Yuk Ji Eun, Lee Jongsun, Jeong Kyoung Yong, Park Kyung Hee, Kim Jung Dong, Kim Ji Tae, Lee Jae Hyun, Park Jung Won
Division of Allergy and Immunology, Department of Internal Medicine, Institute of Allergy, Yonsei University College of Medicine, Seoul, Korea.
Prolagen, Seoul, Korea.
Allergy Asthma Immunol Res. 2021 Jul;13(4):623-637. doi: 10.4168/aair.2021.13.4.623.
Diagnostic tests for allergen sensitization should reflect real exposure. We made 6 new bony fish extracts, which are consumed popularly in Korea, and evaluated their allergenicity and stability.
We manufactured fish extracts from codfish, mackerel, common eel, flounder, cutlass, and catfish. Protein and parvalbumin (PV) were evaluated by Bradford assay, 2-site enzyme-linked immunosorbent assay, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and anti-PV immunoblotting. The immunoglobulin E (IgE) reactivities of the extracts were evaluated with ImmunoCAP and IgE immunoblotting using sera from 24 Korean fish allergy patients, 5 asymptomatic sensitizers, and 11 non-atopic subjects. Stability of the extracts stored in 4 different buffers were evaluated for up to a year.
The protein concentrations of commercial SPT fish extracts varied with up to a 7.5-fold difference. SDS-PAGE showed marked differences in the PV concentrations of commercial SPT reagents. Specific IgE measurements for the following investigatory fish extracts-iCodfish, iMackerel, and iEel-were concordant with that of their corresponding Phadia ImmunoCAP measurements. ImmunoCAP results showed marked IgE cross-reactivity among the fish species, and the overall sensitivity of ImmunoCAP with the investigatory fish extracts for identification of culprit fish species was 85.7%. The protein and PV concentrations in the investigatory extracts were highly stable in saline with 0.3% phenol-50% glycerol at 4°C for up to a year.
The commercial SPT fish extracts exhibited considerable variation in terms of allergenicity, which may impact on diagnostic accuracy. Our new fish extracts have sufficient allergenicity and stability and may be adequate to various clinical applications.
变应原致敏的诊断测试应反映实际接触情况。我们制备了6种在韩国广泛食用的新型硬骨鱼提取物，并评估了它们的致敏性和稳定性。
我们从鳕鱼、鲭鱼、普通鳗鱼、比目鱼、军曹鱼和鲶鱼中制备鱼提取物。通过考马斯亮蓝法、双位点酶联免疫吸附测定、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和抗小清蛋白（PV）免疫印迹法评估蛋白质和小清蛋白（PV）。使用来自24名韩国鱼类过敏患者、5名无症状致敏者和11名非特应性受试者的血清，通过免疫捕获法和IgE免疫印迹法评估提取物的免疫球蛋白E（IgE）反应性。评估了储存在4种不同缓冲液中的提取物长达一年的稳定性。
商业点刺试验（SPT）鱼提取物的蛋白质浓度差异高达7.5倍。SDS-PAGE显示商业SPT试剂的PV浓度存在显著差异。对以下研究用鱼提取物——鳕鱼、鲭鱼和鳗鱼——的特异性IgE测量结果与其相应的法迪亚免疫捕获法测量结果一致。免疫捕获法结果显示鱼类之间存在显著的IgE交叉反应性，免疫捕获法对研究用鱼提取物识别致病鱼类的总体敏感性为85.7%。研究用提取物中的蛋白质和PV浓度在含有0.3%苯酚-50%甘油的盐水中于4℃下长达一年高度稳定。
商业SPT鱼提取物在致敏性方面表现出相当大的差异，这可能会影响诊断准确性。我们的新型鱼提取物具有足够 的致敏性和稳定性，可能适用于各种临床应用。
