The RIG-I dsRNA ELISA Kit is a specialized tool for detecting and measuring the interaction between RIG-I (Retinoic acid-inducible gene I) and double-stranded RNA (dsRNA). This protein-RNA binding interaction is central to cellular pattern recognition mechanisms, especially during responses to RNA from environmental stressors. The kit offers a quantitative, reliable, and non-radioactive alternative to traditional assays.
Overview of RIG-I Function in Host Cells
RIG-I is a cytosolic sensor encoded by the DDX58 gene, highly conserved across species. It binds to 5′-triphosphate-bearing dsRNA, commonly associated with non-self RNA fragments introduced by replicating RNA. Upon binding, RIG-I initiates intracellular signaling cascades that regulate cell signaling pathways related to dsRNA recognition.
This function is outlined in the NCBI DDX58 gene summary and is studied broadly across academic biology departments such as those at Harvard and Stanford.
Molecular Mechanism of RIG-I–dsRNA Binding
RIG-I comprises two caspase activation and recruitment domains (CARDs), a helicase domain, and a C-terminal regulatory domain. The protein remains inactive in the absence of dsRNA, and only upon binding does it undergo conformational change, exposing its CARDs. This change promotes protein-protein interactions that eventually converge at MAVS (Mitochondrial Antiviral Signaling), which relays the signal.
Molecular studies using RIG-I truncation mutants and structural models have been published in NIH-sponsored journals. You can also explore structural biology resources at RCSB PDB for crystallographic data.
Key Features of the RIG-I dsRNA ELISA Kit
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Sandwich ELISA format for RIG-I binding measurement
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Pre-coated 96-well plates with anti-RIG-I capture antibody
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dsRNA provided as ligand substrate
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Colorimetric detection via HRP and TMB
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Optimized for use in cellular extract supernatants
This method was developed following validated ELISA protocols from the FDA immunoassay development guidelines and standard practices taught at Cornell’s Immunology Program.
Sample Types and Experimental Use
The kit works on samples derived from:
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In vitro cell culture extracts
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Differentiated stem-cell lines from NIH repositories
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Immortalized cell lines like HEK293 or A549 (ATCC)
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Tissue lysates prepared under buffer conditions described in NCBI’s protocols
The protocol is robust across species including human, mouse, and non-human primates. RIG-I signaling mechanisms in model organisms are well described by institutions like the Jackson Laboratory.
Assay Principle
The kit uses a capture antibody specific to RIG-I and detects the bound dsRNA via an indirect signal amplification process. Here is the step-by-step principle:
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RIG-I in samples binds to plate-immobilized antibodies.
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Biotinylated dsRNA binds the captured RIG-I.
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Streptavidin-HRP binds to the biotin on the dsRNA.
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TMB substrate reacts with HRP to produce a colorimetric signal.
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Absorbance is read at 450 nm, proportional to RIG-I–dsRNA complex.
This procedure aligns with well-accepted laboratory techniques documented in OpenWetWare ELISA tutorials and NIH guidelines.
Application Areas
This ELISA kit can be applied in various experimental designs, such as:
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Screening RIG-I activators in RNA treatment assays
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Comparing dsRNA recognition across different cell types
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Studying RIG-I’s interaction with exogenous RNA mimetics
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Monitoring RIG-I engagement in co-transfection experiments
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Investigating RIG-I engagement with synthetic RNA or viral byproducts
Several university-led labs have used similar tools to investigate genetic variants of RIG-I in cell-based systems.
Data Reproducibility and Performance Metrics
This ELISA has been tested for:
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Intra-assay CV <10%
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Inter-assay CV <15%
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Sensitivity: ~35 pg/mL
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Detection range: 0.05 – 10 ng/mL
The assay’s specificity has been validated using CRISPR-Cas9–based RIG-I knockout models described by labs at UCSF and Johns Hopkins.
Reagents and Storage
Kit includes:
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Coated microplate (96-well)
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HRP-linked secondary antibody
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Biotin-labeled dsRNA ligand
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Blocking buffer and wash solutions
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TMB substrate
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Stop solution
All components are shipped on ice and stored at 2–8°C. The stability and storage conditions follow NIH reagent handling protocols for molecular kits.
Advantages Over Traditional Methods
| Method | Limitations | ELISA Kit Advantages |
|---|---|---|
| Western blot | Semi-quantitative, low throughput | Quantitative, scalable |
| qPCR | Measures RNA indirectly | Directly measures protein-RNA binding |
| Pull-down assays | Requires native complexes | ELISA works in crude lysates |
The ELISA format avoids radioactive probes and complex detection reagents, making it more accessible for small academic labs. The simplified protocol is compatible with instruments like those listed in NIH lab equipment databases.
Relevant Publications
Frequently Asked Questions
Can this kit be used with non-mammalian samples?
Yes, RIG-I homologs in zebrafish and frogs have been studied using adapted versions of this protocol (source).
Does the kit detect MDA5 or LGP2?
No, antibodies in this kit are specific to RIG-I. To detect MDA5, see reagents listed by BEI Resources.
Can this be used in BSL-2 labs?
Yes. All components are non-hazardous and approved for BSL-2 conditions as per CDC biosafety guidelines.
Summary
The RIG-I dsRNA ELISA Kit offers a robust, scalable platform to study protein–RNA interactions in non-pathogenic systems. It is ideal for quantitative screening, molecular analysis, and binding affinity experiments in the context of environmental RNA triggers and nucleic acid–based investigations.
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