On-Bead Assays

 

The Luminex®  Bead Ligand Binding Assay

This on-bead ligand binding assay enables the cost-effective, time-saving analysis of up to one hundred ligands in the same sample. A mixture of an infrared dye with a red dye is incorporated into polystyrene beads. The concentration ratio of both dyes is varied to produce beads which emitt a well-defined fluorescence spectrum upon excitation. Now each bead can be indentified by its unique colour, and can be coupled with a capture molecule specific for a ligand of interest, e.g. like a protein, peptide, or a transcript. The complex mixture of the beads is quantitatively analyzed in a FACS-like fluorescence-activated bead sorter.

Both the Luminex instrumentation and the software are fully compliant to Good Laboratory Practice (GLP) regulations.

Several application notes and publications demonstrate the universal adaptability of this ligand binding assay to the customer´s needs.

Santaris Pharma A/S is on top to bring the first microRNA based therapeutic into the clinic: a 15mer LNA/DNA chimeric antagomir targeting liver microRNA-122 for Hepatitis C treatment. In the course of a toxicology study in primates, scientists applied the Luminex multiplex bead assay to quantify chemokines and cytokines for immune response monitoring (1). Beads are coated with a primary capture molecule (like an antibody) to immobilize ligands (like chemokines or cytokines) from the liquid phase. A ligand-specific secondary antibody bearing biotins encloses the ligand like a sandwich, final readout is the fluorescence detection of phycoerythrin coupled to streptavidin.

Panomics Inc. uses the Luminex platform for on-bead RNA multiplex quantification. The branched DNA principle is applied (no RNA extraction, no target sequence amplification needed). Beads are coated with capture probes which can immobilize capture extenders from a liquid phase. Target RNAs hybridised to these capture extenders are detected with branched DNA probes bearing numerous biotin labels, final readout is the phycoerythrin fluorescence (2).

Total RNA can also be extracted from clinical specimen, followed by reverse transcription, on-bead qPCR, and fluorescence-activated bead sorting (3).

A recent publication applied the Luminex on-bead ligand binding assay to the detection of immune response to Staphylococcus aureus infection: distinct S. aureus antigens were coupled to the beads, and the amount of ligands (like IgG, IgA, and IgM) in the liquid phase (like serum) was quantified (4).

 

References:
(1) Lanford RE et al (2010), Science 327, 198-201.
(2) Zheng Z et al (2006), Clin Chem 52(7): 1294-302.
(3) Markou A et al (2011), Clin Chem 57(3): 421-30.
(4) Holtfreter S et al (2011), Eur J Clin Microbiol Infect Dis 30: 707-17.

Luminex® is a trademark of Luminex Corp.


 

Systematic Evolution of Ligands by Exponential Enrichment (SELEX)

A well known ligand binding assay to select nucleic acid ligands (DNA or RNA) from a library pool which best bind to a bead-immobilized target, e.g. like a peptide or protein. We almost forgot to add this interesting and also challenging bead assay to our ligand binding assay album. I came across this assay when I browsed through the recent aptamer publications list on Pubmed. There was a paper published by SomaLogic´s founder Larry Gold on the use of aptamers for proteomics capture assays.

Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is a potential method to select small nucleic acid molecules (small RNA or DNA) from a sequence pool to bind to a defined ligand with high affinity. Its basically an alternative to antibody generation. The result of both methods is quite the same: in both cases you receive a capture molecule to bind to a defined ligand both in vitro (for diagnostic purposes) and in vivo (for therapeutic applications by blocking the ligand´s biological activity). Small RNA or DNA molecules capable to bind to a ligand are termed as aptamers.

In case of the SELEX ligand binding assay, the experimental effort is tremendous, that is: 1. the in vitro synthesis of a random sequence pool for the enrichment of high affinity binders (= aptamers); 2. the in vitro synthesis of the target ligand (excellent purity is essential); 3. the selection of small RNA/DNA sequences most suitable to bind to the (immobilized) ligand (usually several successive selection rounds are required to enrich potential binders with the highest binding affinity); 4. and finally, the in vitro characterization of selected small RNA/DNA molecules in terms of sensitivity, selectivity, and binding affinity (say hello to the Biacore); which does not necessarily mean the most potential binders do work in the presence of a biological matrix like serum or plasma. The whole SELEX process unually takes more than a year.

In contrast, the raise of specific antibodies seems to have multiple advantages. Very recent innovations in the antibody technology (e.g. the ISAAC assay, see our paper of the month April 2011) speed up the laboratory effort (accompanied with less human resources and cost of goods).

Although nucleic acid therapeutics (as a well known chemical substance class) are of low toxicity, in most cases do not stimulate the immune system (but the Toll-like receptors), and in the meanwhile have well characterized PK and PD profiles in popular species, aptamers didn´t become fully accepted as a therapeutic agent. Principally because aptamers act like antibodies, and costs for antibody development dropped down in the last years through novel technologies and well developed production capacities, which you barely can tell for the aptamer technology. This is why Biotech companies like Archemix and Noxxon Pharma are still be mired in the aptamer drug business, keeping them in an awkward position. Even though antibodies always bare a risk for in vivo applications, remember the Tegenero/TGN1412  disaster in 2006 (1).

The superior toxicity profile of small RNA or small DNA aptamers is the unique advantage over the antibody technology. This is why we have always been followers of the aptamer approach.

The godfather of SELEX, SomaLogic´s founder Larry Gold, recently advanced the aptamer technology to multiplex biomarker detection in biological samples (2), another try to compete with already established technologies, e.g. like the Luminex® bead assay. Although the aptamer-based proteomics assay involves some cosmic brainchilds like the introduction of Glen Research´s photocleavable linker to nucleic acid synthesis flowcharts. The paper represents great work and reflects a strong background in current proteomics research. But as I said, unfortunately the aptamer-based proteomics research compete with already existing (and distributed) platforms, I´m afraid there is no chance to get in the market with such an assay flowchart outlined in Figure 7 on page 8.

 

References:
(1) Suntharalingam G et al (2006), N Engl J Med 355(10): 1018-28.
(2) Gold L et al (2010), PLoS ONE 5(12): e15004


 

Immuno qPCR Applications on Beads

The sensitive immuno qPCR assay was originally developed in microwell plates which allow for both the ligand immobilization and subsequent qPCR assay. However, hot off the press papers describe an interesting alternative, that is, the immobilization of antibody/ligand complexes on beads, utilizing the globular shape of nanoparticles for increased binding capacity.

Best results were obtained if the ligand (an mycotoxin) is initially captured by a monoclonal antibody in solution, followed by the immobilization of formed complexes (and single antibodies) to protein G covered magnetic beads (1). Another approach uses gold nanoparticles to capture the ligand (a virus core protein), followed by precipitation with magnetic beads covered with another ligand-specific antibody (2).

Please follow also our recent idea to advance this assay to immuno RT-qPCR format using a small RNA tag.

 

References:
(1) Babu D & Muriana PM (2011), J Microbiol Methods, doi:10.1016/j.mimet.2011.05.002
(2) Yin HQ et al (2011), J Virol Methods (2011), doi:10.1016/j.jviromet.2011.05.014

 

Immuno-Capture of Mitochondria by Magnetic Microbeads

Magnetic microbeads are also discovered to be a novel solid phase for the capture of complete and intact cell organelles, e.g. like mitochondria ligands. There are already kits available on the market which facilitate the isolation of intact mitochondria from human tissues. Magnetic microbeads are covered with monoclonal antibodies specific for human TOM22, the 22-kDa translocase of the outer mitochondrial membrane (1). After the binding of mitochondria ligands to the solid phase, the beads are washed, and mitochondria are eluted using a simple PBS/EDTA/BSA buffer (2).

The method was recently applied to study microRNA (miRNA) expression profiles in human mitochondria (3). The authors identified numerous microRNA (miRNA) in the mitochondria, with at least some of them were also found in the cytosol. It was speculated there might be an active import mechanism involved in the transmembrane shuttle of microRNA (miRNA) into the mitochondria (which still remains to be verified). In the light of mitochondria being also a target for disease treatment, this observation opens the opportunity to develop investigational new nucleic acid drugs (IND) like microRNA mimics, or microRNA antagomirs for effective treatment.

 

References:
(1) Yano M et al (2000), Mol Cell Biol 20(19): 7205-7213.
(2) Hornig-Do HT et al (2009), Anal Biochem 189: 1-5.
(3) Barrey E et al (2011), PLoS ONE 6(5): e20220.

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