobenzylcellulose.-Five grams of the nitro compound was sus-pended in 50 ml. of ethanol and heated to near boiling. The mixture was then stirred vigorously and 5 g. of sodium hydrosulfite (Na2S204 2H20) dis-