Oral_Presentation_Submission Level 5.pptx

1. Efficiency of enzyme X assay for detection of
severe strain of Leuser virus infection cases
Presented by Adina Dorobantu

2. Introduction
Leuser virus
- flu-like symptoms
- diagnostic test: ELISA
- expensive
- detects both the mild and severe strain of the virus
Aim: to develop an alternative diagnostic test based on enzyme X, found in higher
serum levels in patients suffering from the severe strain of the virus (Strain 2),
that is:
- rapid
- more specific
- cheaper

3. Introduction
• Recent COVID-19 disease (2020-ongoing)
– infectious respiratory disease caused by
the SARS-CoV-2 virus – rapid spread 
pandemic
• Lead to a global demand of diagnostic tests
to aid in the containment of spread of the
virus; for example: Lateral Flow Tests (LFTs)
(Figure 1)
• General diagnostic test requirements:
• Rapid, cost efficient, reliable and specific
diagnostic tests
• point-of-care testing, screening of
symptomatic or asymptomatic patients
• Quantification of the degree of infectivity
• Prevention, disease progression and spread
management
(Hsieh et al., 2021)
Figure 1. Outline of the principle behind Lateral Flow
Tests (LFTs) (“Lateral Flow Assays: How Does Lateral Flow
Work? - DCN Dx,” 2018)

4. Materials and Methods
• Enzyme assay:
• Buffer preparation: 6.5, 7.5 and 8.5
• Addition of enzyme X substrate
• Incubation at 35 °C, 37 °C and 40°C
• Samples were measured every 4 minutes for a period of 20 minutes
• Absorbance was measured at wavelengths between 400 and 450 nm
• Final parameters:
• pH 8.5
• 37 °C
• Every 4 minutes for a period of 20 minutes
• Abs @ 420 nm
• The optimized enzyme X assay was used for determination of patient Set A (n=20)
and patient Set B (n=40) concentrations.

5. Results
1. Enzyme assay optimization of: pH, temperature, time and
wavelength
• Enzyme X Standard Curve
2. Cut-off determination from known patient Set A samples
3. Classification of strain 1 and strain 2 cases from unknown patient
Set B samples
• Clinical sensitivity and specificity

6. Results
1. Enzyme assay optimization of: pH, temperature, time and
wavelength
• Enzyme X Standard Curve
2. Cut-off determination from known patient Set A samples
3. Classification of strain 1 and strain 2 cases from unknown patient
Set B samples
• Clinical sensitivity and specificity

7. Enzyme assay optimization of: pH, temperature, time and wavelength
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 5 10 15 20 25
Abs
@
420
nm
Time (min)
pH 7.5
35°C 37°C 40°C
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 5 10 15 20 25
Abs
@
420
nm
Time (min)
pH 6.5
35°C 37°C 40°C
Figure 2 and Figure 3. Enzyme assay optimization at pH 6.5 and 7.5 and 3 temperatures: 35 °C, 37 °C and 40°C–
samples measured at every 4 minutes for a period of 20 minutes

8. Figure 4. Enzyme assay optimization at pH 8.5 and 3 temperatures: 35 °C, 37 °C and 40°C– samples
measured at every 4 minutes for a period of 20 minutes
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 5 10 15 20 25
Abs
@
420
nm
Time (min)
pH 8.5
35°C 37°C 40°C
Enzyme assay optimization of: pH, temperature, time and wavelength

9. Enzyme X Standard Curve for Enzyme assay
y = 0.0421x + 0.0159
R² = 0.9913
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0 1 2 3 4 5 6 7 8 9 10
Abs
@
420
nm
Concentration (mg/L)
Enzyme X Standard Curve
Figure 5. Enzyme X Standard Curve
Known
concentration
(mg/L)
Measured
concentration
(mg/L)
QC1 5 3.05
QC2 2.57 1.5
Y 90 0
Table 1. Quality control and enzyme Y
measured concentration (mg/L)

10. Results
1. Enzyme assay optimization of: pH, temperature, time and
wavelength
• Enzyme X Standard Curve
2. Cut-off determination from known patient Set A samples
3. Classification of strain 1 and strain 2 cases from unknown patient
Set B samples
• Clinical sensitivity and specificity

11. Cut-off determination from known patient Set A samples
Figure 6. Cut-off determination from known Patient Set A samples
0
0.5
1
1.5
2
2.5
3
3.5
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Concentration
(mg/L)
Patient A number
Cut-off determination from known Patient Set A samples
Strain 1 Strain 2

12. Cut-off determination from known patient Set A samples
Figure 6. Cut-off determination from known Patient Set A samples
0
0.5
1
1.5
2
2.5
3
3.5
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Concentration
(mg/L)
Patient A number
Cut-off determination from known Patient Set A samples
Strain 1 Strain 2
Cut-off
concentration
= 1.035

13. Results
1. Enzyme assay optimization of: pH, temperature, time and
wavelength
• Enzyme X Standard Curve
2. Cut-off determination from known patient Set A samples
3. Classification of strain 1 and strain 2 cases from unknown patient
Set B samples
• Clinical sensitivity and specificity

14. Classification of strain 1 and strain 2 cases from unknown patient Set B samples
0
1
2
3
4
5
6
7
8
9
10
11
12
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
Concentration
(mg/L)
Patient B number
Cut-off classification of unknown Patient Set B samples
Strain 1 Strain 2
Figure 7. Strain 1 (mild - blue) and Strain 2 (severe - orange) cases in patient Set B samples determined by the
cut-off value
Cut-off
concentration
= 1.035

15. Classification of strain 1 and strain 2 cases from unknown patient Set B samples
0
1
2
3
4
5
6
7
8
9
10
11
12
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
Concentration
(mg/L)
Patient B number
Cut-off classification of unknown Patient Set B samples
Strain 1 Strain 2
Figure 7. Strain 1 (mild) and Strain 2 (severe) cases in patient Set B samples determined by the cut-off value
Cut-off
concentration
= 1.035
False
positives = 6
False
negatives = 0

16. • Clinical sensitivity and specificity was
calculated:
100% sensitivity
70% specificity
Classification of strain 1 and strain 2 cases from unknown patient Set B samples
Post Test Likelihood
PPV 77%
NPV 0
Table 2. Post Test Likelihood – Positive
and Negative predictive values

17. Conclusions
- QC1 (known concentration= 5 mg/L)  3.05
- QC2 (known concentration= 2.57 mg/L)  1.5
• Suggested good accuracy of the enzyme X assay
- Enzyme Y – no absorbance reading  suggested high
specificity of the enzyme assay for enzyme X
- Clinical sensitivity (100%) and clinical specificity (70%)
confirmed and validated enzyme assay reliability
- Values worth investigating in future experiments
and could be considered as relevant for
development of an enzyme X assay-based
diagnostic test for detection of Leuser virus
Limitations:
- Low QC values
- Could be improved by respecting and
limiting the thaw-freeze cycles of the QC
samples which might have an influence
over their activity
- Low sample volume
- Could be improved by additional samples:
QC, patient Set B  to allow triplicates for
increased accuracy

18. References
Hsieh, W.-Y., Lin, C.-H., Lin, T.-C., Lin, C.-H., Chang, H.-F., Tsai, C.-H., … Lin, C.-S. (2021). Development and Efficacy of
Lateral Flow Point-of-Care Testing Devices for Rapid and Mass COVID-19 Diagnosis by the Detections of SARS-CoV-2 Antigen
and Anti-SARS-CoV-2 Antibodies. Diagnostics, 11(10), 1760. https://doi.org/10.3390/diagnostics11101760
Lateral Flow Assays: How Does Lateral Flow Work? - DCN Dx. (2018, February 23). Retrieved March 21, 2022, from DCN Dx
website: https://dcndx.com/lateral-flow-rapid-diagnostic-test/