Lecture 9: Clinical diagnostics (ELISA and PCR)

Question 1

3 ELISA applications

Answer 1

Patient diagnosis for an infectious disease

Population screening for a disease

Checking patients for immunity to a disease

Question 2

ELISA Principle:

Answer 2

Observe for antibodies to see if the person is currently infectious with disease

10 steps

Question 3

10 steps of ELISA

Answer 3

1. Identify the target we want to detect in the patient; diagnostic antibody anti-HCV
2. Identify an antigen with specificity for anti-HCV; HCV antigen
3. Immobilise HCV antigen on a surface. The surface could be glass beads or microtitre plate or synthetic microparticles.
4. Add plasma from the patient to the surface. If the plasma contains anti-HCV it will bind to HCV antigen
5. Incubate
6. Wash the surface to remove unbound proteins

7.Add a second antibody. Here the second antibody is anti-human globulin (AHG) labelled with an enzyme. The enzyme = alkaline phosphatase. AHG binds to the primary antibody.

8.Wash to remove unbound second antibody

9. Add chromogenic substrate. This is a molecule that will be changed by the enzyme to produce a colour change. The substrate here is para-nitro-phenyl- phosphate (pnpp) which will change colour from clear to yellow when changed by the enzyme
10. Measure the intensity of the yellow colour in a spectrophotometer at 405 nm. The intensity is proportional to the amount of anti-HCV

Question 4

PCR principle

Answer 4

A LABORATORY TEST USED TO DETECT BACTERIAL OR VIRAL
GENOME IN A SAMPLE

3 steps in the process
We look for viral RNA of HBV in patient plasma
◦ Detection of viral RNA of HBV is the preferred way to monitor the viral load of a person with HBV

(HBV for example)

Denaturing – when the double-stranded template DNA is heated to separate it into two single strands.

Annealing – when the temperature is lowered to enable the DNA primers to attach to the template DNA.

Extending – when the temperature is raised and the new strand of DNA is made by the Taq polymerase enzyme.

These three stages are repeated 20-40 times, doubling the number of DNA copies each time.

Question 5

PCR applications

Answer 5

Patient diagnosis for an infectious disease

Population screening for a disease

Checking patients for disease progress by monitoring viral load

Question 6

Detection of PCR products

Answer 6

Gel based:
◦ Manual or automated
◦ Uses the principle that smaller fragments of DNA move more quickly through an agarose gel than larger fragments when electrical current is applied

Real time PCR (RT-PCR)
◦ In RT-PCR, no gel is needed, as the intensity of the signal is measured as products are generated.
◦ Fluorescent dyes are incorporated into the nucleotides during the PCR process
◦ The higher the viral load, the more PCR product made.
The more PCR product, the higher the fluorescence.
◦ This makes it a quantitative technique, which makes it ideal to monitor viral load in patients

Question 7

ELISA AND PCR comparison

Answer 7

ELISA
◦ Detects antibody
◦ Lower sensitivity than PCR
◦ Lower specificity than ELISA
◦ Good for disease diagnosis and monitoring immunity in population

PCR
◦ Detects viral genome
◦ Higher sensitivity than ELISA
◦ Higher specificity than ELISA
◦ Good for disease diagnosis and monitoring viral load in patients