Immunoassays used in diagnostic/ pathology labs for detecting infectious diseases, hormones, and cancer biomarkers detection are expensive today mainly due to usage of imported and costly colour reagent component required for testing. Now, Prof. V. Venkataraman and Dr. Venkatesh N.Seshadri from the Department of Physics at IISc, Bangalore, have developed indigenously a Low cost, novel chromogenic reagent called Tetramethylbenzidine (TMB) monoreagent, which makes immunoassays more affordable at diagnostic and research labs. This indigenous TMB monoreagent technology development is said to be first of its kind in India.
3, 3, 5, 5-Tetramethylbenzidine (TMB) reagent is a chromogenic substrate for Peroxidase (HRP) based detection system, widely used in biotechnology applications like ELISA, Dot-ELISA, immunochemistry, western blotting, ELISPOT , microarray and in-situ hybridization in various disciplines of biotechnology such as human diagnostics, veterinary diagnostics, plant pathogenesis & GM crops detection, drug discovery, food pathogen detection and Research & development.
TMB monoreagent composition consists of both TMB substrate and hydrogen peroxide as a co-substrate. Keeping both TMB substrate and hydrogen peroxide together in a single reagent composition for a longer duration and making it robust is said to be a real technical challenge, something like keeping cotton and fire together, Hence, many ELISA kit manufacturers provide both TMB substrate and hydrogen peroxide as a two different reagent components in their ELISA kit and at the time of assay, both the reagent components has to be mixed equally and to be used immediately and cannot be stored for a longer period. Moreover, TMB substrate is hydrophobic molecule, very much sensitive to any contaminants, metal ions, peroxides, humidity, light and atmospheric oxygen, posing a real technical challenge in making it a real workable single and sensitive reagent for immunoassays. Without the appropriate stabilizers , buffer composition and process, TMB substrate is unstable. When taken into aqueous solution, it immediately gets oxidized to blue colour [auto oxidation] in the presence of peroxide, light, environmental conditions etc. It also gets easily precipitated during preparation due to its hydrophobic nature in aqueous solution and therefore TMB reagent loses its identity as “Superior sensitive peroxidase substrate”. The developers have come up with an innovative formulation and process to stabilize both TMB substrate and peroxide together in a single component especially using 100% aqueous environment and thus making the composition highly sensitive and robust with a shelf life of two years.
An important aspect of the TMB reagent produced at IISc is its economic viability. With the new manufacturing techniques, the cost of a litre of TMB is just about 10% of its present cost. The research team attributes the use of indigenous materials during manufacturing for its low-cost. The team asserts that the new TMB manufacturing procedures can be scaled up for commercialization.
The newly developed TMB monoreagent has been evaluated both internally & externally in blood banks for characterizing its sensitivity, specificity, photostability, real time storage stability and batch to batch consistency. In addition the TMB formulation and process has been successfully scaled up for its batch production and its performance characterization in various techniques like ELISA, Dot-ELISA, Western blotting and Immunohistochemistry are well studied. They have used it in the commercial HIV and Hepatitis-B ELISA kits. The kits with this TMB substrate were found to function as efficiently and robustly as any other MNC make.
The current research provides a pathway to bring this tool to the Indian marketplace and make it economically viable.
About the Developers:
Prof. V. Venkataraman is a Professor and Chairman at the Department of Physics at IISc.
Dr. Venkatesh Nagamangalam .Seshadri is a Member of Technical Staff at the RBCCPS, IISc.