Lecture 7: Laboratory Techniques

Question 1

What are polyclonal antibodies

Answer 1

Contains a mixture of antibodies, which bind to the same antigen but may attach to different epitopes of the antigen
Made by different B cells as each different antibody type is made by a different B cell

Question 2

Polyclonal antibody process

Answer 2

1. Inject the antigen into the selected animal (goat, sheep, rat etc)

2.antigen activated B cells

3. Clones of memory B cells
   Clones of plasma B cells
4. Secrete polyclonal antibodies from different B cells
5. Polyclonal antibodies are taken out of the animals body

Lecture slide for picture diagram

Question 3

What are Monoclonal antibodies

Answer 3

Homogeneous population of antibodies which are produced by a single clone of plasma B cells

highly specific for a single epitope

Question 4

Monoclonal antibody process

Answer 4

1. Mouse is injected with single epitope type
2. Spleen cells are collected and form a suspension where B cells are found in this suspension. The B cells fuse with added myeloma cells. This fusion forms hybridomas
3. Culture hybridomas in HAT medium and selection for positive cells
4. Harvest monoclonal cells

Question 5

Differences between Monoclonal and polyclonal antibodies

Answer 5

Monoclonal
1.Production is expensive
2. Production is timely
3. Used as therapeutic drugs
4. Made from the same B cell plasma cell
5.Needs hybridomas
6. Homogenous antibody mixture
7. Interact with the same epitope on the single antigen
8. Less cross reactivity

Polyclonal:
1. Production is cheap
2.Production is LESS timely
3. Used in general research
4. Made from different B cell plasma cell
5. Doesnt need hybridomas
6. Heterogenous antibody mixture
7. Interact with different epitopes on the single antigen
8.High cross reactivity

Question 6

Signals can be detected..

Answer 6

visually
electronically
chemically
physically

Question 7

Labels can be…

Answer 7

Radioactive
Enzyme e.g. HRP, AP
Light producing substance

Question 8

The signal is detectable by..

Answer 8

eye
spectrophotometer
luminometer
Fluorometer

Question 9

Draw sandwhich elisa process

Answer 9

Lecture Slide

Question 10

Non-compettive immunoassay spec/sens level
how many steps?
what is proportional to what

Answer 10

provides the highest level of assay sensitivity and specificity

can be 1 or 2 steps (direct and indirect repectively)
2 step offers the highest sensitivity and specificity (due to the wash steps )

the measured labelled analyte (usually antibody), is directly proportional to the amount of antigen present in the sample
The more antigen that is present, the more labelled antibody will bind

Question 11

Draw a competitive elisa process

Answer 11

Lecture slide

When the antigen level in the sample is high, the level of antibody-bound enzyme-labeled antigen is lower and the color is lighter. Conversely, when it is low, the level of antibody-bound enzyme-labeled antigen is higher and the color, darker. The graph above and to the right illustrates the correlation between absorption and antigen levels in samples.

Question 12

Compettive immunoassay description

Answer 12

unlabelled analyte (usually antigen) in a test sample is measured by its ability to compete with labelled antigen in the immunoassay

the unlabelled antigen blocks the ability of the labelled antigen to bind (because the binding site of the antibody is already occupied)

less label measured in the assay means more of the unlabelled (test sample) antigen is present

the amount of the test antigen is inversely related to the amount of labelled measured

Question 12

Homogenous vs heterogenous assays and example

Answer 12

Heterogeneous
1. Requires one or more steps
Unbound antibodies/analytes are washed away
2.Longer than Homogeneous but more precise
Example - ELISA

Homogeneous
1.Does not require separation of analyte of interest from the biomolecules e.g. labelled antibodies used to detect it
2.Usually used for detection of small simple molecules
Example - Emit

Question 13

Fluorescence Polarisation - FPIA
What is it? Absorbs at what light, releases at what light
Rotation patterns?
Polarised Light pattern?

Answer 13

Fluorescein – fluorescent label

Absorbs light energy at 490nm and releases this energy at 520nm as fluorescent light

Rotation of molecules in solution – large molecules rotate slowly in solution: AgF vs Ab-AgF

Polarised light – light waves that are only present in a single plane of space
Ab-AgF rotates slowly – polarised light same plane
AgF rotates quickly – light released in a different plane of space from that in which it was absorbed = unpolarised

Question 14

Draw the process of Fluorescence Polarisation

Answer 14

Lecture Slide

Question 15

Chemiluminescent Immunoassay: What is it?

Answer 15

the generation of electromagnetic radiation as light by the release of energy from a chemical reaction

Question 16

Chemiluminescent Immunoassay: Draw the process with labels

Answer 16

Magnetic particles are tagged with antibodies of interest. Once the sample (serum or whole blood with antigen of interest) and labelled reporter antibody is added, a complex will form which contains magnetic-bound-antibody and antigen of interest which is then bound to reporter labelled secondary antibody (ALP labelled antibody)

A magnet is applied which then holds on to the complex of interest while being separated from any unbound particles (other components and unbound ALP antibodies). When substrate is added, bound ALP labelled antibodies will fluoresce at 461nm which will detect the presence and quantity of antigen of interest in the sample

Lecture Slide

Question 17

Electrochemiluminescence Immunoassay: What is it?

Answer 17

thegeneration of light when stimulated by electricity in the appropriate chemical environment.

Question 18

Purpose of green and red laser

Answer 18

Red lasers indentify the antigen coated microsphere

green laser detects the presence or absence of a fluorchrome

Question 19

Multiplex assay process diagram and descriptions of steps

Answer 19

1. Patient sample loaded into reaction vessel
   2.Sample diluent and beads are added, mixed and incubated (37degrees)
2. Wash (3X)
   4.Add conjugate and incubate (37degrees)
   5.Wash (3X)
   6.Beads resuspended in wash buffer
   7.Flow based, dual laser detection

Question 20

Turbidimetry and Nephlometry description

Answer 20

based on the scattering or absorption of light by solid or colloidal particles suspended in solution

When light is passed through the suspension, part of incident radiant energy is dissipated by absorption, reflection, and reaction while remainder is transmitted

Question 21

Draw turbidity and nephlometry

Answer 21

Lecture Slide

Question 22

describe turbidity and Nephlometry

Answer 22

Lecture Slide

Turbidimetry: Measures the cloudiness or turbidity of a solution.
the photodetector is placed in direct line with the incident light and the solution. The light source should emit a wavelength in the near ultraviolet range (290–410 nm). The photodetector must be aligned with the incidence source and collect the beam after passage through the solution, therefore measuring a decrease in light intensity that occurs as a result of the combination of reflection, absorption, or scatter of incident light.

Nephlometry: Is defined as the detection of light energy scattered or reflected toward a detector that is not in the direct path of the transmitted light.

Question 23

Factors that impact performance

Answer 23

Calibrations and Controls

Calibrator traceability

Assay interferences