Supplementary Material for: Detection of the Peanut Allergens Ara h 2 and Ara h 6 in Human Breast Milk: Development of 2 Sensitive and Specific Sandwich ELISA Assays

Background: Little is known about breast milk as a vehicle for tolerance development or sensitization to peanuts very early in life. Thus, well-characterized and highly sensitive detection systems for the reliable determination of peanut allergens in breast milk are mandatory. Methods: For the quantification of the marker allergens Ara h 2 and Ara h 6 in the low nanogram per milliliter range in breast milk samples of a German cohort, sensitive and highly specific sandwich ELISAs were optimized and validated. Results: The Ara h 2 ELISA revealed a limit of detection (LOD) of 1.3 ng Ara h 2/mL and a quantification range of 2.3-250 ng/mL, the Ara h 6 ELISA showed an LOD of 0.7 ng/mL and a working range of 1.1-14.4 ng/mL. The assays showed no relevant cross-reactivity against other potentially cross-reactive legume, seed, and tree nut extracts (<0.01%, except for Ara h 1 in the Ara h 2 ELISA <0.1%). Ara h 2 was detectable in breast milk samples from 14/40 (35%) of the participants in concentrations from 2.3 to 184 ng/mL, Ara h 6 appeared in 9/40 (22.5%) of the lactating mothers between 1.1 and 9.7 ng/mL, and 1 highly positive sample with 79 ng/mL. Both allergens appeared at the same time points, but Ara h 6 in lower concentrations than Ara h 2. Conclusions: Sensitive and specific diagnostic tools for the determination of Ara h 2 and Ara h 6 in human breast milk were established. The kinetics of secreted Ara h 2 and Ara h 6 seem to be similar but with a difference in concentration. Follow-up investigations on their tolerogenic or sensitizing properties in breast milk become now accessible.背景：目前对于母乳作为婴幼儿早期花生耐受诱导或致敏媒介的相关机制尚不明晰。因此，开发经过充分验证、高灵敏度的检测体系，以可靠定量母乳中的花生过敏原，是十分必要的。方法：针对德国队列母乳样本中低至纳克每毫升级别的标记性过敏原Ara h 2与Ara h 6的定量需求，本研究优化并验证了灵敏度高、特异性强的夹心酶联免疫吸附试验（sandwich ELISA）方法。结果：针对Ara h 2的ELISA检测限（limit of detection, LOD）为1.3 ng/mL，定量范围为2.3~250 ng/mL；针对Ara h 6的ELISA检测限为0.7 ng/mL，工作范围为1.1~14.4 ng/mL。上述检测方法对其他潜在交叉反应的豆科、籽类及坚果提取物均无明显交叉反应（交叉反应率均<0.01%，仅Ara h 2 ELISA对Ara h 1的交叉反应率<0.1%）。在40名受试者的母乳样本中，14份（35%）可检测到Ara h 2，浓度范围为2.3~184 ng/mL；9份（22.5%）可检测到Ara h 6，浓度范围为1.1~9.7 ng/mL，另有1份样本呈强阳性，浓度达79 ng/mL。两种过敏原可在相同时间点被检出，但Ara h 6的浓度低于Ara h 2。结论：本研究成功建立了可定量人母乳中Ara h 2与Ara h 6的高灵敏度、高特异性检测方法。分泌型Ara h 2与Ara h 6的动力学特征相似，但浓度存在差异。至此，针对母乳中这两种过敏原的耐受诱导或致敏特性的后续研究已具备可行条件。