Generating Antibodies and Aptamers

Antibodies and Aptamers
- Raising antibodies (monoclonal vs polyclonal)
- Generating aptamers (SELEX methodology)
- Sensors utilizing antibodies or aptamers
- Enzyme-based sensors and biological nanopores

Definition: A biomarker is a measurable indicator of a biological state or condition. Biomarkers may be chemical, physical, or biological in nature.
Types of Biomarkers:
- Diagnostic: Indicates presence of a condition
- Monitoring: Reflects the status of a condition
- Pharmacodynamic: Measures response to a therapeutic agent
- Predictive: Indicates the likelihood of an outcome
- Prognostic: Predicts clinical outcomes
- Safety: Assesses toxicity levels
- Risk: Evaluates potential to develop diseases

Definition: A biosensor converts a biological response into a measurable signal.

Definition: Antibodies are Y-shaped protein complexes that are involved in the immune response. They are glycoproteins known as immunoglobulins.
Characteristics:
- Approximately 10^{15} different types exist within the immune system.
- They can bind specifically and with high affinity to an antigen.
- Structure consists of variable (VL/H) and constant (CL/H) domains.
- Visual representation: Diagram of an antibody illustrating Fab and Fc regions.

Each immunoglobulin class serves different functions.
IgG:
- Most abundant immunoglobulin class
- Four subclasses: IgG1, IgG2, IgG3, and IgG4
- An intact IgG monomer weighs around 150 kDa.

Polyclonal Antibodies:
- Produced by animal response to an antigen
- Can bind to various epitopes of the same antigen with different affinities
Monoclonal Antibodies (mAbs):
- Selected to bind to the same epitope with uniform affinity.

Production methods:
- In vivo (using animals) or in vitro (using cell culture)
- In vitro methods such as phage display allow extensive screening for specific antibodies.
- Most clinically used antibodies are produced via phage display, albeit at significant costs.

Definition: A method for selecting monoclonal antibodies.
- Naïve or synthetic DNA encoding mAbs is spliced into a bacteriophage (e.g., M13) genome.
- The displayed mAbs are screened against a target antigen, and repeated selection cycles enhance specificity.

Definition: Aptamers are generally single-stranded DNA (ssDNA) or can be derived from peptides, featuring sequence-dependent 3D structures that bind targets with high affinity.
Characteristics:
- Approximately 30 base pairs (bp) in length, offering around 4^{30} (approximately 1.2 imes 10^{18}) different combinations.
- Created through combinatorial synthesis.

Example Sequence: 5’GGG AGA CAA GGA AAA TCC TTC AAT GAA GTG GGT CGA CA3’
Factors influencing folding: sequence, temperature, ionic strength, counter-ions, and target presence.

Method: Systematic Evolution of Ligands by EXponential enrichment (SELEX)
- Procedure involves screening random DNA against targets through repetitive cycling to enhance selectivity and affinity.
- Notable initial works by Tuerk, C. and Gold, L. (1990), followed by Ellington, A. D. and Szostak, J. (1990).
- Definition of 'nt' as nucleotide.

Definition: An additional negative selection step designed to remove off-target aptamers and improve their selectivity.

Production:
- Antibodies: Raised in animals (polyclonal) or cultured (monoclonal)
- Aptamers: Synthesized on demand through solid-phase synthesis or PCR once the target is known.
Specificity and Affinity:
- Antibodies: Highly specific (especially monoclonals) with high affinity.
- Aptamers: Moderate specificity and affinity.
Target Range:
- Antibodies: Broad (for small and large molecules).
- Aptamers: Narrow, typically focused on small molecules.
Stability:
- Antibodies: Moderately stable but sensitive to denaturation from pH and temperature.
- Aptamers: Highly stable; resistant to pH and temperature, but sensitive to nucleases.
Applications and Assay Development:
- Antibodies: Can be modified with dyes but may lose activity under certain conditions.
- Aptamers: Easily functionalized and adaptable to varied assay conditions.

Utilization: Both antibodies and aptamers can be coupled to sensors through various methods, including:
- Diagnostic lateral flow strips
- Fluorescent dyes for small molecules and proteins
- Nanoparticles and Quantum Dots
- Quantifying assays such as Surface Plasmon Resonance (SPR) and ELISA
- Imaging/tracking applications

Concept: Gold nanoparticles (AuNP) exhibit color changes based on size due to surface plasmon effects, with smaller diameters appearing red and larger appearing blue.
Mechanism: Rationally designed aptamers shield these nanoparticles in the absence of a target molecule.

Components: Descriptions of gold spheres and gold nanoshells, their dielectric core, and metal shell.

Context: Used for cancer imaging, emphasizing theranostic capabilities that include both imaging and treatment processes.

Antibodies: Binding with high affinity to specific antigens.
Aptamers: Short, artificial ssDNA constructs with high specificity and affinity to target analytes.
Both can be integrated with labels or sensors for the detection and quantification of target analytes.