Viral Antigen Detection by Enzyme Immunoassay (EIA)

Lab report 2
Prepared by: Savan Mohammad
Dostan Arkan
Dnya Jumaa
Sariya Atta
Arina Muhsin
Course name: Microbiology 2 laboratory
Section: 5
Instructor name: Peshawa Yunis
Introduction:
Enzyme immunoassays (EIA) detect the virus antigen (Ag) in patient stool
specimens and are easier to implement than electron microscopy or cell culture.
Antigen detection correlates well with clinical disease despite EIA being less
sensitive than molecular methods. Solid-phase enzyme immunoassays have
proved to be useful tools for the direct detection of the antigens of some viruses
directly in clinical specimens. Such assays have been particularly useful in the
diagnosis of viral infections in the gastrointestinal and respiratory tracts. They can
also be used as analytical tools for detecting particular antigens or antibodies in a
certain sample during biomedical research. An EIA uses a sample of your blood or
urine. The sample is exposed to a protein that is known to bind to a very specific
substance, such as an antibody. ELISA is: (i) Simple procedure. (ii) High specificity
and sensitivity, because of an antigen–antibody reaction. (iii) High efficiency, as
simultaneous analyses can be performed without complicated sample pre-
treatment. There are four main types of ELISA: direct ELISA, indirect ELISA,
sandwich ELISA and competitive ELISA. Each has unique advantages,
disadvantages and suitability.

Aim:
The aim of our procedure to detect the presence of antigen and antibodies in our
sample by EIA.
Materials:

1.Sample from patient (Serum or Plasma)

2.Distilled Water
3. Micropipettes and their tips
4.waste Container

5.Incubator
6. Microwell plate reader
7. ELISA kit

Procedure
1. Allow all reagents to reach room temperature for at least 15 minutes before
using.
2. Before using, dilute the wash buffer with distilled water at a rate of 1:40.
3. Add 50ml of HIV1-positive control agent to the second well, HIV-2 positive
control to the third well, negative control to fourth, fifith, and seventh well, and
sample to the final two wells.(the first well is blank)
4. Incubate at 37oC for 5 minutes with the sealing plate membrane sealing the
plate.
5. Remove and discard the plate cover,Take out. Add wash buffer to each well.
6. Add HRP-HIV H2 conjugate to all the wells except blank.
7. Incubate for 5 minutes at 37 oC.
8. Add wash buffer to each well.
9. Add 50ml of substrate A and B to all the wells except Blank.
10. Incubate for 3 minutes in a dark place.
11. Add 50ml stop solution to all the wells except the blank.
12. Read the results, Calibrate the plate reader with the blank well and read the
absorbance at 450 nm if a dual filter instrument is used, set the reference
wavelength at 450/630 nm.Set no blank wells is allowed if use dual wavelength to
detect.Calculate the Cut-off value and evaluate the results.
Results and Discussion
Based on this experiment that we did on the lab, for viral antigens dietician by
enzymes immunoassay we noticed that this principle is used to determine the
amount of antibodies and antigens, so we have micro plate that holds 8 columns
with different numbers that because of we did washing the plates for two times
so it’s one of the reasons that the numbers ( the amount of antigens and
antibodies) decreased. After first washing we added HRP-HIV HZ conjugate that
contained antibodies and enzymes which changed the color to yellow. Finally we
added stop solutions to stop the reaction. The procedure was carried out as
planned, after all the steps done we put the walls inside the ELISA.

Results

A 0.035

B 0.079

C 0.085

D 0.058

E 0.069

F 0.070

J 0.072

H 0.056