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 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.


CONCLUSIONS
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 AbstractA magnetic ELISA with convenient magnetic microplate.
School of Medical Applied Chemistry, Chung Shan Medical University, Taichung, 402 Taiwan ; Department of Medical Education, Chung Shan Medical University Hospital, Taichung, 402 Taiwan.
Hweiyan
H
Tsai
School of Medical Applied Chemistry, Chung Shan Medical University, Taichung, 402 Taiwan.
Yi-Hsuan
YH
Lu
School of Medical Applied Chemistry, Chung Shan Medical University, Taichung, 402 Taiwan.
Huan-Xuan
HX
Liao
School of Biomedical Sciences, Chung Shan Medical University, Taichung, 402 Taiwan.
Shih-Wei
SW
Wu
School of Biomedical Sciences, Chung Shan Medical University, Taichung, 402 Taiwan.
Feng-Yih
FY
Yu
Department of Applied Chemistry, National Chi Nan University, Puli, Nantou, 545 Taiwan.
Chwan Bor
CB
Fuh
Functional magnetic particles; Immunoassay; Magnetic separator