Enhanced sensitivity and precision in an enzyme-linked immunosorbent assay with fluorogenic substrates compared with commonly used chromogenic substrates Yuan Meng a , Katrina High a , Joseph Antonello b , Michael W. Washabaugh a , Qinjian Zhao a, * a Department of Bioprocess and Bioanalytical Research, Merck Research Laboratories, West Point, PA 19486, USA b Department of Vaccine Biometrics Research, Merck Research Laboratories, West Point, PA 19486, USA Received 9 May 2005 Available online 8 August 2005 Abstract Quantitative enzyme-linked immunosorbent assay (ELISA) is a widely used tool for analyzing biopharmaceutical and vaccine products. The superior sensitivity of the ELISA format is conferred by signal amplification through the enzymatic oxidation or hydrolysis of substrates to products with enhanced color or fluorescence. The extinction coefficient for a colored product or the quantum yield of a fluorescent product, coupled with the efficiency of the immobilized enzyme, is the determining factor for the sensitivity and precision of a given ELISA. The enhancement of precision and sensitivity using fluorogenic substrates was demon- strated in a direct-binding ELISA in a low-analyte concentration range compared with commonly used chromogenic substrates. The enhancement in precision was demonstrated quantitatively with lower coefficients of variation in measurements of signal intensities, approximately a five- to six-fold enhancement in signal-to-noise ratio at a given analyte concentration with fluorogenic substrates. Similarly, the amplitude of the enhancement in sensitivity, as reflected by relative limits of detection or quantitation, is approximate- ly two- to five-fold when compared with commonly used chromogenic substrates. Additional advantages of a fluorescence-based ELISA format include the continuous monitoring of initial rates of enzymatic reactions, the measurement of fluorescence changes in the presence of particulate materials, the absence of a quench step, and a larger quantifiable analyte range. Ó 2005 Elsevier Inc. All rights reserved. Keywords: ELISA; Enzyme substrate; Fluorescence; Fluorogenic substrate; Level of detection; Level of quantitation; Z-factor; Immuno assay; Affinity constant determination; Sensitivity; Precision Since the development of the first radioimmunoassay (RIA) 1 in 1959, there has been a constant search for more sensitive, robust, and reproducible immunoassays for life science, research and development, and clinical diagnostics. During recent years, because of progress in genomics and proteomics, more sensitive and robust binding assays are being sought for high-throughput screening and microarray applications. The enzyme- linked immunosorbent assay (ELISA) is replacing the RIA format as the most widely used immunochemical 0003-2697/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.07.026 * Corresponding author. Fax: +1 215 993 3348. E-mail address: qinjian_zhao@merck.com (Q. Zhao). 1 Abbreviations used: RIA, radioimmunoassay; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; AP, alkaline phosphatase; HBsAg, hepatitis B surface antigen; mAb, monoclonal antibody; S/N ratio, signal-to-noise ratio; LOD, limit of detection; LOQ, limit of quantitation; OPD, o-phenylenediamine; 4-MUP, 4-methylumbelliferyl phosphate; TBS, Tris-buffered saline; BSA, bovine serum albumin; %CV, relative coefficient of variation; SD, standard deviation; ICH, International Conference of Harmonization; TMB, 3,3 0 ,5,5 0 -tetramethyl-benzidine; 4-MU, 4-methyl umbelliferone. ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 345 (2005) 227–236 www.elsevier.com/locate/yabio