ELISA, LAT & IHC Flashcards
What are the principles of indirect ELISA?
- bind/coat antigens onto walls of microtitre plate
- wash off excess antigen
- add irrelevant protein
- add patient antibodies
- wash off excess antibody
- add secondary antibody and a substrate
Describe bind/coat antigens onto wells of microtitre plate step in indirect ELISA
- typically 96 well plate
- plate has plastic with high affinity for binding immunoglobulins
- different plates will bind better to different antigens, usually protein binding plates are used
Describe addition of irrelevant protein in indirect ELISA
- protein must not interfere with assay
- gelatine commonly used
- blocks unbound sites of plate
Describe addition of patient antibodies in indirect ELISA
- extract from patient serum beforehand
- if unbound sites not blocked - risk of non-specific binding of patient’s antibody to the plastic
Describe washing off excess antibody in indirect ELISA
- important to note all types of antibodies will be present so need to remove antibodies not specific to desired disease
- antibodies left known as primary antibody
Describe adding secondary antibody and substrate in indirect ELISA
- directed against the 1st antibody
- coupled to enzyme or fluorophore to allow for visual detection
- raised for human IgG - able to recognise and bind to
Describe a positive indirect ELISA result
obvious colour change, usually green
Describe a negative indirect ELISA result
no colour or background colour - antigens can have a slight green hint
Give an example of a common substrate
ABTS - green
How are the colour changes detected in ELISA?
- plate readers common
- human eyes too much error
Describe the example of schistosome and ELISA diagnosis
- soluble egg antigen (SEA) ELISA used
- test validated using eggs present in patient’s faeces/urine that are known to have the disease
- negative control of people with exposure but not infected
- control of avg. blood donors
- negative cutoff value - mean optical density of all tropical negative patients plus 3 standard deviations
Describe improvements to SEA ELISA
- principles of antigen-antibody binding to detect immunodominant antigens in SEA
- stacking gel on top and running gel on bottom
- negative electrode at top - sodium dodecyl sulphate - detergent with negative charge, dragging molecules down to positive electrode
- transfer gels to nitrocellulose paper
- add patient serum to see which antibodies it binds to
What are the principles for a diagnostic test?
- sensitivity
- specificity
- usually a trade off between sensitivity and specificity
Describe sensitivity
- no. people testing positive as a proportion of people that had an infection
- equation is no. true positives / (no. true positives & false negatives)
Describe specificity
- no. people who give negative results as a proportion of total no. people that don’t have the infection
- no. true negatives / (no. true negatives & false positives)
What are the different types of ELISA?
- direct - 1 conjugated antibody
- indirect - 1 conjugated, 1 unconjugated antibody
- sandwich - 2 antibodies used to sandwich the antigen
- competitive
Describe a competitive ELISA
- competitive binding process executed by original sample, add in inhibitory antigen and try to outcompete original antigen
- good for impure samples
- high specificity
Describe LAT
- Latex Agglutination Test
- antigens coated onto latex beads of pre-determined size
- if patient has antibodies against antigen - agglutination of latex beads
- if patient has no antibodies - no agglutination
What are the advantages of ELISA?
- multiple samples/replicates per plate - high throughput
- high sensitivity
- high specificity
- flexibility
- quantitative
What are the disadvantages of ELISA?
- laborious, can take up to 2 days
- specialised equipment
- trained personnel
- cold chain - storage of antibodies
- temporary readouts due to enzyme/substrate reactions
Describe the advantages of LAT
- rapid
- bedside/pen side diagnosis - common in veterinary
- requires limited specialist equipment/training
What are the disadvantages of LAT?
- lower sensitivity
- misinterpretation of results
- qualitative/semi-quantitative
Describe immunohistochemistry (IHC)
- identification of antigens in tissue sections by antibodies
- chromogenic IHC
- fluorescent IHC
Describe chromogenic IHC
- antibody conjugated to an enzyme
- catalyses colour change
- e.g., smooth muscle actin in breast tissue stained using anti-actin antibodies
