Immunoassay

Question 1

What are antibodies?

Answer 1

• 4 polypeptide chains held together by disulphide bonds
• 2 identical heavy chains, 2 identical light chains
• 5 major classes: IgG, IgM, IgA, IgD, IgE
• Immunoassays almost exclusively uses IgG
• Antibodies are not directed against an entire molecule but to short, overlapping sequences called epitopes

Question 2

What parts of antibodies?

Answer 2

• Antigen binding site
• Variable regions
• Constant regions

Question 3

What are Polyclonal Antibodies?

Answer 3

• Produced in animals by injecting them with an antigen and recovering the antibodies they produce as part of their normal immune response.
• Produce a population of antibodies produced by multiple different B-cell lineages. Antibodies are produced to different epitopes of the original antigen. They can recognise multiple epitopes

Question 4

How are Monoclonal Antibodies produced?

Answer 4

• Produced in tissue culture from a cell line created by fusion of an immortal mammalian cancer cell with a single antibody- producing B cell
• Monoclonal antibodies are specific for a single epitope on the antigen
• Alongside immunoassays, also being used therapeutically e.g. cancer treatments

Question 5

What are differences between polyclonal and monoclonal antibodies?

Answer 5

Polyclonal Antibodies

• Recognise multiple epitopes on one antigen
• Binding to multiple epitopes can provide more robust detection/amplify the signal as antigen is bound by more than 1 antibody
• Can get background signal from non-specific antibodies
• Can get batch-to-batch variability.

Monoclonal Antibodies

• Recognise only 1 epitope on an antigen
• Highly specific, less background and less chance of cross-reactivity
• Large amounts of homogenous, specific antibody can be made
• No or low batch-to-batch variability

Question 6

What is Avidity?

Answer 6

The overall strength of binding of an antibody and its antigen and includes the sum of the binding affinities for all the individual sites. Property of the binder (i.e. the antibody)

Question 7

What is Affinity?

Answer 7

The energy of interaction of a single antibody- combining site and its corresponding epitope on the antigen. Property of the substance bound (i.e. the antigen).

Question 8

Describe Antibody-Antigen kinetics?

Answer 8

• The binding of antigen-antibody is an equilibrium reaction
• Binding of antigen and antibody is by non-covalent, reversible association
• Relationship between antibody and antigen can be simply described by the Law of Mass Action

Question 9

What are factors that affecting Antibody-Antigen binding?

Answer 9

• pH
• Ionic
• Strength
• Temperature.

Question 10

What is the Law of mass action equation?

Answer 10

Ka

[Ag] = [Ab] ⇌ [Ag-Ab]

Kd

[Ag] = antigen concentration [Ag-Ab] = complex

[Ab] = antibody concentration

ka = association rate constant

Kd= dissociation rate constant

Question 11

What is a Sandwich Assay (2-site/non- competitive assays) ?

Answer 11

• ‘Capture’ antibody recognises the analyte of interest.
• A second, labelled antibody is added which recognises a different part of the analyte.
• The level of signal is proportional to the concentration of the analyte in the sample.
• Can also be used to detect an antibody in the sample by coating the solid phase with the antigen to which the antibody in the sample will bind.
• Excess reagent

Question 12

What is Competitive Immunoassay?

Answer 12

• Measurement of small molecules where insufficient space for 2 antibodies to bind
• Uses a single antibody, available in limited amounts (limited reagent assays)
• Uses a labelled ‘tracer’ which is made from the target analyte with the addition of a label that can generate a signal.
• The proportion of tracer that binds to the limited antibody sites is indirectly proportional to the concentration of analyte in the sample.
• Can be simultaneous or seuquential

Question 13

What is a simultaneous and sequential competitive immunoassay?

Answer 13

• Simultaneous: Labelled and unlabelled antigen added together and compete for binding of the antibody. Chance of antibody binding the labelled antigen is inversely proportional to the concentration of unlabelled antigen (i.e. the analyte concentration).
• Sequential: Unlabelled antigen is first mixed with excess antibody and binding allowed to reach equilibrium. Labelled antigen is sequentially added and allowed to equilibrate. After separation the bound labelled antigen is determined and used to calculate the unlabelled antigen concentration. In these methods a larger fraction of unlabelled antigen is bound by the antibody especially at low concentrations. Improves the detection limit.

Question 14

What are components of an immunoassay?

Answer 14

• Separation
• Labels
• Signal generation and detection

Question 15

What is the purpose of sepration?

Answer 15

• Ensures that only the bound fractions remain at the signal generation and detection stage.
• Quality of the separation has direct effects on the quality of the assay – Failure to remove unbound labelled antibody/antigen leads to high background signal and therefore reduced assay sensitivity (especially at low concentrations)

Question 16

How does seperation take place?

Answer 16

Surface-coated solid phases

• Attachment of the primary or capture antibody to a solid phase support – E.g. tubes, microwells, beads and particles

Washing - 2 main purposes

• Removal of unbound label
• Removal of substances that could interfere with signal generation

Improves signal:noise ratio

Question 17

What is the difference between heterogenous and homogenous assays

Answer 17

• Most immunoassays are heterogeneous – Rely on separation of antibody-bound from unbound material so that only the bound generates a signal.
• Homegenous - No separation step e.g. washing. The activity of the label attached to the antigen is directly modulated by antibody binding.

Question 18

What is the purpose of a Label?

Answer 18

• Enables quantification of the analyte
• Different labels can affect the analytical detection limit of the assay

Question 19

What are radioactive labels?

Answer 19

• Label the tracer with a radioactive isotope
• Radioactive isotopes are unstable variants of atoms that spontaneously transform to a more stable state emitting energy which can be measured using a scintillation counter
• Common isotopes: iodine(125I,131I) and tritium(3H)
• Being phased outdue to safety concerns
• Can’t be used on automated platforms

Question 20

What are enzyme labels?

Answer 20

• Most commonly used label 

• Use catalytic properties of enzymes to generate coloured, fluorescent or luminescent compounds from the substrate.
• Can enhance the signal from an assay. Amplification through a single enzyme label which produces many detectable product molecules which enhances assay sensitivity 

• Commonly used: alkaline phosphatase, horseradish peroxidase, glucose-6-phosphate dehydrogenase, β- galactosidase 


Question 21

What is a disadvantage of using enzyme labels?

Answer 21

• Main disadvantage of enzyme labelled assays is that they are susceptible to changes/interferences during the signal generation stage of the assay 


Question 22

What is Fluorophores?

Answer 22

• A fluorophore absorbs light at 1 wavelength and reemits light at a longer wavelength
• Can be affected by background fluorescence e.g. from drug metabolites. (Less of a problem in more modern assays which use lanthanide chelate)

Question 23

What is Chemiluminescence?

Answer 23

• Substances that emit light as part of the chemical reaction e.g. acridinium esters, isoluminol
• Sensitive as the only light generated should come from the reaction and ideally no background light
• Better sensitivity than radioactive and fluorophore labels
• The signal can be further enhanced by addition of another chemical that enhances the light output by several magnitudes 


Question 24

How does Detection take place?

Answer 24

• Spectrophotometer - colourimetric
• Luminometer - chemiluminescence
• Fluorimeter - fluorescence
• Nephelometer
• Turbidimeter

Question 25

What is Nephelometry/Turbidimetry?

Answer 25

* **Turbidimetry:** light scattering that occurs with turbidimetry means that the light measured at the detector which is 180° from the incident light beam is reduced when analyte concentration is higher. * **Nephelometry:** detection of light scattered or reflected towards a detector that is not in the direct path of the transmitted light.

Question 26

What is Enzyme-Linked Immunoassay?

Answer 26

**Heterogeneous assay** * One of the reaction components is non-specifically adsorbed/covalently bound to the surface of the solid phase e.g. microtiter plate, magnetic particle, plastic bead (Allows separation of bound and free components) * Add sample and allow it to bind to the solid-phase antibody. * After washing, enzyme-labelled antibody is added and forms a ‘sandwich’ * Wash away unbound antibody and add enzyme substrate. * The amount of product is proportional to the concentration of antigen. * Can use ELISA to detect antibodies in a sample by having the antigen adsorbed onto the solid-phase.

Question 27

What is Enzyme multiplied immunoassay technique (EMIT)?

Answer 27

* Antigen that is covalently attached to enzyme (G6PD) in a position near the substrate binding site * When antibodies against the analyte bind to the enzyme-linked antigen the active site is sterically hindered and enzyme activity in inhibited. * If free antigen is present within the sample it will compete for the antibody, displacing the enzyme- linked antigen and restoring enzyme activity. * Enzyme activity is therefore directly proportional to the amount of free analyte in the sample. * Measure NADP+ spectrophotometrically at 340nm Commonly used for measurement of drugs

Question 28

What is Fluorescence Polarised Immunoassay (FPIA)?

Answer 28

Homogenous fluoroimmunoassay * Uses a fluorescently labelled analogue of the analyte. Different polarisation signal when the labelled analyte is bound to the antibody. Signal is generated when the labelled analyte is bound to the antibody * Analyte in the sample will compete for binding to the antibody * Signal generation is inversely proportional to the analyte concentration of the sample. * Widely used in the measurement of drugs and small molecules * Doesn’t require separation or wash steps

Question 29

What is Microparticle capture enzyme immunoassay (MEIA)?

Answer 29

* Analyte is captured by antibody-coated beads (similar to ELISA except ELISA is coated wells). * Beads incubated with an anti-analyte antibody, labelled with an enzyme and suitable substrate * Beads separated e.g. with a magnet and collected for analysis * The fluorescent or chemiluminescent product is proportional to the amount of analyte in the sample

Question 30

What is Chemiluminescent magnetic microparticle immunoassay (CMIA)?

Answer 30

* Similar to MEIA but uses a chemiluminescent label conjugated to the antibody or antigen that produces light when combined with its substrate * Higher sensitivity than MEIA

Question 31

What is Cloned enzyme donor immunoassay (CEDIA)?

Answer 31

* An enzyme is engineered into 2 inactive fragments: An enzyme donor conjugated to an analogue of theanalyteofinterest and an enzyme acceptor * When the 2 fragments associate the enzyme converts a substrate into a coloured product, however binding of the enzyme donor to the antibody inhibits reassembly and no active enzyme is formed. * If analyte is present in solution it will compete with the enzyme donor-analogue for limited antibody binding sites so that free labelled analogue will bind to the enzyme acceptor generating a colorimetric signal which is directly proportional to the amount of analyte in the sample.

Question 32

What is meant by calibration and standardisation?

Answer 32

* Relationship between the instrument signal and the concentration of analyte in the sample * Established by measurement of samples with known quantities of the analyte- **calibrators** * Where possible calibration material should be traceable to a certified reference material- **standardisation**

Question 33

What can cause Pre-analytical interference?

Answer 33

Pre-analytical * Lipaemia (particularly neph or turb assays) * Haemolysis * Icterus * Hormone binding proteins * Anti-coagulants * Sample storage * Auto-analyte antibodies

Question 34

What is the high dose hook effect?

Answer 34

* Occurs at very high analyte concentrations * Sandwich assays, especially when signal and capture antibody are added simultaneously are particularly vulnerable. * Limited binding sites on the solid phase antibody * Results may be inappropriately low and may even hook back inside the reference range. * Specimens susceptible to hooking should be run on dilution.

Question 35

What are Heterophillic antibodies?

Answer 35

* Natural antibodies and autoantibodies against poorly defined antigens. * Non-competitive interference – bind to conjugate, enzyme or other parts of the detection system * Can act differently in different assays * Can give false positive results by forming a ‘bridge’ between capture and conjugate antibodies in sandwich assays. * False negative results can occur if the heterophilic antibodies block formation of the antibody-antigen complex.

Question 36

How is the effect of Heterophilic Antibodies dampened?

Answer 36

Manufacturer’s try to combat the problem by adding blocking agents into their assays but doesn’t block all

Question 37

How do Blocking agents work?

Answer 37

Heterophilic blocking tubes * Incubate the sample with the reagent which binds to heterophilic antibodies. Sample is then re-assayed to assess whether heterophilic antibodies are present. Result produced is not reportable but gives an idea as to whether the original result was correct.

Question 38

What is Huamn Anti Mouse Antibodies?

Answer 38

* Produced as part of the normal immune response to the administration of foreign protein antigen e.g. mouse monoclonal antibodies or in those working with animals. Monoclonal antibody therapy examples: Infliximab (autoimmune disorders), Herceptin (breast cancer), Avastin (colorectal cancer) * Anti-reagent antibodies, capable of binding to animal immunoglobulins such as those used in immunoassay

Question 39

How can Rheumatoid factor cause interference?

Answer 39

* Rheumatoid factor is an IgM autoantibody found in most patients with rheumatoid arthritis as well as other diseases * Interacts with the Fc region of IgG reagent antibodies * Can cause false positive and false negative results

Question 40

How can Biotin cause interference?

Answer 40

* High levels of biotin (vitamin B7) can be found in dietary supplements and is taken by some patients with MS * Interference in assays using biotin-streptavidin link * Warning has been issued by the FDA about its potential for interference of immunoassay results

Question 41

How is testing for interference conducted?

Answer 41

* **Need to suspect it!:** Do the results fit the clinical picture? * **Linear dilution:** Immunoassay interferences don’t typically dilute linearly * **Test by another method** * **Blocking tubes** * **Polyethylene glycol (PEG) precipitation**