Abstract
BackgroundThe 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.ResultsUsing 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.ConclusionPseudohomogeneous 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 A magnetic ELISA with convenient magnetic microplate.Electronic supplementary materialThe online version of this article (doi:10.1186/s13065-015-0088-1) contains supplementary material, which is available to authorized users.
Highlights
The enzyme-linked immunosorbent assay (ELISA) has been used for diagnosing medical and plant pathologies
A sandwich enzyme-linked immunosorbent assay (ELISA) is a frequently employed bioanalytical assay that involves using an antibody-labeled solid phase to detect the presence of a substance, generally an antigen, in a liquid sample
The ELISA is the simplest method for obtaining excellent results; it is time consuming because the immunoreagents interact only on the contact surfaces
Summary
The enzyme-linked immunosorbent assay (ELISA) has been used for diagnosing medical and plant pathologies. 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 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. We attempted to overcome this limitation using functional magnetic microparticles and an enzyme-conjugated antibody in a magnetic microplate This method has many potential advantages which were reported in previous studies [17], such as (1) the amount of proteins immobilized on the particles is consistent in the same batch, which can be used for performing several reactions. The process of the microplate ELISA will be more adopted in the clinical laboratory than tube-ELISA and the home made magnetic microplate is inexpensive
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