Different types of immunoassay systems

1. By
Gurubarath.M
M.Pharm, department of Pharmacology
PSG College of Pharmacy
JAN 2020
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2. 1. Immunoassay
2. Principle
3. Different types
4. Homogeneous and heterogeneous immunoassay methods
1. Heterogeneous immunoassay
1. Separation techinques
2. Enzyme immunoassay
1. Immuno enzymetric assay (IEMA)
3. Fluorescent immunoassay
1. Particle concentration fluorescence immunoassay (PCFIA)
2. Time resolved fluorescence immunoassay (TRFIA)
2. Homogeneous immunoassay
1. Enzyme immunoassay
1. Enzyme multiplied immunoassay technique (EMIT)
2. Fluorescence immunoassay
1. Substrate labeled fluoro immunoassay (SLFIA)
2. Fluorescence polarization immunoassay (FPIA)
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3.  Biochemical test – measure – concentration or the presence of molecule (analyte) –
use of an antibody or antigen
 “Immuno” – Immune responses that cause body to generate antibodies and
“assay” – test
 Ag-Ab complex (immunocomplex)
 May or may not involve physical separation
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4.  The ability of Ab to recognize and bind to a specific macromolecule – epitope
 Also produce – measurable signal
 Also involve chemically linking Ab or Ag – have detectable label
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RIA
•Radioactive
label
•Radioactive
counter
EIA
•Enzyme label
•Spectro
photometer
FIA
•Fluorescent
label
•Fluorometer
Detection
system

5.  Competitive immunoassays
 Non-Competitive immunoassays
 Heterogeneous immunoassays
 Homogeneous immunoassays
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6.  Immunoassays that require separation of bound Ag-Ab complex – heterogeneous
 Those that do not require separation – homogeneous
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7.  Involves a separation step – free from bound antigen – based on chemical or
immunological differences between Ag and immuno-complex
 Also removes other interfering substances
 More specific and sensitive – less interference
 Large sample size can also be used increasing sensitivity
 Both small and large molecules can be measured
 Requires more manipulations
 Time consuming and labor intensive
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8.  Double antibody method : Second antibody – used to precipitate primary Ag-
Ab complex – centrifuged out of solution
 Coated tube or plates : Reaction tube or microtiter plate – coated with primary
Ab
 Solid phase : Antibody used is bonded to cellulose , Sephadex or Polystyrene
beads
 Adsorption : Free antigen is adsorbed (e.g., Charcoal) – leaving bound antigen
in solution
 Solvent or salt : Antibody is precipitated – bound fraction is counted by adding
salt or solvent e.g., PEG
 Column separation : Ion exchange or gel filtration may be used
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9.  Labor intensive – involves extra step – adding substrate and incubation to
measure product formation
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 IEMA with “sandwich” approach – replacing RIA
 To form Sandwich – Ag should have 2 distinct binding sites for Abs
 Used only to measure large analytes – polypeptide hormones and enzymes e.g.,
Creatine kinase isoenzyme MB (CK-MB) – has 2 subunits
 Can be performed as simultaneous and sequential assay

10.  Fluorescence – a photon excites (excitation wavelength) molecule from
ground state (S0) – to higher electronic state (S1) – and energy is released
as light – returns to ground state at a longer wavelength (emission
wavelength)
 (Excitation wavelength – emission wavelength) – is stoke’s shift
 FIA using fluorometer has advantage over EIA using colorimeter
 It requires removal of endogenous fluorescent compound and interfering
substances
 Allows large sample usage – improves sensitivity and specificity
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11.  Developed by Pandex Laboratories
 Uses antibodies attached to solid phase particles – concentrated on well of
microtiter plate
 Signal is read by front-surface fluorometry
 Ag or Ab is bound to polystyrene latex (0.6-0.8nm) particles – dispersed in solution
 Fluorescein labelled Abs are added – bound to Ag or Ab forming a sandwich
 Filtered through 0.2nm membrane – washed and detected
 Advantages :
wide range of analytes – small and large molecules
good sensitivity (fluo. Label, separation of interferences and conc. of signal)
relatively short incubation time
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12.  Difference in decay time between fluorescent probe and interfering substance
 Most biologicals have short lived fluorescence (1-20ns)
 Long lived fluorescence – rare earth metals such as Lanthanide chelates,
europium and terbium (10-1000mcs)
 DELFIA – manufactured by LKB-Wallac in Finland
 Uses antibody labelled with europium chelate – weakly fluorescent – with
addition of enhancement solution – europium dissociates from chelate –
fluorescence intensified 106 fold
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13.  Eliminates the need for physical separation of bound from free antigen
 To detect changes occurring before and after Immunocomplex, a number of
parameters are used
 Conformational change of enzyme
 Inhibition of enzyme
 Substrate-labelled fluorescence
 Fluorescence energy transfer, protection and polarization
 Fully automated system
 Only small molecules can be measured
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14.  Introduced by Syva corporation – widely used – measure only small molecules
 Change of signal – dependent on enzyme inhibition, activation or
conformational change
 EMIT use an enzyme label – glucose-6-phosphate dehydrogenase (G6PDH),
malate dehydrogenase or lysozyme – coupled to drug derivative
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15.  Release fluoroimmunoassay
 Based on presence of hydrolysable bond between Ag and fluorescent probe
 Developed by Ames Division of Miles Laboratories
 Competitive FIA
 Has ability to measure both small and large molecules
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16.  Fluorogenic substrate – β-galactosyl
umbelliferone is weakly fluorescent –
excitation and emission maxima at 350 and
400nm
 After enzymatic hydrolysis – maxima shifts
to 400 and 450nm
 Fluorescence intensity also increases 10-15
fold
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17.  When a fluorescent molecule is excited with polarized light – resulting
emission depends on the rotational property of molecules
 Small molecules – labelled Ag – rotates faster than large molecule Ag-Ab
complex
 Due to polarized light excitation – polarization signal decreases
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18.  In competitive FPIA – unlabeled Antigen competes for Antibody binding sites with
fluorescent labelled Antigen
 Increased concentration of unlabeled Antigen – Fluorescent labelled antigen
become unbound
 Fluorescence polarization signal decreases
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19.  Immunoassay. A practical guide by Daniel W Chan(1987, Academic press); page
no.1-21
 Heterogeneous and Homogeneous immunoassays for drug analysis by Ricardo
Jorge Dinis-Oliveira (Futurescience.com)
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