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Analytical methods > Immunoassays > Flashcards

Immunoassays Flashcards

1
Q

How are antibodies used in immunoassays?

A

Uses antibodies as their key reagents

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2
Q

What are polyclonal antibodies? how are they produced? what makes them unique?

A

Produced by injecting animals with an antigen and recovering the antibodies they produced.

Produce a population antibodies by multiple different B-cell lineages
- Antibodies are produced to different epitopes of the original antigen

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3
Q

Describe monoclonal antibodies.

A
  • Produced in tissue culture form cell lineage created by fusion of immortal mammalian cancer cells with single antibody-producing B cell
  • Monoclonal antibodies are specific for a single epitope on the antigen
  • Alongside immunoassays, also used therapeutically
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4
Q

Discuss the usage of polyclonal against monoclonal antibodies

A

Polyclonal can recognise multiple epitopes compared to mono which only recognises 1.

Polyclonal produces a more robust detection/amplify the signal due to recognition of multiple epitopes

Monoclonal are highly specific, less background and less chance of cross-reactivity compared to polyclonal

Polycloncal can get batch to batch variability, compared to monoclonal

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5
Q

What is an antigen in immunoassays?

A

Generally the analyte of interest

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6
Q

Define avidity.

A

Overall strength of binding of an antibody and its antigen and includes the sum of the binding affinities for all the individual sites

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7
Q

Define affinity

A

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

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8
Q

Factors affecting binding:

A

pH, ionic strength and temperature

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9
Q

Properties of antibody making it appropriate for clinical assays?

A

Highly specific, sensitive, manufactured on a large scale, can be automated

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10
Q

Describe a sandwich assay (non-competitive assay)

A

Solid phase- antibody coated which is combined with sample containing analyte. 1. Analyte bind to antibody.

  1. Column is washed.
  2. A labelled antibody is added which bind to analyte-solid phase antibody complex
    - Analyte needs to be big enough to have two binding sites for two antibodies
  3. Wash to remove unwanted labelled antibodies
  4. Measure signal, which is directly proportional to conc of analyte
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11
Q

Describe a competitive assay.

A

Combine solid phase with sample containing analyte together with a labelled analyte antagonist.

  1. Give both time to bind to solid phase
  2. Wash
  3. Measure signal, which is indirectly proportional to analyte concentration. So as the signal increases the lower conc of analyte you have
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12
Q

How does washing improve signal:noise ratio?

A

It removes unbound label and substances with the potential to interfere with signal generation

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13
Q

What is meant by homogenous assay?

A

No seperation step aka washing

Activity of the label attached to the antigen is directly modulated by antibody bindign

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14
Q

State labels with examples.

A

Radioactive - label the tracer with radioactive isotope, e.g. iodine (125, 131) and tritium (3H)

Enzyme labels- use catalytic properties of enzymes to generate colour, fluorescent or luminescent compounds. Can enhance signal due to amplification. e.g. alkaline phosphatase, horseradish peroxidase

Fluorophore - Absorbs at 1 wavelength and reemits light at another e.g. DELFIA

Chemiluminescence - substance emits light part of chemical reaction e.g. acridinium ester

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15
Q

Evaluate chemiluminescence

A

Sensititve as the only light generated should come from the reaction and no background

  • Better sensitivity than radiocative and fluorophore labels
  • The signal can be enhanced by addition of another chemical that enhances the light output
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16
Q

Name some common detectors

A

Spectrophotometer - colourimetric

Luminometer -chemiluminescence

Fluorimeter - fluorescence

17
Q

What is the difference between turbidimetry and nephelometry?

A

Turbidimetry - light scattering that occurs with turbidimetry means that the light measured at the detector which is 180 degrees from incident light beam is reduced when analyte conc is higher

Nephelometry: detection of light scattered or reflected towards a detector that is not in the direct path of the transmitted light

18
Q

Describe Enzyme linked immunosorbent assay (ELISA).

A
  1. Add sample and allow it to bind to solid phase
  2. Wash
  3. Add enzyme labelled antibody to form sandwich
  4. Wash
  5. Add enzyme substrate with detectable change
  6. Measure product - amount of product is proportional to conc of antigen
19
Q

Describe enzyme multiplied immunoassay technique (EMIT)

A
  1. Antigen covalently attached to enzyme in a position near substrate binding
  2. When antibodies against the analyte bind to enzyme linked antigen the active site is sterically hindered and no enzyme activity
  3. Presence of antigen will compete for antibody, displacing the enzyme-linked antigen and restoring enzyme activity.
  4. Enzyme activity is directly proportional to the amount of free analyte in the sample
  5. Measure NADP+ at 340nm
20
Q

Describe fluorescence polarised immunoassay (FPIA)

A

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 (analogue) is bound to the antibody

Signal is inversely proportional to analyte conc of sample

21
Q

Microparticle capture enzyme immunoassay (MEIA)

A

Analyte captured by antibody coated beads. Beads incubated with an anti-analyte antibody, labelled with an enzyme and suitable substrate

  • Beads seperated e.g. magnet
  • Fluorescent or chemiluminescent product is proportional to the amount of analyte in the sample

Similar to elisa except with beads

22
Q

Chemiluminescent magnetic microparticle immunoassay (CMIA)

A

Similar to MEIA but uses chemiluminescent label conjugated to the antibody or antigen that produces light

Higher sensitivity than MEIA

23
Q

Cloned enzyme donor immunoassay (CEDIA)

A

Enzyme engineered into 2 inactive fragments

  • an enzyme donor conjugated to an analogue of the analyte of interetes
  • 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 it competes with enzyme donor-analogue for limited antibody binding sites so that free labelled analogue will bind to the enzyme acceptor generating a colorimetric signal directly proportional to amount of analyte

Very similar to EMIT

24
Q

Define calibrators and standardisation

A

Calibrators are established by measurement of samples with known quantities of an analyte

Standardisation - calibration material traceable to a certified reference material

25
Q

Name assay interferances pre-analytical

A

Lipaemia (nephelometry), haemolysis (colour), icterus (colour), hormone binding proteins, anti-coagulants, sample storage, auto-analyte antibodies

26
Q

What is the high dose hook effect?

A

Occurs at very high analyte concentrations.
- Sandwich assay when signal and capture antibody are added together. Not able to form necessary sandwich. So patients with high levels of analyte show low signal - a false negative

27
Q

Can you give an example of high dose hook effect

A

In renal patients with known nephrotic syndrome, where patients albumin levels were seen as low due to hooking.
- Dealth with using both protein and albumin measurements together to detect hooking in albumin results

If hooking happens a dilution may be appropriate to produce a better signal

28
Q

How do heterophilic antibodies interfere with immunoassays?

A

Non-competitive interference - bind to conjugate, enzyme and other parts of the system

  • Try and combat these by blocking agents into their assays but doesnt block all
  • Can produce FP and FN

The heterophilic antibodies can cross-link causing positive interference (meaning antibodies link without having the appropriate analyte present) or block antibody (simply block stationary antibody from binding analyte) causing negative interference

29
Q

How to remove heterophilic antibody interference?

A
  • 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
  • Results produced is not reportable but gives an idea as to whether original results was correct
30
Q

In what scenario is heterophilic blocking tubes necessary?

A

E.g. 60 yo male referred to investigate low serum testosterone and significant elevated LH/FSH with decreased libido and sweating. Also treated with thyroxine and hyperthyroidism.

Intially treated as primary gonadal failure (low testosterone, high LH/FSH), but not the case.

Analytical methods (15 decks)

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