Embed Size (px)
Citation preview
Outline
� Nomenclature
� What is an Antibody
� Monoclonal and Polyclonal antibodies
� Enzyme-Linked ImmunoSorbent Assay ELONA
� Assay formats
� Some examples
� Further examples
� Mass based sensing assay
� Lateral flow assay
Of what we will talk? Nomenclature
Immunosensors: Use antibody as biorecognition element.
Affinity biosensors: Biosensors in which bio-recognition molecules bind analyte molecules leading to formation or dissociation of complex.
Possible receptor molecules: i) antibodies ii) DNA iii) receptors
Design parameters for affinity biosensors
Assay format strongly influenced by:• Nature of analyte• Transduction approach
What are Antibodies?
Antibodies are proteins produced by B cells and that are used by the immune system to identify foreign substances (e.g. bacteria, virus).
Antibodies are specifically binding to an unique part (epitope) of their specific target molecule (antigen).
Antibodies contain a region with high variability, the Fab region, that is involved in antigen recognition and a constant region (fiveisotypes: IgG, IgA, IgD, IgM, IgE are known) the Fc region, that modulate the cellular function of the antibodies.
Synthetic antibodies can exist in the monoclonal or polyclonal form; these differ in their ability of interacting with the antigen.
Basic Structure
1. Variable region (Fab)
2. Constant region (Fc)
3. Heavy chain: contain a
variable domain (VH) and a
constant domain (CH1), a
connection chain, and two more
constant domains (CH2 y CH3).
4. Light chain: with a variable
domain (VL) and a constant
domain (CL)
5. Recognition region
6. Connection region.
Cabohydrate
chain
Monoclonal Antibodies (Mab)
Antibodies produced by theprogeny (cell derived from) of asingle parent B cell.
The produced antibodies will allhave the same specificity(recognise a single epitope ofthe antigen).
Polyclonal Antibodies (Pam)
antigen
When different B-cells produceantibodies against the sameantigen (antigen inoculated inan animal), the producedantibodies will have slightlydifferent specificity and will beagainst different epitopes ofthe antigen.
Polyclonal antibodies (mixtureof monoclonal antibodies).
Antibodies production
Polyclonal Monoclonal
Enzyme-Linked ImmunoSorbent Assay (ELISA)
1. Immobilisation of capture antibodies 2. Addition of samples 3. Addition of detection antibodies (with an
enzyme label) 4. Addition of enzyme substrate for colour
development 5. Measure by optical transducers
How to read?
Multiple-wells spectrophotometer
ELISA: Direct assay
substrate
product
Concentracion
•Used to detect the presence of antigens
in a sample.
•Robust assay
•It can be performed using either MAbs
or Pabs
•Not suitable for small molecules
Primary antibody (biorecognition
element)
Secondary labeled antibody (reporter);
usually Pam against the antigen).
Analyte (antigen)
ELISA: Indirect assay
substrate
product
Concentracion
Primary antibody (target/recognition
element)
•Used for the detection of
antigenes/antibodies in serum
•Robust assay: all reagent are in excess
Secondary antibody (reporter); usually Pam
against the primary antibody.
Antigen (recognition element/target)
Procedures for direct affinity assay
Martin Mak
Interpretation of direct affinity assay
Martin Mak
ELISA: Competitive assay
Concentration
•Sample and marked antigenes are
incubated with the antibodies at the
same time
•Suitable for small molecules
•Low sensitive assay
•Require a fine optimisation of the
concentration of the reagents to be
used
Procedures for competitive affinity assay
Martin Mak
ELISA: Displacement assay
Concentration
•Single step assay
•Low sensitivity high speed
•Requires fine tuning of the reagents concentration to be used.
Interpretation of competitive affinity assay
Martin Mak
Sensitivity in affinity biosensors
Imagine each candle representing an antibody and you are an optical transducer, you can easily detect if one candle light up (direct affinity assay)
You cannot easily detect if one candle is being off (competitive affinity assay)
Beyond ELISA:Other possible assay formats
Membrane-based lateral flow affinity devices
Lateral flow format (1980’S till Today)
• Use capillary forces to allow movement of biomolecules across different
zones: conjugation zone, test zone and control zone.
• Colloidal gold nanoparticles are used as support for bio-recognition molecules
and as labels (give color)
• “One-step” test concept (Whole blood assay)
• Smart use of biochemical interaction for performing different functions.
Membrane-based lateral flow affinity devices: how do they work?
AuNPs work as support for bio-recognition molecule immobilisation and as label (give colour).
The design of the bio-label is crucial for detection to happen.
How can I get my information?
Clearblue Pregnancy tests.Detect the hormone hCG (Human chorionic gonadotropin)
using a combination of different antibodies. Semi-Qualitative evaluation:
Reflectance: The ratio of the total amount of radiation reflected by a surface to the total amount of incident radiation.
Imaging: Combination of digital camera and advance images processing tools.
www.clearblue.com/
Luminex technology: Consist of combining beads-based assay, flow-cytometry and fluorescence detection to perform fast
• The wells used of ELISA assay are replaced by beads• High throughput analysis (up to 100 targets at the time) can be performed in
homogeneous format.• Require lower sample volume.• It is fast and allow significant statistical analysis.
Luminex assay
Polymeric beads containing 2 different fluorophores are used as support.
Direct Assay
Use flow-cytometry for fat high throughput analysis.
Detection principle in Luminex
Flow-cytometry system
Lasers for beads identification
The ratio of the responses from the 2 fluorophores in the beads is used to identify the beads and a result the assay.
Detector for reporter
The fluorescence from the reporters is used to quantify the presence of the different antigens.
Detectors for beads
Electrochemical:
Acustic (QCM):
SPR:
Magnetic:
Other possible transduction approaches
Antibodies immobilisation
Optimal orientation of the antibodies results in:
• Greater affinity for the antigen
• Increased sensitivity of the immunosensor
• High reproducibility in the immobilisation
Ammino linking of antibodies
Early methods of immobilisation using the NH2 and COOH groups of
the antibody for covalent attachment. These groups, being distributed throughout
the molecule surfaces with antibodies generated randomly oriented and low
affinity.
This phenomenon is particularly critical if NH2 groups are used for the
immobilisation because they are preferentially located in the recognition site
NH2
NH2
COOH
NH2
CONH
Clever design of immobilisation
An alternative is to modify the carbohydrate groups of the antibody by oxidation
withperiodate and subsequent introduction of amino groups to achieve a more
effective orientation.
NH2
O O
NH2
1) NaIO42) RNH2/NaBH4
NH2
COOH CONH
Fab immobilisation
+enzymatic
digestion
antibody
(Fab)2 fragment
Fc fragments
(discarded)reducing
agent
Fab fragments-SH
-SH
Preparation of Fab fragment
Advantages
Higher sensitivity; due to the immobilisation of higher number
and better oriented recognition molecules.
Reduced background response: lower unspecific response
due to the absence of the Fc fragment (recognised by several
receptors).
Summary
• We saw how antibodies can be used in biosensing.
• We saw 3 different commercial formats of immunoassays: ELISA, lateral flow strip test and Luminex.
• We saw how different formats or ELISA can be used to detect different targets and how results can be interpreted.
• We saw how optimal orientation of antibodies on surfaces can be achieved.
