PTT 202 Organic Chemistry for

BiotechnologyLecture 7: Immunological Methods

zulkarnainidris@unimap.edu.myZulkarnain Mohamed Idris

Semester 1 2013/2014

Introduction Antibody: protein produced by animal as a result

of the introduction of foreign substance into its tissue (in process known as the immune system).

Antigen: foreign substance/molecule or immunogen that reacts with a antibody.

Hapten: small substance that is not capable to initiate an immune response but may do so when attached to a larger molecule.

Epitope: part of antigen that interacts with an antibody.

Paratope: the complementary portion of the antibody that interacts with an epitope.

Antigen-antibody reactions provide the basis of useful methods of qualitative and quantitative (immunoprecipitation, radioimmunoassay, monoclonal antibodies).

General processes of the immune response

Lymphocytes: small and mononuclear cells primarily associated with the immune processes.T lymphocytes: cells produced in the thymus & responsible for cell-mediated immunity.B lymphocytes: cells produced in the bone marrow & responsible for antibody production.Humoral immunity: mediated by antibodies.Cell-mediated immunity: cells are directly involved in the protective processes.

Kinetics of immune response

Primary response: Type of reaction when an animal encounters an antigen for the first time

Secondary response: Type of reaction when an animal encounters an antigen more than once

Antigen recognition and lymphocyte stimulation

1. Antigen is taken up by macrophages upon entering the tissues.

2. After modification, is exhibited on the outer membrane of the cells.

3. Recognized by lymphocytes via antigen-specific receptors carried on their membranes.

4. T helper cells (Th cells) recognize the antigen and stimulate the specific B, Tc or Td cells.

Structure and antibody roles

Structure and antibody roles

Structure and antibody roles

Structure and antibody roles

Antigen-antibody reactions Similar to the binding of an enzyme to its

substrate. Involves hydrophobic and electrostatic

interactions. Involves no subsequent chemical reaction and

stability depends upon the complementary shape of the antigen and the binding site of the antibody.

Reversible and dissociate dependent upon the strength of binding:

The strength of the binding of antibody to an antigen is referred to its affinity and is defined by the equilibrium constant K:

Production of antibody Polyclonal antibodies: produce by

immunization of an animal involves injection of the pure antigen to stimulate the immune system to produce antibodies. The antiserum contains a heterogeneous mix of antibodies directed against an antigen.

Monoclonal antibodies: produce by immunization of an animal in traditional manner, but instead of allowing the immune system to produce antibodies, the lymphocytes are separated and fused in vitro with myeloma cancer cells growing in cell culture. Contains homogeneous serum containing only one antibody directed against an antigen.

Production of monoclonal antibodies

Effects antigen-antibody complex formation

Immunological methods of analysis

Analytical techniques-Precipitation reactions

Many qualitative and quantitative methods for soluble antigens are based on their precipitation by antibodies.

Immunoprecipitation in solution: the reaction is carried out in an excess of antibody and a calibration is prepared by measuring the turbidity of series of standard solutions of antigen. The formation of antigen-antibody complexes are observed by measuring the apparent absorption of light when incident light is scattered by the complexes or by direct measurement of the scattered light.

Immunoprecipitation in gels: gels are used to stabilize the precipitate, enabling both position and the area of the precipitate to be measured. Antigen is permitted to diffuse into a gel that contains a uniform conc. of antibody and precipitate will form at some point in the conc. gradient of the antigen. The distance between the precipitate and the original starting point of the antigen will be proportional to its initial concentration. Forms the basis of single radial immunodiffusion (SRID) technique (Procedure 7.1).

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Analytical techniques-Precipitation reactions

Immunohistochemistry: antibodies are used to identify the appropriate antigen and the position of this primary antibody will be detected directly (initially labelled) or indirectly using another secondary antibody or molecule to attach to the antibody (Figure 7.8). Samples need to be washed after addition of the primary or labelled antibody to prevent non-specific reactions. Label that have been successfully linked to antibodies include: fluorescent dyes, enzymes, colloidal gold.

Immunoblotting (dot blotting and western blotting): soluble antigens are applied to a nitrocellulose membrane, antibody is then applied to the membrane, the membrane is washed to remove unbound antibody, and the presence or absence of antigen is determined by similar means that employed during immunohistochemistry or autoradiography (Figure 7.9).

Immunohistochemistry

Western blotting technique

Analytical techniques-Immunoassay

Two broad approaches: competitive and non-competitive immunoassays.

Competitive immunoassay: relies on the competition between labelled and unlabelled antigens for a fixed and limited number of antibody-combining sites.

Non-competitive or immunometric immunoassay: employs antibodies in excess and for which there is no competition for binding sites.

Components of immunoassay systems The antibody: both polyclonal and monoclonal

antibodies have been used in immunoassays. It is essential to determine the required optimum antibody concentration (the dilution of antiserum (or titre) which will bind 50% of the labelled antigen in the absence of unlabelled antigen) when using competitive assay. Once the titre of an antiserum is known, its specificity (or ability of an antiserum to recognize only the antigen for which it was generated) may be assessed.

The label: the antibody or antigen needs to be labelled with an appropriate molecule for detection. The molecule should retain high signal efficiency and should have no effect on their subsequent immunoreactivity (e.g: refer to Table 7.3)

Components of immunoassay systems

The choice of labels1. Radioisotopes:

Carbon-14, hydrogen-3 (tritium) and iodine-125 are commonly used radioisotopes.

Carbon-14 and hydrogen-3 are beta-emitting isotopes, while iodine-125 emits both beta particles and gamma radiation.

Carbon-14 and hydrogen-3 are example of internal labels since the radioactive atom replaces an existing atom within the antigen, while iodine-125 as external label because it is necessary to attach the iodine covalently to the antigen.

Advantage of carbon-14 and hydrogen-3: labelled form of molecules is identical to unlabelled antigen, immunoreactivity is maintained.

The choice of labels1. Radioisotopes:

Disadvantage of carbon-14 & hydrogen-3: the efficiency of the measurement of beta emission is less compared to gamma emission of iodine-125.

Disadvantage of iodine-125: the covalent attachment of the isotope to the antigen cause significant structural difference between labelled and unlabelled antigen-loss of immunoreactivity, but due to higher signal measurement, this isotope is prefer to be used for immunoassay system.

2. Enzymes: Are usually associated with solid-phase

antibodies technique known as enzyme-linked immunosorbent assay (ELISA).

The ‘two-site’ assay employing two monoclonal antibodies is the best technique of ELISA (refer to Figure 7.12).

The choice of labels2. Enzymes:

The enzymes commonly used as labels include alkaline phosphatase, horseradish peroxidase and beta-galactosidase.

The enzymes should be capable of being covalently linked to the antigen or antibody without loss of their catalytic activity or immunoreactivity.

The choice of enzyme is governed by the ability of substrate and type of detector.

Enzymes also can be used to amplify the signal from the label using cycling systems.

3. Luminescent labels:• Different types of luminescence depend on the

source of energy used to excite the molecules to higher energy state.

• Radioluminescence immunoassay: occurs when energy is supplied from higher energy particles. Antigen is labelled with radioisotopes.

The choice of labels

3. Luminescent labels:

• Chemiluminescence immunoassay: energy is derived fro a chemical reaction. Antigen is tagged with a molecule such as luminol or acridium ester that emits light with high quantum yield on oxidation.

• Bioluminescence immunoassay: excitation is performed by biological molecule such as enzyme. Antigen is labelled with luciferin which emits light when oxidized by luciferase enzyme.

• Fluorescence/ Photoluminescence immunoassay: excitation is derived from light energy. Antigen is labelled with fluorescein or rhodamine.

*Heterogenous immunoassay- relies upon physical separation of bound and free fraction to assess the relative proportions of each fraction

*Homogenous immunoassay- requires no separation step to observe the ration between labelled and unlabelled antigen and the antibody

Membrane-based immunoassay devices