IMMUNOASSAY

(Antigens and Antibodies as Chemical Reagents)

 Immunoassay Menu
• Allergy • Reproductive
– Total IgE immunoglobulin E – AFP alpha fetoprotein
• Anemia – Estradiol
– Ferritin – FSH follicle stimulating hormone
– Folate – LH luteinizing hormone
– Vitamin B – Progesterone
• Bone – Testosterone
– Vitamin D – Total HCG human chorionic gonadotropin
• Cardiac (enzymes) • Thyroid Function
– BNP brain natriuretic peptide – Free T3 triiodothyronine
– CKMB creatine kinase MB – Free T4 thyroxine
– TnI troponin l – TSH thyroid stimulating hormone
• Immunosuppressant (drugs) – T Uptake
– Cyclosporin
• Tumor Markers
– Tacrolimus
– AFP alpha fetoprotein
• Metabolic – BR breast cancer associated antigen
– Cortisol
– CA cancer antigen (greatest in ovarian)
– Homosysteine
– PSA prostate specific antigen
Antigens
• Antigens (immunogens) – substances that induce an
immune response. The immune response can be
humoral (antibody) or cell mediated.
– Factors Affecting Antigenicity
• Chemical Nature Conformation
• Size Foreignness
• Haptens Genetics
• Complexity Multivalent
(epitopes)
• Antigenic determinant
Antigens
• Chemical Nature
– Proteins, lipids, carbohydrates, nucleic acid
• Size
– 100,000 amu (daltons)
– 10,000 amu
• Complexity
– Homopolymers versus heteropolymers
Antigens
• Antigenic Determinant
– Epitope
– Valency is the number of epitopes on a single antigen.
• Conformation
– Tertiary structure
• Foreignness
– Self from non-self
• Genetics
– Genetic control (experiments with mice)
Lock and Key Model
Antibodies
• Proteins that bind specifically to antigens.
– Produced by a subset of lymphocytes
• B lymphocytes
– Plasma cells (progeny of B cells)
• Responsible for humoral immunity
• Proteins with antibody activity are called
immunoglobulins.
• Glycoproteins (4 to 18%)
• Multiple Myeloma – homogenous /closely related to
normal proteins.
Where are the antibodies?
Antibody Structure
 Antibody Classes

• Heavy and Light Chain

– Heavy Chains Types
• IgG
– mononer
– crosses the placenta binds . Attaches to T-cells
• IgM
– Pentamer
– First responder
• IgA
– Monomer or dimer
– Bodily secretions – saliva, sweat, tears
• IgD
– monomer
• IgE
– Monomer
– Responsible for allergic physiological manifestations
– Light Chain Types
• Kappa and lamda
• Fab and Fc Fragment
• Variable and Constant Regions
Antibodies
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users.rcn.com
Multiple Myeloma
Binding Forces
• Complementarity of fit of the antigenic
determinant to the antigen binding site depends
on:
– The sum of weak non-covalent intermolecular forces
• Hydrogen bonding
• Van der Waals forces
• Hydrophobic interactions
• Electrostatic interaction
– Antigen & antibody must be brought within close
proximity.
– At physiological pH – electrostatic interaction is
strongest force
Antibody Interactions
the strength of binding

• Affinity – closeness of fit. The sum of short

range, non-covalent intermolecular forces.

• Avidity – the measure of binding strength of

antibodies to multiple antigenic determinants
(epitopes).
 Antibody Affinity
Strength of Binding
k1
Ab + L Ab-L
k-1

Ka = k1/k-1 = [Ab-L]/[Ab][L]
Ka is the AFFINITY constant. It is equal to the volume into which one
mole of the binding antibody can be diluted to yield 50% binding of
the ligand.

Low affinity binding antibody concentration – 10-5 to 10-7 moles/liter

High affinity binding antibody – will have binding at 10-8 to 10-11 moles/liter
Antibody Interactions
• Sensitivity (concentration)
– Low affinity - 105 to 107 liters/mole (concentrated solution)
– 10-5 to 10-7 moles/liter

– High affinity - 108 to 1011 liters/mole (dilute solution)

– 10-8 to 10-11 moles/liter

• Specificity/Cross Reactivity
– Binding to chemically related or structurally
related antigens.
• Polyclonal Antibodies
• Monoclonal Antibodies
Monoclonal and Polyclonal Assays
“Antibody specificity”
http://www.youtube.com/watch?
v=0A99pk6kpS4

http://www.youtube.com/watch?v=O-
SrPqJuEVg

http://www.youtube.com/watch?
v=0CK1it7Qltg
Immunoassay
Defined

• Any binding assay in which the binding protein is

an antibody.
– Antibody is an immunoglobulin with a functional domain
F(ab) that binds to an antigen.
– The site on the antigen where the antibody binds is referred
to as the epitope or antigenic determinant.
• Binding of the antibody to the epitope is directly
related to the affinity and avidity of the two
reactants.
• Antigen/Ligand- drugs, hormones, small proteins
 Immunoassays
An assay based on the reversible binding of an ligand to a binding protein.
The ligand competes in proportion to its concentration with a labeled
derivative for limited number of binding sites.

Ligand + Binding Protein Binding Protein-Ligand

Binding Protein = gamma immunoglobulins (IgG)

Ligand = Both analyte and labeled derivative of the analyte

Competitive Binding Immunoassays
Behavior of Competitive-Binding Assays
 Immunoassay
Competitive Binding
Assays

Homogeneous Heterogeneous

ELISA
EMIT FPIA RIA
“Sandwich assay”

Horseradish Horseradish
Peroxidase Peroxidase

Reduced dye to Reduced dye to

Oxidized dye Oxidized dye

Alkaline Alkaline
Phosphatase Phosphatase

p-nitrophenol p-nitrophenol
phosphate phosphate
(intense yellow color) (intense yellow color)
Type of Labels

• Radioisotopes – I125 is the label of choice

• Enzymes – binding modifies enzyme activity
• Fluorescent – compounds that absorb and
emit radiant energy
• Luminogens – emit a photon as a result of
electrical, chemical, or biochemical reaction.
– Luminol – “long emission”
– Acridinium ester – “flash”
 Enzyme Labels for Immunoassay

• Alkaline phosphatase

ALP
p-nitrophenyl phosphate p-nitrophenol
(colorless) Alkaline pH (Intense yellow)

• Horseradish Peroxidase
peroxidase
Reduced dye + H2O2 Oxidized dye + H2O
color
Competitive Binding Assays
Two Formats

• Heterogeneous Immunoassay
– Requires a separation step

• Homogeneous Immunoassay
– Does Not require a separation step
Homogeneous Enzyme Immunoassay

• Enzyme-multiplied Immunoassay
Technique (EMIT)

• Fluorescence Polarization Immunoassay

(FPIA)

• Latex Agglutination Immunoassay

Enzyme- Multiplied Immunoassay Technique (EMIT)

Binding of antibody to an enzyme-labeled antigen changes the enzymatic activity of the label. Low-molecular weight molecules.
Enzyme Labels for Immunoassay
Requirements for Enzyme Label

• Highly specific
• Stable
• Not present in the fluid being analyzed
• Retain activity when conjugated
EMIT

• Advantages
– Sensitive to subnanomolar levels.
– Sensitivity can be increased with a fluorogenic
substrate.
– Easily automated

• Disadvantages
– Subject to interferences
– Unsuitable for high molecular weight compounds
 Fluorescence Polarization Immunoassay (FPIA)

Immunoassay based on the amount of polarized fluorescent light

detected when a fluorophore label is excited with polarized light. The
degree of polarization depends on the rate of rotation.

• NON-POLARIZED LIGHT
(MULTI-DIRECTIONAL)
• POLARIZED LIGHT (UNI-DIRECTIONAL)
• FLUOROPHORE (FLUORESCEIN)
• ABSORB BLUE LIGHT emits GREEN
FPIA
FPIA
 FPIA
• Advantages
– Sensitive to subnanomolar levels
– Easily automated
– May be very stable

• Disadvantages
– Subject to interferences
– Quenching
– Unsuitable for high molecular weight compounds
Heterogeneous Enzyme Immunoassay

Radioimmunoassay (RIA)

Enzyme-linked Immunosorbent Assay

(ELISA)

Immunoradiometric Assay (IRMA)

Separation Techniques

• Solid Phase Attachment

• Double Antibody

• Magnetic Bead
RIA Techniques
The Antigen and a fixed amount of radioactively labeled antigen compete for a
limited number of antibody-binding sites.

Step 1. Ag-I125 + Ab + Ag (patient) =

Ag-I125 + Ab-Ag
Step 2. INCUBATION
Step 3. SEPARATION OF BOUND
AND FREE Ag
Step 4. COUNTING (CPM)
Step 5. CALCULATIONS/PLOTS
RIA Plots
If one counts the radioactivity bound to the antibody
after the separation step, the dose-response curve will have a negative slope. If the
unbound radioactively labeled antigen is monitored, the dose-response curve has a positive
slope.

Bound fraction is inversely

proportional to concentration.
• Ag-I125 + Ab + Ag (sample)

• Ag-I125 + Ab-Ag (sample)

Free fraction is directly

proportional to concentration.
 RIA
• Advantages
– Innovative for it’s time
– Sensitive and specific
– Endocrine Testing

• Disadvantages
– Radioactive waste
– Short Shelf Life
 IMMUNORADIOMETRIC Assay (Irma)

Antigen in the sample competes with antigen attached to a solid surface for the binding sites of a radioactively labeled antibody.

• RADIOACTIVILY LABELED ANTIBODY

• LIGAND BOUND ON SOLID SURFACE
• SAMPLE COMPETES FOR ANTIBODY SITES
• MORE SENSITIVE
• ONE SITE OR TWO SITE IRMA TECHNIQUE
 One-site Irma

sample

• One Antibody System

• CPM in bound directly proportional to antigen
concentration
 Two-Site Irma

• Two Antibody “Sandwich”

 Enzyme-linked IMMUNOSORBENT Assay (ELISA)

• IRMA with enzyme instead of I125

• Enzyme-labeled antibody/sample (Ag)
• Separation of bound/free antibody
• Substrate/cofactor added
• Visualization/monitoring enzyme reaction
• One or two antibody (“sandwich”) technique
http://www.youtube.com/watch?
v=RRbuz3VQ100
http://www.youtube.com/watch?
v=QoK2xBn9fSc&list=PL2xuXsmC3miq
4TnZo-FWdwMnNRKiCD9Hs&index=2

http://www.youtube.com/watch?
v=Tp61S-2F2B4