animal health - immunochemical techniques

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antibody

• immunoglobulin protein molecules capable of specifically recognise/bind to the antigen

antigen

• foreign substance that initiates an immune response

principles of the technique

• antigen + antibody = antigen-antibody complex

antibodies

• immunoglobulins are glycoproteins

• they are produced by B lymphocytes nat in response to an antigen

• IG are abundant in in body fluids of all verts

• are extremely specific + widely use in diagnostics, research + treatment

structure of antibody

• immunoglobulin isotypes (sub classes) - 5 types in mammals

• lgG

• lgM

• lgD

• lgE

• 

antibody classification

• monoclonal v polyclonal

• animal species to raise antibodies

• raised against whole molecule or part of a molecule

monoclonal antibody

• single B cell clone

• highly specific

• tends to be ‘cleaner’

• very consistent from batch to batch

• more likely to get false beg results due to being unable to pick up variation or mutations

polyclonal antibody

• many B cells targeting multiple epitopes

• tends to have more non specific reactivity

• can have very diff affinity batch to batch

• more robust to target changes

polyclonal antibody production

1. inject antigen into rabbit

2. antigen activates B cells

3. plasma B cells produce polyclonal antibodies

4. obtain antiserum from rabbit containing polyclonal antibodies

monoclonal antibody production

1. inject antigen + culture myeloma cells

2. select + grow hybrid cells only

3. separate hybrid cells + allow them to proliferate into clones

4. screen for desired antibody

5. chosen hybridoma is then grown to produce large batches of desires mAB

recombinant antibody production

• generated in vitro using synthetic genes

• essentially produce monoclonal antibodies

• 

techniques available for detecting antigen antibody complexes

• ELIZA

• gel electrophoresis

• western blot

• immunoprecipitation

• spectrophotometry

more techniques available for detecting antigen antibody complexes

• enzyme assays

• immunochemistry

• immunocytochemistry

• flow cytometry

• enzyme linked immunospot ELISPOT

ELISA

• most common used immunological assay

• detects antigen or antibody

• qualitative or quant

• typically bodily fluids used

• sensitive

• specific

• easy

• economical

• can be automated

• safe

types of ELISA

1. direct ELISA

2. sandwich ELISA

3. competitive ELISA

4. indirect ELISA

direct ELISA

• screening antigen

• the sample is immobilized directly on the plate

• conjugated detection antibody binds to the target antigen

sandwich ELIZA

• screening antigen

• 2 specific antibodies (matched pair) are used to sandwich the antigen

1. capture antibody - coat the plate w antibodies which becomes attached to the antigen in the sample

2. conjugated antibody - bind to the capture antibody + antigen complex

competitive ELISA

• screening antigen

• capture antibody is coated on a microplate

• sample (target antigen) + conjugated antigen is added simultaneously into plate

• substrate is added into the reaction plate + terminate w stop reagent

• high level of antigen - low colour

Indirect ELISA

• screening antibody

• a target protein is immobilized on the surface on microplate

• sample (containing antibody) is added onto the plate

• the antigen + antibody complex formed is detected by a conjugated antibody (secondary antibody)

importance of inc control samples

• pos control: known sample, usually come w the kit

• neg control: sample from same species - w no/low target molecule

pos control

• known sample, usually come w the kit

neg control

• sample from same species w no/low target molecule

interpretation of ELISA - qual

• visualise colour change

• amount of coloured product is proportional to amount of enzyme-linked antibody

interpretation of ELISA - quant

• read absorbance with spectrophotometer

• prepare standard curve + read off amount of unknown

quantitative ELISA

• ELISA data can be interpretated in comparison to a standard curve ( a serial dilution of a known purified antigen)

• helps to calculate the concentrations of antigen/antibody

• microtiter plate reader - data

applications of ELISA

• disease diagnosis - detect antigen

• heard health monitoring - detect antibodies following vaccination or prev exposure

• special biomarkers - specific protein e.g. haptoglobin