Changes on glycosylation and serum levels of acute phase protein in severe viral infection.

本研究旨在阐明α_1-酸性糖蛋白作为急性时相蛋白的糖生物学模型在炎症、癌症和病毒感染中的意义。动物AGP的制备：通过硫酸铵沉淀、酸沉淀、离子交换柱层析、凝胶过滤等步骤从腹水中分离纯化猫AGP(FAGP)。纯化的蛋白具有较宽的相对分子质量为40-50 kDa的条带，对硫酸铵和酸性溶液的溶解度较高，等电点小于3.7，表明该蛋白为猫科动物AGP2。抗猫AGP单抗的制备：按常规方法制备。结果共获得8株单抗，其同功型(L链)分别为λ(IgG1b)、λ(IgG2b)和κ(IgA)。在Western印迹分析中，这些单抗在非还原条件下识别fAGP，但在还原条件下不识别fAGP。此外，竞争酶联免疫吸附试验显示，这些单抗识别相同或重叠的表位。凝集素亲和力：猫AGP对ConA、DSA、SAA和UEA-1具有亲和力，具有二天线和三天线结构，糖链上有唾液酸和岩藻糖。抗血清的制备及fAGP的检测：用兔制备抗血清，单克隆试验表明，抗fAGP和猫血清的免疫接受线是连续的。直接结合酶联免疫吸附试验检测范围为0.02g/m1~0.25g/m1，血清FAGP水平为1.37±1.147 mg/ml。

The purpose of this study is to clarify a significance of α_1-acid glycoprotein (AGP) as a model of glycobiology of acute phase proteins in inflammation, cancer and viral infection.1. Preparation of AGP in animals: Feline AGP (fAGP) was purified from ascites by ammonium sulfate precipitation, acid precipitation, ion-exchanged column chromatography, and gel filtration. The purified protein showed a broad band with an M. W. of 40-50 kDa and high solubilities against ammonium sulfate and acid solution with a lower pI than 3.7, indicating that the protein was confirmed as feline AGP.2. Preparation of monoclonal antibodies(Mabs) against feline AGP: Mabs were prepared as the ordinary method. In result, we obtained 8 Mabs of which isotypes (L chains) were IgG1(λ),IgG2b(λ) and IgA(κ). On Western blot analyses, these Mabs recognized fAGP in non-reducing conditions, but not in reducing conditions. In addition, competitive ELISA showed these Mabs recognized the same or overlaped epitope.3. Lectin affinity: Feline AGP had an affinity against ConA, DSA, SAA and UEA-1, having di-antenary and tri-antenary structures, sialic acid and fucose in glycan chains.4. Preparation of antiserum and determination of fAGP: Antiserum was prepared in a rabbit.Ouchterlony test showd a continuous immunoprecipitin line against fAGP and feline serum. Direct binding ELISA using the antiserum with determination range of 0.02 to 0.25 pig/m1 showed serum fAGP levels of 1.37± 1.147 mg/ml.