抗meso-四(α,α,α,α-O-苯乙酰苯)卟啉抗体的酶学性质研究

Studies on Enzymological Properties of Antibody Elicited bymeso-Tetra(α, α, α, α-O-phenylacetyl benzene)porphyrin

利用小分子ｍｅｓｏ－四(α,α,α,α－Ｏ－苯乙酰苯)卟啉作为全抗原免疫小鼠,通过细胞融合等技术制备了单克隆抗体(ＭｃＡｂ)１Ｆ２,利用高效液相色谱(ＨＰＬＣ)、基质辅助激光解吸飞行时间质谱(ＭＡＬＤＩ/ＴＯＦＭＳ)证明纯化得到的单抗是很纯的．ＭｃＡｂ１Ｆ２的保留时间是２．６３ ｍｉｎ,亚型为ＩｇＧ２ａ,相对分子量为１５６ ６７８．８．ＭｃＡｂ １Ｆ２－卟啉复合物形成时,卟啉Ｓｏｒｅｔ带最大吸收峰从４０８ｎｍ红移到４１６ｎｍ,并有增色效应,说明ＭｃＡｂ１Ｆ２－卟啉是刚性且紧密结合．抗体酶稳定性很高,但比活力只有４．６８７ ５ Ｕ/ｍｇ,为天然酶ＨＲＰ的１．８９９%．抗体酶的Ｋｍ＝２０．２９ ｍｍｏｌ/Ｌ,ｋｃａｔ＝３９６．８２ ｍｉｎ－１,ｋｃａｔ/Ｋｍ＝１．９５５ ７×１０４Ｌ·ｍｏｌ－１·ｍｉｎ－１．

meso-Tetra(α,α,α,α-O-phenylacetyl benzene)porphyrin was used as a complete antigen to elicit monoclonal antibody 1F2 through the immunization and cell fusion techniques. McAb 1F2 obtained was demonstrated very pure by HPLC and MALDI/TOFMS. The retention time of McAb 1F2 was 2. 63 min. The subtype of McAb 1F2 was IgG2a. The relative molecular weight was 156 678. 8. When the McAb 1F2-porphyrin was formed, the maximal absorption of the porphyrin soret region had a redshift from 408 to 416 nm and hyperchromical effect, showing that the antigen-antibody combination was rigid and intense , and the abzyme constancy was high. But compared with HRP, the activity of the abzyme was only 4. 687 5 U/mg and 1. 899% of that of HRP. Its Km was 20. 29 mmol/L, kcat 396. 82 min-1, kcat/Km 1. 955 7×104 L.mol-1.min-1.