Project Description

Aim

The aim of our product is two-fold:

1. To be a urinary lateral flow test for a sepsis and acute kidney injury (AKI) biomarker, Cystatin C (Cys C).

2. To improve upon lateral flow assay (LFA) technology by increasing the readout of the test strip, enhancing the detection capabilites of low-concentration biomarkers generally.

Why sepsis?

Sepsis affects millions worldwide, accounting for 20% of global deaths [1]. Low- and middle-income countries (LMIC) bear the highest burden of sepsis, accounting for 85% of all sepsis-related deaths worldwide [2].

Typically, sepsis diagnosis in clinics relies on culturing and Gram-staining of infectious agents to identify the pathogen causing sepsis, and facilitate rapid antibiotic use. However, culturing takes an average of five days, and the Gram-staining method has only 30%-60% sensitivity, which leads to antibiotic misuse and wastes valuable time, decreasing the likelihood of survival [3]. In LMIC, where medical resources are limited, sepsis diagnosis is based on non-specific physiological and mental changes such as hypotension, fever, increased heart rate, and breathing difficulties [4]. Specifically, the most recent and widely used criteria known as qSOFA, developed by the Third International Sepsis Consensus Definitions Task Force, uses altered mental status (GCS < 15), Respiratory rate ≥22, and Systolic BP ≤ 100 as benchmarks for identifying sepsis. However, these symptoms are common to many medical situations and may not reliably identify sepsis.

Early diagnosis of sepsis can significantly reduce the mortality rate [5, 6]. Therefore, it is important to identify sepsis within the first few hours of symptom onset to enhance health service efficiency and survival rate. We therefore decided to make an LFA for the detection of a biomarker that can rapidly diagnose sepsis in resource-poor settings.

Having spoken to clinicians, it has been made clear that there is no ‘perfect’ biomarker for sepsis diagnosis. Even popular diagnostic biomarkers like procalcitonin (PCT) and C-reactive protein (CRP) may be elevated in other conditions and may not always indicate sepsis. With their feedback, we narrowed our scope and concentrated on a specific biomarker known as Cystatin C (Cys C). Cys C is a urinary biomarker that has not received as much attention as other sepsis-related biomarkers, despite it being elevated in patients with sepsis and acute kidney injury (AKI). Cys C is independently predictive of sepsis, AKI, and mortality [7].

There have been several attempts to produce diagnostic tools for sepsis biomarkers, including lateral flow assays [8]. These involve using microchips and biosensors, which are often expensive to produce and require resource-intensive settings for quantitative analysis [10], making them less accessible in LMICs. Therefore, we decided to develop a low-cost point-of-care (POC) test for Cys C using a lateral flow assay (LFA). This test could be on-site and provide rapid results without requiring additional equipment like machine readers.

Biomarker -- The Specifics of Cys C

Cys C is normally not secreted into the urine as it is filtered by the liver back into the blood. Thus, Cys C in the urine can provide an early indication of AKI in critically ill patients, and is also independently predictive of sepsis [9, 10]. Elevated urinary Cys C is independently predictive of mortality, sepsis, and AKI, so has a wide scope of utility. For sepsis prediction, the AUROC score was 0.8 with a cut-off of 0.24ug/ml. for AKI prediction, the AUROC was 0.7 and the cut-off was 0.12ug/ml. For mortality prediction, the AUROC was 0.64 with an optimal cut-off of 0.09ug/ml [10]. Measuring the concentration of urinary Cys C is therefore useful for predicting sepsis, AKI and mortality, so an LFA measuring this would have a wide range of uses.

Our Design

One of our aims is to produce an LFA that is usable in LMIC without the need for any machine detectors. This LFA will be used to measure urinary Cys C. A previous Cys C LFA has been created, which measures urinary Cys C, but requires a machine detector called a colorimetric reader. To bypass the requirement for a machine reader, we needed to make an LFA for Cys C that can be interpreted with the naked eye [11]. We therefore devised a novel way of detecting biomarkers on LFAs that we hope to not only apply to the detection of Cys C, but also to the detection of other biomarkers with lowe cut-offs. Therefore, another, more broad goal of our project, is to improve the capabilities of LFAs to detect low-concentration biomarkers, thus contributing to the advancement of LFA technology in diagnosis.

Improving Readout of LFAs Using Our New Technology

Our LFA has all the familiar aspect of a conventional LFA. A conventional LFA has a region to apply the sample (in our case, urine). Then a region for the sample to capture some kind of secondary conjugate as it flows down the strip, which are often anti-biomarker antibodies conjugated to gold nanoparticles. Then a test line containing immobilised anti-biomarker antibodies which can capture the biomarker-antibody-gold conjugate, giving a colour signal on the test line if there is enough biomarker present to enable the accumulation of gold nanoparticles . (Fig.1)

Due to our goal of improving the signal to biomarker ratio of LFAs, we have kept the basic principles of our LFA the same, but have replaced the antibody-gold nanoparticle conjugate with a few possible alternative solutions. These solutions are all fusion proteins.

Solution 1

To enhance the readout of the test strip, we decided, instead of using gold nanoparticles which are vibrant but do not produce light, to use an enzyme that actually luminesces. The enzyme we decided upon is NanoLuciferase (NanoLuc).

Why NanoLuc?

NanoLuc has been engineered to luminesce more intensely than naturally occurring Luciferases (approx. 150-fold greater luminescence). It has greater thermal stability, broader optimal pH range (pH 7-9), maintains significant activity under more acidic conditions, and is more stable in urea than normal Luciferase (important considering our test is for urine). NanoLuc has been specifically co-evolved with its synthetic substrate furimazine (also called NanoGlo) to give optimal activity. It is also smaller than luciferase (MW= 19kDa compared to 62kDa), making it easier to express. It is not just vibrant, but actually produces light [12, 13], which may be detectable.

In order for Cys C to capture NanoLuc, we knew we needed to use an anti-Cys-C antibody conjugated to Nanoluc. However, antibodies require glycosylation. This means that their expression requires using mammalian systems, which is more expensive. If our LFA is to benefit people in LMIC, we needed a cheaper alternative. We therefore decided to instead conjugate NanoLuc to anti Cys-C nanobodies.

Why Nanobodies?

Nanobodies are small components of antibodies, resembling the binding region to antigens. Nanobodies do not require glycosylation, so are easier and cheaper to express, as a bacterial system can be used instead of a mammalian one. They also are smaller and thus are easier to purify and express compared to antibodies, especially in a conjugate. They possess the same binding affinity as regular antibodies. Nanobodies will work in a wide pH range (pH 3-9), and are less prone to aggregation than mABs [14,15] resulting in a longer shelf life.

After having urinated on the LFA, here is the sequence of events:

1. The urine will make its way to the Nanobody-NanoLuc conjugates on the test strip.
2. The nanobody-nanoluc will bind to Cys C in the urine.
3. The Cys C-Nanobody-NanoLuc complexes will migrate to the immobilised anti-Cys C antibodies on the test line and will be immobilised.
4. The LFA will then be exposed to the Nanoglo assay, which will be used as a substrate by NanoLuc to produce light. If there is a significant accumulation of Cys C on the test line, then the test line will luminesce, and this will be visible to the naked eye. An option we are currently exploring is inserting the LFA into a ‘dark box’, enhancing the ability to view the light emitted on the test strip.

Solution 2

Solution 2 is identical to alternative 1 in all but one aspect: instead of having one NanoLuc conjugated to a nanobody, there will now be two NanoLucs conjugated to the nanobody. We wanted to explore this option to see if ‘stacking’ NanoLucs can lead to an increased signal:biomarker ratio, enhancing our ability to detect low-concentration biomarkers.

Furthermore, it has been reported that NanoLuc dimers, under crystallisation conditions, uses more substrate per NanoLuc, and therefore have a higher activity. We will also test if this is true for our nanobody-NanoLuc- NanoLuc conjugate [16].

Soultion 3 & 4

Finally, we will be conjugating the nanobodies to chromoproteins. Chromoproteins are vibrant proteins, which could perhaps increase the signal on the test line by being more visible to the naked eye. We decided upon two chromoproteins to conjugate to nanobodies: the first being gfasPurple. Due to having a very high extinction coefficient, it is very sensitive to light at a specific wavelength, which leads to a high absorbance (and hence emission) [17].

Separately, we are also conjugating nanobodies to another chromoprotein called TsPurple, because:

1. The chromoprotein is well characterised and familiar to UCL’s Department of Biochemical Engineering, and the Department is accustomed to working with it.
2. Expressing gfasPurple in E. coli halves its growth rate, so we decided to also use TsPurple in case this is an issue [18].

Both chromoproteins give a bright purple colour which is easy to see. Chromoproteins also show more promise than NanoLuc for clinical settings, as biomarker quantification using chromoproteins is more straightforward, due to our ability to use imaging software like ImageJ to analyse the shade of colour, which would be darker the more biomarker there is. For a semi-clinical setting, NanoLuc may be more beneficial, as the light signal would be more visible, but it would probably be more difficult to quantify the light.

Summary

Our project aims to produce a diagnostic LFA for Cys C, which is indicative of AKI, sepsis and mortality. We wish to make our LFA interpretable without machine readers, so we are using different conjugates to achieve this. In doing so, we hope our conjugates will also enhance detection capabilities of low-concentration biomarkers in other diseases, improving LFA technology and advancing the ability to detect diseases with previously unusable biomarkers.

Future Directions

An example of a very useful yet hard to detect biomarker is urinary sTREM-1, which is an early urosepsis and AKI biomarker. Urosepsis has a very high mortality rate, at 20-40%. Urinary sTREM-1 in urosepsis has a cutoff of 30-50pg/ml, and the AUROC is 0.7-0.9 for the diagnosis of AKI. The cut-off of sTREM-1 is very low, so is harder to detect [19]. Perhaps our conjugates could improve LFA technology to the extent where it enables the reliable detection of sTREM-1 for the early diagnosis and prediction of urosepsis and AKI. The reason we are not using sTREM-1 in our project is because there is currently no available nanobody sequence, whereas for Cys C, there is. Producing nanobodies requires phage display. Perhaps in the future, we will have the time and resources to produce an anti-sTREM-1 nanobody, and, combined with our new conjugates, make urinary sTREM-1 detectable using an LFA.

References

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