YEOH HUI SHIH

1
TEAM MEMBERS

• YEOH HUI SHIH INTRODUCTION TO HIV
• RAJAMANI- ELISA
• TIEN WEI PING WESTERN BLOTTING
• YEO HUI YUN OVERVIEW CONCLUSION

2
Retroviruses

• 2 single strands of RNA genome
• Enveloped (membrane-like outer cover)
• Converts viral RNA to DNA by reverse
  transcriptase ( RTase)

HIV ( human immunodeficiency virus) is a
retrovirus that causes AIDS.
3
Human Immunodeficiency Virus

• It is transmitted through these bodily fluids
• Blood
• Semen
• Vaginal fluid and cervical fluid
• Breast milk

HIV can be divided into 2 group

HIV-1 HIV-2 are transmitted by sexual contact,
through blood, and from mother to child. HIV-1
is detected in most of the world. HIV-2 is
detected in West Africa.
4
Human Immunodeficiency Virus

• Antibody Testing
• Most common test used is ELISA (enzyme-linked
  immunosorbent assay).
• indication that the patient might be
  infected - test result indicates most likely
  the patient is not infected .
• HIV Antibodies are developed in blood within 6
  weeks to 3 months. However, in some people it
  might take up to 6 months. The test cannot detect
  recent infections.
• Antigens used for ELISA Testing
• 3 common Ags are p24, gp41, and gp160/gp120

5
ELISA
Positive Control
Negative control
6
ELISA
7
ELISA (Enzyme Linked ImmunoSorbent Assay)

ELISA
ELISA first and most basic test To determine
if individuals are positive for HIV
Negative results
Positive Results
No reaction in sample
Repeat ELISA test
Positive Results
Negative results
Patient is not HIV-infected
Conduct confirmatory test using Western blotting
8
Why we perform ELISA first rather then western
blotting?
ELISA Western blot
Cost US300 gt US400
Time-consuming 3.5h - 4.5h 2 - 3 weeks
Sensitivity 99.0 99.9
Specificity 99.0 99.8
Sensitivity is a likelihood that Abs to HIV are
present. Specificity is a likelihood that Abs to
HIV are not present.
9
Protocol of Western blotting immunoblotting (I)

• Can be divided into 5 steps
• Antigen preparation
• Electrophoresis
• Transfer of proteins to membrane

10
Protocol of Western blotting Immunoblotting (II)
4. Antibody addition 5. Enzymatic or radioactive
detection
11
Western blotting
When a person is infected with HIV for a long
period of time, it will mean that more proteins
are present thus more bands will appear on the
membrane. Bands that are darker than others ?
the more abundant protein in the virus.
12
Band pattern Interpretation
Only positive control gives different bands of
antigen present in a HIV patient. Sample 1 is a
positive control Sample 2 is a negative
control Sample patient A shows negative
result. Sample patient B shows intermediate
results. Sample patient C shows a positive
results.
13
Overview
14
ELISA
Negative results
Positive Results
Repeat ELISA test
Positive Results
Negative results
Conduct confirmatory test using Western blotting
Negative results
Positive Results
Repeat test after 6 months
Indeterminate results
Positive results
The patient is not HIV positive
Cannot be determined. Have to repeat test after
6 weeks
HIV positive
15
Conclusion

ELISA is a screening test in the diagnosis of HIV
infection. If this test is positive, it must be
confirmed with a second test called the Western
Blot which is a more specific test and will
confirm if someone is truly HIV positive, as
there are other conditions which may give a false
positive ELISA screening test.
16
References
http//www.accessexcellence.org/AB/GG/diagram.html
http//www.medicinenet.com/Human_Immunodeficienc
y_Virus_HIV_AIDS/page7.htm http//www.stanford.ed
u/group/SHPRC/ch4_hiv.html http//www.hc-sc.gc.ca
/pphb-dgspsp/publicat/std-mts98hls/std98hte.html
http//www.1uphealth.com/health/hiv_elisa_western_
blot_info.html
17
Thank You