Martsev S P, Preygerzon V A, Mel'nikova Y I, Kravchuk Z I, Ponomarev G V, Lunev V E, Savitsky A P
Department of Protein Chemistry, Academy of Sciences of Belarus, Minsk.
J Immunol Methods. 1995 Oct 26;186(2):293-304. doi: 10.1016/0022-1759(95)00154-3.
Antibodies conjugated with porphyrins and metalloporphyrins have a great potential for applications in fluorescence or phosphorescence immunoassays as well as in photodynamic therapy, radioimaging and internal radiation therapy of cancer. Here we describe how the new preactivated metalloporphyrin, palladium (II) coproporphyrin I-tetra-N-hydroxysuccinimide ester, can be covalently attached to mouse monoclonal and rabbit anti-human ferritin antibodies. The advantages of the proposed reagent over the previously reported carboxylic porphyrins coupled through carbodiimide activation are indicated. Conformational changes in antibodies caused by each of the two methods were assessed from their binding to the antigen (a probe for the antibody Fv domain) and anti-IgG antibodies probing the global conformation of the CH2 domain in the Fc fragment. Porphyrin coupling through carbodiimide activation resulted in a decrease in both functional activities of modified antibodies even at low levels of modification. In contrast, when the N-hydroxysuccinimide (NHS) derivative of porphyrin was used, enhancement of the antigen-binding affinity of porphyrin-antibody conjugates occurred due to an increase in the conformational mobility (flexibility) of the modified antibodies. The stimulatory effect of conjugation was maximal when one porphyrin molecule was coupled to an antibody molecule. Coupling of NHS-activated porphyrin at pH 7.4, 7.8 and pH 8.5 suggested that the high efficiency of the reaction at pH 8.5 could be attributed predominantly to the formation of antibody aggregates, only 50% of which were covalently cross-linked. The lowest percentage of aggregates in porphyrin-antibody conjugates was found at pH 7.4 and a molar reagent-to-protein ratio in the 10:1-40:1 range. Thus, the use of the NHS-activated carboxylic porphyrin provides a mild, simple and convenient procedure for preparing antibody conjugates with enhanced antigen-binding affinity.
与卟啉和金属卟啉共轭的抗体在荧光或磷光免疫分析以及光动力疗法、放射成像和癌症内放射治疗中具有巨大的应用潜力。在此，我们描述了新型预活化金属卟啉——钯（II）粪卟啉I - 四 - N - 羟基琥珀酰亚胺酯，如何共价连接到小鼠单克隆和兔抗人铁蛋白抗体上。指出了所提出的试剂相对于先前报道的通过碳二亚胺活化偶联的羧基卟啉的优势。通过两种方法中每种方法引起的抗体构象变化，是根据它们与抗原（抗体Fv结构域的探针）的结合以及探测Fc片段中CH2结构域整体构象的抗IgG抗体来评估的。通过碳二亚胺活化进行卟啉偶联，即使在低修饰水平下，修饰抗体的两种功能活性也会降低。相反，当使用卟啉的N - 羟基琥珀酰亚胺（NHS）衍生物时，由于修饰抗体的构象流动性（灵活性）增加，卟啉 - 抗体缀合物的抗原结合亲和力增强。当一个卟啉分子与一个抗体分子偶联时，缀合的刺激作用最大。在pH 7.4、7.8和pH 8.5下进行NHS活化卟啉的偶联表明，pH 8.5时反应的高效率可能主要归因于抗体聚集体的形成，其中只有50%是共价交联的。在pH 7.4以及试剂与蛋白质的摩尔比在10:1 - 40:1范围内时，卟啉 - 抗体缀合物中的聚集体百分比最低。因此，使用NHS活化的羧基卟啉为制备具有增强抗原结合亲和力的抗体缀合物提供了一种温和、简单且方便的方法。
