Lec #5 (Wk 3): HIV Detection Tests (DIAGNOSTIC LAB)

Question 1

HIV belongs to the retrovirus class. Which subgroup of retroviruses exactly?

Answer 1

Lentivirus.

Question 2

Which cells does HIV virus target?

Answer 2

Targets cells with CD4 proteins which are:
1- CD4+ T-helper cells.
2- Macrophages.
3- Dendritic cells.

Question 3

When CD4+ levels drop below a critical level in HIV patients, what immunity is lost?

Answer 3

Cell-mediated immunity is lost & the body becomes progressively more susceptible to opportunistic infections.

Question 4

How do we test for HIV?

Answer 4

First you do an antigen/antibody test and the most common one is ELISA.
If you get a negative result here then you are 100% negative since this is a highly sensitive test. If positive then we have to confirm this thus you proceed to a 2nd test:
Serology test. Most common serology test used is Western blotting. If this is positive you are truly positive. If negative, we need to check a 3rd test; Viral load test using real-time PCR.

Question 5

What is ELISA? Advantages?

Answer 5

Enzyme Linked Immunosorbent Assay.

This is the first test used when testing for HIV it is an antigen-antibody test.

Advantages:
- Highly sensitive meaning if you are negative then you are 100% negative.
- Simple methodology.
- Suitable for testing a large number of samples.

Question 6

What are the different types of ELISA done & ingredients needed in all types?

Answer 6

There are 3 different ways;
1- Direct –> looks for HIV antigen.
2- Indirect –> Looks for IgG antibodies against the antigen.
3- Sandwich –> Looks for HIV antigens.

What we need:
A transparent plate with 96 wells which contains something to allow proteins to easily attach to surface.
7 reactants:
1- Patient’s serum sample.
2- Non-specific proteins (Either bovine serum albumin or casein) that only attaches to the well’s surface.
3- Enzyme-linked antibody.
4- Chromogenic substrate.
5- Saline solution & non-ionic detergent (to wash wells between every step)
6- stop solution.
7- two control samples; 1 known to have antigen/antibody. Other doesn’t have antigen/antibody.

Question 7

How do we perform direct ELISA?

Answer 7

This type detects antigens.
1- you take the 96 well plate and put a positive result in 1 of them and a negative result in another and the remaining 94 wells allows u to test for 94 different samples at once!
2- Serum sample is added to the well & incubated. Proteins present in this sample (among them the expected antigen) adhere to the surface of well. The exceeding sample solution is then discarded and the well is washed a few times so that only the attached proteins remain at the surface. but we could have some empty gaps between (since ofc proteins wouldn’t attach to every single corner) and if those are left empty, the enzyme-linked antibody that’s added could bind to these sticky sites & give a false positive result.
3- Thus, Non-specific proteins are added which attach to all the remaining gaps.
4- After incubation period, well is washed again. And the solution containing the enzyme-linked antibody specific for the searched antigen is added, incubation then washed again. if the antigen is present in the sample the enzyme binds to it & cannot be removed by washing. BUT if antigen is absent, the enzyme linked antibody has nothing to attach to and is removed upon washing.
5- Add chromogen & incubate in a dark place.
6- after a while, plate is removed from the darkness and the reaction is stopped with a stop solution.
7- If antigen was present, chromogen would generate a yellow color. The more intense the color is, the greater the concentration is (intensity can be measured using spectrophotometry). No change in color is a negative result.

Nowadays we don’t really use direct ELISA, we use the indirect & sandwich ELISA. Thats because these 2 methods are more sensitive techniques.
Indirect ELISA —> detects IgG antibodies against the antigens.
Sandwich ELISA —> Detects the HIV antigen itself.

notice; intensity of the color can be measured using spectrophotometry AKA ELISA Plate Reader.

Question 8

How do we perform indirect ELISA?

Answer 8

This method looks for IgG antibodies against the HIV antigen.

1- HIV antigen attaches to well’s surface.
2- Primary antibody binds to it (in this case it is the IgG antibody we are looking for in the serum).
3- Secondary enzyme-linked antibody is added and the chromogen is added (Results interpreted the same way; yellow = positive result, blue = negative result).

notice; intensity of the color can be measured using spectrophotometry AKA ELISA Plate Reader.

Question 9

How do we perform sandwich ELISA?

Answer 9

You detect the HIV antigens instead of antibodies against those antigens.
1- Primary antibody attaches to well’s surface.
2- HIV antigen binds to it (the one we are look for in the serum).
3- Secondary enzyme linked antibody.
4- Chromogen is added.

notice; intensity of the color can be measured using spectrophotometry AKA ELISA Plate Reader.

Question 10

What are HIV rapid tests?

Answer 10

This is a simplified version of antibody ELISA tests. They look for HIV antibodies (so just like the ELISA indirect method) in the blood or oral fluid.

How to do it?
1- HIV antigen is fixed on 1 particular strip at 1 end.
2- A sample is placed at the end of the testing stick.
3- When the sample pass over the section with the antigens and it contains antibodies for HIV,m then they will stick to these antigens & change the color.

Question 11

How is the HIV rapid test performed?

Answer 11

1- HIV antigen is fixed on 1 particular strip at 1 end.
2- A sample is placed at the end of the testing stick.
3- When the sample pass over the section with the antigens and it contains antibodies for HIV,m then they will stick to these antigens & change the color.

Question 12

Can we possibly get a false negative result for HIV in ELISA?

Answer 12

Enzyme Linked Immunosorbent Assay is the first step to test for HIV.
Less than 1% of results are false negatives.

Reasons:
1- Window period.
2- Some people don’t develop an immune response to the virus but can still spread it.

Question 13

Can we possibly get a false positive result for HIV in ELISA?

Answer 13

Enzyme Linked Immunosorbent Assay is the first step to test for HIV.
Less than 1% of results are false positive.
Reasons:
- Rheumatological diseases.
- Alcoholic hepatitis
- Malaria

Consequences of false positive:
- Depression / suicide
- Family & emotional issues.

Question 14

What does western blotting mean?

Answer 14

This is an analytical technique for detecting specific proteins.

SNoW DRoP
Southern blotting - DNA
Northern blotting - RNA
Western blotting - Protein

Question 15

How is western blotting done?

Answer 15

1- We separate the proteins according to their size using PolyAcrylamide Gel Electrophoresis (PAGE).

2- Transfer to a nitrocellulose membrane.

3- We block the membrane meaning we want to block any other part of the membrane so that only the proteins are exposed blocking is done using a mild detergent + milk or BSA.

4- We add a primary antibody to bind to the specific protein we are looking for.

5- But now we cannot visualize/see this so we add a secondary (IgG) antibody with horseradish peroxidase attached to it and in the presence of the substrate it will produce chemiluminescence.

https://www.youtube.com/watch?v=faOELRICYqY

Question 16

What are the important genes seen in the HIV virus? Functions?

Answer 16

Gag gene, Pol gene, and env gene.

Gag - codes for structural proteins like p17 (matrix protein) & p24 (capsule protein).

Pol - codes for enzymes like reverse transcriptase, integrase, & protease.

Env - codes for gp160 which is then cleaved using protease enzyme (coded for by the Pol gene) to give gp120 & gp41.

Question 17

Which antibodies are detected by the Western blotting technique for HIV patients?

Answer 17

Antibodies to p24 & p55 are the earliest detected after infection (which declines as disease progresses) by the Western blotting (proteins made by gag gene).

antibodies to gp160, gp120, & gp41 are detected by all HIV patients (proteins made by env genes).

antibodies to p31, p51, & p65 are also commonly detected (proteins made by the pol gene).

Question 18

Which antibodies are first to be detected by western blotting in patients with HIV?

Answer 18

Proteins produced by the gag gene: p24 & p55.

Question 19

What is a positive result for HIV in western blotting? Negative result?

Answer 19

Positive result: any 2 bands to the following: p24, gp41, gp120, & gp160.

Negative results: no bands at all.

Question 20

What does PCR mean?

Answer 20

Polymerase Chain Reaction, this is an in-vitro technique used for the amplification of a region of DNA.

Question 21

What are the reaction components for PCR?

Answer 21

1- DNA template.
2- Primers.
3- Enzyme (DNA polymerase).
4- Buffer.
5- dNTPs.
6- Mg2+.

Question 22

What are the features of the PCR primers?

Answer 22

They are 2: forward & reverse primers.
They are non-complimentary & should contain 40-60% GC rich content for better annealing. They should be 25-35 nucleotides in length & is complimentary to the 3’ end of the target DNA.

Question 23

What enzyme is commonly used for PCR?

Answer 23

Taq polymerase since it is thermostable.
It is stable up to 95 degrees Celsius & has a high processivity.

Question 24

What is the function of Mg2+ in PCR?

Answer 24

This is a cofactor to increase productivity of the polymerase.

Question 25

Function of buffer solution?

Answer 25

To provide optimal activity & stability for the DNA polymerase.

Question 26

What are the stages of PCR?

Answer 26

1- Denaturation; separate both DNA strands by increasing the temperature to 95 degrees.

2- Annealing; where we add the 2 primers at 55 degrees.

3- Extension beyond the 2 primers using dNTPs at 72 degrees.

and the cycle keeps going at 2 to the power of n.
n= no. of cycles.

Question 27

What is reverse transcriptase PCR?

Answer 27

This uses RNA rather than DNA as the initial template. RNA-directed DNA polymerase is used to yield cDNA then we do the normal PCR stages:

1- Denaturation at 95
2- Annealing at 55
3- Extension at 72 degrees

Question 28

What are the applications of PCR?

Answer 28

1- Diagnostics like viral infections, cancer, & prenatal testing for genetic diseases.

2- Forensics like DNA fingerprinting.

3- Detection of drug resistant genes.

4- Genome mapping & gene function determination.

Question 29

What type of PCR does the viral load test use?

Answer 29

Real-time PCR.

This test finds either the RNA of the HIV virus or the DNA of the WBCs infected by HIV. Viral load test isn’t done as frequently as antibody testing bc it requires technical skills & is expensive.

Good thing about viral load test is that it can be done even days after being infected which can pick up the disease even if other tests say negative.

Question 30

What can we use to screen blood or organs for HIV before donation?

Answer 30

Viral load test.

Question 31

What does real-time PCR mean? How is it done?

Answer 31

This is an innovative PCR for the detection & quantification of the PCR products.

1- At the beginning, the reaction mixture contains the denatured DNA, primers, & the dye. The unbound dye molecules weakly fluorescence but is subtracted during computer analysis.

2- After annealing of the primers, a few dye molecules can bind to the double strand. DNA binding results in a dramatic increase of the dye molecules to emit light upon excitation.