immunoassays Flashcards
immunoassays are __
• Assays that employ AB to detect and quantify a specific analyte (usually biomolecule)
• AB against the biomolecule of interest
○ Polyclonal/ monoclonal AB
○ Raised in animal
how to detect Ag-Ab binding in immunoassays
1) MARKER to tag onto AB to facilitate rapid recognition
- radioimmunoassays RIA
- enzyme immunoassays
2) agglutination/ hemagglutination
solid phase enzyme immunoassay
1) AB immobolised on solid surface (plastic surfaces)
a. Incubate with known amt of enzyme-linked antigens
b. Un-linked Antigens
2) Competition binding between enzyme-linked and unlinked antigens for AB 3) Wash away unbound antigens with buffer sol (phosphate buffered saline PBS) a. Add enzyme substrate b. Measure absorbance of coloured product
Determine enzymatic activity
ii. If more enzymatic activity
= More enzyme-linked antigen bind to solid phase AB
=. Less antigens present in sample, insuff competition
Direct ELISA
1) Pt antigen is immobolised on solid surface
2) Wash to prevent false +/-
3) Primary AB conjugate (enzyme linked) added
a. Binds to antigen
4) Wash unbound AB
5) Substrate added, bind to enzyme-linked AB
6) Wash extra substrate
7) Quantitative measure of absorbance of coloured products
Indirect ELISA
1) Pt antigen is immobolised on solid surface
2) Wash to prevent false +/-
3) Primary AB (NOT enzyme linked) added
a. Binds to antigen
4) Wash unbound AB
5) Add 2nd AB conjugate ( enzyme-linked)
6) Substrate added, bind to 2nd enzyme-linked AB
7) Wash extra substrate
8) Quantitative measure of absorbance of coloured products
Sandwich ELISA
1) Captured AB is immobolised on solid surface
2) Wash to prevent false +/-
3) Patient Ag in sample
a. Binds to AB
4) Wash unbound antigen
5) Add AB specific to epitope of antigen
a. Binds to antigen
6) Add 2nd conjugate AB (enzyme-linked) a. Specific to 1st AB Fc domain 7) Wash 8) Substrate added, bind to 2nd enzyme-linked AB 9) Wash 10) Quantitative measure of absorbance of coloured products
Competitive ELISA
1) Captured AB immobilised on surface
2) Co-incubate with:
a. pt sample antigen
b. enzyme linked antigens (fixed amt)
○ Enzyme linked antigen will compete with pt antigen to bind to captured AB 3) Wash 4) add substrate to bind to enzyme-linked AB 5) Detect absorbance
competitive ELISA results
• Higher antigen lvl = more competition to bind to captured AB = less substrate will be acted on for colour change (by enzyme linked antigen)
LIGHTER = HIGHER ANTIGENS IN PT
Advantages of ELISA
○ Specific: high affinity of AB decr cost, less AB needed for test
○ Sensitive: quantity of AB designed for assay
§ Depends on specificity of AB (MONOCLONAL > POLYCLONAL) to epitope but $$$
○ Ease of use: absorbance measurement only require UV spectrophotometer/ fluorescence spectroscopy
○ Safe to use
disadvantage of ELISA
○ Possibility of false positive results
- polyclonal AB bind to other antigens
- inadequate blocking
- inadequate washing (AB or antigens)
- cross reactivity of 2nd AB
○ Possibility of false negative results
§ AB (1st/ 2nd) and conjugate enzymes = PROTEINS
□ May be denatured
□ AB cannot recognise antigens
□ Enzymes cannot react with substrate for color change
Inadequate blocking (gives false +)
□ (captured AB/ antigen onto surface –> insuff antigens bound
□ –> excess binding site will bind to non specific proteins/ impurities from pt sample)
1) Block with bovine serum albumin/ Silent binders 2) Albumin soluble protein bind non specifically on unoccupied antigen binding sites. Better than binding to other proteins 3) Do not contribute to signal 4) Control well also tested with the albumin 5) Cancel out results
agglutination for the test
ability of the viral particles to interact with RBC through viral surface glycoprotein (hemagglutinin)
○ Presence of virus causes clumping of RBC forming lattice ○ Instead of nice full red dot: high surface tension
BUFFER
PHOSPHATE BUFFERED SALINE
PBS
As long as antigen and/or AB is particulate in nature (semi-solid/ solid) being conjugated to a solid particle, agglutination occurs upon Ag-AB binding
1) Ag-AB (soluble): both antigens and AB soluble then not visible, no agglutination
2) Ag-AB : either present in semi-solid/ presented by solid particle
a) Visible by clumping
3) AG-AB: both AG, AB are particulate (presented by solid particle)
a) agglutination occurs, turbidity more intense than (2)
Direct agglutination
latex on AB
• Latex particles (solid particle) coated with AB for hCG
• Binds to hCG in urine (if sufficient lvl of hormone)
• Aggregates = +ve test
