March of Progress

45 years ago, when I was a master’s student, schistosomiasis was detected by immunodiagnosis: a patient’s antibody to the parasite was detected on frozen sections using a second, fluorescently-labeled antibody. Fluorescence rapidly faded and quantitation was based on brightness from – to +++, which was open to personal interpretation. I aimed to develop quantitative assays using “purified” antigens and, after a few years’ work, Bas Ploem and I implemented assays based on schistosome protein antigens coupled to agarose beads – the DASS system. A variation on this theme, this time in collaboration with Han Streefkerk, was our quantitative immunoperoxidase assay, marking the arrival of enzyme-linked immunosorbent assays (ELISAs). The perceived impact of these assays was not too high; in 1975, at a presentation that I gave on ELISA, a famous malariologist remarked, “Remember young man: her first name was Eliza, but her family name was Doolittle.” So much for the rapid acceptance of novel technologies…

As antibody detection in schistosomiasis reflects active infections poorly, if at all, I began to focus on detection of schistosome antigens in the circulation of the host. Initial assays were not sensitive, detecting only heavy experimental infections, and therefore not useful for human infection diagnosis. Over the years, we painstakingly transformed these assays into highly sensitive and specific tests by evaluating immunoassays, inspired by hybridoma technology’s ability to generate thousands of specific and reproducible monoclonal antibodies. Assays became more and more sensitive and we conducted large-scale immunoepidemiological studies, even demonstrating schistosome antigen in a 5,000 year-old Egyptian mummy. Today, a lateral flow assay based on upconverting phosphorescence labels can detect just one pair of worms in a urine sample.

To study the unusual characteristics of glycan moieties, Hans Kamerling, Hans Vliegenthart and I used nuclear magnetic resonance (NMR). This led to the study of antigen synthesis, biomolecular interactions (both surface plasmon resonance and fluorescence correlation spectroscopy), and crystallization studies on Lewis X-antibody interactions, but also to the purchase of our first mass spectrometer – an ion trap instrument – some ten years ago.

Working at a university medical center, I realized that there was a huge and largely unexploited potential for the application of mass spectrometry (MS) in clinical studies and, ultimately, in the clinic. I also realized that we must switch from our initial approach, which was targeted to particular antigens, parasite life-cycle stages, or defined cell populations, to less targeted discovery studies using multi-sample cohorts. With Rob Tollenaar, I began to develop reliable and robust MS-based assays to define serum profiles and enable detection of colorectal and breast cancer. Our interest and infrastructure quickly grew and the medical center now houses a special unit for research on (glyco)proteomics, metabolomics and MS imaging; we work in close collaboration with clinical colleagues, using a suite of MS systems and a dedicated NMR metabolic profiler platform. Conducting large-scale studies with thousands of samples has made us painfully aware of the importance of sample preparation standardization – something we have heavily invested in.

If I extrapolate my experiences as a medical parasitologist, it seems we are halfway up the slope – but with a long way to the summit. There is now a rapidly growing interest, that is matched (and initiated) by increasingly sophisticated MS instruments, in the quantitative measurement of multiple proteins or metabolites in patient body fluids.

While huge steps have already been made in this area, much work remains, such as the improvement of inter-laboratory reproducibility. And although specific algorithms for complex data analysis are rapidly becoming more powerful, the concept of (ultimate) multi-parameter measurement versus unimarker measurements still largely needs to be integrated into clinical diagnostic procedures. For the analysis of translational modifications and, in particular, glycosylation, the field is still wide open: despite significant progress, the analysis of (aberrant) plasma protein glycosylation is still largely unexplored.

It is interesting to see how, in conjunction with MS, immunoaffinity procedures are increasingly being used to highlight low abundant peptides and proteins. Likewise, I believe MS-analysis will significantly contribute to the further immunoassay optimization.