Introduction

VeriFied is a homogeneous enzyme fragment complementation assay (EFCA) based on the interaction of nanobodies with human serum albumin (HSA), which is found in bloodstains. The assay has two bifunctional fusion proteins, each with a different nanobody and a complementary half of a luciferase enzyme, NanoLuc, attached by a peptide linker. Upon interaction of both nanobodies with HSA, the enzyme fragments are brought to a close proximity of each other. This results in complementation of the enzyme and therefore, the restoration of enzyme activity and a bioluminescent signal. The signal is dependent on the binding ability of the nanobodies. As bloodstains age, oxidation-mediated structural changes in HSA disrupt epitope integrity and therefore also the binding of the nanobodies, leading to a decrease in signal intensity.

Enzyme fragment complementation assay

EFCAs are commonly used to detect protein-protein interactions both in vivo and in vitro. The technology is based on split-enzyme engineering where the reporter enzyme is split into two or more complementary fragments that are inactive on their own but able to reconstitute into an active whole enzyme [1]. In homogeneous in vitro applications, low intrinsic affinity of the enzyme fragments is essential to avoid analyte-independent complementation and to establish a high signal-to-noise ratio [2]. The intrinsic affinity should be substantially lower than that of the interacting proteins of interest (K_D in μM) [3]. Furthermore, the accuracy and analytical sensitivity of the assay are dependent on the relative concentrations of assay components, which need to be optimized experimentally [4].

The homogeneous system offers a platform for a rapid mix-and-read application, suitable for an on-site solution. Additionally, the bioluminescent signal provides fast quantitative data that could be either used to compare bloodstains and rule out the irrelevant ones or possibly connect the signal intensity to the bloodstain age.

Biomarker for estimating the age of bloodstain

Our aim was to design our test while taking into consideration the real-world application. We began the design by going through several possible biomarkers and assessing their suitability for an on-site test that would be easy-to-use, noncontaminating, nondestructive and nonprofiling. We evaluated the biomarkers based on their abundance, degradation time and method, possible ways of detection and interindividual variability.

In crime scene investigation, the available sample volume is often small and the samples should be kept intact for further downstream forensic analysis. For an on-site and easy-to-use test, the required processing of the sample and the complexity of the test equipment should be minimal. Therefore, we sought to find an abundant biomarker that could be tested from a whole blood sample and would be detectable even in small sample sizes. In addition, for further forensic analysis integrity of the genetic information is crucial. The test should not contaminate the crime scene with foreign genetic material.

Several biomarkers have been studied for bloodstain age determination. The most researched ones are hemoglobin and genetic markers such as DNA [5], different RNAs [6]. Other studies include hormones related to the circadian rhythm [7], metabolomics [8], plasma proteins [9] and enzyme activity. Although the existing studies show promise, none of the developed methods we found fully met the objectives we had identified. Therefore, we decided to make an effort towards biomarker discovery and developed a method based on human serum albumin oxidation.

Due to the uncontrolled nature of the environment and the lack of circumstantial information, crime scenes present a challenge for diagnostics. Without contextual data, it is difficult to predict how the biomolecular composition of bloodstains change over time. Factors such as bloodstain size, surface properties like porosity, light exposure, temperature, humidity, microbial activity, and the presence of cleaning agents are expected to affect the natural aging of bloodstains.

Human serum albumin

In comparison to other proposed biomarkers, we chose HSA due to its high abundance in blood, the possibility of an immunological approach, and moderate inter-individual variability, more notably the variability related to sex [10]. Ruling out the identification of sex with the assay is an ethical consideration but also helpful in standardising the results, and in cases where the parties are unidentified.

Our hypothesis is that in dried bloodstains the degradation of HSA happens due to oxidation. Most notable structural changes happen in domain I due to the oxidation of the free thiol in cysteine 34. Other significant impairments happen in surface exposed methionines. After identifying the specific areas susceptible for oxidative damage, we faced a challenge: how to determine a standard when the original volume of the sample is not known? Rather than us using a separate biomarker for the standard, Professor Urpo Lamminmäki suggested that we take advantage of the divergent oxidative modifications and measure two qualities from the same biomarker. This way we can use the other result as a point of comparison, both as the standard, and the control.

VeriFied includes two separate assays, meaning two fusion protein pairs (Figure 1.). One where both of the nanobodies target stable areas on the HSA structure and another, where one nanobody targets a stable area and the other targets an unstable area. The stable-stable pair measures the total amount of HSA and the signal from the stable-unstable pair correlates with the amount of reduced HSA. Comparing the two, we can determine the amount of oxidized HSA in the sample and thus evaluate the age of the bloodstain.

Figure 1. The mechanism behind VeriFied assay. VeriFied consists of a pair of bifunctional proteins. The nanobody part of the proteins bind to human serum albumin, while the NanoLuc fragment part produces a bioluminescent signal when it meets its complementary fragment. One of the nanobodies target a stable epitope of human serum albumin, while the other targets an oxidation prone epitope (1). NanoLuc fragments bind to each other and produce a bioluminescent signal when both of the nanobodies bind to human serum albumin (2). When HSA is oxidized, the oxidation prone epitopes have degraded, which in turn inhibits nanobody binding and reduces produced signal (3). VeriFied test includes a control, in which both nanobodies of the pair bind to stable epitopes. Thus, the control produces a luminescent signal even with oxidized human serum albumin (4).

The structure of nanobodies

Nanobodies, discovered in 1993 by Hamers-Casterman et al., are small heavy-chain variable domains (VHHs), originally sourced from camelid antibodies [11]. Uniquely among mammals, camelids possess homodimeric heavy-chain antibodies (HCAbs), meaning the antibodies consist of only the heavy-chain [12]. Compared to conventional antibodies that are formed from heavy- and light-chains, the homodimeric nature of HCAbs allows the isolation of only the VHHs (Figure 2.), which work as functional binding units capable of binding various antigens with high affinity [12].

Figure 2. Differences between nanobodies and antibody fragments. Yellow indicates variable domains, blue indicates constant domains. Fab: fragment antigen-binding, scFv: single-chain variable fragment.

Benefits of using nanobodies

The single domain nature of nanobodies in addition to an extra disulfide bond, accounts for a more rigid structure with a higher thermal stability when compared to multidomain antibody fragments [13]. Additionally, the surfaces of nanobodies are more rich in hydrophobic amino acid residues compared to conventional antibody fragments, which increase their solubility and reduce risk of aggregation [13]. This risk of aggregation and the relatively more complex structure of multidomain antibody fragments leads to difficulties in using bacterial expression systems in their production despite continued attempts and improvements [14]. Functional nanobodies with their simpler and more stable structure can instead usually be expressed in high yields in bacterial systems such as E. coli [15]. Additionally, the small size of nanobodies (12-15 kDa) makes them ideal for homogeneous immunometric tests using enzyme fragment complementation, since they are less likely to cause issues related to steric hindrance than other, larger antibody fragments.

Origin of our nanobodies

When looking for anti-HSA nanobodies, we found a nanobody library by Shen et al. 2021 that contains ~70 high affinity anti-HSA nanobodies with varying epitopes [16]. The exact epitopes of the nanobodies were not detailed in the study, so we used protein-protein docking software to analyze the epitopes based on cross-link mass spectrometry data provided by the article. In addition to the nanobodies from the article by Shen et al. 2021, we used the nanobody ALB8 (PDB:8Z8V), which was sourced from PDB and had a structure solved by X-ray crystallography. We chose suitable nanobodies to use in our project based on predicted epitope stability and steric hindrance.

To emit a signal using enzyme fragment complementation, two nanobodies are required, one for each enzyme fragment. These two nanobodies must not compete for the same epitope and steric hindrance should not stop them from binding or the enzyme complementation can not work. To gather accurate information about the age of the bloodstain, we need to measure the amount of oxidized HSA, but we also need to measure the total HSA in the sample to use as a reference to calculate the ratio of oxidized to reduced HSA. A simple illustration of nanobody mediated EFCA can be seen in Figure 3.

Figure 3. Cartoon of enzyme fragment complementation assay. The heart indicates human serum albumin with domains I, II, and III shown in different colors, the blue shoulders indicate two nanobodies with different epitopes, the yellow hands indicate the NanoLuc enzyme fragments. Therefore, one arm from shoulder to hand represents a nanobody-NanoLuc fragment fusion protein.

For the test measuring oxidized HSA, the two nanobodies consist of a nanobody with an oxidization stable epitope, and another with an epitope unstable to oxidation based structural changes. The other test, measuring total HSA in the sample, consists simply of two nanobodies with oxidation stable epitopes. To improve the odds of finding a working pair of nanobodies for each pair, we chose at least two nanobodies to represent each role. Since both tests need at least one nanobody with an oxidation stable epitope, this nanobody can be the same for both tests.

Nanobodies with epitopes that are more stable to oxidation

Nanobodies with oxidation prone epitopes

Choosing split enzyme

Linker

Proteins

Potential pairs

Periplasmic expression in E. coli BL21(DE3)pLysS

Sequences

Plasmids