2. • Leuser Virus has re emerged in South East Asia.
• Along with the historically mild strain 1, a second fatal strain is ravaging the
rural communities.
• The current standard for testing is an ELISA-based blood test, however it is
unable to differentiate this second fatal strain.
• Early research has shown that the second strain only, causes yellowing of
the eyes and an increase in enzyme X serum levels.
• Consequently we have developed an assay for this enzyme, to improve
diagnostics and allow for differentiation of the two strains, which will
prevent the unnecessary and costly treatment of strain 1.
• Samples from a deceased patient with strain 2 have also been investigated
to find out more about the pathology of the virus, however this
presentation will focus on the development of the assay only, including:
• Optimisation of parameters
• Determination of cut off points and performance characteristics
• Comparison with the current standard of testing (ELISA)
Introduction
4. Assay optimisation
• Jon Darkrows testing unknown enzymes simulation
programme was used to determine the optimal pH,
temperature and incubation time of the assay method.
• Each parameter was investigated individually, by keeping
the other parameters constant and observing the
relationship between changing the parameter and
changes in enzyme activity
Fig (I) John Darkrows testing unknown enzymes simulation programme.
Optimal pH -
• Assay ran in triplicates Physiological
temperature (37C)
• 500mg/dL substrate concentration
• Integer intervals between pH 2 and pH
12
Optimal temperature –
• Assay ran in triplicates
• Optimal pH as previously
determined
• 500 mg/dL substrate concentration
• 5C intervals between 15C and
55C
Data used to
confirm the optimal
assay duration for
the remaining assay
runs.
Data used to
determine the
optimal assay
duration for
the remaining
assay runs
5. Serum samples from previously diagnosed Patient set A were ran on
the optimised assay to produce triplicate absorbance values:
1. A solution of 0.1 M Tris HCl pH 8 assay buffer and 10mM enzyme X
substrate was prepared
2. 1ml was added to 20 test tubes, each labelled with a set A patient,
A1-A20, which were placed in a water bath at 40oC for five minutes
3. 0.1 ml of sample serum from each patient was added to it’s
labelled test tube and the tubes were left to incubate for 10
minutes.
4. The tubes were then removed from the water bath and 1ml sodium
hydroxide was added immediately and mixed.
5. Absorbance of each test tube was read at the optimal wavelength
(which couldn’t be optimised for) and recorded
Determination of clinical cut off points
Figure (III) Samples were incubated in a 40C
water bath. Image taken from www.jsr.kr.
Figure (II) Patient serum sample.
6. • To calculate the enzyme concentration a standard curve of
enzyme X concentration against assay absorbance was
made from research data, and this was used to convert the
absorbances.
• Possible cut off points were determined at different
enzyme X concentrations that were higher than the
majority seen in strain 1 patient samples (A1-A10) and
lower than the majority seen in strain 2 patient samples
(A11-A20).
• 1ml serum samples from undiagnosed patient set B were
ran on the assay as previously described to determine their
enzyme x concentrations
• Diagnosed with the different cut off points.
• These diagnosis's were compared to the known diagnosis of
each patient, to calculate the specificity and sensitivity of
each cut off point.
• For this presentation only the chosen cut off point will be
discussed.
y = 0.2108x + 0.4198
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
Absorbance
(Au)
Enzyme X concentration (mg/L)
Standard curve of enzyme X concentration
against absorbance
Fig (IV) Standard curve of enzyme X concentration against
absorbance. Used to convert Set A sample absorbances.
7. Comparison with the ELISA standard
As the ELISA is the current standard test for the diagnosis of Leuser Virus, serum samples from Patient Sample
Set C were tested using the enzyme X assay method, as previously described, and the ELISA method, as follows,
for comparison.
1. 100 ml of capture antibody was transferred to each well, incubated for a week
at 4C and removed from by inverting, washing with 300 ml wash buffer and
blotting the wells three times.
2. 300 ml blocking buffer was added to each well, incubated at room temperature
for an hour, then removed as previously described.
3. 100 ml of a negative control sample not containing Leuser virus antigen,
positive control sample containing the antigen, reference sample, patient C1
sample, patient C2 sample were added to labelled wells, incubated for an hour
at room temperature, then removed as previously described.
4. This was repeated with detection antibody
5. 100 ml streptavidin-HRP, diluted 1:40 in reagent dilutent, was transferred to each well, incubated for 20 minutes at room
temperature and removed as previously described.
6. 100 ml of mixed 1:1 substrate solution A to B was added to each well and incubated for 20 minutes
7. The reaction was stopped by adding 50 µL of stop solution.
8. The plates were then read at 450 nm in a plate reader.
Fig (V) 96 well plate for ELISA.
9. Enzyme optimisation
Fig (VI) Graph showing the activity of enzyme X at pH2-pH7
0.00
100.00
200.00
300.00
400.00
500.00
600.00
2 3 4 5 6 7 8 9 10 11 12
Enzyme
X
Concentration
(mg/dL)
pH
Effect of pH on Enzyme X activity
Optimal pH was
found to be pH 8
10. Enzyme optimisation
Fig (VII) Graph showing the activity of enzyme X between 15-55 C
0.00
100.00
200.00
300.00
400.00
500.00
600.00
2 3 4 5 6 7 8 9 10 11 12
Average
Poduct
Concentration
(mg/dL)
pH
Effect of pH on Enzyme X activity
0.00
100.00
200.00
300.00
400.00
500.00
600.00
15 20 25 30 35 40 45 50 55
Average
Product
Concentration
(mg/dL)
Temperature (C)
Effect of temperature on Enzyme X activity
40
Optimal temperature was found to be 40C
Fig (VI) Graph showing the activity of enzyme X at pH2-pH7
11. Enzyme optimisation
0.00
100.00
200.00
300.00
400.00
500.00
600.00
2 3 4 5 6 7 8 9 10 11 12
Average
Poduct
Concentration
(mg/dL)
pH
Effect of pH on Enzyme X activity
0.00
100.00
200.00
300.00
400.00
500.00
600.00
15 20 25 30 35 40 45 50 55
Average
Product
Concentration
(mg/dL)
Temperature (C)
Effect of temperature on Enzyme X activity
Fig (VIII) Enzyme simulation programme runs for pH optimisation at
physiological (37oC) temperature and 500 mg/dL substrate concentration.
Optimal
incubation
duration
was found
to be 10
minutes
Fig (VII) Graph showing the activity of enzyme X between 15-55 C.
Fig (VI) Graph showing the activity of enzyme X at pH2-pH7.
Fig (IX) Enzyme simulation programme runs for temperature
optimisation at pH 8 and 500 mg/dL substrate concentration.
12. Determination of cut-off points and
performance characteristics
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
3.60
3.80
4.00
4.20
4.40
4.60
4.80
5.00
5.20
5.40
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Enzyme
X
Concentration
(mg/L)
Pateint Number
Concentration of enzyme X in Set A Patient
Samples
Figure (x) Graph showing the determination of the clinical cut off point from
patient set A sample enzyme concentrations.
Table (I) Comparison of patient set B sample assay and confirmed
diagnosis’s used to calculate specificity and sensitivity.
The enzyme X
concentration that was
selected as the clinical cut
off value for the assay was
2.30 mg/dL, as it gave the
highest sensitivity of 100%,
and a relatively high
specificity of 90%.
SampleLabel
Assay Diagnosis (Strain) Confirmed Diagnosis
(Strain)
TP/FP/TN/FN
Patient B1 2 2 TP
Patient B2 2 1 FP
Patient B3 1 1 TN
Patient B4 2 2 TP
Patient B5 2 2 TP
Patient B6 1 1 TN
Patient B7 1 1 TN
Patient B8 2 2 TP
Patient B9 2 2 TP
Patient B10 1 1 TN
Patient B11 2 2 TP
Patient B12 2 2 TP
Patient B13 1 1 TN
Patient B14 1 1 TN
Patient B15 2 2 TP
Patient B16 1 1 TN
Patient B17 2 2 TP
Patient B18 2 2 TP
Patient B19 1 1 TN
Patient B20 2 2 TP
Patient B21 1 1 TN
Patient B22 2 2 TP
Patient B23 2 2 TP
Patient B24 1 1 TN
Patient B25 2 1 FP
Patient B26 2 2 TP
Patient B27 1 1 TN
Patient B28 2 2 TP
Patient B29 2 2 TP
Patient B30 1 1 TN
Patient B31 2 2 TP
Patient B32 1 1 TN
Patient B33 2 2 TP
Patient B34 1 1 TN
Patient B35 1 1 TN
Patient B36 1 1 TN
Patient B37 2 2 TP
Patient B38 1 1 TN
Patient B39 1 1 TN
Patient B40 2 2 TP
Assay cut
off point
2.30 mg/dL
Sensitivity calculation –
20/20 x 100
Sensitivity = 100 %
Specificity calculation –
18/20 x 100
Specificity = 90%
13. ELISA Enzyme X Assay
Average Patient C1 Result 0.748 AU at 450 nm 4.90 mg/dL Enzyme X concentration
Standard Deviation +/- 1.27 x 10 ֿ ² AU +/- 5.90 x 10 ֿ ² mg/dL
Clinical cut off value 0.808 AU at 450 nm 2.30 mg/dL Enzyme X concentration
Diagnosis (Negative, Positive – strain
undifferentiated, Strain 1, Strain 2)
Negative Strain 2
ELISA Enzyme X Assay
Average Patient C2 Result 1.13 AU at 450 nm 1.10 mg/dL Enzyme X concentration
Standard Deviation +/- 4.81 x 10 ֿ ² AU +/- 1.48 x 10 ֿ ¹ mg/dL
Clinical cut off value 0.808 AU at 450 nm 2.30 mg/dL Enzyme X concentration
Diagnosis (Negative, Positive - strain
undifferentiated, Strain 1, Strain 2)
Positive – strain undifferentiated Strain 1
Comparison to the ELISA standard
Table (II) – Comparison of ELISA for patient C1 diagnosis and Enzyme X Assay for diagnosis.
Table (III) – Comparison of ELISA for patient C2 diagnosis and Enzyme X Assay for diagnosis.
14. Conclusions
• In summary, this work provides proof of principle to validate a full scale clinical evaluation of the
enzyme X assay for the diagnosis of ‘yellow eye’ Leuser Virus.
• The parameters of the assay for this investigation should be kept to a pH of 8, temperature of 40oC
and an incubation time of 10 minute. Further investigation is needed to determine the optimal
wavelength of this assay, as the clinical investigation was limited by its inability to determine this.
• The clinical cut off point of this assay should be kept at 2.30 mg/dL enzyme X concentration to insure
100% sensitivity and keep specificity as high as possible.
• The new assay overcomes important barriers in the diagnosis of the yellow eye strain, currently
limited by the standard ELISA test, which is not able to differentiate between this and strain 1.
• Although the ELISA was found to be more specific, the 100% sensitivity of the new assay is highly
desired for diagnostics, as ‘yellow eye’ is fatal and so a false negative result will lead to the patients
death as they will not receive vital treatment.
• Further investigation is needed to confirm either the ELISA, negative, or Enzyme X Assay, strain 2,
diagnosis of patient C1, in order to calculate specificity and sensitivity of the ELISA in diagnosing
undifferentiated Leuser Virus for comparison.
