1. Immunochemical Methods and Biosensors for pollutants determination (General principles and application) 

2. antigen – antibody complex.
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. Immunize with Antibodies formed DNP None BSA Anti-BSA DNP-BSA Anti-DNP
Hapten - DNP
Protein Carrier -
Bovine Serum Albumin
Immunize with Antibodies formed
DNP None
BSA Anti-BSA
DNP-BSA Anti-DNP
Anti-BSA
Anti-DNP-BSA

6. ANTIBODY (immunoglobulin)
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

7. Antibodies: production and labelling
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 recognize multiple antigenic sites
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
Enzyme-Linked Immunosorbent Assay

15. immobilisation surface
Direct competitive immunoassay (I)
Specific Ab Ag E
antigen- enzyme conjugate
immobilisation surface
Coating
Incubation E E E S E
Enzym. reaction
Product
measurement P
Affinity reaction

16. Direct competitive immunoassay (II)
I. No analyte - high detection signal E
II. Analyte present - detection signal reduced E

18. FREE Ag and SPECIFIC Ab ADDITION
Indirect competitive ELISA format
ENZYMATIC REACTION S P
SECONDARY LABELLED Ab
FREE Ag and SPECIFIC Ab ADDITION
ANTIGEN COATING
BLOCKING
The enzymatic product concentration is inversely proportional to the analyte (standard or sample) amount

19. enzyme produces colour
ELISA SANDWICH FORMAT
Y Y Y
Y Y Y
enzyme produces colour
Antibody
2nd antibody with enzyme Y
Y Y Y
Y Y Y
Antibody/Antigen

20. signal/concentration curve
Functional concentration range
Signal (enzyme activity)
Antigen concentration

21. SPECTROPHOTOMETER ADAPTED FOR ELISA PLATES
ELISA PLATE WASHER
ELISA PLATES
SPECTROPHOTOMETER ADAPTED FOR ELISA PLATES

22. Lateral Flow Strips (Dipsticks) Immunochromatography (Lateral Flow)
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 Predator support Sample pad analyte
Ab-colloidal gold

24. QUALITATIVE TEST: Analyte absent in the sample
Test line
Sample pad
Analyte
Ab-colloidal gold

25. If the analyte is ABSENT in the sample the line will be colored
Test line
Sample pad
Analyte
Ab-colloidal gold

26. Analyte PRESENT in the sample
Test line
Sample pad
Analyte
Ab-colloidal gold

27. Test line
Sample pad
Analyte
Ab-colloidal gold

28. Test line
Sample pad
Analyte
Ab-colloidal gold

29. If the analyte is present in the sample the line will be not colored
Test line
Sample pad
Analyte
Ab-colloidal gold

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

32. biosensor
Recorder
Biological component
Signal
transducer
Analyte

33. What do they have in common?
Biosensor
Small molecules / olfactory membrane / nerve cells / brain
Visible light / rods and cones / nerve cells / brain
Nose
Eye
Analyte / bioreceptor / transducer / processor

36. Staphylococcus aureus
gram-positive, non spore-forming bacterium
able to synthetise:
Enterotoxins: A, B, C, D, E (thermostable);
Coagulase;
Thermonuclease.
ng of enterotoxins are sufficient to cause toxinfection in immuno-compromised subjects.

38. DEVELOPED TEST: Conventional ELISA Proteina A
Conventional ELISA S. aureus
ELISA/AMPLI S. aureus
ELIMC S. aureus
ELIME S. aureus

39. Specific antibody (MAb o PAb) Secondary antibody-AP
Spectrophotometric ELISA AP
p-NPP
p-NITROPHENOL
Protein A/S.aureus
Human IgG
Specific antibody (MAb o PAb)
Secondary antibody-AP

40. ELISA Protein A MAb PAb LOD Sensitivity 0.6 ng/mL 7.6 ng/mL LOD
(a – d) x c + 1 b
+ d
MAb
IgG mg/mL
MAb :10000
Ab2-AP :1000
PAb
LOD
Sensitivity
0.6 ng/mL
7.6 ng/mL
LOD
Sensitivity
0.07 ng/mL
0.6 ng/mL
y = <x0> + 3s
IgG mg/mL
PAb :10000
Ab2-AP :1000
Sensitivity was calculated as tha amount of protein A needed to produce a 25% increase in the signal

41. ELISA S.aureus LOD Sensitivity 2 106 cell/mL 9 106 cell/mL LOD
MAb
PAb
IgG mg/mL
IgG mg/mL
MAb :10000
PAb :10000
Ab2-AP :1000
Ab2-AP :1000
No cross-reactivity

42. AMPLI Q INT FORMAZAN NADPH Acetaldehyde NADH NAD+ Ethanol
Alkaline phosphatase Pi
Acetaldehyde
INT
NADH
Alcohol deydrogenase
Diaphorase
NAD+
FORMAZAN
Ethanol
DAKO, Handbook for AmpliQ, 1997

43. ELISA S.aureus AMPLIQ
Because a new lot of PAb showed lower affinity for Protein A/S. Aureus, next experiments were carried out using only Mab.
MAb
LOD
LOD
PAb
6 104 cell/mL
7 102 cell/mL
IgG mg/mL
IgG mg/mL
Sensitivity
Sensitivity
MAb :10000
PAb :10000
2 105 cell/mL
6 103 cell/mL
Ab2-AP :1000
Ab2-AP :1000

44. Magnetic Beads Good results in immunological field Ø 1-5 µm
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 AP
p-NITROPHENOL
Microtitre
ELISA
p-NPP

46. + ELIME (Enzyme Linked ImmunoMagnetic Electrochemistry) AP
a-naphthol AP
+ NaH PO 2 3
a-naphthyl phosphate
DPV
Potential range mV
Scan speed mV/s
Pulse width ms
Modulation time 60 ms
Interval time s +
Selectivity Ag-Ab;
Sensibility of electrochemical detection;
Possibility of concentrating magnetic particles on the electrode surface.

47. Electrochemical measurement
Addition of
Enzymatic substrate
for
Electrochemical
measurement
Magnetic tube

48. ELIMC S.aureus ELIME S.aureus MAb LOD 1 104 cells/mL MAb LOD
IgG 0.5 mg/mL
Mab :50000
Ab2-AP 1:300
LOD
Sensitivity
1 104 cells/mL
2 105 cells/mL
MAb
IgG 1.2 mg/mL
MAb :1000
Ab2-AP :100
LOD
Sensitivity
1 103 cells/mL
2 104 cells/mL

49. Mab ELIME
Mab ELIMC
Pab ELISA AmpliQ
Mab ELISA AmpliQ
Pab ELISA S.a
Mab ELISA S.a
Pab ELISA prot A
Mab ELISA prot. A
Sensitivity
LOD
0.6 ng/mL
0.07 ng/mL
2 10 6
cell/mL 4
6 10
7 10 2
1 10 3
9 10 5
7.6 ng/mL
22 h
4 h
Analysis Time

50. Air samples Sample 1 Sample 2 660 cell/m3 ± 15% 11700 cell/m3 ± 11%
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
Sample 2
660 cell/m3 ± 15%
11700 cell/m3 ± 11%

51. Immunoassay test products validated by OSW

52. Anticholinesterase activity measurement by an enzyme biosensor:
application in water analysis

53. Acetilcholine Choline + Acetic ac. Choline + O2 + H2O Betaine + H2O2
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
Acetilcholine
Choline + Acetic ac.
Inhibited by
pesticides
Choline oxidase
Choline + O2 + H2O
Betaine + H2O2
Not Inibited
Electrode
H2O2 O2
+ 2H e-

54. Trasmission of the nervous impulse
Acetilcholinesterase

55. Inhibition measurements
I%=[( E0-Ei)/E0]•100
inhibited enzyme (E1)
Non inhibited enzyme (E0)
Time (min)

56. Inhibition of AChE with Paraoxon1 2 3 4
TIME (minutes)
I (nA) 6
Inhibition of AChE with Paraoxon
Blank
2 ppb
6 ppb
10 ppb

57. HEAVY METALS DETERMINATION BASED ON THE USE OF INVERTASE ENZYME

58. REACTIONS E1= Invertase E2 = Glucose Oxidase Sucrose E1 + H2O
D-Glucose + D-Fructose
D-Glucose E2
+ O2
Gluconic acid + H2O2
Electrode
O2 + 2H++ 2e-
H2O2

59. I1 I2 INHIBITION MEASUREMENTS Current Sucrose INV B Sucrose
INV + Inhibitor I2 A
Reaction Time
Time

60. FIA Calibration with sucrose 10mM20 40 60 80
[Hg2+] (ppb)
Inibition %
100
FIA Calibration with sucrose 10mM

61. 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

62. THANKS!!!!