The study on the development of precise assays for human hemoglobin

人血红蛋白精密检测方法的开发研究

• 批准号: 02671071
• 负责人: ITOH Yoshihisa
• 金额: $ 0.77万
• 依托单位: Jichi Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02671071/
• 关键词: Hemoglobin; Urine; a latex agglutination test; radioimmunoassay; ラテックス; 凝集反応

The present study was undertaken to establish clinical usefulness of urinary hemoglobin under pathlogic conditions. Hemoglobin (Hb) was purified from erythrocytes and its rabbit antibody was thus prepared. Using purified Hb as standard and rabbit antibody, a manual latex agglutination test and a radioimmunoassay of a double antibody system were established. Based on previous experiences, Hb was obtained by isoelectric focusing : hemolysate was at first obtained by hemolyzing with distilled water containing a equal volume of toluene. Hb rich fractions were thus separated on isoelectricfocusing under pH 6-8 and sucrose density gradient from 5 t6 50%. A highest peak from PH 7.02-7.08 was collected and used as standard and immunogen. Rabbit antibody was prepared according to established methods previously reported.A latex agglutination test was thus established in the same manner as previously reported. There was no positive reaction in 14 normal urine. In evaluating pathologic urine, the result by the test showed a good coincidence with that by a dipstick test. Provided urine osmolarity was low, the present test could more sensitively detect minute amounts of Hb. The merit of the test based on antigen-antibody reaction was that it can detect Hb without interference of ascorbic acid and myoglobin.Since the variation of microhemoglobulinuria may be of clinical significance, a double antibody radioimmunoassay was establised. The assay was quite sensitive to detect about 10 ng/ml of Hb and intra-assay precision in purified Hb was satisfactory. however, precision, recovery and a dilution test were all poor in evaluating urinary Hb. This was explained by generation of methohemoglobin upon exposure to air. A further study is needed to prevent Hb from being degenerated.

本研究旨在确定病理条件下尿血红蛋白的临床应用价值。从红细胞中提纯血红蛋白（Hb），制备了Hb的兔抗体。以纯化血红蛋白为标准品，用兔抗建立了乳胶凝集法和双抗体放射免疫分析法。根据以前的经验，Hb是通过等电聚焦获得的：首先通过用含有等体积甲苯的蒸馏水溶血获得溶血产物。因此，在pH 6-8和5至650%的蔗糖密度梯度下，等电聚焦分离富含Hb的组分。收集pH 7.02-7.08的最高峰，并用作标准品和免疫原。按已建立的方法制备兔抗体，建立乳胶凝集试验。14例正常人尿液中均无阳性反应。在评价病理性尿时，本试验与试纸条试验的结果有较好的一致性。如果尿渗透压较低，本试验可以更灵敏地检测微量Hb。本试验以抗原抗体反应为基础，其优点是不受抗坏血酸和肌红蛋白的干扰，可测定Hb，尿微量血红蛋白的变化可能有临床意义，因此建立了双抗体放射免疫分析法。该方法对10 ng/ml左右的血红蛋白具有很高的灵敏度，对纯化的血红蛋白具有良好的批内精密度。但精密度、回收率和稀释试验均较差。这是通过暴露于空气后生成甲氧血红蛋白来解释的。防止血红蛋白变性需要进一步研究。

项目成果

Y. Itoh et al.: "A fundamental study on a radioimmunoassay for urinary hemoglobin AO."

Y. Itoh 等人：“尿血红蛋白 AO 放射免疫测定的基础研究。”

伊藤 喜之: "尿中ヘモグロビンA_0のRadioimmund assay法の開発と基礎的研究"

伊藤义之：“尿血红蛋白A_0放射免疫测定方法的发展与基础研究”

佐藤 喜久: "尿中ヘモグロビンA_0のRadisimmunoassay法の開発と基礎的研究"

佐藤佳久：“尿血红蛋白A_0放射免疫测定方法的发展及基础研究”

Y.Itoh: "Development of a manual latex agglutination test for urinary hemoglobin"

Y.Itoh：“尿血红蛋白手动乳胶凝集试验的开发”

Y. Itoh et al.: "Development of a manual latex agglutination test for urinary hemoglobin"

Y. Itoh 等人：“尿血红蛋白手动乳胶凝集试验的开发”