ELISA, LAT & IHC

Question 1

What are the principles of indirect ELISA?

Answer 1

• 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

Question 2

Describe bind/coat antigens onto wells of microtitre plate step in indirect ELISA

Answer 2

• 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

Question 3

Describe addition of irrelevant protein in indirect ELISA

Answer 3

• protein must not interfere with assay
• gelatine commonly used
• blocks unbound sites of plate

Question 4

Describe addition of patient antibodies in indirect ELISA

Answer 4

• extract from patient serum beforehand
• if unbound sites not blocked - risk of non-specific binding of patient’s antibody to the plastic

Question 5

Describe washing off excess antibody in indirect ELISA

Answer 5

• 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

Question 6

Describe adding secondary antibody and substrate in indirect ELISA

Answer 6

• 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

Question 7

Describe a positive indirect ELISA result

Answer 7

obvious colour change, usually green

Question 8

Describe a negative indirect ELISA result

Answer 8

no colour or background colour - antigens can have a slight green hint

Question 9

Give an example of a common substrate

Answer 9

ABTS - green

Question 10

How are the colour changes detected in ELISA?

Answer 10

• plate readers common
• human eyes too much error

Question 11

Describe the example of schistosome and ELISA diagnosis

Answer 11

• 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

Question 12

Describe improvements to SEA ELISA

Answer 12

• 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

Question 13

What are the principles for a diagnostic test?

Answer 13

• sensitivity
• specificity
• usually a trade off between sensitivity and specificity

Question 14

Describe sensitivity

Answer 14

• no. people testing positive as a proportion of people that had an infection
• equation is no. true positives / (no. true positives & false negatives)

Question 15

Describe specificity

Answer 15

• 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)

Question 16

What are the different types of ELISA?

Answer 16

• direct - 1 conjugated antibody
• indirect - 1 conjugated, 1 unconjugated antibody
• sandwich - 2 antibodies used to sandwich the antigen
• competitive

Question 17

Describe a competitive ELISA

Answer 17

• competitive binding process executed by original sample, add in inhibitory antigen and try to outcompete original antigen
• good for impure samples
• high specificity

Question 18

Describe LAT

Answer 18

• 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

Question 19

What are the advantages of ELISA?

Answer 19

• multiple samples/replicates per plate - high throughput
• high sensitivity
• high specificity
• flexibility
• quantitative

Question 20

What are the disadvantages of ELISA?

Answer 20

• laborious, can take up to 2 days
• specialised equipment
• trained personnel
• cold chain - storage of antibodies
• temporary readouts due to enzyme/substrate reactions

Question 21

Describe the advantages of LAT

Answer 21

• rapid
• bedside/pen side diagnosis - common in veterinary
• requires limited specialist equipment/training

Question 22

What are the disadvantages of LAT?

Answer 22

• lower sensitivity
• misinterpretation of results
• qualitative/semi-quantitative

Question 23

Describe immunohistochemistry (IHC)

Answer 23

• identification of antigens in tissue sections by antibodies
• chromogenic IHC
• fluorescent IHC

Question 24

Describe chromogenic IHC

Answer 24

• antibody conjugated to an enzyme
• catalyses colour change
• e.g., smooth muscle actin in breast tissue stained using anti-actin antibodies

Question 25

Describe fluorescent IHC

Answer 25

• antibody tagged with a fluorophore
• e.g., corneal epithelium stained using anti-cytokeratin 3 & 12 antibodies

Question 26

What are the applications of ELISA, LAT & IHC?

Answer 26

• veterinary and medical diagnostics
• extensively used in disease staging and prognosis
• transfusion medicine
• forensic & autopsy pathology applications
• fundamental and applied research
• vaccine development/response