Bernhisel-Broadbent J, Dintzis H M, Dintzis R Z, Sampson H A
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland.
J Allergy Clin Immunol. 1994 Jun;93(6):1047-59. doi: 10.1016/s0091-6749(94)70054-0.
When attempting to generate mouse monoclonal antibodies to hen's egg ovalbumin, injection of commercially purified ovalbumin resulted in monoclonal antibodies, which when assayed against commercially purified ovalbumin (Gal d I) or ovomucoid (Gal d III), appeared to be specific to both. With the use of high-performance liquid chromatography (HPLC)-repurified ovalbumin and ovomucoid in assay procedures, monoclonal antibodies generated by commercially purified ovalbumin were found to be specific for ovomucoid only. To clarify this phenomenon, mice were serially injected with commercially purified ovalbumin or HPLC-repurified ovalbumin. It was found that most of the antibody response to commercially purified ovalbumin was directed against the minor (< 1%) ovomucoid contaminant and that HPLC-repurified ovalbumin failed to produce antibodies to ovomucoid. Commercially purified ovomucoid resulted in only minimal amounts of antibodies to ovalbumin. Thus when commercially purified ovalbumin is used both for immunization and immunoassay, most of the antibodies produced are actually against the small amount of ovomucoid contaminant, and not ovalbumin. To determine whether ovomucoid is the major antigenic and allergenic egg white protein in human beings, one group of 18 children with egg allergy were skin prick tested with half-log dilutions of egg white extract and diethylaminoethyl cellulose (DEAE)-repurified ovomucoid, ovalbumin, and lysozyme. Ovomucoid mean wheal diameters were significantly greater than wheal diameters in response to ovalbumin, lysozyme, and egg white extract at the three most concentrated of five dilutions tested: 0.01, 0.03, and 0.1 mg/ml (p < 0.01). Serum ovomucoid-specific IgE and IgG antibody concentrations to DEAE-repurified ovomucoid were significantly greater than that to DEAE-repurified ovalbumin (p < 0.05). In a second study, 10 patients with egg allergy and persistent egg hypersensitivity were compared with 11 patients with egg allergy in whom clinical tolerance to egg developed. IgE antibodies to repurified ovomucoid were significantly greater in patients with persistent egg hypersensitivity compared with patients in whom clinical tolerance developed at the time of both initial and follow-up food challenges. In contrast, there were no significant differences in IgE antibody concentrations to repurified ovalbumin in either group at any time. These results suggest that ovomucoid is the immunodominant protein fraction in egg white and that the use of commercially purified ovalbumin has led to an overestimation of the dominance of ovalbumin as a major egg allergen and antigen in human beings.
在尝试制备针对鸡卵清蛋白的小鼠单克隆抗体时，注射商业纯化的卵清蛋白可产生单克隆抗体。当用这些单克隆抗体检测商业纯化的卵清蛋白（Gal d I）或卵类粘蛋白（Gal d III）时，它们似乎对两者都具有特异性。在检测过程中使用高效液相色谱（HPLC）再纯化的卵清蛋白和卵类粘蛋白后，发现由商业纯化的卵清蛋白产生的单克隆抗体仅对卵类粘蛋白具有特异性。为了阐明这一现象，给小鼠连续注射商业纯化的卵清蛋白或HPLC再纯化的卵清蛋白。结果发现，对商业纯化卵清蛋白的大多数抗体反应是针对少量（<1%）的卵类粘蛋白污染物，并且HPLC再纯化的卵清蛋白未能产生针对卵类粘蛋白的抗体。商业纯化的卵类粘蛋白仅产生少量针对卵清蛋白的抗体。因此，当使用商业纯化的卵清蛋白进行免疫和免疫检测时，产生的大多数抗体实际上是针对少量的卵类粘蛋白污染物，而非卵清蛋白。为了确定卵类粘蛋白是否是人类主要的蛋清抗原性和致敏性蛋白质，一组18名对鸡蛋过敏的儿童用蛋清提取物以及二乙氨基乙基纤维素（DEAE）再纯化的卵类粘蛋白、卵清蛋白和溶菌酶的半对数稀释液进行皮肤点刺试验。在测试的五种稀释液中最浓的三种：0.01、0.03和0.1mg/ml时，卵类粘蛋白的平均风团直径显著大于对卵清蛋白、溶菌酶和蛋清提取物的风团直径（p<0.01）。针对DEAE再纯化的卵类粘蛋白的血清卵类粘蛋白特异性IgE和IgG抗体浓度显著高于针对DEAE再纯化的卵清蛋白的浓度（p<0.05）。在第二项研究中，将10名对鸡蛋过敏且持续存在鸡蛋超敏反应的患者与11名已产生对鸡蛋临床耐受性的鸡蛋过敏患者进行比较。在初次和后续食物激发试验时，与已产生临床耐受性的患者相比，持续存在鸡蛋超敏反应的患者中针对再纯化卵类粘蛋白的IgE抗体显著更高。相比之下，两组中针对再纯化卵清蛋白的IgE抗体浓度在任何时候均无显著差异。这些结果表明，卵类粘蛋白是蛋清中的免疫优势蛋白组分，并且商业纯化卵清蛋白的使用导致高估了卵清蛋白作为人类主要鸡蛋过敏原和抗原的优势地位。
