Immunoassay Presented by Dr Reza Javadi MD APCP

Immunoassay Presented by : Dr Reza Javadi MD , AP/CP 2016

Ag-Ab reaction For efficient interaction to occur between the antigen and the antibody: the epitope must be readily available for binding. If the target molecule is denatured, e. g. through fixation, reduction, osmolalaity changes, p. H changes, temperature, chemical agents, the epitope may be altered and this may affect its ability to interact with an antibody.

Characteristics of a Good Antigen Include: 1. Areas of structural stability and chemical complexity within the molecule. 2. Lacking extensive repeating units. 3. A minimal molecular weight of 8, 000– 10, 000 Daltons, although haptens with molecular weights as low as 200 Da have been used in the presence of a carrier protein. 4. The ability to be processed by the immune system.

Characteristics of a Good Antigen Include: 5. Immunogenic regions which are accessible to the antibodyforming mechanism. 6. Structural elements that are sufficiently different from the host. 7. For peptide antigens, regions containing at least 30% of immunogenic amino acids: K, R, E, D, Q, N. 8. For peptide antigens, significant hydrophilic or charged residues.

Class/ Subclass Heavy Chain Light Chain Molecular Weight (k. Da) Structure Function lg. A 1 lg. A 2 a 1 a 2 l or κ 150 to 600 Monomer to tetramer Most produced lg; protects mucosal surfaces; resistant to digestion; secreted in milk. lg. D d l or κ 150 Monomer Function unclear; Works with lg. M in B-cell development. mostly B cell bound lg. E e l or κ 190 Monomer Defends against parasites; causes allergic reactions lg. G 2 a lg. G 2 b lg. G 3 lg. G 4 g 1 g 2 g 3 g 4 l or κ 150 Monomer Major lg in serum; good opsonizer; moderate complement fixer (lg. G 3); can cross placenta lg. M µ l or κ 900 Pentamer First response antibody; Strong complement fixer; Good opsonizer

Monoclonal vs. Polyclonal 2 nd Ab : Mouse / goat / rabbit anti- 1 st Ab 1 st Ab : Mouse / goat / rabbit anti-human

Antigen-Antibody Interaction The specific association of antigens and antibodies is dependent on hydrogen bonds, hydrophobic interactions, electrostatic forces, and van der Waals forces. These are all bonds of a weak, non-covalent nature-Like antibodies.

Antigen-Antibody Interaction Antigens can be multivalent, either through multiple copies of the same epitope, or through the presence of multiple epitopes that are recognized by multiple antibodies. Interactions involving multivalency can produce more stabilized complexes, however multivalency can also result in steric difficulties, thus reducing the possibility for binding.

Antigen-Antibody Interaction All antigen-antibody binding is reversible, however, and follows the basic thermodynamic principles of any reversible bimolecular interaction: where KA is the affinity constant, Ab and Ag are the molar concentrations of unoccupied binding sites on the antibody or antigen respectively, and Ab–Ag is the molar concentration of the antibody-antigen complex.

Antigen-Antibody Interaction Affinity describes the strength of interaction between antibody and antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody “arm” interacts through weak non-covalent forces with antigen at numerous sites; the more interactions, the stronger the affinity.

Antigen-Antibody Interaction Avidity is perhaps a more informative measure of the overall stability or strength of the antibody-antigen complex. It is controlled by three major factors: 1. antibody-epitope affinity; 2. the valence of both the antigen and antibody; 3. and the structural arrangement of the interacting parts.

Polyclonal vs monoclonal Polyclonal antibodies often recognize multiple epitopes, making them more tolerant of small changes in the nature of the antigen.

Monoclonal vs polyclonal Because of their specificity, monoclonal antibodies are excellent as the primary antibody in an assay, or for detecting antigens in tissue, and will often give significantly less background staining than polyclonal antibodies.

1. Non-Labeled Immunoassay ( e. g. : Nephelometry – IEP – SRID ) 2. Labeled Immunoassay (conjucated) ( e. g. : RIA- EIA- FIA- CLIA )

EIA Homogeneous EIA refers to any antigen - antibody reaction that does not require a separation step. EMIT SLFIA ARIS Heterogeneous EIA needs physical separation (washing) of free from bound ligands ELISA ELFA

EMIT( Homogenous)

ELFA(SPR and Strip)

Principle of method

Solid Phase technology

Enzyme –labeled antibody

Non competitive

Competition Assays Insulin –Estrogen T 3, T 4. T 3 Ru High amount of Ag in sample reduce the amount of labeled Ag that can bind

Sandwich ELISA

Sandwich method Tumor markers –Interleukins HBSAg, TSH, Ferritin, PTH

Calibrators are materials used to standardized the instrument or other assay method each time testing is performed. Controls are materials intermixed with patient samples and assayed in order to ascertain the reliability of the assay.

Interfering substances Hemoglobin , bilirubin , and other substances can interfere with some assays. The potential interferences afforded by such substances should be specified in the package insert.

Method HCV ADALTIS • • • Controls and calibrators 200 micro L Sample 200 micro L 1 st incubation 45 min Temp 37 C Wash step 5 cycles Enzyme conjugate 100 micro L 2 nd Incubation 45 min Temp 37 c Wash step 5 cycles TMB /H 2 o 2 100 micro L 3 rd incubation 15 min Rt Sulphidric acid 100 micro L Reading OD 450 nm

Result • • Wavelength 450 nm 630 nm Calibrators 1 Conc: 200 ABS : 1. 13 Calibrator 2 Conc: 20 ABS: 0. 206 Calibrator 3 Conc: 2 ABS : 0. 054 PC Conc: 103 ABS: 0. 639 NC conc: 9. 9 ABS: 0. 121 Test

ELISA WASHER and INCUBATOR

Automation

Software

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