Seguela P, Geffard M, Buijs R M, Le Moal M
Proc Natl Acad Sci U S A. 1984 Jun;81(12):3888-92. doi: 10.1073/pnas.81.12.3888.
Antibodies against gamma-aminobutyric acid (GABA)-glutaraldehyde-lysine were obtained by using a procedure based upon (i) a high yield of coupling of GABA to protein carriers, (ii) the reduction of the resulting immunoreactive double bonds, and (iii) a protocol of alternative immunizations using different immunogens having in common only the GABA-glutaraldehyde-lysine segment. This strategy led to the use of the resulting GABA antiserum without further purification. Specificity controls have been carried out with a radiolabeled ligand, [3H]GABA-glutaraldehyde- prolylphenylalanyl -lysine , which mimicked the structure of the immunogen and the fixed hapten in the tissue. Displacement curves showed that the nearest coupled analogs, beta-alanine and glycine, cross-react poorly with GABA, requiring 175-fold or 795-fold higher concentrations, respectively. Immunocytochemical results indicated that the localization obtained with this GABA antiserum largely corresponds with that reported after glutamate decarboxylase immunocytochemistry. The approach may have general applicability to other small molecules such as amino acids.
通过以下步骤获得了抗γ-氨基丁酸(GABA)-戊二醛-赖氨酸抗体:(i)使GABA与蛋白质载体高效偶联;(ii)还原产生的免疫反应性双键;(iii)使用仅具有GABA-戊二醛-赖氨酸片段的不同免疫原进行交替免疫的方案。该策略使得所得的GABA抗血清无需进一步纯化即可使用。已使用放射性标记配体[3H]GABA-戊二醛-脯氨酰苯丙氨酰-赖氨酸进行特异性对照,该配体模拟了免疫原的结构以及组织中的固定半抗原。置换曲线表明,最接近的偶联类似物β-丙氨酸和甘氨酸与GABA的交叉反应性较差,分别需要高175倍或795倍的浓度。免疫细胞化学结果表明,用该GABA抗血清获得的定位与谷氨酸脱羧酶免疫细胞化学后报道的定位基本一致。该方法可能对其他小分子如氨基酸具有普遍适用性。