Immunochemical Methods and
Biosensors for pollutants
determination
(General principles and application)
Danila Moscone
Department of Chemical Science and Technology
University "Tor Vergata"
Rome, Italy
danila.moscone@uniroma2.it
Immunoassays (IAs) are techniques based on
the formation of a thermodynamically stable
antigen – antibody complex.
These methods already play an important role,
especially in clinical chemistry, being used for
the fast and safe detection of proteins,
hormones, and pharmaceutical agents.
 Immunoassays become important
when:




Fast measurement and evaluation are required
Highest possible detection strength is required
Large numbers of samples are to be expected
Only complex and expensive analytical methods
are otherwise available.
 The greatest potential for the use of immunoassays in
environmental analytical chemistry is in SCREENING
i.e., for the selection of contaminated and
uncontaminated samples for further validation
analysis.
Terminology
Antigen:
Original - Substance able to generate
antibody.
More general - Substance that can be
recognized by antibody or T cells
Immunogen: Substance able to generate an immune
response
Hapten:
Non-immunogenic substance. Usually low
molecular weight. Induces antibody
formation when coupled to a larger “carrier”
molecule. Can bind antibody
Hapten - DNP
Protein Carrier Bovine Serum Albumin
Immunize with
DNP
BSA
DNP-BSA
Antibodies formed
None
Anti-BSA
Anti-DNP
Anti-BSA
Anti-DNP-BSA
Antibody structure
ANTIBODY (immunoglobulin)
.
A biological molecule (protein) that specifically recognizes a foreign
substance (antigen) as a means of natural defence
Antigen binding sites
Light
Chain
Heavy
Chain
Antibodies: production and labelling
PRODUCTION
•Animals have a large number of antibody producing cells, all
producing a different antibody. When an animal (rabbit) is injected
with antigen, proliferation of the corresponding antibody
producing cell is promoted. Blood from the rabbit contains
antibodies, originating from different cells with slight variations.
LABELLING
Radio-isotopes, Enzymes, Fluorescent, probes (Quantum dots), Chemiluminescent probes, Metal tags
Antibodies
Polyclonal
Antibodies that are collected
from sera of exposed animal
recognize
multiple antigenic sites
of injected biochemical.
Monoclonal
Individual B lymphocyte hybridoma
is cloned and cultured.
Secreted antibodies are collected
from culture media
recognize
ONE antigenic site
of injected biochemical
Antigen-antibody Interactions
Features of the AntigenAntibody Interaction
•Reversibility
Non-covalent Interactions
•Affinity
Measure of the strength of the binding
Ease of association or dissociation
•Avidity
Increase in affinity due to multivalent
binding
The summation of multiple affinities
Non-covalent binding
Affinity and Avidity
Antibody-based
assays
ELISA
Enzyme-Linked
Immunosorbent Assay
immobilisation surface
Specific Ab
antigen- enzyme conjugate
E
Ag
Coating
Incubation
E
E
E
E
E
S
Enzym. reaction
P
Affinity reaction
Product
measurement
I. No analyte - high detection signal
E
E
E
E
E
II. Analyte present - detection signal reduced
E
E
E
Indirect competitive ELISA format
FREE Ag and
SPECIFIC Ab
ADDITION
SECONDARY
LABELLED
Ab
ENZYMATIC
REACTION
ANTIGEN
COATING
BLOCKING
The enzymatic product concentration is inversely
proportional to the analyte (standard or sample)
amount
ELISA SANDWICH FORMAT
YYY
Antibody
YYY
2nd antibody with enzyme
YYY
enzyme produces colour
YYY
YYY
Antibody/Antigen
YYY
Signal (enzyme activity)
signal/concentration curve
Functional
concentration range
Antigen concentration
ELISA PLATES
ELISA PLATE WASHER
SPECTROPHOTOMETER
ADAPTED FOR ELISA PLATES
Lateral Flow Strips
(Dipsticks)
Apply sample solution, upon application
of sample biochemicals dissolve
Positive: no antigen
Immobilised
Antibody area
Control area
Negative: antigen present
• Immunochromatography (Lateral Flow)
• Biochemical components are separated across an absorbent
membrane into discrete distinct regions.
QUALITATIVE TEST
Test line
Predator
support
Sample pad
analyte
Ab-colloidal gold
QUALITATIVE TEST: Analyte absent in the sample
Test line
Sample pad
Analyte
Ab-colloidal gold
If the analyte is ABSENT in the
sample the line will be colored
Test line
Sample pad
Analyte
Ab-colloidal gold
Analyte PRESENT in the sample
Test line
Sample pad
Analyte
Ab-colloidal gold
Test line
Sample pad
Analyte
Ab-colloidal gold
Test line
Sample pad
Analyte
Ab-colloidal gold
If the analyte is present in the
sample the line will be not colored
Test line
Sample pad
Analyte
Ab-colloidal gold
 We can use these immunochemical
elements to assemble a special kind of
biosensors called
Immunosensors
Analyte
Biological
component
Signal
transducer
Recorder
What do they have in common?
Biosensor
Analyte / bioreceptor / transducer / processor
Nose
Small molecules / olfactory membrane / nerve cells / brain
Eye
Visible light / rods and cones / nerve cells / brain
Staphylococcus aureus
gram-positive, non spore-forming bacterium
able to synthetise:
Enterotoxins: A, B, C, D, E (thermostable);
 Coagulase;
 Thermonuclease.
100-200 ng of enterotoxins are sufficient to
cause
toxinfection
in
immuno-compromised
subjects.
DEVELOPED TEST:
Conventional ELISA Proteina A
Conventional ELISA S. aureus
ELISA/AMPLI S. aureus
ELIMC S. aureus
ELIME S. aureus
Spectrophotometric ELISA
p-NITROPHENOL
AP
Secondary
antibody-AP
Specific antibody
(MAb o PAb)
Protein
A/S.aureus
Human IgG
p-NPP
ELISA Protein A
2,40
ABS @ 405 nm
ABS @ 405 nm
0,90
0,60
0,30
1,60
0,80
0,00
0,00
0,1
1,0
10,0
100,0
0,01
0,10
protein A (ng/mL)
IgG
10 mg/mL
MAb
1:10000
Ab2-AP
1:1000
(a – d)
1+
LOD
0.6 ng/mL
10,00
100,00
protein A (ng/mL)
y=
MAb
1,00
x
b
c
y = <x0> + 3s
+ d
PAb
LOD
0.07 ng/mL
Sensitivity
Sensitivity
7.6 ng/mL
0.6 ng/mL
IgG
10 mg/mL
PAb
1:10000
Ab2-AP
1:1000
Sensitivity was calculated as tha amount of protein A needed to produce a 25% increase in the signal
ELISA S.aureus
2,50
1,20
ABS @ 405 nm
ABS @ 405 nm
2,00
0,90
0,60
1,50
1,00
0,30
0,50
0,00
0,00
1,e+6
1,e+7
1,e+8
1,e+3 1,e+4 1,e+5 1,e+6 1,e+7 1,e+8
[S.aureus] (cell/mL)
MAb
IgG
[S.aureus] (cell/mL)
LOD
LOD
2 106 cell/mL
2 104 cell/mL
10 mg/mL
Sensitivity
MAb
106
IgG
10 mg/mL
PAb
1:10000
Sensitivity
1:10000
9
Ab2-AP
PAb
cell/mL
2
1:1000
No crossreactivity
105
cell/mL
Ab2-AP
1:1000
AMPLI Q
I
NADPH
N N
+
Br
N N
Alkaline phosphatase
NO2
Pi
Acetaldehyde
INT
NADH
Alcohol deydrogenase
Ethanol
Diaphorase
NAD+
FORMAZAN
H2N N CH N NH
DAKO, Handbook for AmpliQ, 1997
4
3,00
3
2,50
ABS @ 490 nm
ABS @ 490 nm
ELISA S.aureus AMPLIQ
2
1
2,00
1,50
0
1,00
1,e+3 1,e+4 1,e+5 1,e+6 1,e+7 1,e+8
1,e+3 1,e+4 1,e+5 1,e+6 1,e+7 1,e+8
[S.aureus] (cell/mL)
MAb
IgG
10 mg/mL
MAb
1:10000
Ab2-AP
1:1000
[S.aureus] cell/mL
PAb
LOD
LOD
6 104 cell/mL
7 102 cell/mL
IgG
10 mg/mL
Sensitivity
Sensitivity
PAb
1:10000
2 105 cell/mL
6 103 cell/mL
Ab2-AP
1:1000
Magnetic Beads
Magnetic particles are particles constituted from a dispersion of magnetic
material (Fe2O3 and Fe3O4) and then covered with a thin shell of polymer which
contains the magnetic material and also serves to define a surface area for the
absorption or coupling of a large variety of other molecules.
Good results in immunological field
Ø 1-5 µm
Measurements on real samples
ELIMC (Enzyme Linked ImmunoMagnetic Colorimetry)
All reactions were carried out in eppendorf tubes
OH
No intermediate washings
+
NaH2 PO3
NO2
p-NITROPHENOL
AP
Microtitre
ELISA
p-NPP
O
HO P ONa
O
NO2
ELIME (Enzyme Linked ImmunoMagnetic Electrochemistry)
a-naphthol
OH
AP
+ NaH2PO3
ONa
O P O
O
a-naphthyl phosphate
DPV
+
•Selectivity Ag-Ab;
•Sensibility of electrochemical
detection;
•Possibility of concentrating
magnetic particles on the
electrode surface.
Potential range 0-600 mV
Scan speed
Pulse width
100 mV/s
50 ms
Modulation time 60 ms
Interval time
0.16 s
Addition of
Enzymatic substrate
for
Electrochemical
measurement
Magnetic tube
ELIMC S.aureus
ELIME S.aureus
4,0
0,8
0,6
14,0
0,4
0,2
4,0
12,0
0,0
3,0
1,e+4
1,e+5
2,0
10,0
Current (µA)
ABS @ 405 nm
3,0
1,0
2,0
1,0
8,0
0,0
103
104
6,0
4,0
0,0
1,e+4
1,e+5
1,e+6
1,e+7
2,0
[S. aureus] cells/mL
0,0
1,e+3
MAb
IgG 0.5 mg/mL
Mab
1:50000
Ab2-AP
1:300
1 104 cells/mL
2 105 cells/mL
1,e+5
1,e+6
1,e+7
[S. aureus] cells/mL
LOD
Sensitivity
1,e+4
MAb
IgG 1.2 mg/mL
MAb
Ab2-AP
1:1000
1:100
LOD
1 103 cells/mL
Sensitivity
2 104 cells/mL
LOD
Sensitivity
Analysis Time
Mab ELISA prot. A
0.6 ng/mL
7.6 ng/mL
22 h
Pab ELISA prot A
0.07 ng/mL
0.6 ng/mL
22 h
Mab ELISA S.a
2 106 cell/mL
9 106 cell/mL
22 h
Pab ELISA S.a
2 104 cell/mL
2 105 cell/mL
22 h
Mab ELISA AmpliQ
6 104 cell/mL
2 105 cell/mL
22 h
Pab ELISA AmpliQ
7 102 cell/mL
6 103 cell/mL
22 h
Mab ELIMC
1 104 cell/mL
2 105 cell/mL
4h
Mab ELIME
1 103 cell/mL
2 104 cell/mL
4h
Air samples
Two air samples from hospital rooms
Sampling carried out by a SAS air-sampler.
Flow rate 35 litri/min, for 30 minuts, collin
30 ml of buffer
Sample 1
660 cell/m3 ± 15%
Sample 2
11700 cell/m3 ± 11%
Immunoassay test products
validated by OSW
Anticholinesterase activity
measurement by an enzyme biosensor:
application in water analysis
This method is a fast, cheap, and good analytical choice to measure
the total anti-ChE charge in the sample, an important toxicological
index defined as the amount of compounds which causes a % of ChE
inhibition equivalent to that produced by a known amount of a
pesticide (e.g. Paraoxon) taken as reference compound.
Acetilcholinesterase
Choline + Acetic ac.
Acetilcholine
Inhibited by
pesticides
Choline oxidase
Choline + O2 + H2O
H2O2
Electrode
Not Inibited
Betaine + H2O2
O2 + 2H+ + 2e-
Trasmission of the nervous impulse
Acetilcholinesterase
Inhibition measurements
I%=[( E0-Ei)/E0]•100
Non inhibited enzyme (E0)
inhibited enzyme (E1)
Time (min)
Inhibition of AChE with Paraoxon
6
Blank
2 ppb
6 ppb
4
10 ppb
2
0
0
1
2
3
TIME (minutes)
4
HEAVY METALS DETERMINATION
BASED
ON THE USE OF INVERTASE ENZYME
REACTIONS
E1= Invertase
Sucrose
E1
E2 = Glucose Oxidase
+ H2O
D-Glucose + D-Fructose
D-Glucose
E2
+ O2
Gluconic acid + H2O2
Electrode
H2O2
O2 + 2H++ 2e-
INHIBITION MEASUREMENTS
Sucrose
I1
INV
B
Sucrose
INV + Inhibitor
I2
A
Reaction Time
Time
FIA Calibration with sucrose 10mM
80
60
40
20
0
0
20
40
60
[Hg2+] (ppb)
80
100
Biosensors applied to the determination
of pollutants in real samples
From: S. Rodriguez-Mozaza, M. J. L´opez de Aldaa, M.-P. Marcob, D. Barcel´oa,, Talanta 65 (2005) 291–297