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Aptamers are short single-stranded DNA (ssDNA), RNA or synthetic XNA molecules, which are used as a class of affinity binders recognizing target molecules with a very high affinity and specificity. The aim of this study was to generate and characterize ssDNA aptamers for the detection of Newcastle disease virus (NDV). These aptamers were selected using systematic evolution of ligands by exponential enrichment (SELEX) in combination with quantitative high-throughput DNA sequencing. After three rounds of selections, a highly enriched ssDNA pool was sequenced, and the results were analyzed using FASTAptamer Toolkit. Sequencing reads were sorted by copy numbers and clustered into groups, according to their sequence homology. Top aptameric sequences were used to develop a sandwich enzymatic linked aptamer assay (ELAA) for rapid and sensitive detection of NDV in farm samples. The selected aptamers have an affinity within the nanomolar range, and a high specificity with no cross-reactivity towards other avian viruses. Following optimization of the sandwich ELAA method, the results demonstrated that both selected aptamers Apt_NDV01 and Apt_NDV03 with dissociation constant values of 31 nM and 78.1 nM, respectively, showed the highest specificity and affinity for NDV detection. The ELAA results were verified by quantitative real-time PCR, demonstrating strong concordance, and showing outstanding accuracy for detection of NDV in field sample. In summary, combination of SELEX with high-throughput DNA sequencing allowed rapid screening and selection of aptamers. The selected aptamers allowed recognition of NDV with high affinities. This is the first report that uses a validated sandwich ELAA for rapid and specific detection of NDV in poultry samples.

The diagnosis of malignant melanoma currently relies on clinical inspection of the skin surface and on the histopathological status of the excised tumor. The serum marker S100B is used for prognostic estimates at later stages of the disease, but analyses are marred by false positives and inadequate sensitivity in predicting relapsing disorder.To investigate SOX10 as a potential biomarker for melanoma and vitiligo.In this study we have applied proximity ligation assay (PLA) to detect the transcription factor SOX10 as a possible serum marker for melanoma. We studied a cohort of 110 melanoma patients. We further investigated a second cohort of 85 patients with vitiligo, which is a disease that also affects melanocytes.The specificity of the SOX10 assay in serum was high, with only 1% of healthy blood donors being positive. In contrast, elevated serum SOX10 was found with high frequency among vitiligo and melanoma patients. In patients with metastases, lack of SOX10 detection was associated with treatment benefit. In two responding patients, a change from SOX10 positivity to undetectable levels was seen before the response was evident clinically.We show for the first time that SOX10 represents a promising new serum melanoma marker for detection of early stage disease, complementing the established S100B marker. Our findings imply that SOX10 can be used to monitor responses to treatment and to assess if the treatment is of benefit at stages earlier than what is possible radiologically.

The objective of this study was to develop and apply a novel multiplex panel of solid-phase proximity ligation assays (SP-PLA) requiring only 20 μL of samples, as a tool for discovering protein biomarkers for neurological disease and treatment thereof in cerebrospinal fluid (CSF). We applied the SP-PLA to samples from two sets of patients with poorly understood nervous system pathologies amyotrophic lateral sclerosis (ALS) and neuropathic pain, where patients were treated with spinal cord stimulation (SCS). Forty-seven inflammatory and neurotrophic proteins were measured in samples from 20 ALS patients and 15 neuropathic pain patients, and compared to normal concentrations in CSF from control individuals. Nineteen of the 47 proteins were detectable in more than 95% of the 72 controls. None of the 21 proteins detectable in CSF from neuropathic pain patients were significantly altered by SCS. The levels of the three proteins, follistatin, interleukin-1 alpha, and kallikrein-5 were all significantly reduced in the ALS group compared to age-matched controls. These results demonstrate the utility of purpose designed multiplex SP-PLA panels in CSF biomarker research for understanding neuropathological and neurotherapeutic mechanisms. The protein changes found in the CSF of ALS patients may be of diagnostic interest.

The quality of DNA-labeled affinity probes is critical in DNA-assisted protein analyses, such as proximity ligation and extension assays, immuno-PCR, and immuno-rolling circle amplification reactions. Efficient, high-performance methods are therefore required for isolation of pure conjugates from reactions where DNA strands have been coupled to antibodies or recombinant affinity reagents. Here we describe a universal, scalable approach for preparing high-quality oligonucleotide-protein conjugates by sequentially removing any unconjugated affinity reagents and remaining free oligonucleotides from conjugation reactions. We applied the approach to generate high-quality probes using either antibodies or recombinant affinity reagents. The purified high-grade probes were used in proximity ligation assays in solution and in situ, demonstrating both augmented assay sensitivity and improved signal-to-noise ratios.

Despite intense interest, methods that provide enhanced sensitivity and specificity in parallel measurements of candidate protein biomarkers in numerous samples have been lacking. We present herein a multiplex proximity ligation assay with readout via realtime PCR or DNA sequencing (ProteinSeq). We demonstrate improved sensitivity over conventional sandwich assays for simultaneous analysis of sets of 35 proteins in 5 µl of blood plasma. Importantly, we observe a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that higher levels of multiplexing are possible. We used ProteinSeq to analyze proteins in plasma samples from cardiovascular disease (CVD) patient cohorts and matched controls. Three proteins, namely P-selectin, Cystatin-B and Kallikrein-6, were identified as putative diagnostic biomarkers for CVD. The latter two have not been previously reported in the literature and their potential roles must be validated in larger patient cohorts. We conclude that ProteinSeq is promising for screening large numbers of proteins and samples while the technology can provide a much-needed platform for validation of diagnostic markers in biobank samples and in clinical use.