Introduction to Immunoanalysis Lecture 1 PDF

Introduction to Immunoanalysis Lecturer: Caroline Browne LECTURE 1 BIOT6002 Learning Objectives 1. Describe and draw the basic structure of Immunoglobulin G (IgG) 2. Define: a) An antibody b) An antigen c) A paratope d) An epitope e) A hapten 3. Understand the bas...

Introduction to Immunoanalysis Lecturer: Caroline Browne LECTURE 1 BIOT6002 Learning Objectives 1. Describe and draw the basic structure of Immunoglobulin G (IgG) 2. Define: a) An antibody b) An antigen c) A paratope d) An epitope e) A hapten 3. Understand the basic interaction between an antibody and antigen 4. Give examples of immunoassay applications Immunoanalysis ▪ Immunoanalysis ▪ Analytical methods using high affinity specific binding proteins ▪ Immunoassays (also known as ELISA, EIA) ▪ Assays using Antibodies (Ab) as specific binding reagents ▪ Affinity, specificity & variety of Ab binding reactions - ideal tools for routine analysis/research ▪ Applications ▪ Clinical diagnostics ▪ Food & environmental testing ▪ Forensics ▪ Pharmaceutical development/testing ▪ Research Antibodies The presence of immunogens or antigens (Ag) triggers the immune system of the host into producing antibodies (Ab) Ab a protein that recognises the Ag naturally produced by the hosts’ plasma cells The Ab-Ag complex initiates other immune reactions to destroy the invading pathogen or foreign molecule. Note: Plasma cells - differentiated B-lymphocyte, commonly referred to as white blood cells. Types of Ab - immunoglobulins (Ig) Antibody Properties Structure IgA Found in mucous, saliva, tears, and breast milk. Protects against pathogens IgD Found on immature B cells. Activates basophils and mast cells. IgE Protects against parasitic worms. Responsible for allergic reactions. IgG Secreted by plasma cells in the blood. Able to cross the placenta into the foetus. IgM May be attached to the surface of a B cell or secreted into the blood. Responsible for early stages of immunity. IgG - Predominantly used in immunoassays 150,000 Dalton or 150 kDa in size 2 x identical Light polypeptide chains 215 amino acid residues 1 variable (VL) & 1 constant (CL) region 2 x identical Heavy polypeptide chains 420 amino acid residues 1 variable (VH) & 3 constant (CH) regions Chains are linked by disulphide bonds or bridges Definitions – Ag and Epitope Antigen (Ag) Substance which elicits an immune response & reacts with Ab Epitope (Antigenic Determinant) Region of Ag that interacts with Ab Journal of immunology research, volume: 2017, issue: 1, first published: 28 december 2017, DOI: (10.1155/2017/2680160) Definitions - AA and Paratope Protein Antigen (AA) Continuous: stretch of AAs directly linked in the sequence of protein Discontinuous: AAs are separate from one another in the primary linear sequence Paratope (Antigen Binding Site) Composed of 50 AA residues across both variable segments of H & L chains of antibody Journal of immunology research, volume: 2017, issue: 1, first published: 28 december 2017, DOI: (10.1155/2017/2680160) Definitions - Hapten Hapten A molecule of low molecular weight that is non-antigenic. Can be conjugated to large protein (e.g. bovine serum albumin BSA) to produce an immunogen used to generate Ab against the hapten Diagnostic tests - Basic principles – ELISA or EIA Group Activity - Applications ▪ Clinical diagnostics ▪ Food & environmental testing ▪ Forensics ▪ Pharmaceutical testing ▪ Research Applications – Clinical diagnostic tests Frew, E., et al. Sci Rep 11, 23009 (2021). https://doi.org/10.1038/s41598-021-02128-y PMCID: PMC8626481  Applications – clinical diagnostic applications Hormone testing human chorionic gonadotropin (Hcg) Hormone testing Troponin 1 https://web.archive.org/web/20180809113913/http://primeglobalcomms.com/ Applications – Food & environmental testing Pork extract in food products Hendrickson et al., 2021 Food Chemistry doi.org/10.1016/j.foodchem.2021.129927. Residual Serratia marcescens endonucleases – common pathogen in hospitals. Applications - Forensics Historical doping in cycling and horse racing – Erythropoietin (EPO) testing kits commonly used a first screen Applications – Pharmaceutical testing Screening for “Drugs of abuse” THC Cocaine Opiates Week 6 20 Fentanyl Ethanol Consumption 15 Cathinones (g/kg) 10 Benzodiazepines 5 Barbiturates 0 0 50 100 150 BEC (mg/dL) Blood ethanol concentrations (BEC) correlated with ethanol consumption (g/kg) following six weeks in intermittent intake. Campanile, Lucki and Browne unpublished.  Future Applications – Multiplex plates and Biochips Multiplex plates Biochips - contain up to millions of biosensors or microarrays – takes advantage of new developments in microfluidics and bioengineering.  Lucki, Sonkusale and Browne – Funded Grant program Design of microneedle biosampling device: hollow chitosan microneedles used for interstitial fluid (ISF) and 3D electrochemical Paper Analytical Device (3D ePAD) multiplexed detection of biomarkers). Learning Objectives 1. Give examples of a range of applications of immunoassays 2. Describe and draw the basic structure of IgG 3. Define what is: a) An antibody b) An antigen c) A paratope d) An epitope e) A hapten 4. Understand the basic interaction between antibody and antigen