Just Published in ScienceAdvances!

We’re proud to share that Hzymes' dsRNA ELISA Kit played a supporting role in a recent breakthrough study on ligand-free mRNA purification.

The paper — titled

“The dsRNA content in feed and eluate samples was quantified using a dsRNA ELISA kit (Hzymes Biotech Co. Ltd, Wuhan, China).”

Product Highlights

High Sensitivity | Limit of detection as low as 0.001pg/ul; Limit of quantification as low as 0.01pg/μl

High Specificity | Specific to dsRNA with no affinity to other nucleic acid fragments (dsDNA, ssDNA, ssRNA)

User friendly and labor free | Ready-to-use with pre-coating plates

High Versatility | Four quantitative standards ensure accurate quantification in different applications (UTPN1-Me-PUTP, PUTP, 5-OMe-UTP)

High Accuracy | Identify dsRNA structure > 60bp in sequence-in-dependent patterns

Kit Performance

Stable, Accurate, Sensitive: Hzymes has something to say about dsRNA quantitative detection.

dsRNA is one key mandatory inspection item for quality control of mRNA drug substance (DS).

mRNA vaccine delivers molecules of anigen-encoding mRNA into immune cells, which use the designed mRNA as a blueprint to build foreign protein that would normally be produced by a pathogen (such as a virus) or by a cancer cell. The mRNA is delivered by a co-formulation of the RNA encapsulated in lipid nanoparticles that protect the RNA strands and help their absorption into the cells ^[1-2].

The production of dsRNA byproducts has been suggested to occur predominantly through two distinct mechanisms ^[3].

ELISA method is more suitable for dsRNA quantitative detection.

In the USP "Analytical Procedures for mRNA Vaccine Quality (Draft Guidelines)" 2nd Edition, the recommended dsRNA detection methods are dot blot and ELISA.

Taking into account the current four dsRNA detection strategies (Table 1), developing a dsRNA ELISA assay kit that combines specificity, sensitivity, and versatility would better meet the requirements of large-scale production and technological advancements in the mRNA field.

Table 1. Comparison of four dsRNA detection methods

Hzymes developed the dsRNA ELISA kit, which is stable, accurate and sensitive

During the development of a dsRNA ELlSA quantitative assay kit, the first challenge lies in the selection, preparation, and quantification of standards. While some ELlSA kits use poly(C) as a standard material, the antibody's recognition response differs between poly(C) and dsRNA (figure 1) ^[4], leading to inaccurate quantification of dsRNA in sample. Therefore, it is essential to prepare dsRNA standards (Figure 2) that are randomly sequenced. highly pure （Figure 3). and accurately quantified for reliable dsRNA quantification ^[5].

Figure 1. dsRNA antibodies' different recognition response between L-dsRNA (poly(A) poly(U) and poly(l) poly(C)).

Figure 2. Confirmation of Hzymes dsRNA standards by S1 nuclease and RNaselll digestion

Figure 3. Agarose gel electrophoresis and Capillary electrophoresis (CE) analysis of Hzymes dsRNA standards

Apart from dsRNA, the mRNA transcript itself can also result in immunogenicity. The body recognizes mRNA as foreign material and attempts to clear it ^[6], impacting mRNA stability and efficacy. The introduction of chemically modified nucleotides can effectively reduce mRNA auto-immunogenicity ^[7], as highlighted by the Nobel Prize in Physiology or Medicine awarded to Katalin Karikó (left) and Drew Weissman (right) in 2023. TLR7 and TLR8 binding in the GU-rich region of mRNA underscores the importance of modifying uridine residues with compounds like pUTP, N1-Me-pUTP, and 5-OMe-UTP to inhibit pattern recognition receptors (PRRs) from recognizing mRNA ^[8-9], Different types of modifications on dsRNA lead to significant differences in antibody recognition (Figure 4) emphasizing the need for matching standards of the same modification type to ensure accurate quantification.

Figure: 2023 Nobel Prize in Physiology or Medicine awarded to Katalin Karikó (left) and Drew Weissman (right)

Figure 4. Different antibody recognition towards different modified dsRNA by Hzymes dsRNA ELISA kit (left) and other dsRNA ELISA kit (right)

While antibody recognition of dsRNA is influenced by the sequence and nucleotide composition to some extent, optimization of antibody selection and concentration combinations can minimize the impact of these factors on measurements (figure 5). This optimization ensures that measurements are minimally affected by dsRNA sequence and length variations.

Figure 5. Antibody selected by Hzymes recognition of dsRNA with different sequence and length.

Based on the prepared dsRNA standards and selected antibodies, Hzymes developed the dsRNA ELlSA detection kit. The assay kit demonstrates specific recognition of dsRNA with no cross-reactivity towards ssRNA, dsDNA, or ssDNA (Figure 6). It exhibits excellent stability, robustness against interference (Figures 7-9) and overall superior performance (Table 2) Provided with pre-coated plates, this kit offers a convenient, accurate, and stable tool for quantifying dsRNA, providing you with are liable dsRNA quantification solution.

Figure 6. Hzymes dsRNA ELISA kit recognizes dsRNA specifically

Figure 7. Stability of standard curve measurements of Hzymes dsRNA ELISA kit

Figure 8. Stability of sample measurements by Hzymes dsRNA ELISA kit

Figure 9. Anti-interference performance of Hzymes dsRNA ELSA kit

Table 2. Summary of Hzymes dsRNA ELISA kit (Cat No. HBP003800-01) performance

References

[1] Kowalski Ps, Rudra A, Miao L, Anderson DG. Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery. Mol Ther. 2019 Apr 10:27(4):710-728.

[2] Rein Verbeke. ne Lentacker, Stefaan C, De Smedt, Heleen Dewitte, Three decades of messenger RNA vaccine development, Nano. Today, Volume 28, 2019, 100766, ISSN 1748-0132.

[3l Bijoyita Roy. Monica Z, Wu, Understanding and Overcoming the lmmune Response from synthetic mRNAs. Genetic Engineering & Biotechnology News. 2019 Dec 19; 39(12):56-58.

[4] Schonborn J, Oberstrass J, Breyel E, Tittgen J, Schumacher J, Lukacs N. Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts, Nucleic Acids Res, 1991 Jun 11:19(11):2993-3000.

[5] Nwokeoji AO, Kilby Pl, Portwood DE, Dickman M. Accurate Quantification of Nucleic Acids Using Hyperchromicity Measurements in Conjunction with UV Spectrophotometry, Anal Chem, 2017 Dec 19:89(24):13567-13574.

[6] Chaudhary, N., Weissman, D. & Whitehead, K.A. mRNA vaccines for infectious diseases: principles, delivery and clinical translation. Nat Rev Drug Discov. 2021.20,817-838.

[7] Mu X, Greenwald E, Ahmad s, Hur s. An origin of the immunogenicity of in vitro transcribed RNA. Nucleic Acids Res. 2018 Jun 1:46(10):5239-5249.

[8] Kariko K, Muramatsu H, Welsh FA, Ludwig J, Kato H, Akira s, Weissman D. lncorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol Ther. 2008 Nov;16(11):1833-40.

[9] Vaidyanathan S, Azizian KT, Haque AKA, Henderson JM, Hendel A, Shore S, Antony JS, Hogrefe Rl, Kormann SD, Porteus MH, McCaffrey AP, Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce lmmunogenicity without HPLC Purification. Mol Ther Nucleic Acids.2018 Sep 7:12:530-542.

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