biosensors lecutre 13

immunosensors

what are immunosensors

type of immunoassay

• biosensors that use antibodies or antigens as bioreceptors

• much more sensitive and specific than other biosensors

gold standard biosensors for medical diagnostics

ELISA

SPR

Antigens

generally any foreign substance in the body

include “not-self” molecules and cells

• Foreign proteins

• viruses

• environmental pollutants

• bacteria and parasites

• foreign transplanted tissue

• cancerous cells

scientifically any molecule that a specific antibody can bind to

Antibodes

a protein molecule created by the immune system

what happens when a foreign molecule invades the body

when an Antigen invades the body, the immune system recognizes and creates an antibody molecule that is a perfect fit for the foreign molecule (lock-and-key)

details on antibodies

• over 80% of human antibodies are in the IgG class

• they are shaped like a Y

• they are found in the blood, lymph, and intestine

molecular weight on IgG molecules

150,000 Da

application of antibodies

the high specificity of antibodies makes them an excellent tool for detecting and quantifying a broad array of targets, from drugs to serum proteins to microorganisms

Antibodys specificity

an antibodies specificity results from antigen-binding sites

• regions of the antibody that have unique patterns of amino acids that can only bind to target antigens with a molecular sequence that provides complementary charges and noncovalent bonds

Epitopes

• specific regions of an antigen to where antibodies bind

• a single antigen can have multiple epitopes for different, specific antibodies

affinity

a measure of the binding strength between an antibodies binding site and an epitope

how are polyclonal antibodies (pAbs) formed

becuause most antigens are complex structures with multiple epiptopes, they result in the production of multiple antibodies in the lab animal

how to pAbs bind

pAbs may bind to different antigens that have identical epitopes

• can possibly cause false positive results

Monoclonal antibodes (mAbs)

all antibodies must bind to a single epitope with high affinity

• better antibody specificity and affinity

monoclonal antibody characteristics

• expensive production

• long production time

• large quantities of specific antibodies

• recgonize a single epitope on an antigen

properties of polyclonal antibodies

• inexpensive production

• rapid production

• large quantities of nonspecific antibodies

• recgonize multiple epitopes on an antigen

how are monoclonal antibodies produced

in vitro using tissue-culture techniques

antibody fragments

antibodies have two pairs of polypeptide chains

• heavy chains and light chains

what are polypeptide chains

linear organic polymers consisting of a large number of amino acid residues bonded together in a chain

hypervariable region of antibodies

top portion of both heavy and light chains

• depends on the target antigen

constant regions of antibodies

identical remainder of the antibody

pros of small fragments

• potentially more stable

• easier to immobilize on solid surfaces

• better reactivity with target antigens

Enzyme Linked Immunosorbent Assay (ELISA)

method that has been used to detect antigens or antibodies in the blood serum, or to measure the amount of various other proteins such as hormones, toxins, allergens

4 types of ELISA methods

• direct

• indirect

• sandwich

• competitive

two most commonly used ELISA methods

indirect and sandwich

direct ELISA protocol

• place substrate in ELISA plate well to allow proteins to stick to surface

• insert a blocking protein to prevent false positives

• insert specific detection antibody solution (antibody + enzyme)

• rinse out unbound antibodies

• insert substrate to react with enzyme

• rinse

advantages of direct ELISA

• minimum procedure

• avoids cross-reactivity from a secondary antibody

disadvantages of direct ELISA

requires labeling of all primary antibodies

indirect ELISA protocol

• place substance in ELISA plate well to allow antigens to stick to surface

• insert a blocking protein or detergent to eliminate false positives

• insert antibody to stick to antigen

• rinse our unbound antibodies

• insert specific detection antibody solution to react with other antibody

• rinse our unbound antibodies

• insert substrate to react with enzyme

• rinse

advantages or indirect ELISA

• secondary antibodies are capable of signal amplification

• many available antibodies can be used for different assays

• unlabeled primary antibodies retain maximum immunoreactivity

disadvantages of indirect ELISA

cross reactivity may occur

sandwich ELISA protocol

• place antibody in ELISA plate well then rinse out excess

• insert a blocking protein or detergent to eliminate false positives

• insert substance to attach to antibody

• rinse

• insert specific detection antibody solution to react with other antibody

• rinse our unbound antibodies

• insert substrate to react with enzyme

• rinse

advantages of sandwich ELISA

• sensitive

• highly specific

• antigen does not need to be purified prior to use

disadvantages or sandwich ELISA

antigens must contain atleast 2 antibody binding sites

competitive ELISA protocol

• mix samples with a reagent

• coat ELISA plate with antibody

• insert blocking protein

• insert sample and enzyme conjugated antigen

• insert substrate to react with enzyme

• rinse

advantages of competitive ELISA

• crude or impure samples may be used

• high reproducibility

disadvantages or competitive ELISA

lower sensitivity and specificity

reactive functional groups of antibodies

Amine group (NH2)

Carboxyl group (COOH)

Hydroxyl group (OH)

2 Physical absorption mechanisms

1. hydrophobic interactions

2. electrostatic interactions (van der waals)

hydrophobic interactions

nonpolar substances tend to clump together rather than distributing in a water medium becuase this allows them to have minimal contact with water

van der walls interactions

the attractive or repulsive interaction between objects having electric charges

advantages of physical absorption

simplest approach

disadvantages of physical absorption

• uncontrollable

• antibodies can be immobilized in a randomly oriented manner, denatured, or displaced in later steps by washing

Covalent binding

the covalent coupling of capture antibodies ensures robust immobilizations and can improve density and orientation outcomes at the substrate

groups for covalent binding

1. Amine and carboxyl groups

2. Thiol groups

3. Carbohydrate groups

amine and carboxyl groups

ubiquitous throughout an antibodies structure and are common at the antibodys surface

EDC/NHS

can be employed as a covalent attachment method for immobilization of proteins and also as a method for preparing substrates

• couples antibodies amine/carboxyl groups to surface carboxyl/amine groups

what is used for silanization

APTES

TESPSA

GOPS

what is plasma treatment used for

plasma treatment can be used to activate the polymer substrate surface, increasing hydrophilicity and reducing denaturation of bound proteins