Abstract
ObjectiveImmunoassay usually deal with the antibody labeling with various reporter molecules, one such useful reporter molecule is horseradish peroxidase (HRPO). Conjugating enzyme with antibody without losing its enzymatic activity is a challenging task. Our aim is to modify existing classical method of conjugating antibodies with HRP to enhance immunoassay techniques with better sensitivity. We used chemicals such as sodium meta periodate to generate aldehyde group by oxidation of carbohydrate moieties on HRPO. The activated form of HRPO is lyophilized and then mixed with 1 mg/ml concentration of antibodies to be conjugate.ResultsAfter confirming chemical modification of conjugates via UV-Spec and SDS-PAGE independent molecules were used for conjugation and HRP–antibody conjugate. Finally, enzymatic activity of HRP–antibody conjugate was confirmed by performing direct ELISA. Functional properties were analyzed using ELISA with dilution of 1:5000, whereas the conjugate prepared by existing method of conjugation worked with as low dilution of 1:25 with a p value highly significant (< 0.001) for classical verses modified method of conjugation preparation. Collectively, this study showed the enhanced ability of antibody to bind more number of HRPO with an additional step of lyophilization in the regular conjugation protocol. Future exploration are necessary on wide range of IgG antibodies.
Highlights
After confirming chemical modification of conjugates via ultraviolet–visible spectroscopy (UV-Spec) and SDS-PAGE independent molecules were used for conjugation and Horseradish peroxidase (HRP)–antibody conjugate
In UV-spec the wavelength scan of the conjugate was performed at a range of 280–800 nm.The result obtained was compared with the results of unconjugated horse radish peroxidase (HRPO) and unconjugated antibody alone
Due to the modification of HRPO during the conjugation procedure, there was a shift in the absorption which resulted in a small peak at 430 nm when compared to the peak of HRPO alone which confirms the efficient chemical modification leading to conjugation (Fig. 1)
Summary
The conjugate obtained by the modified method of existing classical protocol was taken for further experiments. In support to the above results, SDS-PAGE was performed to confirm the conjugation of HRPO to antibodies. Conjugate resulted from both classical and modified method (Lane-1 and 2) are treated along with HRPO and dengue antibodies for heat denaturation at 95 °C, alongside non-reducing conjugates Whereas lower molecular size HRPO (Lane-4) reached almost at the end of the gel and antibodies (Lane-3) are denatures and shown mobility according to its molecular size (Fig. 2). This indicated that the conjugation of the antibody to HRPO was done efficiently. In conjunction with collision theory, molecules must collide to react and rate of reaction is proportional to number of reacting molecules present in the
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