• 江蘇省人民醫(yī)院，南京醫(yī)科大學(xué)第一附屬醫(yī)院肝臟外科（南京210029）;

【摘要】目的報道一種用雙功能螯合劑BAT與鼠單克隆抗體Fab’片段行點特異性交聯(lián)的新方法。
方法通過位于Fab’鉸鏈區(qū)、遠(yuǎn)離抗原結(jié)合位點的活性巰基（-SH）直接與BAT相交聯(lián)。用簡單的兩步法制備B43單抗的Fab’片段。首先單抗用胃蛋白酶處理, 產(chǎn)生穩(wěn)定的F(ab’)2 片段，然后 F(ab’)2經(jīng)半胱氨酸還原后產(chǎn)生Fab’片段，最后Fab’片段直接與BAT交聯(lián)。
結(jié)果每個Fab’分子平均含1.8個-SH基團， 74%的Fab’片段可與BAT生成交聯(lián)物，平均每個Fab’分子攜帶1.28個BAT。經(jīng)檢測，F(xiàn)(ab’)2、Fab’ 及 Fab’-BAT 均保持著良好的免疫活性。
結(jié)論該交聯(lián)法簡單、高效，為雙功能螯合劑BAT與鼠單抗Fab’片段的交聯(lián)提供了一個新途徑。

引用本文： 李君,王學(xué)浩,成峰,王曉明,陳兆雷,沈喜平. 雙功能螯合劑BAT與鼠IgG1 Fab’ 片段的點特異性交聯(lián)方法的研究. 中國普外基礎(chǔ)與臨床雜志, 2005, 12(3): 268-270. doi: 復(fù)制

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12. SmithJones PM, Fridrich R, Kaden TA, et al. Antibody labeling with copper67 using the bifunctional macrocycle 4[(1,4,8,11tetraazacyclotetradec1yl)methyl]benzoic acid [Ｊ]. Bioconjug Chem, 1991; 2(6)∶415.
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  1. 1. Kaklamani V, O’Regan RM. New targeted therapies in breast cancer [Ｊ]. Semin Oncol， 2004; 31(2 Suppl 4)∶20.
  2. 2. Sanz L, Blanco B, AlvarezVallina L. Antibodies and gene therapy: teaching old ‘magic bullets’ new tricks [Ｊ]. Trends Immunol, 2004; 25(2)∶85.
  3. 3. Merdan T, Callahan J, Petersen H, et al. Pegylated polyethylenimineFab’ antibody fragment conjugates for targeted gene delivery to human ovarian carcinoma cells [Ｊ]. Bioconjug Chem, 2003; 14(5)∶989.
  4. 4. Von Rothenburg T, Schaffstein J, Ludwig J, et al. Imaging osteomyelitis with Tc99mlabeled antigranulocyte antibody Fab’ fragments [Ｊ]. Clin Nucl Med, 2003; 28(8)∶643.
  5. 5. Liu X, Zhen Y. Antitumor effects of monoclonal antibody Fab’ fragmentcontaining immunoconjugates [Ｊ]. Chin Med Sci J, 2002; 17(1)∶1.
  6. 6. Hashida S, Imagawa M, Inoue S, et al. More useful maleimide compounds for the conjugation of Fab’ to horseradish peroxidase through thiol groups in the hinge [Ｊ]. J Appl Biochem, 1984; 6(1-2)∶56.
  7. 7. Smith A, Alberto R, Blaeuenstein P, et al. Preclinical evaluation of 67Culabeled intact and fragmented anticolon carcinoma monoclonal antibody MAb35 [Ｊ]. Cancer Res, 1993; 53(23)∶5727.
  8. 8. McCall MJ, Diril H, Meares CF. Simplified method for conjugating macrocyclic bifunctional chelating agents to antibodies via 2iminothiolane [Ｊ]. Bioconjug Chem, 1990; 1(3)∶222.
  9. 9. Anderson CJ, Schwarz SW, Connett JM, et al. Preparation, biodistribution and dosimetry of copper64labeled anticolorectal carcinoma monoclonal antibody fragments 1A3F(ab’)2 [Ｊ]. J Nucl Med, 1995; 36(5)∶850.
  10. 10. Riddles PW, Blakeley RL, Zerner B. Reassessment of Ellman’s reagent [Ｊ]. Methods Enzymol, 1983; 91（1）∶49.
  11. 11. Meares CF, McCall MJ, Reardan DT, et al. Conjugation of antibodies with bifunctional chelating agents: isothiocyanate and bromoacetamide reagents, methods of analysis, and subsequent addition of metal ions [Ｊ]. Anal Biochem, 1984; 142(1)∶68.
  12. 12. SmithJones PM, Fridrich R, Kaden TA, et al. Antibody labeling with copper67 using the bifunctional macrocycle 4[(1,4,8,11tetraazacyclotetradec1yl)methyl]benzoic acid [Ｊ]. Bioconjug Chem, 1991; 2(6)∶415.
  13. 13. Parham P, Androlewicz MJ, Brodsky FM, et al. Monoclonal antibodies: purification, fragmentation and application to structural and functional studies of class I MHC antigens [Ｊ]. J Immunol Methods, 1982; 53(2)∶133.