Affinity Measurement of Lactoferrin (LF)-Anti-LF Immunoglobulin in Yolk (IgY) Complexes by Competitive Indirect Enzyme-Linked Immunosorbent Assay (CI-ELISA)

以間接型競爭性ELISA法(CI-ELISA)測定乳鐵蛋白(LF)－抗乳鐵蛋白蛋黃抗體(IgY)複合物之親合力

杜豔櫻(Yann-Ying Tu) ； 馬嘉軉(Chia-Yu Ma) ； 何學斌(Shyue-Bin Ho) ； 陳昭誠(Chao-Cheng Chen) ； 張鴻民(Hung-Min Chang)

《藥物食品分析》 14卷4期 (2006/12) Pp. 379-384

摘要

以競爭性ELISA法(CI-ELISA)來測定牛乳乳鐵蛋白(LF)抗原與蛋黃中抗LF特異性抗體間之解離常數(Kd)與親合常數(Ka)，而同時以兔與雞之抗血清為對照組。於溶液狀態下，抗原-抗體混合物中抗原濃度增加時則ELISA數值降低，說明溶液中游離之LF分子與固定於盤中LF對於抗LF特異性IgY抗體間之競爭更增加。由Klotz Plots法計算得知LF對抗LF特異性IgY抗體間之Kd及Ka值分別約為2.6×10^(-8) M及0.5×10(-8) M^(-1)。1500倍稀釋後，粗IgY、雞抗血清及兔抗血清之Kd數值皆非常接近於親合性膠體純化之抗LF特異性IgY抗體，顯示溶液狀態下之競爭性ELISA法對於抗原-IgY抗體平衡親合力之數值測定，可直接與含IgY樣品檢測。

關鍵字

解離常數 ； 親合常數 ； 間接型競爭性ELISA ； 蛋黃中抗體 ； 乳鐵蛋白

並列摘要

Competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) was employed to perform the affinity measurement of dissociation constant (Kd) and affinity constants (Ka) for bovine milk lactoferrin (LF) and IgY (immunoglobulin in yolk) specific against LF using antisera from rabbit and hen as references. In liquid phase equilibrium measurements by CI-ELISA, the elevation in antigen level in antigen-antibody mixtures decreased the ELISA values, suggesting the increased competition of free LF in solution with that coated on plate for LF-specific IgY purified by immunoaffinity chromatography (purified IgY). From the Klotz plots of the binding of LF to purified IgY, Kd and Ka were determined to be about 2.6×10^(-8) M and 0.5×10^(-8) M^(-1), respectively. The Kd values of 1500-fold diluted crude IgY, diluted sera from hen and rabbit were determined to be very close to that of purified IgY, revealing that CI-ELISA under liquid phase equilibrium by CI-ELISA was appropriate for the affinity measurement of IgY samples.

並列關鍵字

dissociation constant ； affinity constant ； indirect competitive ELISA ； immunoglobulin in yolk ； lactoferrin