Proximity Ligation Assay

Proximity ligation assay (PLA) was developed and first described by Fedriksson and colleagues in 2002. PLA is a unique method in which single-stranded oligonucleotides are conjugated to affinity binders of proteins, followed by amplification of the signal by DNA polymerization and hybridization of complementary oligonucleotides labeled with fluorogenic or chromogenic readout.
PLA allows the detection and evaluation of individual proteins in diverse sample types and applications. PLA brings together the specificity of ELISA and sensitivity of PCR.

How PLA Works
PLA works via the simultaneous and close binding of affinity probe pairs onto a target protein that results in an amplifiable detection signal in the form of a DNA hybrid product:

  • The target protein is bound by two proximity probes. Consequently, the oligonucleotides are brought in proximity
  • A connector oligonucleotide can hybridize to both oligonucleotides, creating a template for ligation
  • Only the newly created DNA-molecule can now be amplified and read out by PCR
    PLA can use either monoclonal or polyclonal antibodies, as well as a combination of the two. For antibody/antigen interaction, there are two alternative approaches.

The method
Samples-PLA can be performed on many different samples including protein suspensions (e.g. cell lysates, conditioned media, blood serum or plasma) or fixed tissues (e.g. cell culture slides or tissue sections).
Affinity Probes– The probes that bind specifically to your protein of interest are usually either purified antibodies or specially designed DNA aptamers. They are capable of binding two different sites on the target antigen.
Ligation Arms- The short sequences of DNA attached to the affinity probes. Ligation arms can be directly attached to the affinity probe or indirectly using a secondary antibody with the ligation arm conjugated.
Connector Oligonucleotide & DNA Ligase– The connector oligo is a short sequence of DNA that hybridizes to the two ligation arms and guides their free ends together where they can ligate to each other to form a ligation product.
Amplify & Detect-After the amplification of your DNA ligation product, there are three possible detection methods for PLA.

Types of PLA

The homogenous PLA: Complete PLA is performed in the homogenous solution. The two proximity probes (3’ and 5’) are incubated with the target antigen and a connector oligonucleotide which can hybridize to both proximity probes if the probes bind to adjacent epitopes on the target antigen. After a ligation step a DNA molecule is generated, which can be amplified and detect real-time PCR amplification.

Solid phase PLA: Uses capture antibody to immobilize target protein onto a solid phase. The sample is combined with a capture antibody and this complex is then incubated with the proximity probes and thus the target antigen is sandwiched between the proximity probes and the capture antibody. After wash steps, the ligation and qPCR steps are carried out.

In situ proximity ligation assay: Uses rolling circle amplification (RCA) for the detection of individual proteins and protein-protein interaction in cell lines and tissues. Cell or tissues are fixed on a slide and proximity probes are added causing binding of two probes to the same protein complex in the sample. The addition of two connector oligonucleotides hybridizes oligonucleotides conjugated to the antibodies. Ligase is added to this complex causing ligation and this seals the gap to form a circular DNA molecule which is then amplified by an isothermal amplification method known as RCA. Amplification occurs and the amplified product is then detected through hybridization of fluorescence – labeled oligonucleotide complementary to a tag sequence in the RCA product.

Detection methods:
 qPCR Detection – in this method amplification and detection occurs simultaneously
 Fluorescence detection – in this method the amplification and detection steps are performed separately
 Blot detection – this method involves the attachment of separated proteins onto a solid-phase support by standard SDS-PAGE and Western blot methods

Advantages of PLA:

  • Fast, ultra-sensitive and highly convenient assay
  • Uses the specificity of ELISA & the sensitivity of PCR, hence a reliable assay
  • Can be developed for diagnosis of pathogens and proteins
  • Useful method for the validation of potential biomarkers for clinical diagnostic needs
  • PLA may help with the development of mutation – specific targeted therapies

Limitations of PLA:

  • Highly dependent upon the quality of the antibody used in the probe
  • Variation in the PLA results due to antibody performance variation from batch to batch
  • Background signal due to nonspecific ligation of oligonucleotides
  • Covalent conjugation of oligonucleotides to antibodies can be difficult and time consuming

To overcome the complexity of conjugating antibodies to oligonucleotides, Innova Biosciences offer Thunder-Link® PLUS, an easy-to-use kit that enables rapid conjugation of antibodies to oligonucleotides, with high recovery of materials and a superior clean-up procedure.
The kit is quick and simple to use, overcoming time consuming and lengthy protocols associated with standard conjugation methods.

  • Quick and easy to use – save time, no specialist knowledge required
  • High levels of antibody and oligo recovery – save precious reagents
  • 30 minutes activation, 60 minutes conjugation
  • Use your own oligo and antibody, at your desired ratio – flexible
  • Stringently QC tested – consistent high quality, excellent batch-to-batch reproducibility
  • Unidirectional chemistry – no risk of cross-linking
  • Covalent bond – highly stable conjugates Suitable for single-stranded oligos of 10-120 bases, double-stranded oligos up to 80 base pairs – covers the majority of sequences
  • Linking chemistry works at both 5’ and 3’ end – provides ability to combine with other modifications
  • Post-conjugation clean-up step – no interference from unbound oligo
  • Positive control antibody and oligo included – enables confirmation of protocol success
  • Wide range of target proteins – also applicable to antibody fragments and small proteins

 

Our Thunder-Link® PLUS oligo conjugation system enables antibody-oligo conjugates to be generated easily and efficiently and are applicable to assay formats, including proximity ligation assays, proximity extension assays, and electrochemical proximity assays.

 

Fig 1: Thunder-Link® PLUS process diagram

To find out more about this product please click here. We have an expert technical support team that are happy to help, contact us here.

Custom Services
Optimizing your antibody/oligonucleotide conjugates can be time consuming and expensive, especially when using traditional multi-step conjugation techniques. Our antibody/oligonucleotide micro-optimization is a unique service performed by our experienced scientists. We use unique conjugation that can enhance the performance of even the most temperamental of antibodies. To find out more, click here.

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