Detection of rabbit IgG by using functional magnetic particles and an enzyme-conjugated antibody with a homemade magnetic microplate
Background The enzyme-linked immunosorbent assay (ELISA) has been used for diagnosing medical and plant pathologies. In addition, it is used for quality-control evaluations in various industries. The ELISA is the simplest method for obtaining excellent results; however, it is time consuming because...
Saved in:
Published in | BMC chemistry Vol. 9; no. 1; p. 8 |
---|---|
Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
Cham
Springer International Publishing
22.02.2015
Springer Nature B.V |
Subjects | |
Online Access | Get full text |
Cover
Loading…
Summary: | Background
The enzyme-linked immunosorbent assay (ELISA) has been used for diagnosing medical and plant pathologies. In addition, it is used for quality-control evaluations in various industries. The ELISA is the simplest method for obtaining excellent results; however, it is time consuming because the immunoreagents interact only on the contact surfaces. Antibody-labeled magnetic particles can be dispersed in a solution to yield a pseudohomogeneous reaction with antigens which improved the efficiency of immunoreaction, and can be easily separated from the unreactive substances by applying a magnetic force. We used a homemade magnetic microplate, functional magnetic particles (MPs) and enzyme-labeled secondary antibody to perform the sandwich ELISA successfully.
Results
Using antibody-labeled MPs enabled reducing the analysis time to one-third of that required in using a conventional ELISA. The secondary antibody conjugated with horseradish peroxidase (HRP) was affinity-bound to the analyte (IgG in this study). The calibration curve was established according to the measured absorbance of the 3, 3′, 5, 5′-tetramethybezidine–HRP reaction products versus the concentrations of standard IgG. The linear range of IgG detection was 114 ng/mL–3.5 ng/mL. The limit of detection (LOD) of IgG was 3.4 ng/mL. The recovery and coefficient of variation were 100% (±7%) and 116% (±4%) for the spiked concentrations of 56.8 ng/mL and 14.2 ng/mL, respectively.
Conclusion
Pseudohomogeneous reactions can be performed using functional MPs and a magnetic microplate. Using antibody-labeled MPs, the analysis time can be reduced to one-third of that required in using a conventional ELISA. The substrate–enzyme reaction products can be easily transferred to another microplate, and their absorbance can be measured without interference by light scattering caused by magnetic microbeads. This method demonstrates great potential for detecting other biomarkers and in biochemical applications.
Graphical Abstract
A magnetic ELISA with convenient magnetic microplate. |
---|---|
Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 1752-153X 1752-153X 2661-801X |
DOI: | 10.1186/s13065-015-0088-1 |