immunoassays Flashcards
definition of immunoassay
assays that employ antibodies to detect and quantify a specific analyte (usually a biomolecule)
what are the key components in immunoassays
antibodies against the biomolecule of interest
- polyclonal or monoclonal antibodies may be used and they usually raised in an animal
what does anti-mouse insulin goat antibody mean?
mouse insulin is injected into the goat and hence is the antigen
goat produced antibodies that targets the mouse insulin (so anti-mouse)
what are the means of detection of antigen-antibody binding in immunoassays?
which means of detection are used in commercialised imunoassays?
- marker
- radioimmunoassays using radioactive labels
- enzyme immunoassays using enzymes - agglutination/hemagglutination
EIA and agglutination
state the principle of solid phase enzyme immunoassays ( steps)
- antibodies immoblised on solid surface such as the wells
- then incubate with known amount of enzyme-linked antigens together with sample-containing antigens (this is obtained from the user)
- there is now a competition between enzyme-linked and unlinked antigen to bind to the antibodies
- after incubuation, wash away bound antigens (linked and unlinked) with buffer solutions such as phosphate buffered saline (PBS). this is known as the washing step.
- add enzyme substrate and determine enzymatic activity by measuring absorbance of coloured product formed
what is the importance of washing step?
if not, the unbound antigen will lead to inaccurate results collected?
what happens if there are more free antigens than the enzyme linked antigens?
greater the amount of antigen present in the sample, the less enzyme-linked antigen will bind to the solid phase –> less enzymatic activity will be detected
name the types of enzyme-linked immunosorbent assays (ELISA)
- direct ELISA
- indirect ELISA
- sandwich ELISA
- competitive ELISA
describe direct elisa
- antigen from patient sample is fixed and immobilised onto the solid surface
- (optional) washing step –> to wash off unbound antigen
- enzyme-linked antibodies present in the ELISA will bind to the antigen
- washing step –> to wash off unbound enzyme-linked antibodies
- substrate specific to the enzyme is added. then gives off coloured product
colour produced is directly proportional to the amount of antigen that is present in the sample
describe indirect ELISA
employs antigens to detect the presence of a specific antibody in a sample
- antigen from sample immobilised on surface
- add in a primary antibody that is not enzyme linked –> purpose is to bind to the antigen on the surface
- may have washing stage to wash of antibody
- secondary enzyme-linked antibody added –> bind to the FC domain of primary antibody
- may have washing stage
- substrate specific to the enzyme is added. then gives off coloured product
colour produced is directly proportional to the amount of antigen that is present in the sample
describe sandwich elisa
employs antibodies to detect the presence of a particular antigen in a sample
1. immobilised primary antibody on the solid surface that is specific to one of the epitope of antigen
2. washing stage
3. introduce antigen from patient
4. washing stage
5. introduce secondary antibody that is specific to another epitope of antigen
6. washing stage
7. introduce enzyme-linked antibody that is specific to the FC domain of the secondary antibody
8. washing stage
9. substrate specific to the enzyme is added. then gives off coloured product
colour produced is directly proportional to the amount of antigen that is present in the sample
describe competitive ELISA
similar to sandwich ELISA
1. immobilised antibody on the solid surface that is specific to antigen
2. washing stage
3. introduce antigen from patient
4. washing stage
5. introduce lab developed enzyme-linked antigen (inhibitor antigen)
(they compete with antigen in sample for antibody binding)
6. washing stage
9. substrate specific to the enzyme is added. then gives off coloured product
colour produced is directly proportional to the amount of antigen that is present in the sample
if there are more antigen in patient sample, these antigen will preferentially bind to antibody instead of the lab developed enzyme-linked antigen and hence less colour is observed
compare the difference between direct, indirect, sandwich and competitive ELISA (look at which is immobilised on the surface, how many antigens, how many antibodies)
direct: antigen immobilised on surface
indirect: antibody
sandwich: antibody
competitive: antibody
direct: 1 antigen, 1 enzyme-linked antibody, 1 substrate for enzyme
indirect: 1 antigen, 1 enzyme-linked antibody, 1 antibody, 1 substrate for enzyme (2 Ab)
sandwich: 1 antigen, 1 enzyme-linked antibody, 2 antibody, 1 substrate for enzyme (3 Ab)
competitive: 1 antigen, 1 enzyme-linked antigen, 1 antibody, 1 substrate for enzyme (2 Ag)
advantages of ELISA (3)
- specific and sensitive (dependent on the specificity of antibodies used, polyclonal or monoclonal –> more specific than polyclonal)
results can be quantitative, semi-quantitative or qualitative - easy to use –> absorbance measurement only need UV spectrophotometer
- safe
disadvantages of ELISA (2)
- possibility of false positive
due to use of polyclonal Ab, inadequate blocking, inadequate washing, cross reactivity of secondary Ab - possibility of false negative
due to denaturation of antibodies (pri or sec) or conjugated enzymes which are proteins in nature
elaborate on use of polyclonal antibodies leading to false positive results
cross react with multiple epitopes as some epitopes might not even come from the antigen but a look alike antigen
elaborate on use of inadequate blocking leading to false positive results
not alot of antigens in the sample, but the amount of captured antibodies is alot and hence are there are many antigen binding sites available. these excess antibodies may end up binding non-specifically and bind to impurities present in the biological samples
to counteract this: introduce blocking agent BSA - bromide serum albumin
principle of hemagglutination/agglutination
- antigen/antibody need to be particulate in nature (semi-solid/solid by being cojugated to a solid particle)
- agglutination then occurs upon Ag-Ab binding –> which is visible as cloudiness (semi-solid) / turbidity (solid)
- if antigen and antibody is soluble, antigen-antibody binding is not visible because there is no agglutination
types of hemagglutination/agglutination
- passive hemagglutination/ direct agglutination
- hemagglutination inhibition/ inhibition of agglutination
describe direct agglutination/ passive hemagglutination
agglutination test only works with particulate antigens/antibodies.
so if the antigen/antibody is soluble, you can coat the antigen/antibody on a solid particle, for agglutination to be visible to naked eye as cloudiness.
describe hemagglutination inhibition/ inhibition of agglutination
soluble antigen in sample inhibits agglutination of antigen-coated solid particles by competitively binding to antibodies –> hence, no agglutination means there are sufficient amount of soluble antigen present in the sample
blood group A, B, AB, O antigens and antibodies
group O rbc type express NO antigen, with BOTH anti A and anti B antibodies in plasma
group A rbc type express antigen A, with anti B antibodies in plasma
group B rbc type express antigen B, with anti A antibodies in plasma
group AB rbc type express both antigen A and B, with NO anti A and anti B antibodies in plasma
ABO blood type principles
antibodies against A or B antigens will cause Ab-Ag binding and the clustering of rbc that are visually seen as hemagglutination
rationale to extend ABO blood type to develop assay for diagnosis/quantification of some enveloped virus in sample
ability of these viral particles to interact with red blood cells through a viral surface glycoprotein called hemagglutinin
presence of virus will cause clumping of rbc (hemagglutination) forming a lattice instead of a nice full red dot
principle of pregnancy test
direct agglutination (passive hemagglutination)
1. latex particles are coated with an antibody for hCGv (anti-hCG antibodies)
2. introduce urine (where hCG - human chorionic gonadotropin in biological fluid is present). if urine contains sufficient hCG, agglutination occurs - positive test appears as aggregates
3. appears visually as cloudiness. the more agglutination (more binding), the more turbidity (intensity can be measured using UV absorbance)
3. assay time: 3 minutes
principle of pregnancy test
inhibition of agglutination
1. introduce latex particles coated with anti-hCG antibody (antibodies that bind to hCG) and standard amount of hCG coated on the particulate. this coated particulate is the agglutinator (supplied together in the commercial kit)
2. agglutination between particulate hCG antibody and particulate antigen
3. sample urine containing hCG is then added (soluble antigen)
4. competitively bind to the particulate antibody
5. if there is sufficient hCG in urine, agglutination will not occur –> low scattering with low absorbance –> positive results –> person is pregnant
list the commercialise immunoassays
- ovulation prediction kit - detect presence of LH in urine
- pregnancy test kit - detect presence of hCG in urine
- HIV test - detect presence of HIV capsid/envelop protein (p24) antigen OR HIV antibodies in blood
- covid-19 antigen rapid test - detect presence of SARS-CoV-2 nucleocapsid proteins in respiratory sample
- immunoassays for drugs of abuse (DOA) - detect presence of multiple DOA in urine
what does direct ELISA detect?
antigen
what does indirect elisa detect?
antibody
what does sandwich elisa detect?
antigen
what does competitve elisa detect?
antigen
