Immunochemical Methods and Biosensors for pollutants

1
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_at_uniroma2.it

2
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.

3

• 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.

4
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
5
Protein Carrier - Bovine Serum Albumin
6
Antibody structure
ANTIBODY (immunoglobulin) A biological molecule
(protein) that specifically recognizes a foreign
substance (antigen) as a means of natural defence
.
7
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), Chemi-luminescent probes,
Metal tags
8
Antibodies
Polyclonal
Monoclonal
Individual B lymphocyte hybridoma is cloned and
cultured. Secreted antibodies are collected from
culture media
Antibodies that are collected from sera of
exposed animal
recognize multiple antigenic sites of injected
biochemical.
recognize ONE antigenic site of injected
biochemical
9
Antigen-antibody Interactions
10
Features of the Antigen-Antibody 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

11
Non-covalent binding
12
Affinity and Avidity
13
Antibody-based assays
14
Enzyme-Linked Immunosorbent Assay
ELISA
15
Direct competitive immunoassay (I)
Incubation
Coating
E
E
E
S
Enzym. reaction
Product measurement
P
Affinity reaction
16
Direct competitive immunoassay (II)
I. No analyte - high detection signal
II. Analyte present - detection signal reduced
17
(No Transcript)
18
Indirect competitive ELISA format
The enzymatic product concentration is inversely
proportional to the analyte (standard or sample)
amount
19
ELISA SANDWICH FORMAT
Y Y Y
Antibody
2nd antibody with enzyme
Y Y Y
Antibody/Antigen
20
signal/concentration curve
21
(No Transcript)
22
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.

23
QUALITATIVE TEST
Test line
analyte
Ab-colloidal gold
24
QUALITATIVE TEST Analyte absent in the sample
Test line
25
If the analyte is ABSENT in the sample the line
will be colored
Test line
Sample pad
26
Analyte PRESENT in the sample
Test line
Sample pad
27
Test line
28
Test line
Sample pad
29
If the analyte is present in the sample the line
will be not colored
Sample pad
30
(No Transcript)
31

• We can use these immunochemical elements to
  assemble a special kind of biosensors called
• Immunosensors

32
biosensor
Analyte
33
What do they have in common?
Biosensor
Analyte / bioreceptor / transducer / processor
34
(No Transcript)
35
(No Transcript)
36
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.

37
(No Transcript)
38
DEVELOPED TEST
Conventional ELISA Proteina A
Conventional ELISA S. aureus
ELISA/AMPLI S. aureus
ELIMC S. aureus
ELIME S. aureus
39
Spectrophotometric ELISA
40
ELISA Protein A
PAb
y ltx0gt 3s
IgG 10 mg/mL
 
PAb 110000
Ab2-AP 11000
Sensitivity was calculated as tha amount of
protein A needed to produce a 25 increase in the
signal
41
ELISA S.aureus
MAb
PAb
IgG 10 mg/mL
IgG 10 mg/mL
MAb 110000
PAb 110000
Ab2-AP 11000
Ab2-AP 11000
No cross-reactivity
42
AMPLI Q
NADPH
Alkaline phosphatase
Pi
INT
Acetaldehyde
NADH
Alcohol deydrogenase
Diaphorase
FORMAZAN
NAD
Ethanol
DAKO, Handbook for AmpliQ, 1997
43
ELISA S.aureus AMPLIQ
MAb
PAb
LOD
LOD
6 104 cell/mL
7 102 cell/mL
IgG 10 mg/mL
IgG 10 mg/mL
Sensitivity
Sensitivity
MAb 110000
PAb 110000
2 105 cell/mL
6 103 cell/mL
Ab2-AP 11000
Ab2-AP 11000
44
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
45
ELIMC (Enzyme Linked ImmunoMagnetic Colorimetry)
All reactions were carried out in eppendorf tubes
No intermediate washings
p-NITROPHENOL
Microtitre ELISA
p-NPP
46
ELIME (Enzyme Linked ImmunoMagnetic
Electrochemistry)
a-naphthol

NaH
PO
2
3

a-naphthyl phosphate
DPV Potential range 0-600 mV Scan speed
100 mV/s Pulse width 50 ms Modulation
time 60 ms Interval time 0.16 s

• Selectivity Ag-Ab
• Sensibility of electrochemical detection
• Possibility of concentrating magnetic particles
  on the electrode surface.

47
Addition of Enzymatic substrate
for Electrochemical measurement
Magnetic tube
48
ELIMC S.aureus
ELIME S.aureus
49
(No Transcript)
50
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
51
Immunoassay test products validated by OSW
52
Anticholinesterase activity measurement by an
enzyme biosensor application in water analysis
53
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
Betaine H2O2
Choline O2 H2O
Not Inibited
Electrode
H2O2
O2
2H 2e-
54
Trasmission of the nervous impulse
55
Inhibition measurements
I( E0-Ei)/E0100
Non inhibited enzyme (E0)
Time (min)
56
(No Transcript)
57
HEAVY METALS DETERMINATION BASED ON THE USE OF
INVERTASE ENZYME
58
REACTIONS
E1 Invertase E2 Glucose Oxidase
Sucrose
H2O
E1
D-Glucose D-Fructose
D-Glucose
E2
O2
Gluconic acid H2O2
Electrode
O2 2H 2e-
H2O2
59
INHIBITION MEASUREMENTS
Current
I1
Sucrose
INV
B
Sucrose
INV Inhibitor
I2
A
Reaction Time
Time
60
(No Transcript)
61
Biosensors applied to the determination of
pollutants in real samples
From S. Rodriguez-Mozaza, M. J. Lopez de Aldaa,
M.-P. Marcob, D. Barceloa,, Talanta 65 (2005)
291297
62
THANKS!!!!