Immuno. III - Week 7 (Immunoassay)

Question 1

Define Immunoassay

Answer 1

A test that uses antibody and antigen complexes as a means of generating a measurable result

Question 2

Define polyclonal antibodies

Answer 2

a heterogenous mixture of antibodies which bind to the same antigen but may attack to different epitopes of the antigen

Question 3

How are polyclonal antibodies made?

Answer 3

Produced by sheep, rabbits, and goats as a defence mechanism when exposed to an antigen

Question 4

Define Monoclonal antibodies

Answer 4

A homogenous population of antibodies which bind to a specific single epitope and are produced by single clone of plasma B cells

Question 5

How are monoclonal antibodies made?

Answer 5

Hybridoma and mouse myeloma cell lines
Hybridoma tumor cells produce many copies of the same antibody

Question 6

What are the four immunoassay methodologies?

Answer 6

1. Non competitive
2. Competitive
3. Homogenous
4. Heterogenous

Question 7

Describe the non competitive immunoassay process

Answer 7

1. 1 to 2 steps
2. Analyte in between two highly specific antibody reagents
3. the labelled antibody is directly proportional to the amount of antigen present in the sample

Question 8

Describe the Competitive immunoassay process

Answer 8

1. 1 to 2 steps
2. Unlabelled analyte (patient sample) and labelled antigen compete in the sample.
3. Unlabelled analyte (patient sample) blocks labelled antigen from binding to the sample
4. More unlabelled analyte (patient sample) measured means less label antigens

Question 9

Describe Heterogenous Immunoassay Methology

Answer 9

1. 1 or more steps
2. More precise than homogenous
3. Example ELISA

Question 10

Describe Homogenous Immunoassay Methology

Answer 10

1.Used to detect small simple molecules
2. Does not require separation of analyte of interest from biomolecules
3. Example Emit

Question 11

List five detection techniques of an immunoassay

Answer 11

1. Fluorescence Polarisation (FPIA)
2. Chemiluminescent immunoassay (CMIA)
3. Electrochemiluminescenty immunoassay (ECLIA)
4. Multiplex
5. Turbidimetry and Nephlometry

Question 12

Describe the principle of Fluorescence Polarisation Immunoassay

Answer 12

1. Antigen has Fluorescent label called Fluorescein
   2.Light energy absorbed at 490 nm
2. energy released at 520 nm
3. Presence of antibody binds to labelled antigen, rotating slowly leading to polarised light in same plane
4. Lack of antibody rotates quickly leading to unpolarised light released in a different plane

Question 13

Describe the principle of Chemiluminescent Immunoassay

Answer 13

1. Labels used: Patented acridinium derivative - Chemiflex, Patented acridinium ester - AE
2. Generation of electromagnetic radiation as light by the release of energy from a chemical reaction

Question 14

Describe the principle of Electrochemiluminescence Immunoassay

Answer 14

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

Question 15

Factors that impact performance if detection techniques

Answer 15

1. Calibrations and controls
2. Calibrator traceability
3. Assay interferences

Question 16

Describe the multiplex assay procedure

Answer 16

1. Beads with attached antibodies mixed with sample.
2. Conjugate added
3. Bead resuspended in wash buffer

Question 17

Define Turbidimetry and Nehplometry

Answer 17

Scattering or absorption of light by solid or colloidal particles suspended in solution