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

Retroviruses 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.

It is transmitted through these bodily fluids: 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.

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

ELISA Negative control Positive Control

Patient c Positive control Patient B Patient A Negative control

ELISA (Enzyme Linked ImmunoSorbent Assay) Patient is not HIV-infected ELISA – first and most basic test To determine if individuals are positive for HIV To determine if individuals are positive for HIV ELISA Negative results Positive Results No reaction in sample Conduct confirmatory test using Western blotting Repeat ELISA test Positive Results Negative results

Why we perform ELISA first rather then western blotting? ELISA Western blot Cost ~ US$300 > US$400 > US$400 Time- consuming 3.5h - 4.5h 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.

Can be divided into 5 steps: 1.Antigen preparation 2.Electrophoresis 3.Transfer of proteins to membrane Protocol of Western blotting & immunoblotting (I)

Protocol of Western blotting & Immunoblotting (II) 4. Antibody addition 5. Enzymatic or radioactive detection

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.

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.

ELISA Negative results Positive 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 Repeat ELISA test Positive Results Negative results

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.

References: IDS/page7.htm

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