Anti-Oligonucleotide Antibodies



Anti-Sense Oligonucleotide Antibodies


ASO Therapy Diagram 
Figure 1. Antisense oligonucleotide mechanism
Anti-sense oligonucleotide antibodies developed by Rockland Immunochemicals, Inc. are useful to support characterization studies of Anti-Sense Oligonucleotides (ASO) also known as anti-sense drugs. Anti-oligonucleotide antibodies exhibit sequence or modification-specific binding properties and are intended for applications including ELISA

ASO are short fragments of nucleic acid complementary to a specific mRNA, or other types of nucleic acids, that can impede the expression of a gene, for instance by inhibiting transcription and translation of the corresponding protein. The development of ASO technologies as therapeutic agents has led to the first regulatory approval of an anti-sense oligonucleotide as a therapeutic. Consequently, several clinical trials are currently underway for many other oligonucleotides as anti-sense drugs proposed to treat a variety of diseases including cancer, HIV/AIDS Duchenne muscular dystrophy and Cytomegalovirus retinitis.

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Types of Nucleic Acids Used for Antibody Development


Rockland has experience successfully working with diverse nucleic acids chemical structures for antibody development. Rockland works closely with clients to properly understand the biochemical properties of the target nucleic acids and subsequently design appropriate antibody generation strategies. 

  • Conventional oligonucleotides (12-15 bases)
  • DNA-RNA hybrids
  • PS and PdiS modified backbone ASO
  • Single stranded oligonucleotide mixtures (cocktails)
 
  • Single stranded RNA (ssRNA)
  • Double stranded RNA (dsRNA)
  • Nucleotides
  • Nucleosides
 



ASO Antibody Development


Starting with a target nucleic acid molecule and the desired application for the intended antibody, Rockland’s scientists can design and conduct all the necessary steps for the generation of sequence specific or conformation specific anti-ASO antibodies. Rockland has a demonstrated history of generating antibodies to ASO that exhibit high specificity and sensitivity. Antibodies can be developed as polyclonal, conventional monoclonal or recombinant monoclonal formats. The choice of clonality is dependent on client specific needs and timelines of the client.

For polyclonal antibodies (pAb) Rockland will recommend a host, immunization protocols and testing strategies to generate a volume of antiserum that satisfies the client’s reagent needs. Additionally, we perform downstream processing to purify antibody, enhance specificity by cross adsorption (if needed), and validate antibody performance by pre-determined criteria.

For conventional monoclonal antibody (mAb) and recombinant monoclonal antibodies (rAb), recommendations are made for generation of clonal cell lines or clonal selection, respectively, resulting in antibody reagents with the desired performance characteristics including sensitivity, specificity and affinity.

Oligo Timeline

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Antibodies Recognize Specific ASO Modifications


Like many other technologies anti sense oligo nucleotide design has evolved over time to include several generations of development intended to improve in vivo distribution, availability, delivery and for the ability of the ASO to suppress the target gene. Notably, second and third generation ASOs are characterized by chemical modification of backbones that improve the pharmacological properties of these compounds (see figure below). The phosphorothioate (PS) backbone (also referred to as thiophosphate backbone) was one of the first modifications incorporated into ASO technology. This modification, the replacement of a non-bridging oxygen atom with a sulfur atom in the phosphate backbone, is commonly used in anti-sense technology.

Oligo 2a
Figure 2a. Unmodified phosphodiester (PO) backbone 1


Advantages of designing an ASO with a phosphorothioate backbone include that the modified backbone provides resistance against nuclease degradation, favors trafficking and enhances uptake. However, data has shown that this modification alone is not sufficient to guarantee clinical efficacy and therefore nucleotides with 5’-phosphate stabilization, sugar modifications and conformational constraints have been further developed to include:

Oligo 2b   Oligo 2c 2
 Figure 2b. Phosphorothioates (PS)    Figure 2c. Phosphorodithioates (PdiS)


Methylation of the 2’ hydroxyl group on the ribose moiety of either PO, PS or PdiS forms of the backbone can also utilized in the design of the immunogen. This is reported to result in enhanced stability to various nucleases. For more information on chemical modification to the oligonucleotide backbone see Evers et al2.

Oligo 2d
Figure 2d. Additional sugar-phosphate backbone modifications.


Additional modifications include fluoro, amino, and thio substitutions (see Vorobyeva et al.3 for more information).



Sequence Specific Antibodies to Oligonucleotides Support ASO Studies


antibody dnaSeveral properties of ASO, including pharmacokinetics, pharmacodynamics and tissue specificity, can be conveniently studied using customized antibodies developed to exhibit high specificity and sensitivity for the intended nucleic acid sequence. Rockland’s antibody development capabilities include our proven ability to generate critical antibody reagents for the preclinical detection and characterization of specific ASO under consideration as drug candidates. Such antibodies exhibit sequence or modification-specific binding properties depending on the intended use and are suitable for standard applications including ELISA and immunohistochemistry (IHC).




Conjugation of ASO and Other Nucleic Acids to Carrier Proteins


Nucleic acids by themselves are notoriously poor immunogens and are difficult to use unconjugated as the analyte in immunoassays. However, by utilizing optimized methods for conjugation developed at Rockland these limitations can be overcome with great success. Rockland’s nucleic acid to protein (e.g. carrier protein or antibody) conjugation technology provides for efficient conjugation of ASO and other DNA or RNA oligonucleotides to immunogenic carriers. These oligonucleotide:carrier conjugates are effective when applied to the generation and purification of antibodies that specifically recognize nucleic acid-derived molecules. These oligonucleotide:carrier conjugates may also be used in screening assays during development and clonal selection.

Rockland utilizes multiple conjugation methods resulting in efficient presentation of the oligonucleotide to the host immune system. Proper presentation results in antibodies that specifically recognize sequence or backbone specific nucleic acids (e.g. phosphorothioate) and/or compositions (i.e. RNA-DNA hybrids).


SYBR

Figure 3. Electrophoretic mobility shift assay (EMSA) showing fluorescence staining for DNA (left) and protein (right) of agarose gels loaded with ASO-protein conjugate (lane 8) and ASO only (lane 11) at equivalent amounts. Protein conjugated ASO co-localize with both stains and is retained in the well while unbound oligo can be seen migrating to the bottom of the gel (red circles).




ASO Antibody Characterization and Validation


The specificity and sensitivity of ASO antibodies produced at Rockland can be characterized by a variety of immunoassays including ELISA, Western Blot and/or IHC. When producing fit-for-purpose antibodies, the performance of the antibody in the assay for the intended use of the antibody is considered when antibody development and immunogen design are planned. Below by example data is shown for ELISA of antisera screening during generation of anti-ASO antibody. Pre-immune serum (data shown) and non-specific anti-oligonucleotide antibody control (data not shown) showed no response.


ASO Antisera titration3

Figure 4. Antisera titration from rabbits immunized with ASO. ELISA plates were coated with carrier-conjugated ASO, blocked with ELISA Microwell Blocking Buffer with Stabilizer (Azide and Mercury Free) - MB-064-0100 and antisera to ASO were applied at various logarithmic serial dilutions along with pre-immune serum. ASO specific antibodies were detected with Anti-GOAT IgG (H&L) (RABBIT) Antibody Peroxidase Conjugated - 605-4302 secondary antibody followed by the addition of TMB ELISA PEROXIDASE SUBSTRATE - TMBE-100.


Final antibody validation is performed according to mutually agreed upon conditions and is immunoassay specific in design. Typically ELISA, immunohistochemistry and western blotting are validation assays used to determine the specificity, sensitivity and reproducibility of anti-sense oligonucleotide antibodies.




Related Products and CRO, CMO and RMS Services


Rockland delivers products, services and solutions to the research, diagnostic and therapeutic sectors of the life science community. Catalog reagents are used in academic, government, private, biotechnology and pharmaceutical research laboratories. Contract Research Organization (CRO), Contract Manufacturing Organization (CMO), and Raw Material Supplier (RMS) services are also provided by Rockland Immunochemicals, Inc. as a solutions provider:

   


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1 "File:Phosphodiester Bond Diagram.Svg - Wikimedia Commons". Commons.Wikimedia.Org, 2018, https://commons.wikimedia.org/wiki/File:Phosphodiester_Bond_Diagram.svg. Accessed 8 Mar 2018.

2 Evers, Melvin M. et al. "Antisense Oligonucleotides In Therapy For Neurodegenerative Disorders". Advanced Drug Delivery Reviews, vol 87, 2015, pp. 90-103. Elsevier BV, doi:10.1016/j.addr.2015.03.008.

3 Vorobyeva, Maria et al. "Key Aspects Of Nucleic Acid Library Design For In Vitro Selection". International Journal Of Molecular Sciences, vol 19, no. 2, 2018, p. 470. MDPI AG, doi:10.3390/ijms19020470.

  TrueBlot IP/Western Blot Mouse Rabbit Goat


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