Rothenberg S P, Marcoullis G P, Schwarz S, Lader E
J Lab Clin Med. 1984 Jun;103(6):959-72.
Antiserum to cobalamin was raised in rabbits by immunization with the monocarboxyl derivative of cyanocobalamin coupled to albumin. The antiserum was treated to remove transcobalamin II and transcobalamin I. The partially purified antibody bound free cyano[57Co]cobalamin, but not the vitamin precoupled to the transcobalamins. Cyano[57Co]cobalamin bound by the antiserum eluted from Sephadex G-200 as a single peak with a mol wt of 160,000 and was precipitated by goat anti-rabbit gamma globulin, indicating that the vitamin was bound to an IgG immunoglobulin. Unlabeled cyanocobalamin and hydroxocobalamin competitively inhibited the binding of cyano[57Co]cobalamin to this antibody. Neither adenosylcobalamin, in a similar concentration range, nor cyanocobinamide at a concentration 30-fold greater than the tracer cobalamin competed appreciably with the binding of cyano[57Co] cobalamin. The association constant for the interaction of the antibody with cyanocobalamin and cyanocobinamide was estimated to be 8.6 X 10(9) and 9.6 X 10(6) L/mol, respectively. The association constant for adenosylcobalamin, methylcobalamin, and hydroxocobalamin was indirectly determined, and values of 2.5 X 10(5), 1.7 X 10(9), and 2.3 X 10(9) L/mol, respectively, were obtained. Photolysis in the presence of potassium cyanide rendered each of the three cobalamins equivalent to cyanocobalamin in immunoreactivity. The mean concentration of cobalamin in normal human sera and cobalamin-deficient sera measured as cyanocobalamin by radioimmunoassay using this anticobalamin antibody was significantly lower than the concentration measured in the same extracts by competitive ligand-binding radioassays using intrinsic factor and transcobalamin I. These findings, although indirect, support the proposition that there may be factor(s) in normal and cobalamin-deficient sera that falsely elevate the concentration of true cobalamin if the radioassay uses R protein as the binder. However, the lower concentration of serum cobalamin measured by radioimmunoassay compared with the intrinsic factor radioassay also indicates that this "purported" factor(s) reacts to some extent with intrinsic factor but not with the cobalamin antibody.
通过用与白蛋白偶联的氰钴胺单羧基衍生物免疫家兔，制备了抗钴胺素抗血清。对该抗血清进行处理以去除转钴胺素II和转钴胺素I。部分纯化的抗体可结合游离的氰基[57Co]钴胺素，但不结合与转钴胺素预偶联的维生素。抗血清结合的氰基[57Co]钴胺素从Sephadex G - 200上洗脱时为单一峰，分子量为160,000，并且可被山羊抗兔γ球蛋白沉淀，这表明该维生素与IgG免疫球蛋白结合。未标记的氰钴胺和羟钴胺竞争性抑制氰基[57Co]钴胺素与该抗体的结合。在相似浓度范围内，腺苷钴胺和浓度比示踪钴胺素高30倍的氰钴胺酰胺均未与氰基[57Co]钴胺素的结合发生明显竞争。抗体与氰钴胺和氰钴胺酰胺相互作用的缔合常数估计分别为8.6×10(9)和9.6×10(6) L/mol。间接测定了腺苷钴胺、甲基钴胺和羟钴胺的缔合常数，分别得到2.5×10(5)、1.7×10(9)和2.3×10(9) L/mol的值。在氰化钾存在下进行光解使三种钴胺素中的每一种在免疫反应性上等同于氰钴胺。使用这种抗钴胺素抗体通过放射免疫测定法测定的正常人血清和钴胺素缺乏血清中钴胺素的平均浓度，显著低于使用内因子和转钴胺素I通过竞争性配体结合放射测定法在相同提取物中测得的浓度。这些发现虽然是间接的，但支持这样的观点，即如果放射测定法使用R蛋白作为结合剂，正常血清和钴胺素缺乏血清中可能存在某些因子会错误地提高真正钴胺素的浓度。然而，与内因子放射测定法相比，放射免疫测定法测得的血清钴胺素浓度较低也表明这种“所谓的”因子在一定程度上与内因子反应，但不与钴胺素抗体反应。
