A stable and highly sensitive 3,3′,5,5′-tetramethylbenzidine-based substrate reagent for enzyme-linked immunosorbent assays

• Published in: Journal of immunological methods 2000-01, Vol.233 (1), p.47-56
• Main Authors: Frey, Andreas, Meckelein, Barbara, Externest, Dörthe, Schmidt, M.Alexander
• Format: Article
• Language: English
• Subjects: 3,3',5,5'-tetramethylbenzidine; Animals; Benzidines; Biological and medical sciences; Chromogenic Compounds; Drug Stability; Enzyme-Linked Immunosorbent Assay - methods; Enzyme-Linked Immunosorbent Assay - statistics & numerical data; Evaluation Studies as Topic; Female; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Horseradish Peroxidase; Immunoassay; Immunoglobulin A - analysis; Immunoglobulin G - analysis; Indicators and Reagents; Mice; Mice, Inbred BALB C; Molecular immunology; Ovalbumin - immunology; Sensitivity; Sensitivity and Specificity; Substrate reagent; Substrate Specificity; Techniques
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(99)00166-0

3,3′,5,5′-Tetramethylbenzidine (TMB) is a widely used chromogen for horseradish peroxidase-based detection systems because it yields reaction products with high absorption coefficients and lacks carcinogenicity. Unfortunately, TMB is labile and poorly soluble in aqueous buffers and such solutions must be freshly prepared before each experiment. Moreover, substrate depletion can occur under assay conditions. To overcome these problems we have developed a two-component TMB substrate system which has a lower detection limit and is more sensitive than many commercially available TMB reagents when compared in microtiter plate enzyme-linked immunosorbent assays. Both components of the substrate system are stable for at least 1 year at 4°C and the usual discoloration of TMB stock solutions is prevented by the addition of a stabilizer that decomposes upon mixing of the two components.