An Aptamer Based Assay for the Early Detection of Lung Cancer

Bob Bruce20th DEC 2011

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Introduction

What is an Aptamer?

Aptamers are oligonucleic acid or peptide molecules that bind to a specific target molecule

How are they selected? They have been engineered through a process known as SELEX (systematic

evolution of ligands by exponential enrichment)

What is SELEX?• The process begins with the synthesis of a very large oligonucleotide library of

randomly generated sequences of fixed length flanked by constant ends that serve as primers.

• The sequences in the library are exposed to the target ligand – such as a protein or a small organic compound - and those that do not bind the target are removed.

• The bound sequences are eluted and amplified by PCR to prepare for subsequent rounds of selection to identify the tightest-binding sequences

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Advantages of Aptamers over Antibodies in Diagnostic Assays

O’Sullivan CK. Aptasensors – The Future of Biosensing? Analytical and Bioanalytical Chemistry 2002: 372(1): 44-48.

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Outline of Apatmer Capture Assay

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More detailsB PCB

B+ +

B

B

-Removal of unbound protein-Biotin labeling of protein

B

B

BCleaving off of protein:aptamer complex

B

-Binding to a new strepavidin plate- Washing

B

Disruption of protein:aptamer interaction

L B

Luminex assaybead hybridizationProtein binding to biotinylated

aptamer followed by binding to streptavidin plate

ProteinsAptamers with photocleavable biotin

Streptavidin plate

Steps 1-12

Steps 13-21

Steps 22-29

Steps 30-41

Steps 46-66

Steps 42-45

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Areas where the Assay could be Improved?

• Currently the assay takes two days to complete. • One way to simplify it would be to recast it in a format similar to an ELISA. • Incubate the serum sample with a bead-antibody multiplex, followed by incubation

with biotinylated aptamers. • Another variant is to conjugate aptamers to beads and incubate with serum sample

followed by detection with a biotinylated polyclonal antibody capable of binding to all analytes in the assay.

• After incubation with SAPE, the assay could be read in the Luminex. • There are advantages to either strategy

– It would streamline the assay to less than a day

– Reduce the large variances that have been observed

– Aptamer-bead conjugates more consistent.

– Aptamers are valuable IP asset.

– Polyclonal antibody to multiple analytes would reduce inconsistencies that may arise from lot to lot variation of multiple antibodies.

• However, conjugation of aptamer to beads may affect aptamer binding to analyte.

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Experimental Details for Antibody Aptamer Hybrid Assay

● Two concentrations of antibody and aptamer were used.

● EGFR was incubated with beads conjugated to EGFR antibody for 2 hours at RT.● Biotinylated detection antibody or biotinylated aptamer was added and incubated for

1 hour at RT.● Plate was then incubated for 30 min. with SAPE.● Plate was read on Luminex with antibody detection read at normal PMT and aptamer

detection at high PMT.● Results demonstrated comparable performance between equimolar amounts of

antibody and aptamer.● Antibody samples displayed better linearity and better sensitivity at low concentrations

than aptamer samples.

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Results of Antibody Aptamer Hybrid Assay

Eqimolar concentrations of antibody and aptamer performed comparably

Aptamer assay showed:•Decreased sensitivity•Higher background•Increased variability

(ng/ml) EGFR Aptamer 0.33nM Avg Std. Dev CV EGFR Antibody 0.33nM Avg Std. Dev CV

40 8735 8795.5 6772.5 8101.00 1150.91 0.14 7117.5 7125 7186 7142.83 37.57 0.01

10 3404 4193 3779 3792.00 394.66 0.10 4254 4023 4307 4194.67 151.01 0.04

2.5 946 1328 1179 1151.00 192.53 0.17 1553.5 1451.5 1376.5 1460.50 88.84 0.06

0.625 396.5 387 330 371.17 35.97 0.10 365.5 393 489 415.83 64.84 0.16

0.156 169.5 176 177.5 174.33 4.25 0.02 155 124 145.5 141.50 15.88 0.11

0.039 131.5 180 122.5 144.67 30.93 0.21 42 42.5 49 44.50 3.91 0.09

0.0097 106.5 90.5 106 101.00 9.10 0.09 34 29 31 31.33 2.52 0.08

Blank 113 108.5 105.5 109.00 3.77 0.03 17.5 18 17.5 17.67 0.29 0.02

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Conclusions

• This is a promising assay that could lead to saving lives by detecting lung cancer in it’s early stages when it can be effectively treated.

• This assay utilizes aptamers which have practical advantages over antibodies in terms of synthesis, storage and modification with reporter molecules.

• They also have a sensitivity greater than that of antibodies. • However, in it’s current form the assay is not practical to use in a clinical setting. • To be a practical clinical assay, it must be run in a day with a low variance between

sample replicates. • Future development work will focus on

-Shorter assay time.

-Aptamer conjugations and titrations.

-Biotinylated polyclonal antibody to multiple analytes.

-Assay optimization. Buffers Incubation times and temperatures Washing.