The main purpose of this research project was to develop a quantitative serological diagnostic enzyme assay to detect and differentiate between the two strains of Yellow Eye Disease. The research optimized an enzyme assay that achieved a 97.5% diagnostic success rate compared to the current ELISA method. Investigation into the pathogenesis through urinalysis, haemolysis assays, and H&E staining indicated hepatic malfunction and haemolysis in diseased patients. The results provide a foundation to improve both treatment and diagnosis of the more severe strain two of the virus.

1. Development of a diagnostic enzyme assay to detect and diagnose
Yellow Eye Disease alongside an investigation into the pathogenesis
Adam Boulger
BMS 1
27040016

2. Introduction to Leuser Virus
Currently ravaging rural communities in South-East Asia
First investigated in the 1970’s due to animal epidemics.
Only caused mild symptoms in humans.
OLD Strain one
non-fatal, mild symptoms & no
treatment required
NEW Strain two
rapidly fatal & treatment necessary
(fairly effective)
RECENTLY mutated to form two strains;

3. Current Diagnostic Options
• Unspecific ELISA-based blood test which cannot detect
between strain one and strain two
• Consequence: Health agencies stating treatment &
containment is becoming unsustainable.
• Research discovered high serological levels of enzyme X in diseased
strain one patients, not present in strain two.
• Bisswanger’s landmark research states enzymes assays can be used
to ‘detect the presence and determine the concentration’ of an
enzyme
• Based upon this, purpose was to develop a quantitative serological
diagnostic enzyme assay.
• An investigation into the pathogenesis also occurred to improve
treatments.
What was the main purpose of this research project?

4. Research Project Experimental Methodology
• Urinalysis, haemolysis and H&E staining followed standard practice.
• Patient Samples provided by Public Health England
• Optimised Enzyme Assay (EA)
• ELISA Assay
• Urinalysis
• Haemotoxylin & Eosin Staining
• Haemolysis Assay

5. Optimised Enzyme Assay
• Following optimization based experiments analysing
the variables of; temperature, pH, water-bath
incubation duration and wavelength to measure
absorbance.
• It was decided that the optimum conditions for the
enzyme assay were.
• 311.5K, pH 8, 1200s and 415nm respectively.
Principles of this Enzyme Assay
• 10mM enzyme X substrate assay buffer solution was heated to
311.5K for 300s. After, 100μl of patient serum sample was
added and incubated for 1200s and the absorbance read at
415nm.
Materials:
SA Buffer – 43529
SA Enzyme X substrate P4744
Patient Sample provided by Public Health England

6. ELISA Principles
25 microlitres of samples added to plate in
duplicates alongside 100ul detection antibody and
followed standard incubations. 100 microlitres poly-
HRP streptavidin was added and then the plate was
washed. 100 microlitres TMB substrate solution was
added and then stop solution (H2SO4) was added
and the plate read at 450nm
Materials:
TF – Plates – 88-50650-22
AL – Detection Antibody – DIA HE1-01
TF – Poly-HRP streptavidin – N200
SA – Wash Buffer – N301
Samples (patient, control and blank)
provided by Public Health England

7. Determination of an enzyme assay cut off point
Graph produced using
manipulated data.
Statistical normal
distribution was plotted
against enzyme
concentration (mg/L).
Graphical manipulation of
a scatter graph was
performed to produce
bell-curves which were
extrapolated with a
polynomial trendline until
cross over point (cut off
point – 1.69mg/L)

8. Investigation into Proof of Principle
Analysis of 40 unknown
patient samples occurred
using optimised
methodology.
Patient samples were
plotting on a scatter graph
and analysed.
New method displayed
93% success rate, with 1
false negative and 2 false
positives.
True diagnostic success
rate of 97.5%

9. Investigation into Proof of Principle
Analysis of 40 unknown
patient samples occurred
using optimised
methodology.
Patient samples were
plotting on a scatter graph
and analysed.
New method displayed
93% success rate, with 1
false negative and 2 false
positives.
True diagnostic success
rate of 97.5%

10. Comparison between enzyme assay and current diagnostic
ELISA
Currently; ELISA
diagnosed both
as positive,
requiring
treatment.
Future: EA
diagnosed C1 as
positive for strain
two and C2 as
negative for
strain two.

11. Haemolysis Assay to further investigate mechanisms of
Leuser virus
Major haemolysis
of RBC found in
diseased strain
two patients.
Result of 1.49
absorbance and
the cut off
haemolysis level
was 0.997.

12. Urinalysis and H&E staining
Urinalysis was performed due to
indicative nature. Results present
abnormal bilirubin, urobilinogen and
blood
Following the urinalysis, the indicators
link to hepatic malfunction.
H&E stain displays morphological loss
of hepatocytes and hepatic vessels
alongside an increased density of
nuclei.

13. Haemolysis Assay
Identified major
haemolysis which
directly links to
hepatic malfunction.
Diseased strain two
patient shows severe
breakdown of
erythrocytes, links to
high levels of
bilirubin and
urobilinogen.

14. Conclusion of Results and Indicative Discussion
• Optimised enzyme performed better compared to the current diagnostic
ELISA. Has a true success rate of 97.5% alongside 90% specify and 95%
sensitivity
• Urinalysis indicates hepatic, renal and haemolysis
• H&E indicates morphological loss & increased density of nuclei
• Haemolysis assay indicates major haemolysis of erythrocytes

15. Report Conclusion and Futuristic Scope
This report established foundations to improve both treatment and
diagnosis of strain two Leuser Virus.
• Future treatment research should focus on the viral genomic with
mechanism investigation, transmission mechanisms and enzyme X
proteonomics.
• Future diagnostic research should focus on further optimisation, PCR
investigation and the potential for futuristic approaches such as Crispr
and controlled non-disease viral integration