ANSC20003 Chap.8 ELISA and Antibody Based Diagnostics
ELISA and Antibody Based Diagnostics
Three things a diagnostic test can measure
A diagnostic test has two jobs: to pick up what is abnormal, and to mark out where normal stops. In animal health the measurable quantities fall into three groups: a physiological or production parameter such as a white cell count or milk volume, the pathogen itself, and the host's antibody response to it.
Antibody-based methods sit in the third group, and their strengths and weaknesses all follow from that choice.
The structural facts an assay depends on
Antibodies are produced by B cells and are built from heavy and light chains, each with variable and constant domains, with the variable domains supplying specificity. The binding site is formed from both chains, while the heavy chain determines the isotype.
Recognition works on three-dimensional structure through complementary charge, hydrophobicity, conformation and hydrogen bonding, so a diagnostic antigen has to be correctly folded to retain its epitope.
Antibodies are large and hydrophilic and cannot cross cell membranes, so they reach only exposed epitopes.
The isotype is the clock
A primary exposure induces IgM with some IgG following, while a secondary exposure produces a faster and higher IgG response. IgM therefore points at a very recent infection and IgG at a past or chronic one.
Three properties make antibody results awkward: a response takes one to two weeks to become detectable, so an early negative is uninformative; antibody persists for months or years after clearance, so a positive is evidence of exposure; and cross-reaction with antigenically similar proteins is possible.
Two assays, one sequence
An indirect assay coats the well with antigen and measures antibody in the sample, reporting a titre.
A sandwich assay coats the well with a capture antibody and measures antigen, reporting a concentration. Everything else is shared: block, add sample, add an enzyme-conjugated antibody, add substrate and read the colour, washing between every step. In a sandwich assay the capture and detection antibodies must bind different parts of the antigen so they do not compete, which is why monoclonals are usually used there.
What this chapter covers
- 01
The three quantities a diagnostic test can measure, and what each answers
- 02
Antibody structure, isotypes, and the paratope and epitope relationship
- 03
Why an assay antigen must be correctly folded and why antibodies reach only exposed epitopes
- 04
Response kinetics, and what an isotype says about the timing of an infection
- 05
The indirect and sandwich assays, step by step, and what each one reports
- 06
Choosing between them from infection dynamics rather than from the laboratory
- 07
Monoclonal against polyclonal antibodies, and the other antibody-based methods
Interpreting a negative result four days into illness
- 1Explain why a negative is expected at this stage.
- 1State what the result does establish.
- 1Name a method that fits the timepoint and say why.
Key terms
- Epitope
- The small patch on an antigen that one particular antibody or lymphocyte receptor reads.
- Paratope
- The part of an antibody that binds the antigen, formed from both the heavy and the light chain.
- Antibody titre
- The measured level of a specific antibody in a sample, and the output of an indirect plate assay.
- Monoclonal antibody
- An identical antibody preparation recognising a single epitope, derived from one immortalised B cell.
- Lateral flow test
- A strip assay in which capillary movement carries labelled antibody and bound analyte to a test line, giving a visible, non-quantitative result.
ELISA and Antibody Based Diagnostics FAQ
What does a positive serological result actually establish?
That the animal has been exposed to the agent, or to something antigenically similar. It does not establish current infection, because antibody persists for months or years after the infection has cleared, and it does not establish that the animal is shedding.
How can a test tell a recent infection from an old one?
By isotype. A primary exposure induces IgM first with some IgG following, so IgM indicates a very recent infection in the order of weeks. A secondary exposure produces a faster and higher IgG response, so IgG points to a past or chronic infection.
What decides whether to run an indirect or a sandwich assay?
The infection dynamics rather than the laboratory. Whether the target is antibody or the pathogen, whether the pathogen is detectable directly and in which fluid, how long antibody remains detectable in that species for that agent, and whether the test will run in the field or in a specialised laboratory.
Why are monoclonal antibodies preferred in a sandwich assay?
Because the capture and detection antibodies have to bind different parts of the antigen without competing for the same epitope. Monoclonals recognise a single epitope each, which makes that separation controllable, and they also give consistency between production batches.
Exam move
Learn the two assay sequences as one sequence with a single substitution, since that is how they are most reliably recalled under time pressure. Attach the three awkward properties of antibody results to worked scenarios rather than memorising them as a list, because questions here are almost always scenarios.
Practise saying what a result does not establish, since both the mid semester and the final papers ask for that explicitly, and keep the one to two week detection window as an exact figure you can quote.
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