Immunology practical Flashcards
(14 cards)
DO CASE 3; doggo
answers in book
Name the main problem with blue tongue diagnostics
there is significant cross-reactivity (when the epitope is shared or similar to BT) with BT and epizootic haemorrhage disease virus (EHD) which leads to potential false positives.
Discuss indirect ELISA
Indirect ELISA; there is lack of specificity due to the reactions with ag impurities or closely related diseases which can give non specific reactions (EHD and BT) therefore indirect ELISA can’t distinguish bw true anti-BTAb and anti-EHD ab bound to ag the greater the colour change the more ab present in the sample
List the steps for non competitive indirect ELISA for an animal which is reactivity (4)
steps for non competitive indirect ELISA animal is reactivity 1) coat wells with ag, there is extra step which, there is a block solution to stop it from bonding to anything apart from the wells and wash 2) serum from the animal is added and the ab will bind to ag if there are present in the well and wash 3) add conjugate ab which is conjugated (combines) to an enzyme its an anti species ab (from an animal which has ab for the specific ag you inject ab into another animal which will think the ab are foreign and this will cause ab to be created) and wash 4)Add the substrate, they will be cleaved (broken apart) and there will be a colour change to green because ab are present, for the colour change everything MUST be present
List the steps for a nonreactor animal for non competitive indirect ELISA
steps for a nonreactor animal for non competitive indirect ELISA 1. coat the wells with ag and wash 2. add serum form animal, there is no BT virus so the ab don’t bind and will be washed away and wash 3. there is nothing for he conjugate to bind to and therefore it will be washed away and wash 4. there is no colour change
Briefly discuss competitive ELISA
competitive ELISA will reduce the issue of non-specific reactions by using a highly specific mouse indicator monoclonal Ab (mAb) that only recognises a specific BT Ag to compete with anti-BT Ab in the test serum. The higher the ab concentration in the sample the weaker the signal, the weaker the colour signal
List the steps for competitive ELISA (4)
competitive ELISA step 1) coat the wells with ag and wash step 2) Ab which are competitive bind to the ag and the unbound ab will be washed away add the animals serum and wash step 3) Add conjugate, it won’t able to bind and will be washed away and wash step 4) add substrate, if there is no colour change this means there are ab present in the serum if the conjugate and ab is present= colour change because there was no ab from the serum to compete for the binding site
what isotopes do ELISAs test for
isotopes which ELISA test for IgM (usually 1st to appear) IgG (appears 14 days after the infection and when the host is infected with same pathogen)
What does it mean if you do an ELISA and there is no IgM or IgG
What does it mean if you do an ELISA and there is no IgM or IgG there has been n exposure or its very early in the infection stage
What does it mean if you do an ELISA and there is only IgM and no IgG
What does it mean if you do an ELISA and there is only IgM and no IgG its only early in the infection stage
What does it mean if you do an ELISA and there is IgE
What does it mean if you do an ELISA and there IgE Its a hypersensitivity or parasite pathogen and there would be IgM first and then a class switch to IgE
What does it mean if you do an ELISA and there is only IgG
What does it mean if you do an ELISA and there is only IgG its a secondary immune response, persistent infection, late in the infection stage or the animal has been vaccinated
Determine if the following is positive or negative for BTV 1. non competitive ELISA;+++, competitive ELISA - 2. non competitive ELISA;-, competitive ELISA + 3. non competitive ELISA;++, competitive ELISA ++
- non competitive ELISA;+++, competitive ELISA - BTV is +ve 2. non competitive ELISA;-, competitive ELISA + BTV is -ve 3. non competitive ELISA;++, competitive ELISA ++ BTV is -ve but has been injected with similar virus like EHD
What does a positive and negative result look for the snap test
