Fujiwara K, Araki M, Kitagawa T, Inoue Y
Faculty of Pharmaceutical Sciences, Nagasaki University, Japan.
Histochemistry. 1993 Jun;99(6):477-83. doi: 10.1007/BF00274101.
Antiserum was produced in rabbits against the polyamine spermidine (Spd) conjugated to bovine serum albumin (BSA). The reactivity of the serum to Spd and a variety of structurally related compounds was quantified by a new immunocytochemical model system incorporating an enzyme-linked immunosorbent assay (ELISA) binding test. This is based on the principle of coupling these compounds to the wells of microtiter plate activated with poly-L-lysine and glutaraldehyde and incubating the wells by the indirect immunoperoxidase method. The antiserum showed a 25% cross reaction with spermine (Spm), putrescine (Put), and cadaverine (Cad), and a 1% cross reaction with 1,3-diaminopropane (Dap), but no cross reaction with monoacetyl polyamines and amino acids. The antibody binding was inhibited most effectively by absorption of the antiserum with N1-acetylspermidine and Spd in the ELISA inhibition test. Also, immunoblot analysis of the antiserum with nitrocellulose paper gave completely identical results to the ELISA binding tests. Spd-like immunoreactivities in human melanoma BD and neuroblastoma IMR 32 cell lines are presented as examples of the staining pattern obtained with the antiserum. Absorption of the serum with N1-acetylspermidine and Spd was demonstrated to abolish the immunostaining reaction. The immunohistochemical model is simple: amines and amino acids are bound in the same way as in aldehyde-fixed tissues and, in comparison to immunoblot analysis, the immunoreactivity can be more easily and accurately quantified by assay with the antibody. The model should prove useful in assessing the specificity of other antisera.
用与牛血清白蛋白（BSA）偶联的多胺亚精胺（Spd）在兔体内制备抗血清。通过结合酶联免疫吸附测定（ELISA）结合试验的新型免疫细胞化学模型系统，对血清与Spd及多种结构相关化合物的反应性进行定量。这基于将这些化合物偶联到用聚-L-赖氨酸和戊二醛活化的微量滴定板孔中，并通过间接免疫过氧化物酶法孵育孔的原理。抗血清与精胺（Spm）、腐胺（Put）和尸胺（Cad）有25%的交叉反应，与1,3-二氨基丙烷（Dap）有1%的交叉反应，但与单乙酰多胺和氨基酸无交叉反应。在ELISA抑制试验中，用N1-乙酰亚精胺和Spd吸收抗血清能最有效地抑制抗体结合。此外，用硝酸纤维素纸对抗血清进行免疫印迹分析，结果与ELISA结合试验完全相同。以人黑色素瘤BD细胞系和神经母细胞瘤IMR 32细胞系中的Spd样免疫反应性为例，展示了用抗血清获得的染色模式。用N1-乙酰亚精胺和Spd吸收血清可消除免疫染色反应。该免疫组织化学模型很简单：胺类和氨基酸的结合方式与醛固定组织相同，与免疫印迹分析相比，通过抗体检测可以更轻松、准确地定量免疫反应性。该模型在评估其他抗血清的特异性方面应会证明很有用。
