Masood Kamali's projects – Proximity assays for proteome analyses and biomarker validation

Illustration of part of the PLA process
The reporter DNA oligonucleotides
are joined by enzymatic ligation
to form a PCR template for
quantitative amplification

Masood Kamali-Moghaddam, Radiosa Gallini, Liza Löv, Felipe Oliveira, Qiujin Shen, Agata Zieba

Using various proximity assays, specific proteins as well as their interactions and modifications, can be analyzed by translating detection reactions to reporting DNA sequences. In these methods protein-binding reagents are modified by conjugation to DNA oligonucleotides. When two or more of these modified binders recognize a target molecule or a pair of interacting proteins, the free ends of the attached oligonucleotides are brought in proximity and can subsequently be joined by DNA ligation. The ligation products are amplified by PCR enabling sensitive detection.

PLA in solution, on a solid phase or in situ

Illustration of the PLA process
Either on a solid support or in
solution the target molecule is
recognized by several binders
equipped with ssDNA molecules
that will subsequently be hybridized
to a connector oligonucleotide
upon multiple recognition events

The PLA technique can be carried out in solution – requiring very small amounts of materials to be tested – or on a solid phase whereby the target molecules to be detected can be first immobilized via affinity probes, while other materials are removed by washes.

In a yet another format of PLA (in situ PLA) that can be used for protein analyses in cells and tissues the oligonucleotides are designed to guide circularization of two accessory linear DNA molecules. The DNA molecules that form by ligation are amplifiable by rolling circle amplification and visualized using epi-fluorescence or confocal microscopes.

The combination of the use of two or more binding reagents and efficient DNA amplification provides high sensitivity and specificity of detection, surpassing conventional protein detection methods. PLA can therefore provide a powerful molecular tool for protein measurements at extremely low concentrations.

Further improvements of protein detection

We continuously improve methods for sensitive proteome analyses, aiming for further improved sensitivity of detection and for simultaneous detection of proteins in highly multiplexed formats. In addition to sensitive detection of soluble proteins, different variants of the technology have been used to establish assays for detection of immune complexes, aggregated proteins, fusion proteins and micro vesicles.

Using PLA to identify biomarkers of neurodegenerative disorders

In the field of neurodegenerative disorders we have developed a sensitive assay for specific detection of protein oligomers that plays a central role in diseases such as Alzheimer, prion and Parkinson diseases. Using in situ PLA, we have established extremely specific and sensitive assays to study protein interactions and posttranslational modifications such as phosphorylation of Tau protein, which plays a central role in development of Alzheimer’s disease.

In addition, we have developed a multiplex PLA in which up to 47 proteins are analyzed simultaneously using very small amount of patient samples. The multiplex PLA has, for instance, been used to screen blood samples from patients with chronic pain, and cerebrospinal fluid samples from patients with amyotrophic lateral sclerosis, and we have identified several biomarker candidates in the latter disease.

4PLA can be used to detect prostasomes in blood

We have also developed a version of PLA (4PLA) in which requirement of simultaneous binding of five different antibodies allows specific detection of more complex target molecules. Using this sensitive assay form we have for the first time been able to detect prostasomes in blood plasma – establishing these as a member of a new class of biomarkers generally referred to as microvesicles/exosomes.

4PLA-based detection of prostasomes revealed elevated levels of these microvesicles in samples from prostate cancer patients, and the analysis also demonstrated that the concentration of prostasomes better reflects disease aggressiveness than the currently used PSA test. 

Currently, we utilize multiplex proximity assays to identify and characterize a large number of classes of microvesicles originating from different organs – such as prostate, lung and breast – to establish tools for characterization of microvesicles in order to trace them to their originating cells/tissues. This proteomic characterization will also facilitate establishment of new sensitive and reliable diagnostic and prognostic tests using this novel class of biomarker candidates.

Flow cytometry-based PLA

Using multiplex in situ PLA we have established a unique method for multicolor, specific and sensitive detection of microvesicles via flow cytometry, which allows identification of different microvesicles originating from different organs and/or cells in complex matrices such as blood plasma.

The flow cytometry-based PLA has also been used to establish sensitive assays for detection of fusion proteins such as BCR-ABL in chronic myeloid leukemia. Since the detection is carried out in intact cells, the method allows simultaneous immunofluorescence staining in order to identify cell populations that are expressing the fusion protein.