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

本研究的目的是阐明α_1-酸性糖蛋白（AGP）作为炎症、癌症和病毒感染急性期蛋白糖生物学模型的意义。动物AGP的制备：采用硫酸铵沉淀法、酸沉淀法、离子交换柱层析法和凝胶过滤法从腹水中纯化猫AGP （fAGP）。纯化后的蛋白具有较宽的条带，分子量在40 ~ 50 kDa之间，对硫酸铵和酸溶液具有较高的溶解度，pI值小于3.7，表明该蛋白为猫科动物AGP.2。抗猫AGP单克隆抗体的制备：采用常规方法制备单克隆抗体。结果，我们获得了8个同型（L链）为IgG1（λ）、IgG2b（λ）和IgA（κ）的单抗。在Western blot分析中，这些mab在非还原条件下识别fAGP，而在还原条件下不识别。此外，竞争性ELISA结果显示，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，具有双触角和三触角结构，唾液酸和集中在聚糖链上。抗血清的制备及测定：制备兔抗血清。体外试验显示对fAGP和猫血清有连续的免疫沉淀线。采用直接结合ELISA法，测定范围为0.02 ~ 0.25猪/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.