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Dot-blot ELISA for the detection of IgM RF and IgA RF
N Banchuin1, K Janyapoon, L Parivisutt
1Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Bangkok, Thailand.
Insights
A novel dot-blot ELISA effectively detects rheumatoid factor (RF) IgM and IgA in rheumatoid arthritis patients. This method shows high accuracy and concordance with traditional ELISA, offering a reliable diagnostic tool.
Area of Science:
- Immunology
- Biochemistry
- Medical Diagnostics
Background:
- Rheumatoid factor (RF) is a key biomarker in rheumatoid arthritis (RA).
- Traditional ELISA methods for RF detection can be time-consuming and require specialized equipment.
- There is a need for rapid and accessible RF detection assays.
Purpose of the Study:
- To develop and validate a dot-blot enzyme-linked immunosorbent assay (ELISA) for detecting immunoglobulin M (IgM) RF and immunoglobulin A (IgA) RF.
- To assess the diagnostic performance of the developed dot-blot ELISA for rheumatoid arthritis.
Main Methods:
- A dot-blot ELISA was established using normal rabbit IgG as the antigen immobilized on nitrocellulose membranes.
- Standardized incubation times and room temperature conditions were employed for serum, conjugate, and substrate.
- The assay's stability was evaluated by storing pre-dotted membranes for up to 6 weeks.
Main Results:
- The dot-blot ELISA detected IgM RF in 31/51 RA patients (1:800 dilution) and IgA RF in 27/51 RA patients (1:100 dilution).
- Only 3/68 healthy individuals showed positive IgM RF, and none showed positive IgA RF.
- High concordance (Kappa ≥ 0.78) was observed between the dot-blot ELISA and microtitre plate ELISA results for both IgM RF and IgA RF.
Conclusions:
- The developed dot-blot ELISA is a sensitive and specific method for detecting IgM RF and IgA RF.
- This assay demonstrates significant agreement with conventional ELISA, indicating its potential as a reliable diagnostic tool for rheumatoid arthritis.
- The assay's stability and room temperature conditions suggest its utility in various laboratory settings.
Abstract:
Dot-blot ELISA was developed for the detection of IgM RF and IgA RF. Normal rabbit IgG (NRIgG), concentration 100 micrograms/ml, was used as the antigen for dotting on the 0.45 microns pore size nitrocellulose membrane. Serum, conjugate and substrate incubation conditions were at room temperature for 1 hour, 1 hour and 3 minutes, respectively. The membrane with NRIgG dot could be sotred for 6 weeks before use in the assay. Positive results of IgM RF, at the serum dilution 1:800, were found in 31/51 patients with either classical or definite rheumatoid arthritis and 3/68 normal healthy individuals. Positive IgA RF, at the serum dilution 1:100, was found in 27/51 of the former and none of the latter. Significant concordance with high agreement index was found between the results of the dot-blot ELISA developed and those obtained from ELISA performed in microtitre plate (Kappa greater than or equal to 0.78 for IgM RF and 0.83 for IgA RF, p less than 0.001).
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