AMITY INSTITUE OF VIROLOGY

AND IMMUNOLOGY

WEEKLY REPORT THREE

SUBMITTED TO: SUBMITTED BY:

Dr. Shweta Dubey Mahima Chauhan

Amity Institute of Virology and MSc. Immunology

Immunology
Roll No. – A12137518001
  Test for the detection of Anti-HCV
antibodies and the viral antigen of the
HEPTITIS C virus in serum :

Monolisa HCV Ag-Ab Ultra V2 is a qualitative enzyme immunoassay fo

the detection of infection by hepatitis C virus (HCV) based on the
detection of anti-HCV antibodies and capsid antigen in serum.

The HCV is an enveloped RNA positive sense virus belonging to the

Flaviviridae family. The HCV is recognized as being the main cause of
non-A and non-B viral hepatitis. HCV infection is characterized by an
acute and chronic form that may lead to cirrhosis and hepatocellular
carcinoma.

 PRINCIPLE:

This test is based on the use of a slid phase prepared with purified
antigens: two recombinant proteins from the non structural region
(NS3 and NS4) and a peptide from the structural region (capsid) of the
hepatitis C virus and a monoclonal antibody against the hepatitis C
capsid. The liquid phase comprises two conjugates . The first conjugate
(R6) consists of a biotinylated monoclonal mouse antibody against the
hepatitis C capsid .

 PROCEDURE:
1. Establish the sample distribution and identification plan.
2. Prepare the diluted washing solution R2 and the antigen
positive control working solution (R5a+R5b)
3. Distribute in the well in the following order:
-100ul of conjugate 1(R6) in each well.
- 50ul of negative control (R3) in well A1.
- 50ul of positive control (R4) in wells B1, C1, D1.
- 50ul of the working positive control antigen solution (R5a +
R5b) in well E1.
- 50ul of the first sample in well F1.
- 50lul of the second sample in G1 etc.
4. Cover the plate with adhesive film.
5. Incubate the microplate for 90 mins at 37 degree C.
6. Remove the adhesive film, Aspirate the contents of all the wells
in a liquid waste container and add a min. of 370ul of washing
solution into each well. Aspirate again and repeat washing a min.
5 times.
7. Distribute quickly 100ulthe solution of conjugate2 (R7) into
each well within the plate. .
8. Cover the plate with new adhesive film. Incubate for 30mins at
37degree C
9. Remove the film, empty all the wells by aspiration and wash
min of 5 times as described above.
10. Prepare enzymatic development solution (R8+R9).
11. Distribute 80ul of prepared enzymatic solution in all wells .
Allow the reaction to develop in dark for 30 mins at room
temp.
12. Add 100ul of stopping solution (R10) using the same sequence
 13. Wait atleast 4 mins after stopping solution addition and within
30 mins read the optical density at 450-620nm using a plate
reader.

 TEST VALIDATION CRITERIA:

This test is validated if the conditions below are respected:

1) For negative control R3:

The measured absorbance value must be less than 60% of the cut

off:

O.D. < cut off *0.6

2) For the antibodies positive control R4 :

0.800 </= Mean O.D. R4 </= 2.700

3) For the working solution R5 :

O.D. > 0.500

 CALCULATION AND INTERPETATION OF RESULTS :

The cut-off is determined with the R4 positive control:

Calculate the mean measured absorbance value for the positive

control R4.
Calculate the cut off value:

CO = Mean OD R4 /5

The presence or absence of anti-HCV antibodies or HCV capsid

antigen is determined by comparing the registered absorbency to
the calculated cut-off value for each sample.

The following ratio is calculated for each sample:

Ratio- OD of the sample/CO value

Samples with optical density lower than the cut-off value are
considered to be negative (ratio<1).

Samples with optical density greater or equal to the cut off are
considered to be initially positive . They should be retested in
duplicate before final interpretation.

 Test for the detection of the surface

antigen of the HEPATITIS B virusin human
serum :
 Monolisa HBs Ag ULTRA is a qualitative one step enzyme
immunoassay technique of sandwich type for the detection of the
surface antigen of the (hepatitis B virus (HBs Ag) in the human serum.

The detection of HBs Ag in the serum indicates an infection caused by

the hepatitis B virus . It is the first marker to appear and may be
observed 2 or 3 weeks before the clinical and biological symptoms of
the disease. Its period of presence may be very short (a few days ) or
very long (several years ) . HBs Ag persisting beyond 6 months in the
serum denotes chronic hepatitis .

 PRINCIPLE:

This test is a qualitative one step enzyme immunoassay based on

the principle of sandwich type using monoclonal antibodies and
polyclonal antibodies selected for their ability to bind themselves to the
various subtypes of HBs Ag.

The solid phase is coated with monoclonal antibodies. The conjugates

are based on the use of monoclonal antibodies from mouse and
polyclonal antibodies from goat against the HBs Ag . These antibodies
are bound to the peroxidase.

 PROCEDURE:

1. Establish the sample distribution and identification plan.

2. Prepare the diluted washings solution R2

 3. Prepare the conjugate R6+R7 working solution.

4. Distribute in the wells in the following order:

- 100ul of negative control (R3) in wells A1, B1 , C1 and D1

- 100ul of positive control (R4) in well E1.

- 100ul of first unknown sample in well F1 and etc.

5. Dispense 50ul of conjugate solution (R6+R7)into all wells .

Homogenize the reaction mixture

6. Cover the plate with adhesive film.

7. Incubate the microplate for 1 hour and 30 mins at 37 degree C.

8. Remove the adhesive film . Aspirate the contents of all wells

into a liquid waste container and add min. 370ul of washing

solution a min. of 4 times. Dry the strips by turning them upside

down on absorbent paper.

9. Prepare the development solution (R8+R9)

10. Dispense into each well 100ul of prepared development

. Allow the reaction develop in dark for 30 mins. At room temp..

solution .

11. Add 100ul of stopping solution (R10) by using the same

Sequence . Homogenize the reaction mixture.

 12. Wait atleast 4 mins before taking the reading at 450-620nm
using a plate reader.

 TEST VALIDATION CRITERIA:

This test is validated if the conditions below are respected:

1. For the negative control R3:

O.D. R3 </= 0.080

2. For the positive control R4:

O.D. R4 >/= 1.000

 CALCULATION AND INTERPRETATION OF RESULTS:

The cut-off is determined with negative control R3:

Calculate the mean measured absorbance value for negative
control R3

Calculate the cut off value: mean OD R3 + 0.050

The presence or absence of HBs Ag is determined by

comparing the registered absorbency to the calculated
cutoff value for sample.
The following ratio is calculated for each sample :

Ratio= OD of the sample /Co value

Samples with an optical density value lower than the cut off
value are considered to be negative

Samples with optical density values greater than or equal to

the cut off are considered to be initially positive. . They
should be retested in duplicate before final interpretation.