Lecture 9: Lab Techniques

Question 1

What is immunoelectrophoresis

Answer 1

Combines electrophoresis and immunoprecipitation for identifying and characterizing proteins within complex mixtures.

• An antigen mixture is first separated into its component parts by electrophoresis and then tested by double immunodiffusion.
• Protein components are separated by size and electrical charge.

• The test helps in the identification and approximate quantisation of various
proteins present in the serum.

• Can be used to detect normal as well as abnormal proteins, such as myeloma proteins in human serum.

Question 2

Immunoelectrophoresis process and results

Answer 2

1. Sample first separated into component parts by electrophoresis.
2. Antibodies then placed in trough.
3. Time allowed for antibodies and antigens to
   diffuse into the gel.

Results:
- The presence of elliptical precipitin arcs
represents antigen-antibody interaction.
- The absence of the formation of precipitate
suggests no reaction.

• Nature/identity of antigens can then be determined
  ➢ Different antigens (proteins) can be identified based on the intensity, shape, and position of the precipitation lines.

Serum protein components can be separated into five major fractions by size and electrical charge: serum albumin, alpha-1globulins, alpha-2 globulins, beta 1 and 2 globulin, and gamma globulins.

Question 3

Immunoblotting types for detection of..

• specific DNA sequence
• RNA
• specific proteins in a protein mixture

Answer 3

• specific DNA sequence (Southern blot)
• RNA (Northern blot)
• specific proteins in a protein mixture (Western Blot)

Question 4

Process of western blotting

Answer 4

Western Blot:

• Antigens separated by electrophoresis
• Gel transferred to carrier membrane (eg nitrocellulose sheet)
• The proteins adhere to the membrane in the same pattern as they have been separated due to interactions of charges.
• The proteins on this immunoblot are then identified by staining with appropriately labeled antibodies.

Question 5

Nucleic acid amplification (PCR) process and purpose

Answer 5

Because significant amounts of a sample of DNA are necessary for molecular and genetic analyses, studies of isolated pieces of DNA are nearly impossible without PCR amplification.

There are three main stages:
• Denaturing – 1 min 94 degrees. 
double-stranded
template DNA is heated to separate it into two
single strands.

• Annealing – 45 seconds at 54 degrees.
DNA primers to attach to the template DNA.

• Extending – 2 mins at 72 degrees. The new strand of DNA is made by the Taq polymerase enzyme.

Question 6

Purpose of flow Cytometry

Answer 6

analyse characteristics of cells or particles.

• Flow cytometry can be used for:

• Cell counting.
• Cell sorting.
• Determining cell function.
• Determining cell characteristics.
• Detecting microorganisms, such as bacteria, fungus or yeast.

Question 7

Describe How Flow Cytometry works

Answer 7

Its working depends on the light scattering features of the cells under investigation, which may be derived from dyes or monoclonal antibodies targeting either extracellular molecules located on the surface or intracellular molecules inside the cell

• Sheath fluid focuses the cell suspension, causing cells to pass through a laser beam one cell at a time. Forward and side scattered light is detected, as well as fluorescence emitted from stained cells.

Question 8

What does forward scatter measure

What does side scatter measure?

Answer 8

Forward scatter (FSC) measures size

* Side scatter (SSC) measures internal complexity

Question 9

Define Gold Standard Test

Answer 9

A gold standard test is usually the diagnostic test or benchmark that is the best available under reasonable conditions. Other times, a gold standard is the most
accurate test possible without restrictions.

Question 10

Define Specificity

And

What does specificity of 90% mean

Answer 10

a measure of the incidence of negative results in persons known to be free of a disease, that is a ‘true negative’ TN

90% of the non diseased people dont have the disease. 10% of the people who dont have the disease would have been diagnosed as having the disease based on results (false pos)

Question 11

Define Sensitivity

What does a sensitivity of 90% mean

Answer 11

a measure of the incidence of positive results in patients known to have a condition that is ‘true positive’ (TP)

A sensitivity of 90% implies that only 90% of the people known to have the disease would be diagnosed as having it on the basis of the test alone, 10% would be false negatives

Question 12

Define Positive Predictive value (PPV)

Answer 12

The proportion of positive results in statistics and diagnostic tests that are true positive results.

Question 13

Define Negative Predictive value (NPV)

Answer 13

The proportion of negative results in statistics and diagnostic tests that are true negative results.