Kumagai Y, Okumura K, Tada T
Mol Immunol. 1984 Jun;21(6):545-59. doi: 10.1016/0161-5890(84)90071-3.
A T-cell hydridoma, 7C3-13-Ag6, which produces a (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific suppressor T-cell factor associated with an I-J determinant, was utilized to study the hapten-binding receptor of T-cells. This hybridoma had been shown to express NP-binding receptor molecules on the cell surface with heteroclitic fine specificity for a cross-reactive hapten, (4-hydroxy-5-iodo-3-nitrophenyl) acetyl (NIP). The stoichiometric analysis of the hapten binding by 7C3-13-Ag6 cells was performed by the measurement of direct binding of highly radioactive haptens to the cell surface. The affinity constant (Ka) of the receptor for N125IP-epsilon-aminocaproic acid (N125IP-cap), as calculated from a Hill plot, was 5.75 X 10(7) M-1 [Hill coefficient (a) = 0.86; expression of receptor sites per cell = approximately 1 X 10(3) on average]. The receptor molecule was specifically affinity labeled with photoreactive nitroaryl azide derivatives of N125IP (510-570 Ci/mmole). The specificity of photoaffinity labeling was demonstrated both by competitive inhibition of labeling with NIP- or NP-cap and by differential photoaffinity labeling based on the reversibility of hapten-receptor interaction. The gel electrophoretic analysis of the photoaffinity-labeled molecule indicated that the hapten-binding receptor of 7C3-13-Ag6 has a mol. wt of 28,000 +/- 3000 and an isoelectric point of 5.6-5.7. No immunoglobulin determinants were detected on the molecule. A comparative immunoprecipitation analysis of the membrane lysate of 7C3-13-Ag6 with monoclonal anti-I-J reagents identified a separate I-J molecule of 25,000 +/- 1000 mol. wt that is distinct from the photoaffinity-labeled hapten-binding molecule.
一种T细胞杂交瘤7C3 - 13 - Ag6，它产生与I - J决定簇相关的（4 - 羟基 - 3 - 硝基苯基）乙酰基（NP）特异性抑制性T细胞因子，被用于研究T细胞的半抗原结合受体。已证明这种杂交瘤在细胞表面表达NP结合受体分子，对交叉反应性半抗原（4 - 羟基 - 5 - 碘 - 3 - 硝基苯基）乙酰基（NIP）具有偏侧性精细特异性。通过测量高放射性半抗原与细胞表面的直接结合，对7C3 - 13 - Ag6细胞的半抗原结合进行了化学计量分析。根据希尔图计算，受体对N125IP - ε - 氨基己酸（N125IP - cap）的亲和常数（Ka）为5.75×10⁷ M⁻¹[希尔系数（a） = 0.86；平均每个细胞受体位点的表达量约为1×10³]。受体分子用N125IP的光反应性硝基芳基叠氮衍生物（510 - 570 Ci/mmole）进行特异性亲和标记。通过用NIP或NP - cap进行标记的竞争性抑制以及基于半抗原 - 受体相互作用可逆性的差异光亲和标记，证明了光亲和标记的特异性。对光亲和标记分子的凝胶电泳分析表明，7C3 - 13 - Ag6的半抗原结合受体的分子量为28,000±3000，等电点为5.6 - 5.7。在该分子上未检测到免疫球蛋白决定簇。用单克隆抗I - J试剂对7C3 - 13 - Ag6的膜裂解物进行的比较免疫沉淀分析鉴定出一个分子量为25,000±1000的单独的I - J分子，它与光亲和标记的半抗原结合分子不同。
