Enzyme Immunoassay for the

Determination of Hepatitis B
Virus “e” Antigen in Human
Plasma / Sera (HBeAg)

Enzyme Linked Immunosorbent Assay

 Objective:
To determine of hepatitis B virus e Ag in
human plasma and serum

Introduction:
HBeAg stands for Hepatitis B e-antigen.
This antigen is a protein from the hepatitis B virus
that circulates in the infected blood when the virus
is actively replicating.
The presence of HBeAg suggest that the person is
infectious and is able to spread the virus to other
people.
 Components of kit
u Microplate(Coated)
u Negative control
u Antigen Positive control
u Antigen calibrator
u Wash buffer concentrate
u Enzyme conjugate
u Substrate
u Stopping solution (Sulphuric acid)
 Principle

Anti HCV
 Assay Procedure
1. Well No. Reagent added Quantity
A1 Blank
B1 Negative Control 100μl
C1 Negative Control 100μl
D1 Negative Control 100μl
E1 Antigen Calibrator 100μl
F1 Antigen Calibrator 100μl
G1 Antigen Positive Control 100μl
H1 Sample 100μl

2. Cover and incubate the microplate for 60 min at 37 °C.

Procedure………

3. Wash the microwells using ELISA washer, 4-5 washing

cycles with 350 µl / well of washing solution with a soaking
time of 20 - 30 sec between cycles.

4. Dispense 100 µl enzyme conjugate in all wells, except for

A1 (blank).

5. Incubate the microplate for 60 min at 37°C.

6. Wash the microwells same as step 3.

7. Add 100µl of substrate in all the wells except A1(Blank)
Procedure………

8. Incubate the plate for 20 min in dark.

9. Add 100µl of stopping solution (Sulphuric

acid) in all the wells.

10. Read the plate using Elisa reader at 450nm,

taking 630nm as reference wavelength.
 Important Notes

1. Ensure that no finger prints are present on the bottom

of the microwell before reading at 450nm. Finger prints
could generate false positive results on reading.

2.Reading should be performed immediately after the

addition of stop solution and no longer than 20 min
afterwards. Some self-oxidation of the chromogen can
occur leading to a higher background.
 HBe Ag
Calculation of the cut-off

The results are calculated by means of a cut-off

value determined with the following formula:

1. Cut-off (Co) = Mean negative control+ 0.1

2. Sample
(Co) Cut-off
 Interpretation of Results

u Results are interpreted as follows:

Sample/Co Interpretation
< 0.9 Negative

0.9 - 1.1 Equivocal

> 1.1 Positive

Example: Calculations
Well No. Reagent added OD Values
A1 Blank
B1 Negative Control
C1 Negative Control
D1 Negative Control
E1 Antigen Calibrator
F1 Antigen Calibrator
G1 Antigen Positive Control
H1 Sample

1. Cut-off (Co) = Mean negative control + 0.1

2. Sample =
(Co) Cut-off value
Result Interpretation:
Sample/Co Interpretation
< 0.9 Negative
0.9 - 1.1 Equivocal
> 1.1 Positive

Report:
Sample is found to be _______for the presence of HBeAg,
since the Sample/Co value was found to be _____which is
______than .
THANKS