Eriksson S, Vehniäinen M, Jansén T, Meretoja V, Saviranta P, Pettersson K, Lövgren T
Department of Biotechnology, University of Turku, Tykistökatu 6, FIN-20520 Turku, Finland.
Clin Chem. 2000 May;46(5):658-66.
Recombinant Fab fragments are attractive as reagents for novel sandwich immunoassays, but no such assays have been described. We developed a dual-label two-site immunoassay based entirely on recombinant Fab fragments and compared it to the same assay with intact monoclonal antibodies.
The capture Fab fragment, which binds free prostate-specific antigen (PSA) and PSA in complex with alpha(1)-antichymotrypsin on an equimolar basis, is site-specifically biotinylated and attached to the solid phase in streptavidin-coated microtitration wells. The Fab fragment that detects only free PSA is site-specifically labeled with a fluorescent europium chelate, and the Fab fragment that detects both free and serpin-complexed PSA in an equimolar fashion is labeled with a fluorescent terbium chelate. Time-resolved fluorescence is used to measure both europium and terbium signals in one well.
The detection limits of the assay (mean + 3 SD of zero calibrator) were 0.043 and 0.28 microgram/L, respectively, for free and total PSA. The within-run and day-to-day CVs were 2-11% and 4-10%, respectively. Mean recoveries were 93% and 98% in female and male sera, respectively. Compared with the commercial ProStatus PSA Free/Total Assay, the intercepts of the regression equations (r >0. 99) were not significantly different from zero, and the slopes were 0.95-1.01. In one female serum sample, PSA was falsely increased with the monoclonal assay but was undetectable with the recombinant assay.
The performance of this novel assay based on recombinant components is comparable to a conventional assay based on monoclonal antibodies. The more complete control of the essential characteristics of site-specifically derivatized recombinant Fab fragments will be valuable for the design of miniaturized and multianalyte assay concepts where correct antibody orientation, density, and capacity as well as uncompromised binding affinity are required.
重组Fab片段作为新型夹心免疫测定的试剂很有吸引力，但尚未见此类测定的报道。我们开发了一种完全基于重组Fab片段的双标记两点免疫测定法，并将其与使用完整单克隆抗体的相同测定法进行比较。
捕获Fab片段能以等摩尔比结合游离前列腺特异性抗原（PSA）和与α1-抗糜蛋白酶结合的复合PSA，对其进行位点特异性生物素化，并附着于链霉亲和素包被的微量滴定孔的固相上。仅检测游离PSA的Fab片段用荧光铕螯合物进行位点特异性标记，以等摩尔方式检测游离和丝氨酸蛋白酶抑制剂复合PSA的Fab片段用荧光铽螯合物进行标记。采用时间分辨荧光法在一个孔中测量铕和铽的信号。
该测定法（零校准品的平均值+3标准差）对游离PSA和总PSA的检测限分别为0.043和0.28μg/L。批内和批间变异系数分别为2% - 11%和4% - 10%。在女性和男性血清中的平均回收率分别为93%和98%。与商业化的ProStatus PSA游离/总PSA测定法相比，回归方程（r>0.99）的截距与零无显著差异，斜率为0.95 - 1.01。在一份女性血清样本中，单克隆测定法使PSA出现假升高，但重组测定法未检测到。
这种基于重组成分的新型测定法的性能与基于单克隆抗体的传统测定法相当。对位点特异性衍生的重组Fab片段基本特性的更全面控制，对于设计需要正确抗体方向、密度、容量以及不降低结合亲和力的小型化和多分析物测定概念将具有重要价值。
