TOPIC -Identification of Japanese B Encephalitis and Dengue through Enzyme Linked Immunosorbent Assay (ELISA )

1. TOPIC -Identification of Japanese B Encephalitis and Dengue
through Enzyme Linked Immunosorbent Assay (ELISA )
Reg no. – 02191 of-2020-2021
Roll- PG/ VUOGP57/ BIT IV S No.
043
Subject – M.Sc in Biotechnology

2. Aim of the Project:-
Through this ELISA procedure we can detect the positive and negative
results of the JAPANESE B ENCEPHALITIS and DENGUE disease.
Introduction:-
ELISA is a plate- based assay techniques designed for detecting and
quantifying peptides proteins, antibodies and hormones. There are mainly
Japanese B Encephalitis and Dengue are detected through ELISA.
Japanese B Encephalitis is a mosquito born viral disease and is an important
cause of seasonal viral encephalitis.The JE virus belongs to family
Flaviviridae genus Flavivirus.
Dengue viruses, transmitted by Aedes aegypti and Aedes aldopictus
mosquitoes. There are four types of dengue virus (DEN-1,DEN-2, DEN-3,
DEN-4)

3. • Coating- Polystyrene plate is
treated with a solution of either
antigen or antibody.
• Blocking – An unrelated protein –
based solution is used to cover all
unbound sites on the plate.
• Detection – Enzyme conjugated
antibody or antigen binds
specifically to the target antigen
or antibody.
• Read results – Substrate is added
and the signal produced by the
enzyme – substrate reaction is
measured.

4. ELISA TYPES:-
Competitive ELISA

5. Japanese B Encephalitis
IgM antibodies in the patients serum/CSF captured by anti-human IgM coated
on the solid surface.
Some reagents in kit : -
Anti- human IgM coated wells, Sample diluent for JE IgM,Wash buffer
concentrate, JE antigen, Anti JE Monoclonal antibody,Avidin-HRP, Liquid TMB
substrate,Stop solution, JE IgM positive and negative control.
Procedure:-
1. Add diluted sample 50 microlitre each well and 50 microliter positive &
negative control.
2. Incubation for 1 hours at 37°C at incubator.
3. Wash the plate five times with wash buffer.
4. Add 50 microliter of JE antigen to all each well of the plate.

6. 5.Incubation for 1 hours at 37° C at incubator.
6. Wash the plate five times with wash buffer.
7. Add 50 microliter Monoclonal antibody to all each well of the plate.
8. Incubation for 1 hours at 37°C at incubator.
9. Wash the plate five times with wash buffer.
10.Add 50 microliter of Avidin- HRP to each well of the plate.
11.Incubation for 30 minutes at 37°C at incubator.
12.Wash the plate five times with wash buffer.
13.Add 100 microliter of TMB- substrate to each well of the plate.
14.Incubation for 10 minutes at room temperature (in dark place).
15.Add 100 microliter stop solution at each well of the plate.
16. Measure the absorbance at 450 nm within 10 minutes.

7. Fig- Transmission process of
Japanese B Encephalitis disease

8. Dengue
Antibody Detection through STANDARD E ELISA kit and Antigen Detection
through Dengue NS1 antigen kit:-
STANDARD E Dengue IgM Capture ELISA contains a microplate, which has
been pre-coated with monoclonal Human IgM on well. On the other side
Dengue NS1 ELISA is called is a solid phase Enzyme Linked Immunosorbent
Assay based on antigen sandwich capture principle.
In this kit some reagents are used for ELISA procedure.Each ELISA kit have
different procedure according to the identification of each different disease.
So this Dengue Antibody detected ELISA procedure we are collected the
sample which is added to the microwells through added different types of
reagents and then it is allowed to washing with appropriate dilution of wash
buffer after that the samples are incubated at incubator for some times , at
last in each well added substrate and then stop solution which is give some
colours then the OD of the colours are measured and detect the result of the
virus.

9. Fig- Transmission process of Dengue disease

10. Materials and methods:-
A total number of Japanese B Encephalitis were 42 and a total sample
number of Dengue were 293, these total samples were collected from
patients between March to May 2022, in the Microbiology department
collection room of Bankura Sammilani Medical College and hospital. The
total female patients were 150 and the total male patients were 185 out of
335.
We were seperated patients sample (blood/CSF) , then centrifuged it the
serum were separated from blood into the another vials. Then in the
experimental day we were take out the sample and allowed for experiment.

11. Results and discussion:-
Reference Serum Testing:-
The 311 serum specimens were classified by reference laboratory testing for
Japanese B Encephalitis and Dengue (IgM & NS1). All samples were tested by
JE detect kit and Dengue( IgM &NS1) detect kit and equivocal result were
retested. Of the 18 JE sample reference,all were negative with no equivocal
result. Of the 293 Dengue (219 for Dengue IgM and 74 for Dengue NS1),total
7 patients sample had positive ( 6sample for Dengue IgM &1 sample for
Dengue NS1) results and remaining number of the patients sample had
negative results by Dengue kit.
Reference CSF Testing:-
The 24 CSF samples were classified by reference laboratories as JE, 23
patients samples had negative results and 1 patients had positive results.

12. Results for Japanese B Encephalitis Results for Dengue IgM
Results for Dengue NS1

13. Calculation of the results for Japanese B Encephalitis :-
Quality control:- Each kit contains one vial of positive control and one vial of
negative control. These work as marker of kit performance.
1. If OD of negative control is more than 0.18 or 2. If OD of the positive control
is less than 5 times the OD of negative control ,in both the situation, the test
should be considered as invalid.
Interpretation of Results:-
A . If OD value of sample tested is less than OD of Negative control by a factor 3.0
( sample OD < negative control OD ×3.0), the sample should be considered as
“Negative “. B. If OD value of sample tested exceeds OD of negative control by a
factor 5.0( sample OD >negative control OD ×5.0),the sample should be
considered as” Positive”. C. If OD value of sample tested exceeds OD of negative
control by a factor 3.0( sample OD> negative control OD ×3.0), but is less than
OD of negative control by a factor 5.0( sample OD <negative control OD ×5.0),
the sample should be considered as “Equivocal “.

14. Fig- Japanese B Encephalitis
Fig – Dengue IgM
Fig – Dengue NS1

15. Conclusions:-
The developed ELISA assay provides a convenient and specific method for
the large scale determination of JEV antigen in infected mosquito samples
with high sensitivity and specificity.
On the other side the Dengue results indicate that the DENV ELISA is
dengue group specific and can be used to differentiate dengue infection
from other circulating Flavivirus infections.This ELISA can also be used to
distinguish between primary and secondary dengue fever on the basis of
IgM/IgG ratios.

16. Refference:-
1. Kilks SC, Nimmanitya S,Nisalak A, Burke DS, Evidence that maternal
dengue antibodies are important in the development of dengue
haemorrhagic fever in infants, Am J Trop Med Hyg Jan 38(2):411-
419,1988.
2. Ludolfs D.et.al., Serological differentiation of infections with dengue virus
serotypes 1 to 4 by using recombinant antigen, J ClinMicrobial,
40(11):4317-4320,2002.
3. Matthew T.R et al.Dengue virus pirates human platlets, Blood 126(3):286-
287,2015.
4. Guzman M.G et al. Dengue: A continuing global threat, Nat Rev Microbial,
8:S7-S16, 2010.

17. Thank you