Search research articles

ABOUT JoVE

Overview Leadership Blog JoVE Help Center

AUTHORS

Publishing Process Editorial Board Scope & Policies Peer Review FAQ Submit

LIBRARIANS

Testimonials Subscriptions Access Resources Library Advisory Board FAQ

RESEARCH

JoVE Journal Methods Collections JoVE Encyclopedia of Experiments Archive

EDUCATION

JoVE Core JoVE Business JoVE Science Education JoVE Lab Manual Faculty Resource Center Faculty Site

Terms & Conditions of Use

Search research articles

Related Experiment Videos

The use of tetramethylbenzidine for solid phase immunoassays.

J L McKimm-Breschkin¹

¹CSIRO Division of Biomolecular Engineering, Parkville, Australia.

Journal of Immunological Methods

|December 31, 1990

Summary

This study introduces a faster, more sensitive solid-phase immunoassay using horseradish peroxidase (HRPO) and 3,3

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by

Same author

Resistance to neuraminidase inhibitors conferred by an R292K mutation in a human influenza virus H7N9 isolate can be masked by a mixed R/K viral population.

mBio·2013

Same author

Biochemical Methods for the Characterization of Influenza Viruses with Reduced Sensitivity to 4-Guanidino-Neu5Ac2en.

Methods in molecular medicine·2011

Same author

Virological Methods for the Generation and Characterization of Influenza Viruses with Reduced Sensitivity to 4-Guanidino-Neu5Ac2en.

Methods in molecular medicine·2011

Same author

Evaluation of neuraminidase enzyme assays using different substrates to measure susceptibility of influenza virus clinical isolates to neuraminidase inhibitors: report of the neuraminidase inhibitor susceptibility network.

Journal of clinical microbiology·2003

Same author

Antiviral activity and structural characteristics of the nonglycosylated central subdomain of human respiratory syncytial virus attachment (G) glycoprotein.

The Journal of biological chemistry·2001

Same author

Resistance of influenza viruses to neuraminidase inhibitors--a review.

Antiviral research·2000

Area of Science:

Biochemistry
Immunochemistry
Assay Development

Background:

Solid-phase immunoassays are crucial for detecting various analytes.
Traditional methods using horseradish peroxidase (HRPO) with substrates like 4-chloro-1-naphthol or diaminobenzidine have limitations in sensitivity and color stability.
Alkaline phosphatase-based assays offer high sensitivity but can be less rapid.

Purpose of the Study:

To develop a rapid and highly sensitive solid-phase immunoassay.
To enhance color stability in HRPO-based immunoassays.
To compare the sensitivity of the new assay with existing methods.

Main Methods:

A solid-phase immunoassay was developed using horseradish peroxidase (HRPO) and 3,3',5,5'-tetramethylbenzidine (TMB).
A key step involved incubating filters in 1% dextran sulphate in a pH 5 buffer before TMB reaction.
Assay sensitivity was evaluated and compared to HRPO with other substrates and alkaline phosphatase.

Main Results:

The HRPO/TMB system demonstrated superior sensitivity and more stable color development compared to HRPO/4-chloro-1-naphthol or HRPO/diaminobenzidine.
The assay sensitivity was found to be equal to or greater than that achieved with alkaline phosphatase.
Incubation with dextran sulphate was identified as an essential step for optimal performance.

Conclusions:

The optimized HRPO/TMB immunoassay offers a rapid, sensitive, and stable alternative for solid-phase detection.
This method provides a valuable tool for various diagnostic and research applications.
The inclusion of dextran sulphate significantly improves assay performance.

Related Experiment Videos