Skip to main content
To KTH's start page To KTH's start page

On the application and validation of multiplexed affinity assays

Time: Fri 2020-10-02 13.00

Location:, (English)

Subject area: Biotechnology

Doctoral student: Tea Dodig-Crnković , Affinity Proteomics, Science for Life Laboratory, SciLifeLab

Opponent: Dr Norman Leigh Anderson, SISCAPA Assay Technologies, Washington DC, USA

Supervisor: Universitetslektor Jochen M. Schwenk, Science for Life Laboratory, SciLifeLab, Affinity Proteomics; Mun-Gwan Hong, Science for Life Laboratory, SciLifeLab; Professor Peter Nilsson, Science for Life Laboratory, SciLifeLab, Affinity Proteomics

Export to calendar


Proteins are essential macromolecules that carry out complex functions in human cells, tissues, and organs. They regulate a diverse set of biological processes and protect against pathogens. However, dysregulation or malformation of proteins can cause disease. By characterizing proteins in health and disease, we can gain insights into disease aetiology and identify druggable targets to treat disorders. By bringing protein discoveries from the research lab into clinical practice, protein assays have been and will continue to be important tools for enabling and improving medical decision-making.

The work presented in this thesis concerns both exploratory and targeted affinity-based assays applied for the study of proteins. High-throughput and multiplexed suspension bead arrays have been the primary technology for measuring proteins with antibodies in samples such as human blood. Identification and validation of protein-protein interactions that may provide novel insights into the druggable proteome have also been carried out. Throughout the projects, methods for validating the observations have been pursued and include replication in independent sample sets, as well as the assessment of antibody selectivity via other proteomics assays or orthogonal methods such as genetic associations.

In Paper I, we used multiplexed exploratory antibody arrays comprising almost 1.500 affinity binders to study proteins that circulate in plasma. Here, the focus was to determine the longitudinal variability of proteins. We analysed samples from 101 clinically healthy individuals, collected each third month for one year. The protein data provided insights into inter-individual diversity and the unique molecular fingerprint of each participant. We found that 49% of the studied proteins were stable across one year, as these had low variability in each individual. Eight modules, each containing 11-242 proteins, were found to co-vary across one year. We also found genetic variations to influence 15 of the detected protein profiles and confirmed selected indications in an independent set of 3.000 subjects. In summary, we observed the existence of individual-specific protein profiles and found that short-term and continuous changes occurred in almost every participant.

In Paper II, we investigated blood-derived serum and plasma to identify age-associated proteins. We started from a large set of exploratory antibody bead arrays to screen 156 individuals aged 50-92 years. We found protein profiles of the histidine-rich glycoprotein (HRG) to be significantly associated with age. This association was further corroborated by the analysis of >4.000 individuals from eight additional and independent sets of blood samples. We further validated the HRG protein profiles by sandwich assays and protein microarrays developed in-house. Comparing genetic data and HRG profiles obtained by two independent antibodies, we observed strong but inverse associations to the genetic variants for two anti-HRG antibodies.

In Paper III, we applied multiplexed assays for the detection of autoantibodies against cancer-testis antigens (CTAs) in 133 non-small cell lung cancer (NSCLC) patients. We found reactivity against 29 unique CTAs exclusively in cases, compared to 57 matched controls with benign lung diseases. The presence of six CTAs was further confirmed in an independent set of 34 NSCLC cases. Analysis of longitudinal samples from seven patients demonstrated that the presence of CTA autoantibodies was stable over time for each of the individuals.

In Paper IV, we developed a novel multiplexed sandwich-immunoassay for the detection of interaction partners to G-protein coupled receptors (GPCRs). This pharmaceutically important family of membrane proteins is believed to be regulated by another group of receptor activity-modulating proteins (RAMPs) by the formation of protein complexes. We studied cell lysates expressing combinations of 23 GPCRs with three RAMPs. We confirmed most of the previously reported interaction pairs and additionally found evidence for 15 new GPCR-RAMP complexes. All interactions were validated using epitope tags that were engineered onto the proteins. Selected complexes were further validated by in situ proximity ligation assays performed in cell membranes.

In summary, the work included in this thesis describes the use of multiplexed affinity-based assays for research within plasma proteomics and the interrogation of protein complexes. The work highlights the method’s potential for the identification of circulating proteins that may aid and add to the current knowledge about human health and disease.