Robinson M K, Babcock L S, Horn P A, Kawabata T T
Procter & Gamble Company, Cincinnati, Ohio 45253, USA.
Fundam Appl Toxicol. 1996 Nov;34(1):15-24. doi: 10.1006/faat.1996.0171.
An intranasal (i.n.) dosing model was developed in mice as a potential alternative to more difficult, time-consuming, and costly guinea pig intratracheal (GPIT) or mouse intratracheal models for assessment of the respiratory immunogenicity of detergent enzymes. Using a benchmark enzyme, Alcalase (protease subtilisin Carlsberg), studies were conducted to standardize the model in terms of mouse strain, dosing and serum harvest regimen, and the primary immunoglobulin endpoint to use. The primary assay endpoint selected was the enzyme-specific IgG1 titer determined by an Alcalase-specific ELISA. This is not the primary allergenic antibody in mice (IgE is); however, IgG1 is coregulated with IgE via the IL-4/TH2 pathway and may have a role in mediating allergic-type responses. BDF1 mice (C57B1/6 x DBA/2) were selected as representative of high responder strains, with high response associated with the H-2b (C57B1/6) parent. The dosing regimen used for most studies incorporated three i.n. exposures (Days 1, 3, and 10) and bleeding of the animals on Day 15. The animals were anesthetized and then immunized by allowing them to inhale 5-microliters aliquots of dosing solution into each nostril at each immunization. Positioning of the animals with their heads down (vs up) may have allowed more of the dosing solution to remain in the nasal region for a slightly longer period of time, but did not change the eventual GI tract migration and excretion of each dose. The presence of a detergent matrix in the enzyme dosing solution enhanced the IgG1 response. Immunizing with enzyme plus detergent gave highly consistent dose-response curves for Alcalase when evaluated over many studies. An enzyme-specific allergic antibody (IgE) response was weak and inconsistent under the dosing regimen used to generate the IgG1 response, but was stronger with longer-term dosing, consistent with the delay in IgE vs IgG1 responses seen in some other studies. Using IgG1 as a surrogate for allergic sensitization, we have preliminary data showing similar differential potencies between Alcalase and other test enzymes as detected in previous GPIT tests. On the basis of these data, we believe the i.n. immunization/IgG1 response model is a robust technique that may be useful in determining the relative immunogenicities of detergent enzymes and other proteins.
已在小鼠中建立了一种鼻内给药模型，作为豚鼠气管内给药（GPIT）或小鼠气管内给药模型的潜在替代方法，后两种模型评估洗涤剂酶的呼吸道免疫原性更困难、耗时且成本更高。使用一种基准酶碱性蛋白酶（枯草杆菌蛋白酶卡尔伯格）进行研究，以便在小鼠品系、给药和血清采集方案以及要使用的主要免疫球蛋白终点方面对该模型进行标准化。选择的主要测定终点是通过碱性蛋白酶特异性酶联免疫吸附测定（ELISA）测定的酶特异性IgG1滴度。这不是小鼠中的主要变应原抗体（IgE才是）；然而，IgG1通过白细胞介素-4/辅助性T细胞2（IL-4/TH2）途径与IgE共同调节，可能在介导过敏样反应中发挥作用。BDF1小鼠（C57B1/6×DBA/2）被选为高反应品系的代表，高反应与H-2b（C57B1/6）亲代相关。大多数研究使用的给药方案包括三次鼻内暴露（第1、3和10天），并在第15天对动物进行采血。将动物头部向下（而非向上）定位可能会使更多给药溶液在鼻腔区域停留稍长一段时间，但并未改变各剂量最终在胃肠道的迁移和排泄情况。酶给药溶液中洗涤剂基质的存在增强了IgG1反应。在多项研究中评估时，用酶加洗涤剂进行免疫可得到碱性蛋白酶高度一致的剂量反应曲线。在所采用的用于产生IgG1反应的给药方案下，酶特异性变应原抗体（IgE）反应较弱且不一致，但长期给药时反应更强，这与其他一些研究中观察到的IgE与IgG1反应的延迟一致。使用IgG1作为过敏致敏的替代指标，我们有初步数据表明，在之前的GPIT试验中检测到的碱性蛋白酶与其他测试酶之间存在类似的差异效价。基于这些数据，我们认为鼻内免疫/Igg1反应模型是一种可靠的技术，可能有助于确定洗涤剂酶和其他蛋白质的相对免疫原性。
