Proximity Ligation qPCR Immunoassay

 

Another interesting ligand binding assay which allows for the real-time quantification of protein or peptide ligands is the antibody-based proximity ligation immunoassay. Invitrogen already makets some kits for Applied Biosystem´s Real-Time qPCR 5´-exonuclease platform (be aware only the company´s names are different, not the company itself). IP and trademarks with regards to the proximity ligation immunoassay are owned by Olink Bioscience AB, Sweden.

This immunoassay originally was designed as a classic liquid phase ligand binding assay, which comprises two monoclonal antibodies recognizing different epitopes of the target ligand (1), generally of the protein or peptide type. Both antibodies bear different DNA oligonucleotides, which are brought – upon binding to the target ligand – to close proximity to each other. If a complementary DNA oligonucleotide bridge is added to the antibody/ligand complex, a double-stranded DNA ligase can covalently link the two oligonucleotide tags. The ligation product afterwards can easily be detected in a real-time 5´-exonuclease assay (qPCR assay).

The proximity ligation immunoassay has recently been applied to the detection of proteins in situ (2, 3, 4), or to study protein/protein interactions (5). The latter one was advanced to the early diagnosis of prostate cancer from plasma samples (6).

It remains fascinating which of the two immuno qPCR variants comes off as the winner in the “best-sensitivity-to-lowest-cost-of-goods-ratio” challenge. Both assays are available in the liquid phase and the solid phase immunoassay format. Both inventors (and distributors) claim to have superior immunoassay sensitivity, but to our knowledge so far, there is no published study which compares both assay principles face-to-face for the same target ligand. Also, both immunoassays are of the sandwich type, meaning that there are two antibodies involved in the ligand binding events (ah, binding to different epitopes anyhow). Both immuno qPCR assays come in a single-tube format, with the only difference the ligation-based immuno qPCR assay can be performed in commonly used 96-microwell optical plates (the other one uses flat-bottom plates).

Well, as long as we don´t check it out for our own, and without having the possibility to compare to each other, I would say, the proximity ligation immunoassay MUST have an increased sensitivity, because only ligation products will be detected in the subsequent real-time qPCR assay. Furthermore, the proximity ligation immunoassay goes without intermediate washing steps of the plate, because unbound antibodies, or even unligated oligo tags should not interfere with the immunoassay, but may be responsible for the excellent assay sensitivity. On the other hand, it is well known that 5´-phospates (necessary for the ligation) are not stable in biological liquids; the ligation product yield also depends on the hybridization effeciency of the bridging oligo, and of the DNA ligase activity as well.

Above all, the proximity ligation assay makes use of two distinct monoclonal antibodies, which often are not available for the same target ligand. And if so, it does not necessarily mean the DNA tags of both antibodies are in proximity to each other to enable the ligation reaction. It is obvious that small molecules are not accessible to the proximity ligation immunoassay (but also not for the immuno qPCR assay, I guess).
And finally, the proximity ligation assay seems not to be the right tool to investigate immunogenicity of investigational new drugs (IND) like peptides, proteins, nucleic acids, and alike. Antibody detection assays (ADA) usually are applied to immobilized antigen. For the immuno qPCR assay, e.g. only one DNA-tagged anti-IgG antibody allows for positive readout. I already mentioned the proximity ligation assay doesn´t get along with only one antibody.

As I said, it remains exciting.

See also our post on how the immuno qPCR assay can be of increased sensitivity by using a RNA tag instead of DNA.

References:
(1) Gullberg M et al (2004), Proc Natl Acad Sci USA 101(22): 8420-4.
(2) Söderberg O et al (2006), Nat Methods 3: 995-1000.
(3) Gullberg M & Andersson AC (2009), Olink´s advertising Nature Methods feature
(4) Leuchowius KJ et al (2911), Curr Protoc Cytom, Chapter 9, Unit 9.36
(5) Laulier C et al (2011), Cancer Res 71(10): 3590-602.
(6) Tavoosidana G et al (2911), Proc Natl Acad Sci USA 108(21): 8809-14.

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