Proof of Concept

We performed enzymatic assays on our nanobody-NanoLuciferase (NB-NL) conjugate and compared its bioluminescence to that of NL. The experiment, in brief:

1. Multiple dilutions of NB-NL and NL were created, as detailed in the enzymatic assay protocol. The same molar concentrations of NL were also tested.
2. A 1:1 volume ratio of diluted proteins and NanoGlo (NG) was added to each well to give the following concentration of conjugate: 2.5e-8 M, 5e-9 M, 2.5e-9 M, 1.25e-9 M. For each concentration, three repeats were performed. After adding the substrate, we quickly covered the plate with tin foil and used a plate reader to take readings for the duration of an hour, with data collected every 30 seconds.
3. Two controls are designed to prove that the bioluminescence detected by the plate reader was as a result of the enzyme using the NG substrate:

Data Analysis

At 2.5e-8 M, the results indicated that NB-NL exhibited lower bioluminescence than NL (Figure 1). This suggests that the attachment of the NB-(GGGGS)₃ linker to NL reduced its bioluminescent capacity.

Fig. 1: (left): The mean bioluminescence curve for [NL]=2.5e-8M when NG is added, (average mean over 3 wells). (right): The mean bioluminescence curve for [NB-NL]=2.5e-8M when NG is added, (average mean over 3 wells). NOTE: all concentrations are molarities after addition of NG or binding buffer. Error bars display 5% error.

However, when we observed the wells containing the NB-NL conjugates, the bioluminescence (blue light) was clearly visible, indicating that NB-NL is bioluminescent enough to be detected by the naked eye (Fig. 2). This led us to theorise that NB-NL's bioluminescence wasn’t impaired but instead, it could be that NB-NL depletes the substrates so rapidly that by the time bioluminescence was read by the plate reader, most substrate was consumed, preventing detection of the initial signal. This is supported by the Promega NanoGlo handbook, which notes that excess NL can hinder detection by quickly exhausting the substrate. To test this theory, we examined lower concentrations of NB-NL: 5e-9 M (5-fold dilution), 2.5e-9 M (10-fold dilution), and 1.25e-9 M (20-fold dilution).

Fig. 2: Moments after NG introduction, we observed that the wells in row 7 (containing NB-NL conjugates at two different concentrations (7CDE = 2.5e-8M, 7FGH = 8e-8M)) exhibited significant bioluminescence.

At 5e-9 M, 2.5e-9 M, and 1.25e-9 M, NB-NL displayed a much higher bioluminescence than NL (Figure 3, 4, 5). Therefore, we concluded that the NB-(GGGGS)3 linker attachment to NL did not impede the bioluminescence of the NL, and instead the NB-NL was more enzymatically active than NL.

As this result was unexpected, we repeated the enzymatic assay of NB-NL at above concentrations and the same trends were obtained. In addition, comparing to the control groups at 5e-9 M, the bioluminescence of NB-NL (Fig. 3) was much higher which proved that bioluminescence of NB-NL was achieved due to the presence of the substrate.

Fig.6.(left) The bioluminescence curve for [NB-NL]=5e-9M when binding buffer is added instead of NG, averaged over 3 wells. (right) The bioluminescence curve when there is no NB-NL and only NG and binding buffer added, averaged over 3 wells. All graphs have 5% error bars.

Summary

In summary, NB-NL showed greater bioluminescence than NL, suggesting that the NB fusion enhances NL's activity. The highest luminescence was observed at 5e-9 M, leading us to conclude that the concentration of NB-NL must be fine-tuned to enable a sustained high luminescence by slowing down substrate depletion. The enhanced activity may be due to the larger size of NB-NL, increasing collisions with the substrate, but this requires further investigation.

We performed enzymatic assays on our nanobody-NanoLuciferase-NanoLuciferase (NB-NL-NL) conjugate and compared its bioluminescence to that of NB-NL. We reasoned that attaching two NL molecules together in NB-NL-NL would double the bioluminescence compared to NB-NL. However, there may also be structural or enzymatic complications that impede bioluminescence.

We created dilutions of NB-NL-NL and compared their bioluminescence graphs to those of NB-NL with double the molarity. For example, we created a dilution of NB-NL-NL at 2.5e-9M, and compared the bioluminescence of that to NB-NL at 5e-9M. This ensured that there were an equal number of NL domains between the samples, enabling us to attribute any differences in bioluminescence to the structural differences between NB-NL and NB-NL-NL.

The experiment, in brief, was as follows. Unless specially mentioned, the procedures were the same as the enzymatic assay for NB-NL.

• The following dilutions of NB-NL-NL was prepared: 2.5e-9 M, 1.25e-9 M, and 6.25e-10 M. The bioluminescence from these were to be compared to the following dilutions of NB-NL respectively: 5e-9M, 2.5e-9M and 1.25e-9M.
• For control, we added 102 μl of binding buffer instead of NG into 100 μl of NB-NL-NL. This was used to account for background luminescence, and to prove that NB-NL-NL’s reaction with NG creates the bioluminescence detected by the plate reader.

Data Analysis

Firstly, compared with the control, NB-NL-NL at 2.5e-9 M had much higher bioluminescence, confirming that the conjugate produces light in response to NG addition (Fig.7).

Fig.7: The bioluminescence graph for [NB-NL-NL]=2.5e-9M when exposed to NG (left), and the control graph without any NG (right). All graphs display the average mean data from 3 wells and have 5% error bars.

Comparing NB-NL-NL at half the molar concentration of NB-NL gave interesting results. For example, despite NB-NL-NL at concentration 2.5e-9 M having the same number of NL domains as NB-NL at concentration 5e-9 M, the bioluminescence was much lower. This was true for all concentrations tested (Fig. 8, 9, 10).

In summary, though the number of NL units between compared concentrations of NB-NL and NB-NL-NL are the same, the bioluminescence of NB-NL-NL was lower. This could be because NB-NL-NL has an extra domain than NB-NL. This extra domain could cause the active site of the NL on the end to be blocked by the NB.

While rationally plausible, it seems that within NB-NL, no such blockage of the active site occurs, and instead, NB-NL has a higher bioluminescence compared to NL at the same concentrations. Indeed, it has been reported that the makeup and length of the linker used in a fusion protein alters the enzymatic activity of that fusion protein [1]. It’s possible that the linker is too short in NB-NL to cause NB to block NL’s active site. While in NB-NL-NL, a ring-like structure may be formed that may block both the first and second NLs’ active site.

Summary

Overall, our enzymatic assays show that NB-NL is a promising reporter, with greater activity than NL. This is a surprising finding that warrants further investigation. Since we only tested one type of NB (an anti-cys C nanobody) [2], further research is needed to explore NL's bioluminescence when conjugated to other nanobodies. NB-NL-NL, despite having two NL domains, showed reduced bioluminescence compared to NB-NL, suggesting that tandem NL fusions are not a viable option as a reporter.

Having proven that nanobody-NanoLuciferase (NB-NL) maintained significant bioluminescent activity, we designed an ELISA experiment to investigated the affinity of NB-NL to sepsis biomarker, Cystatin C (Cys C).

Experimental Design

Firstly, we immobilised different concentrations of Cys C to the wells of a 96-well plate (1000, 100, 10, 1, 0.1 ng/mL). We used a fixed volume and concentration of the NB-NL conjugate (1E-8 M, as informed by the enzymatic assay) and NanoGlo (NG), the substrate of NL. Therefore, our test wells contained immobilised Cys C, NB-NL, and NG.

Three controls were designed as follows:

1. Wells containing immobilised Cys C, NG, and NL
This group was used to test the non-specific binding between NL and immobilised Cys C. If a significant difference is observed between the mean bioluminescence signal between these wells and the test wells, this proves that the binding of NB to immobilised Cys C is responsible for a higher bioluminescent signal.
2. NB-NL and NG, without immobilised Cys C
This group was used to test the non-specific binding of NB-NL to the empty wells. If there is a significant difference is observed between the mean bioluminescence of these wells and the test wells, this suggests the binding of NB to immobilised Cys C is responsible for a higher bioluminescent signal.
3. Wells only containing 1000 ng/mL immobilised Cys C
This group was used to measure the background luminescence.

We designed the wells and carried out bioluminescence measurements in accordance with the ELISA protocol. Blocking steps ensured that there was no non-specific binding of the proteins (NL or NB-NL) to the bottom of the wells, so the only way a protein can be immobilised in the well is via Cys C. Washing steps ensured no unbound protein was left present in the well. Together, they ensured that the only enzymes present in the well were those bound to Cys C.

Data were gathered from the plate reader and analysed in Microsoft Excel. All P values were calculated using the Mann-Whitney U test, a non-parametric version of the T-test that does not assume a normal distribution.

Data Analysis

The graph below (Fig.1) shows the bioluminescence curve of NB-NL in the presence of NG and immobilised Cys C. The concentration of Cys C solution used in immobilisation was 1000 ng/mL, and we also tested the bioluminescence signal for lower concentrations (from 10-fold dilutions down to 0.1 ng/mL Cys C).

Fig.1: The bioluminescent signal curve for NB-NL+1000 ng/mL Cys C+NG. The bioluminescence was measured every 30 seconds for 30 minutes. All bioluminescence values shown in the graph are averaged over 3 identically prepared samples (mean). The error bars are 5% error.

Below is the bioluminescence curve for the NB-NL + NG only control, the immobilised Cys C-only control, and the Cys C +NG +NL control (Fig.2). Comparing any of these to the test curve in Fig.1, it seems these graphs have a lower bioluminescence, indicating the NB immobilised NL to the well by binding to Cys C.

Fig.2(left) NB-NL + NG only control (middle) immobilised Cys C-only control (right) Cys C, NG, and NL control.

Statistical Analysis

Table 1: P values obtained for comparison of the test wells with the control wells.

From Table 1 column 2, all p values obtained were significant (alpha=0.05). This indicated that for all concentrations of Cys C within a 1000-0.1ng/ml range, there was a significantly higher bioluminescence of the wells containing NB-NL and NG than those not containing it.

From Table 1 column 3, all p values within the Cys C concentration range 1000-10 ng/ml were significant (p < 0.05). This showed that bioluminescence was significantly higher when Cys C was immobilized in the wells, indicating that Cys C is necessary for capturing the NB-NL conjugate within the wells.

From Table 1 column 4, there is a significant difference (p < 0.05), demonstrating that NB-NL was immobilised in the wells significantly more than non-specific binding of NL to Cys C.

Summary

From this analysis, we concluded that NB-NL is immobilised via its NB domain to Cys C at the bottom of the well, giving a noticeable increase in the bioluminescence. This confirms that both the NL and NB domains in the NB-NL are functional. Our data strongly suggest that the NB in NB-NL retains its Cystatin C-binding ability, with a limit of detection of 10 ng/mL concentration of Cys C. This confirms NL as a reporter for use in biomarker detection via fusion with a nanobody.

Experimental Design

One of our aims is to assess whether using two tandem NanoLuciferases fused to an NB as a reporter would give a larger bioluminescent signal than an NB fused to one NL. Given the lower bioluminescence of NB-NL-NL compared to NB-NL, we saw less potential for using NB-NL-NL in biomarker detection, than we did with NB-NL. Nonetheless we carried out an ELISA test for NB-NL-NL. For test wells, we added 5e-9 M NB-NL-NL and NG, to immobilised Cys C.

Two controls were designed as follows:

1. Wells containing NB-NL-NL, NG, without Cys C
This group was used to test the non-specific binding between NB-NL-NL and the wells. If a significant difference is observed between the mean bioluminescence signal between these wells and the test wells, this proves that the binding of NB to immobilised Cys C is responsible for a higher bioluminescent signal. If it didn’t, then it either means that NB-NL-NL is not bioluminescent enough to pick up the difference between the two different types of wells, or the NB’s binding capabilities to Cys C are impaired, or both.
2. Wells only containing 1000 ng/mL immobilised Cys C
This group was used to measure the background liminescence.

We did not create control containing NL-NL, NG, and Cys C because NL-NL was not expressed. For more details, please visit the Engineering Page.

Data Analysis

Below (Fig.3, left) is a bioluminescent graph of NB-NL-NL when immobilised by Cys C (where 1000 ng/mL of Cys C was used for immobilisation). Fig.3 (right) is the bioluminescence graph for the control NB-NL-NL+NG only. The bioluminescence level shown in these two graphs does not seem to be different. However, this must be statistically tested.

Fig.3 (Left) The bioluminescence graph for NB-NL-NL in the presence of NG, when a solution of 1000 ng/mL of Cys C was used for Cys C immobilisation. (Right) The bioluminescence graph for the NB-NL-NL+NG only control.

Statistical Analysis

Table 2: All P values obtained for comparison of the test wells with the control wells.

From table 2 column 2, all p values except for one were not significant ( > 0.05 = alpha). However, when 1000 ng/mL Cys C was used for immobilisation, there was a significant difference (P=0.028 < 0.05), indicating that NB-NL-NL gives a discernible bioluminescent signal at this concentration of Cys C. From table 2 column 3, all P values for the test samples within the Cys C concentration range 1000-10 ng/mL were not significant (>0.05=alpha). Overall, we believed that NB maintained some of its Cys C binding capabilities, but the detection of Cys C is greatly impeded by the tandem NL.

Summary

In conclusion, our findings demonstrate that NB-NL can still bind to Cys C and can still produce bioluminescence, making it a promising reporter for the detecting Cys C. This also suggest that NL can be fused to other nanobodies to achieve similar results. Thus, we have successfully shown that our anti-Cys C NB-NL conjugate can be used as a reporter for Cys C detection. We hope these findings will be useful for the prevention of deaths associated with delayed diagnoses of sepsis and AKI. We also hope these findings could be applied to the detection of other biomarkers, improving diagnostic technology overall.

Additionally, using a nanobody instead of an antibody, we have reduced the production cost, since nanobodies don’t have glycosylation and can be expressed in E. coli rather than the more expensive mammalian systems. We look forward to seeing what future iGEM teams will do with our findings, and we hope that the experiments outlined in this section will inspire the use of NB-NL conjugates for the low-concentration biomarker detection helping drive down costs, accelerate diagnosis, and save lives.