sRAGE project

Before reading

There are a few terms that I would like to explain to you before you start reading the text below:
–antibodies: antibodies are proteins that are used by our immune system to specifically bind harmful substances, such as bacterial cells and viruses, after which these can be neutralized an destroyed. Based on their capability to only bind a specific target substance and to not bind to the thousands of substances that are also present in our bodies, antibodies are being used in medical sciences, for example, for detecting and enriching specific protein targets of interest.
–biomarkers: biomarkers are measurable parameters that, for instance, can be used to tell whether you are healthy, sick, or whether a treatment is effective (or not). Think about tests that measure glucose in urine to diagnose diabetes or the well-known pregnancy test that measures the hormone ‘HCG’ in urine. For these examples, glucose in urine represents a biomarker for diabetes and HCG in urine represents a biomarker for pregnancy.
–mass spectrometry (MS): mass spectrometry is an analytical technique that can be used to detect various compounds such as drugs, lipids, and proteins. MS instruments, so-called mass spectrometers, separate these compounds on the basis of their mass (weight) and thereby can detect multiple compounds at the same time. Basically, mass spectrometers are very sensitive balances which you can use to simultaneously measure several compounds at a molecular level.

My soluble Receptor for Advanced Glycation End-products (sRAGE) project is carried out under the umbrella of the Dutch Biomarker Development Center (BDC) which is a multi-center research consortium that aims at translating promising biomarkers to actual clinical tests. In this project we wanted to focus on the most promising biomarker for the disease ‘chronic obstructive pulmonary disease’, which is a chronic lung disease and the current cause of death worldwide number 3. There are no disease-specific biomarkers for this disease available in hospitals yet, and many researchers and doctors believe that sRAGE may become the first one.

Quite some clinical research has been done in the past years on the sRAGE protein, and corresponding projects were all carried out using the same commercial sRAGE tests. There were, however, some rumors going around that the reliability of these tests was insufficient. These rumors inspired us to develop a novel method for measuring sRAGE in human blood for which we wanted to employ mass spectrometry as detection technique. Unlike the techniques that are mostly used in medical laboratories, mass spectrometry measures a target of interest directly hence we know for sure that what we measure is sRAGE. The mostly-used methods, however, have an indirect detection principle and typically measure an electric current or the formation of a color which is proposed to reflect the presence of a target of interest in a sample. To this regard, it should be noted that neither one of these detection principles is better than the other. Corresponding techniques should, in turn, be considered as being complementary, which means that they are different from each other but make a good combination.

The main challenge in my sRAGE project was the fact that sRAGE is present in very small amounts in our blood. To illustrate this challenge, the protein called ‘albumin’ which represents around 50% of all proteins in our blood, outnumbers sRAGE by a factor of 10 million (!). Measuring sRAGE directly without first removing highly abundant proteins like albumin is simply impossible, even with the most advanced analytical techniques that are available these days. We therefore developed a method for fishing sRAGE out of blood before detecting it by mass spectrometry. Well, we developed three of them:

• We initially started using antibodies to enrich sRAGE from blood. In order to do so, we needed to bind antibodies to a ‘support’, just like you need to attach a hook to a line when you go fishing. Magnetic beads are the mostly-used supports in our field of research which, in combination with a magnet, allow for very easy enrichment of protein targets from sample materials like blood or urine. We thus developed and optimized a magnetic beads-based method for sRAGE, but this method was, to put it simply, too expensive. Beads are already quite expensive but we also needed to use high amounts of antibodies which are far more expensive than beads. In an attempt to come up with a cheaper alternative, we recalled that antibodies passively bind to some plastic surfaces, for example to those of certain sample plates that are both cheap and present in every medical laboratory. We starting to test these plates in combination with sRAGE-binding antibodies accordingly, and we soon discovered that these plates were good alternatives to magnetic beads. We also discovered that we needed ten times less antibody per sample as compared to our optimized beads-based method, hence we stopped using magnetic beads and switched completely to plastic plates.

• Antibodies are considered to be the workhorses of (bio)medical experiments, but these proteins do have some limitations. For example, antibody production is an expensive and complex process, and antibody manufacturers have difficulties to guarantee a consistent quality of these type of proteins. Several researchers tried to develop robust alternatives in consequence, which resulted in quite a number of innovative antibody alternatives. For our sRAGE project, we started collaborating with the company Avacta, a spin-off company from the University of Leeds (West Yorkshire, England), with the aim of developing alternative binders, so-called ‘affimers’, for sRAGE. This collaboration led to a set of sRAGE-specific affimers which we could use as alternatives to antibodies in the plastic plate-based method that is mentioned above.
• sRAGE is a peculiar protein as it holds a strong negative charge on one side of the protein and a strong positive charge on the other side of the protein. Negative charges are quite common among proteins, but strong positive charges are not. We thus wanted to investigate whether we could make use of sRAGE’s peculiar charge distribution, and we tried to enrich this protein by ‘ion exchange’. This may sound fancy, but ion exchange is nothing more than a technique for binding negatively charged substances with a positively charged surface or, conversely, binding positively charged substances with a negatively charged surface. Opposites attract when it comes to charges. Fortunately, we could pull it off as we developed a third method for enriching sRAGE from blood.

All three methods were thoroughly tested following regulatory guidelines, and we could prove that all three of them meet with the quality requirements relevant for analytical techniques to be used for clinical purposes. We also compared them with each other and with the commercial sRAGE tests which had been used for all clinical studies on sRAGE thus far. The latter comparison in particular revealed some remarkable differences. The sRAGE levels reported by the commercial tests were 2 to 4 times lower than those reported by our methods. We were actually able to find a potential explanation for this difference, which likely arose from a too low amount of antibody that is used by these commercial tests. To this regard, the manufacturer in question is currently working on an improved method for measuring sRAGE, and thus can use our findings to develop a new, robust test.

We furthermore applied our methods to large numbers of patient samples to study important clinical questions. Most of these projects are still ongoing, but one of them already led to an important finding. Specifically, we found that cigarette smoking just before blood sampling leads to around 25% lower sRAGE levels that will be measured. It is therefore essential to take blood samples in a standardized way, potentially by asking people to stop smoking at a fixed time before they give blood. Such standardization has not been applied in any of the clinical studies on sRAGE thus far, so we need to study this phenomenon further and find out whether previously reported associations are still valid.