Institute of Chemistry of Organometallic Compounds (ICCOM), National Research Council of Italy (CNR), Sesto Fiorentino, Italy.
Fischer analytics GmbH, Bingen am Rhein, Germany.
Methods Mol Biol. 2024;2821:157-163. doi: 10.1007/978-1-0716-3914-6_12.
Antibodies from sera of a multiple sclerosis (MS) patient subpopulation preferentially recognize the hyperglucosylated adhesin protein HMW1ct(Glc) of the pathogen Haemophilus influenzae. This protein is the first example of an N-glucosylated native antigen candidate, potentially triggering pathogenic antibodies in MS. Specific antibodies in patients' sera can be isolated exploiting their biospecific interaction with antigens by affinity chromatography. Herein, the proteins HMW1ct and HMW1ct(Glc) were first immobilized on appropriately functionalized supports and further used to purify antibodies directly from MS patients sera. We describe a protocol to obtain an antibody fraction specifically recognizing the glusosylated residues on the HMW1ct(Glc) adhesin protein depleting antibodies to the unglucosylated HMW1ct sequence. Different elution solutions have been tested to recover the purified antibody fraction, strongly bound to the immobilized HMW1ct(Glc) adhesin protein.
多发性硬化症 (MS) 患者亚群血清中的抗体优先识别病原体流感嗜血杆菌的高葡聚糖化粘附蛋白 HMW1ct(Glc)。该蛋白是首例 N-葡聚糖化天然抗原候选物，可能在 MS 中引发致病抗体。可以通过亲和层析利用患者血清中特异性抗体与抗原的生物特异性相互作用来分离这些抗体。在此，首先将 HMW1ct 和 HMW1ct(Glc) 蛋白固定在适当功能化的载体上，并进一步用于直接从 MS 患者的血清中纯化抗体。我们描述了一种从 MS 患者血清中获得专门识别 HMW1ct(Glc)粘附蛋白上葡糖基化残基的抗体的方法，该方法可以去除针对未葡糖基化 HMW1ct 序列的抗体。已经测试了不同的洗脱溶液以回收与固定化 HMW1ct(Glc) 粘附蛋白强烈结合的纯化抗体。
