AUGUSTIN R, HAYWARD B J
Immunology. 1962 May;5(3):424-60.
Cocksfoot and Timothy pollen extracts are each found to contain at least fifteen components antigenic in rabbits. Most of these can also be allergens for man, but only a few are regularly so. These principal' allergens have now been isolated in highly purified form. Procedures are given for a simple method of preparing extracts for clinical purposes and for the partial separation, concentration and purification of the allergens by means of differential extractions of the pollens and by means of ultrafiltration, isoelectric precipitation and salt fractionations (at acid and neutral pH) of the extracts. Isoelectric precipitations gave highly pigmented acid complexes, two of which moved as single sharp peaks at pH 7.4 in free electrophoresis, but proved to be hardly active by skin tests. Acid NaCl fractionation of the remainder resulted for Cocksfoot and Timothy in the isolation of a nearly white powder (T21.111121112 = T21B) which was weight for weight 1000–10,000 times as active as the pollen from which it had been derived. The powders have retained their activity for 7 years. By gel diffusion tests, they were found to contain two antigens (one in each preparation) which were immunologically partially related, but the Timothy preparation contained in addition the innermost' twin' antigens specific for Timothy that we had discovered previously in the crude extracts by gel diffusion methods. Skin reactions could be elicited in hay-fever subjects by prick tests with concentrations of 10–10 g./ml., which is equivalent to intradermal injections of 10–10 mg. and represents a 300-fold purification with respect to the concentrates of crude pollen extracts prepared by ultrafiltration and dialysis. Fractionation on DEAE-cellulose of one of the highly purified Timothy preparations (T21.11112112 = T21A) and other, crude Timothy and Cocksfoot extracts resulted in considerable and reproducible separation of the various antigens, with no indication of the continuous dissociation into smaller fragments reported in recent publications. The immunologically related antigens of Cocksfoot and Timothy were among a number not retained by the DEAE-cellulose at pH 7.4–8.5. These proved to be skin reactive in all the patients tested. When this fraction was prepared from T21.11112112(= T21A) only one main line (plus a faint subsidiary line) was found in the gel diffusion tests. At pH 6.4 the important dissimilar Cocksfoot and Timothy allergens were eluted; the fraction derived from T21A contained only the special innermost' twin antigens in roughly a further ten-fold purification over and above that achieved in the acid NaCl fractionation.
鸭茅和梯牧草花粉提取物均被发现至少含有15种在兔体内具有抗原性的成分。其中大多数成分对人类也可能是过敏原，但只有少数成分通常如此。这些“主要”过敏原现已被高度纯化。文中给出了一种简单的临床用提取物制备方法，以及通过花粉的差异提取、提取物的超滤、等电沉淀和盐分级分离（在酸性和中性pH值下）对过敏原进行部分分离、浓缩和纯化的方法。等电沉淀产生了颜色很深的酸性复合物，其中两种在自由电泳中于pH 7.4时以单一尖锐峰移动，但经皮肤试验证明几乎没有活性。对其余部分进行酸性氯化钠分级分离，鸭茅和梯牧草分别得到一种近乎白色的粉末（T21.111121112 = T21B），其重量活性是其来源花粉的1000至10000倍。这些粉末已保持其活性7年。通过凝胶扩散试验发现，它们含有两种抗原（每种制剂一种），在免疫上部分相关，但梯牧草制剂还含有我们先前通过凝胶扩散法在粗提物中发现的梯牧草特有的“最内层”“孪生”抗原。用浓度为10⁻¹⁰克/毫升的提取物进行点刺试验，可在花粉症患者中引发皮肤反应，这相当于皮内注射10⁻¹⁰毫克，相对于通过超滤和透析制备的粗花粉提取物浓缩物而言，实现了300倍的纯化。对一种高度纯化的梯牧草制剂（T21.11112112 = T21A）以及其他粗提的梯牧草和鸭茅提取物在DEAE - 纤维素上进行分级分离，导致各种抗原得到显著且可重复的分离，没有近期出版物报道的持续解离成更小片段的迹象。鸭茅和梯牧草的免疫相关抗原在pH 7.4 - 8.5时未被DEAE - 纤维素保留的成分之中。这些成分在所有受试患者中均被证明具有皮肤反应性。当仅从T21.11112112（= T21A）制备该级分时，在凝胶扩散试验中仅发现一条主线（加上一条微弱的副线）。在pH 6.4时，重要的不同鸭茅和梯牧草过敏原被洗脱；从T21A得到的级分仅含有特殊的“最内层”孪生抗原，其纯化程度比酸性氯化钠分级分离所达到的程度大约又提高了10倍。
