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
Passive hemagglutination
latex on antigen
• Agglutination test only works with particulate antigens
• Erythrocytes coated with soluble Ag
• Coated RBC in agglutination test for AB to the soluble Ag
Hemagglutination inhibition
latex on RBC
add pt antigens to block RBC-latex binding to AB
• Soluble Ag (in sample) inhibits agglutination of Ag-coated RBC by AB
• +ve = lack agglutination
1) Antigen coated onto latex particle (semi-solid) -- fixed amt 2) Add pt sample with antigen (soluble) 3) Compete binding for AB a. Interferes with agglutination i. Lower turbidity = more pt antigen in sample
EG preg test
1) Latex particles coated with anti-hCG AB + hCG (agglutinator) added — standard amt
a. Agglutination occurs between anti-hCG AB + Ag
2) Sample urine with hCG added
a. Prevents agglutinator from binding
b. Decr absorbance
other egs
1) Ovulation prediction kit: LH in urine
2) Pregnancy kit: hCG in urine
3) HIV test: HIV capsid or envelop proteins antigens// HIV AB in blood
4) Covid-19 antigen rapid test: SARS-CoV 2 nucleocapsid proteins in respiratory sample (nasal/ throat swab)
5) Drugs of abuse in urine sample
a) Multi-drug rapid test
b) 5-10 drugs
Require further confirmatory test afte
HbA1c
• Glycated hemoglobin (sugar molecule attached to hemoglobin)
• Glycation of hemoglobin takes place non-enzymatically
○ condensation reaction (glucose + amino end of Beta chain in Hb)
high levels of HbA1c
• Higher blood glucose levels = higher Hba1c
• Longer periods of high bgl = higher Hba1c
• Glycated Hb remains throughout RBC lifespan (20days~3mnths) –> Hba1c for LONG-TERM monitoring
IMMUNOASSAY for HbA1c
Semi-quantitative/ quantitative assay for HbA1c in blood
Based on latex immunoagglutination inhibition method
Agglutinator
synthetic polymer contain multiple copies of immunoreactive portion of HbA1c
Latex coated with anti-HbA1c mouse monoclonal AB
INSULIN
• Hormone that regulates blood glucose by promoting shift of glucose from circulatory –> intracellular compartment
feature of insuline
• 110 aa (MW similar to cytokine)
• 2 chains joined
○ α, β chain
• Produced by Islets of Langerhans, β cells
production of insulin
Islets of Langerhans, β cells
1) Pre-proinsulin produced
○ α (21 aa) + β chain (30 aa) + terminal signal sequence (23 aa)
○ 2 disulfide bonds between α, β chain
2) Form proinsulin by:
a. Remove signal peptide
b. When enter secretory vesicles
3) Produce mature insulin
a. Carboxypeptidase cleavage of proinsulin releases C peptide by proteases
final insulin
A chain (21) + B chain (30) + 2 disulfide link
mature insulin may form
○ 6 Mature insulin (histidine molecules) interact with Zn2+ ions
Hexamer crystal insulin ready to be secreted
Different host produces slightly different insulin (porcine–prok, bovine - beef)
○ Porcine: 1 aa variation at B30
○ Bovine: 3 aa variation A8, A10, B30
§ Can lead to immune response (lipoatrophy
- loss of fatty tissues locally// local allergic rxn)
○ Need to purify and remove proinsulin!
Production: proinsulin method - recombinant human insulin production
Production of recombinant human insulin
via proinsulin method
1) Methionine codon ATG start codon
a) Chemically synthesised and attached to 5’ end of proinsulin cDNA
2) Chemically cleavage by cyanogen bromide (CnBr) to remove Met residue from proinsulin pp chain
3) Proinsulin chain subjected to folding process
a) Allow intermolecular disulfide bond formation
4) C-peptide cleaved by enzymes
a) Trypsin, carboxypeptidase
b) Yields human insuli
insulin hexamer dissociates when
Dissociates into monomer at site of injection
When injected conc < physiological levels
Rapid-acting insulin eg
LISPRO
ASPART
GLULISINE
onset: 15-30min
duration: 3-5hr
modifications of rapid insulin
Prevent dimerisation/ hexamerisation
• [lispro] Reversal of Lys 29 and Pro 28 in b chain
- Resemble insulin-like growth factor IGF1 that poorly associates
• [aspart] Substitute Pro28 –> Asp in b chain
- Reduce association, non-hexameric
•[glulisine] Substitute Asn 3 –> Lys and Lys 29 –> Glu B chain
- Reduce association, non-hexameric
Intermediate-acting insulin eg
- NPH, neutral protamine hagedorn
- regular
onset: 1-4hr
duration: 10-24hr
intermediate modification: create insulin crystals
• Insulin + Protamine + Zn in formulation
• Isophane insulin, cloudy suspension
• Protamine: highly basic, 30 aa peptide
• Co-crystallized w/ zinc in neutral pH (phosphate buffer)
• Neutral crystalline insulin suspension
intermediate can be mixed with
– Can be mixed with regular/ rapid-acting insulin (cover post-meal hypergly)
70/30 or 50/50 (NPH/regular)
Long-acting eg
glargine
detemir
onset:1-2hr
duration: 20-24hr
modification to glargine/ lantus
insulin crystals form in sc inj
• a chain: Substitute Asn21 –> Gly
• b chain: add 2 Arg residues on C terminal
• Structural modification change pI (5.4 –> 6.7)
• Glargine more soluble at acidic pH, less soluble at neutral pH (~~7.4) ○ Is stablised and soluble in formulation of pH 4 (acidic) ○ Less soluble in pt sc (neutral pH)
detemir (levemir)
3-4hr
6-24hr
Myristic acid binds to serum albumin
• B chain:
• Thr B30 deleted
• C14 FA (lipophilic chain, more hydrophobic) covalent attach to LysB29
○ Modification result in binding to serum albumin
§ site of inj
§ At walls of blood vessels when enter circ (arterial end of cap network)
Slow dissociation into blood, prolonged release
