Single Molecule Array Assay For Phosphorylated Alpha-synuclein Detection In Cerebrospinal Fluid
Currently, the diagnosis of Parkinson’s disease (PD) is a challenge given the variety and severity of symptoms that patients may experience. Additionally, the disease has overlapping symptoms and pathology with other neurodegenerative diseases. Thus, there is a great need for highly sensitive biomarkers for the early detection and monitoring of PD, which can lead to timely intervention and improved disease management.
There is a small protein called alpha-synuclein (α-syn) that has been associated with PD. In healthy brains, α-syn helps neurons to function optimally. In PD, α-syn abnormally aggregates and forms clumps within neurons called Lewy bodies (LB) and Lewy neurites (LN). These are a pathological hallmark of PD and are harmful to the brain because they can damage brain cells. A-syn is built-up by 140 amino acids and like all proteins, it is subject to modifications at different amino acids (residues). One of these modifications that is of particular interest for PD is called phosphorylation, which can occur at different residues (Figure 1). Phosphorylation can significantly affect the ability of a-syn to clump together into LB or LN.
Figure 1. Alpha-synuclein structure. Black dots represent phosphorylation sites. The green dot indicates Serine 129.
In patients with PD, the majority of α-syn in LB is phosphorylated at the 129th residue, which is the amino acid called serine (pS129-α-syn). Therefore, measuring pS129-α-syn in patient samples is of special interest. For this, researchers use a technique called enzyme-linked immunosorbent assay (ELISA), but this has not led to conclusive results yet, probably because of limited sensitivity of the method. Recent advances have led to the development of a new ultrasensitive type of ELISA called single molecule array (SIMOA), which can detect even the lowest levels of proteins in human samples with improved accuracy.
The SIMOA assay uses two antibodies: (1) a capture antibody to “catch” the target molecules from a biological fluid sample and (2) a detector antibody to detect the target molecules. This forms a complex with the target molecules sandwiched between the antibodies. As suggested by its name, the SIMOA bead technology makes use of tiny magnetic beads that are coated in capture antibody. When added to a sample, target molecules are caught by the capture antibody on the beads. A magnet is used to hold the beads in one place and any other molecules are washed away before the detector antibody is added. Together, the beads coated with capture antibody, the target molecules, and the detector antibody form what is referred to as an immunocomplex (Figure 2).
Figure 2. Formation of an immunocomplex. Courtesy of Quanterix.
There are several advantages of using SIMOA technology over traditional methods used to quantify proteins. Most importantly, the SIMOA allows a digital quantification, where the captured pS129-α-syn molecules are counted (hence the name single molecule). Traditional ELISA use color-changes which require a certain quantity of captured pS129-α-syn molecules before a signal is produced. Because of this, SIMOA can detect much lower levels of protein and thus requires smaller sample volumes for protein quantification. The other main advantage of the SIMOA is that it eliminates interferences from other substances present in the patient sample. Together, these improvements make the SIMOA an ultrasensitive detection method.
Many biofluids can be used to quantify proteins, including blood and saliva. For neurodegenerative diseases, cerebrospinal fluid (CSF) is most commonly used. CSF is located around the central nervous system (CNS), which includes the brain and spinal cord, and transports waste from the CNS to the blood. Because of how close it is to the brain, changes that occur in the brain due to disease can be detected in CSF too.
Therefore, we aim to develop and validate a SIMOA bead-based assay to detect pS129-α-syn in human cerebrospinal fluid (CSF).
To begin with, pairs of commercially available antibodies were tested in different combinations and orientations (i.e. testing antibodies for their ability to either catch or detect the target molecule) using an ELISA method. We evaluated pairs of antibodies using a range of parameters, including specificity for pS129-α-syn, dynamic range (the ratio between signals produced when there was no pS129-α-syn and the signals produced with varying concentrations of pS129-α-syn present in a sample), and the minimum and maximum concentrations of pS129-α-syn that each pair could detect and quantify. Five promising combinations of antibodies were then tested on the SIMOA SR-X platform (Quanterix), a semi-automated benchtop system to see whether they are suitable for ultrasensitive pS129-α-syn measurement. Once we selected the best antibody pair, we performed some optimization steps to fine-tune our assay. Reagent preparation, initial testing of antibody pairs on the SIMOA, and optimization were carried out according to the manufacturer’s protocols and recommendations. We then validated our in-house assay according to previously outlined parameters including parallelism, spike-recovery, and precision. These measures tell how reliable and precise an assay is.
In conclusion, SIMOA assays offer superior performance in terms of sensitivity, reproducibility, and elimination of matrix effects. We developed an in-house SIMOA assay for pS129-α-syn that is well-suited for pS129-α-syn analysis in CSF samples.
Camilla Christina Pedersen, BA, MSc, is a PhD student at the Centre for Movement Disorders, Stavanger University Hospital, Norway and the Department of Chemistry, Bioscience and Environmental Engineering, University of Stavanger, Norway. She presented her work as part of a guided poster tour at the WPC 2023 in Barcelona.
Ideas and opinions expressed in this post reflect that of the author solely. They do not necessarily reflect the opinions or positions of the World Parkinson Coalition®