1. TMB Elisa Protocol
TMB substrate mainly consists of 3,3′,5,5′-Tetramethylbenzidine(non-carcinogenic HRP substrate) and becomes the charge transfer complex(CTC) upon oxidation due to loss of electrons. The max absorption peak is 652nm(blue). The added acid substance results in the protonation which forces the separation of the CTC and also generates TMB(colourless) and TMB+ (yellow, max absorption peak: 450nm). Meanwhile, HRP enzyme is inactive in acetic condition and the visualization stops.
2. Analyzing Data between OD652 and OD450
Seen from tables below, OD652 data are positively correlated to OD450 data. (The relative error between OD620 and OD652 is 10%.)
Table 1. Comparison between monitored OD620(blue) and OD450 after stopping the reaction.
Table 2. Visualization control of some items.
3. Notes for Visualization Control and Accurate Calculation
- Read TMB+ at 450nm in a microplate reader with resolution ratio of 3.0. The linearity is quite well under resolution ratio of 2.2(2X relation). For the quantitative detection, the visualization shouldn’t be too dark. Please refer to data provided by Thermo FC microplate reader.
- Use professional curve fitting software. Recommended software is available on FineTest e-resource center. MMF four-parameter function is recommended for the parabola.
- The concentration of TMB+ is higher if the visualization is too dark(OD450>3.5). The solubility is much less than TMB. Gradually, TMB+ forms crystals and precipitates at the bottom of the well(black needle-like particle). Read the O.D. absorbance immediately otherwise the yellow will fade. A number of black crystals will appear at the bottom of the well after 12h due to the dark visualization. The yellow is unavailable as a result.
- Notes: Sensitivity of microplate readers among different vendors is also varied.
REFERENCES
[1] Integration and Quantitative Visualization of 3,3',5,5'-Tetramethylbenzidine-Probed Enzyme-Linked Immunosorbent Assay-like Signals in a Photothermal Bar-Chart Microfluidic Chip for Multiplexed Immunosensing, PMID: 34734693.
[2] Rationale of 3,3',5,5'-Tetramethylbenzidine as the Chromogenic Substrate in Colorimetric Analysis, PMID: 32794705.