Immunochemical Techniques

Question 1

Immunochemical techniques definition

Answer 1

Biochemical techniques that employ antibodies or antibody related reagents for the selective determination of an analyte in a sample
Based on antigen-antibody rxns

Question 2

2 classifications of immunochemical techniques

Answer 2

1. assays using non labelled reagents - immunoelectrophoresis
2. assays using labelled reagents - enzyme immunoassays

Question 3

Advantages of immunochemical techniques

Answer 3

1. highly specific as Ag & Ab binding
2. sensitive
3. easily automated
4. good reproducibility
5. can be qualitative & quantitative
6. wide scope - all clinical disciplines, research, pharma etc

Question 4

Immunoassays using labelled reagents can be one of two things…

Answer 4

1. Homogenous
2. Heterogenous

Question 5

Homogenous immunoassay

Answer 5

Does not require separation of bound Ab-Ag from the free components

Question 6

Heterogenous immunoassay

Answer 6

Does require the separation of bound Ab-Ag complex from free components

Question 7

Heterogenous immunoassays can be either….

Answer 7

Non competitive or competitive

Question 8

Immunoassay requirements

Answer 8

• Antibodies - polyclonal/monoclonal
• Signal generating labels
• Separation matrices

Question 9

What does the success of the immunoassay depend on

Answer 9

The use of the right antibody

Question 10

Examples of signal generating labels

Answer 10

Radioisotopes, enzymes, fluorescent tags/probes, chemi luminescent probes

Question 11

Steps in assay formulation of heterogenous non competitive (sandwich IA)

Answer 11

1. Excess of primary antibody immobilised to solid support
2. Wash to remove unbound or loosely bound Ab
3. Add sample/control/standard. Analyte binds to the primary Ab. Fractional occupancy is directly proportional to the analyte.
4. Wash to remove unbound material
5. Add excess of secondary antibody, labelled w a reporter. This antibody directed against a different epitope on the analyte
6. Wash to remove unbound secondary antibody
7. Measure the signal. Signal directly proportional to concentration of analyte

Question 12

Steps in assay formulation of heterogenous competitive

Answer 12

• limited fixed amount of antibody immobilised to a solid support
• sample/standard/control is co-incubated w a limited fixed amount of reporter labelled analyte
• competition between sample/standard/control analytes & labelled analyte for fixed limited number of antigen binding sites on the antibody
• law of mass action - the entity present in highest conc has higher occupancy
• wash to remove unbound material
• measure the signal
• measured signal is indirectly proportional to the conc of test analyte

Question 13

Steps in assay formulation of homogeneous competitive

Answer 13

• limited fixed amount of Ab immobilised to a solid support
• sample/standard/control is co-incubated w a limited fixed amount of reporter labelled analyte
• on incubation the sample/standard/control analyte competes w the labelled analyte for the fixed limited number of antigen binding sites on the Ab
• law of mass action - entity present in highest conc has higher occupancy
• in homogenous assay formulations the binding event between the Ab & labelled antigen results in modulation of the signal - e.g: inactive label when binds or activate signal when binds
• measure signal - directly proportional to test analyte

Question 14

5 reporter labels for IA

Answer 14

1. radioactive labels
2. enzyme labels
3. fluorescent labels
4. luminescent labels
5. miscellaneous (e.g; DNA probes)

Question 15

Advantages of using enzyme labels in immunoassays

Answer 15

• Enzymes are specific in their action
• enzyme substrate rxns produce easily observable, measurable colour

Question 16

what are the most commercially available enzyme immuno assays

Answer 16

enzyme linked immunosorbent assays (ELISAs)

Question 17

Sandwich ELISA method steps

Answer 17

1. Plate coated w capture antibody
2. Sample added, if antigen present it binds to capture antibody
3. Detecting antibody added, bonds to antibody
4. Enzyme linked secondary antibody added & binds to detecting antibody
5. Substrate added & converted by enzyme into a detectable form

Question 18

Requirements of ELISA

Answer 18

1. Immobilised test sample on a solid support - either non specifically (via adsorption to the surface) or specifically (use of capture Ab)
2. Detection Ab added - can be either covalently linked to enzyme or is a secondary Ab added that is linked to the enzyme
3. Wash w detergent to remove unbound material
4. Add substrate to react which produces a visible signal/colour

Question 19

Label - Substrates - Advantages - Disadvantages: Horse radish peroxidase (HRP)

Answer 19

Label: HRP
Substrates: TMB, DAB, ABTS-photometric
Advantages: Cheap, stable, strong signal
Disadvantages: Some chromogenic substrates are mutagenic

Question 20

Label - Substrates - Advantages - Disadvantages: Alkaline phosphatase

Answer 20

Label: Alkaline phosphatase
Substrates: p-nitrophenol phosphate-dephosphorylates molecules
Advantages: v stable, sensitive, safe
Disadvantages: Source material (calf intestine) expensive

Question 21

Label - Substrates - Advantages - Disadvantages: B-galactosidase

Answer 21

Label: B-galactosidase
Substrates; ONPG, CRPG
Advantages: no inherent enzyme activity, good detection limits
Disadvantages: Cost

Question 22

IAs with Fluorescent labels (IFA) - what is fluorescence

Answer 22

Molecules absorb radiant energy & electrons are re distributed

Question 23

IAs with Fluorescent labels (IFA) - Principle of Fluorescence

Answer 23

Light emitted (lambda em) at lower energy and a longer wavelength than the original absorbed energy (lambda exc)

Question 24

IAs with Fluorescent labels (IFA) - What is the difference betweeen lambda exc and lambda em known as

Answer 24

The stokes shift

Question 25

IAs with Fluorescent labels (IFA) - What does a larger Stokes shift mean

Answer 25

Better discrimination & more precise detection

Question 26

IAs with Fluorescent labels (IFA) - what is the quantum yield

Answer 26

The ratio of absorbed to emitted light energy

Question 27

IAs with Fluorescent labels (IFA) - what does the quantum yield depend on

Answer 27

1. Temperature 2. Polarity & pH of solvent 3. Concentration of fluorophore 4. Other quenching effects - molecular O2

Question 28

IAs with Fluorescent labels (IFA) - what are the antibodies conjugated to

Answer 28

Fluorochromes (fluorescent dyes)

Question 29

Luminescent labels - chemiluminescence

Answer 29

Light emission that occurs during the course of a chemical rxn

Question 30

Luminescent labels - bioluminescence

Answer 30

Type of chemiluminescence found in biological systems, due to presence of specific catalytic proteins

Question 31

What are the two major types of application of luminescent labels

Answer 31

a) the use of a component of the chemi/bio luminescence reactions as a label b) the use of the rxn itself to detect a conventional enzyme label