Horseradish Peroxidase Immunoassays Patrick A. Williams Ian M. - PowerPoint PPT Presentation

Horseradish Peroxidase Immunoassays Patrick A. Williams Ian M. Klink Brandon D. Shaw Chemical Engineering Senior Design Project

Overview I. Purpose II. Immunoassay Purpose Purpose III. Market Immunoassay Immunoassay Market Market IV. Characteristics of Our Immunoassay Characteristics Characteristics V. Design Options Design Options Design Options VI. Fabrication Fabrication Fabrication VII. FDA Approval FDA Approval FDA Approval Financial Risk Risk Financial VIII. Financial Risk

Objectives & Goals o Produce a quality diagnostic test o Provide leading immunoassays for bacterial detection Purpose Purpose Purpose Purpose Immunoassay Immunoassay o Target rural hospital market initially Market Market o Make Money Characteristics Characteristics Design Options Design Options Fabrication Fabrication FDA Approval FDA Approval Financial Risk Risk Financial

Product Immunoassay Kit o Analytical test Purpose Purpose Purpose Purpose o Targets specific molecule Immunoassay Immunoassay o Results obtained by signal Market Market Characteristics generation Characteristics Design Options Design Options Fabrication Fabrication FDA Approval FDA Approval Financial Risk Risk Financial

Why S. aureus immunoassay? Staphylococcus aureus o Major cause of hospital infections Purpose Purpose Purpose Purpose o 8 common infections Immunoassay Immunoassay o Can cause deadly complications Market Market o Hospital Infections 4 th leading cause Characteristics Characteristics of US deaths Design Options Design Options Fabrication Fabrication FDA Approval FDA Approval Financial Risk Risk Financial www.ulb.ac.be/sciences/biodic/ ImBacterie2.html

Why S. aureus immunoassay? Common Conditions Purpose Purpose Purpose Purpose Infection Description Immunoassay Immunoassay Folliculitis inflammation of the follicles Market Market Boils painful, pus-filled inflammation of the skin and subcutaneous tissue Sties Inflammation of one or more sebaceous glands of an eyelid Characteristics Characteristics Impetigo a contagious skin infection Design Options Design Options Abscesses A localized collection of pus in part of the body, formed by tissue disintegration and surrounded by an inflamed area Fabrication Fabrication Staphylococcal pneumonia inflammation of the lungs Osteomyelitis onset after surgery FDA Approval FDA Approval Toxic shock syndrome acute infection associated with tampon use during menstruation Financial Risk Risk Financial

S. aureus immunoassay o Early detection yields better treatment o Sanitation control Purpose Purpose Purpose Purpose Immunoassay Immunoassay o Can be modeled and adapted to Market Market other immunoassays Characteristics Characteristics Design Options Design Options Fabrication Fabrication FDA Approval FDA Approval Financial Risk Risk Financial

Immunoassay

Components Antibody o Immune system vesicle Purpose Purpose Antigen Immunoassay Immunoassay o Molecule being detected Market Market Characteristics Characteristics Enzyme Design Options Design Options o Oxidizing agent using in signal generation Fabrication Fabrication FDA Approval FDA Approval Substrate Financial Risk Risk Financial o Signal generator in conjunction with enzyme

Background o Antibody Polyclonal Purpose Purpose • Same form of antibody with o Chains Variable Immunoassay Immunoassay Immunoassay Immunoassay varying binding specificities and o Regions Region affinities for the targeted antigen Market Market Light Chain o Types o Polyclonal Characteristics Characteristics Monoclonal o Monoclonal Constant Design Options Design Options Region • Arises from a single cell and have Heavy Fabrication Fabrication Chain FDA Approval FDA Approval Financial Risk Risk Financial

Background o Which antibody for our assay? o Immunoglobulin G (IgG) Purpose Purpose o Why IgG? Immunoassay Immunoassay Immunoassay Immunoassay Market Market o Widely available Characteristics Characteristics o Higher antigen affinity compared to Design Options Design Options other antibodies Fabrication Fabrication FDA Approval FDA Approval Financial Risk Risk Financial o Structure of IgG

Background o Antigen — Protein A o Source - Staphylococcus aureus Purpose Purpose Immunoassay Immunoassay Immunoassay Immunoassay Market Market Characteristics Characteristics Design Options Design Options Fabrication Fabrication FDA Approval FDA Approval Financial Risk Risk Financial

Background o Immunoassays o Immunometric Purpose Purpose o Enzyme linked immunosorbent assay Immunoassay Immunoassay Immunoassay Immunoassay Market Market E E Characteristics Characteristics E Design Options Design Options E E Fabrication Fabrication E E E FDA Approval FDA Approval Financial Risk Risk Financial Antigen Enzyme Solid phase Wash to remove coated with labeled unbound labeled antibody antibody antibody

Background o Immunoassays Purpose Purpose Immunoassay Immunoassay Immunoassay Immunoassay S S Market Market E E S Characteristics Characteristics S E E Design Options Design Options S S E E Fabrication Fabrication S FDA Approval FDA Approval S Financial Risk Risk Financial Enzyme Catalyzed Reaction to Produce Signal

Immunoassay Operation o Equilibrium test o Approximately Purpose Purpose 3 hours Immunoassay Immunoassay Market Market o Working Range Characteristics Characteristics Design Options Design Options o Pico-molar Fabrication Fabrication FDA Approval FDA Approval Financial Risk Risk Financial

o Laboratory operation Purpose Purpose Immunoassay Immunoassay Immunoassay Immunoassay Market Market Sample Conjugated Chromogenic Characteristics Characteristics HRP Reagent Wash Wash Stop Spectrophometer λ =403 nm Design Options Design Options 96 Well Plate Fabrication Fabrication FDA Approval FDA Approval Financial Risk Risk Financial

Market

Market o Target market segment o Rural hospitals Purpose Purpose o Why rural hospitals? Immunoassay Immunoassay o Untapped market Market Market Market Market o Technology — Cost barrier Characteristics Characteristics o Automated throughput vs. manual Design Options Design Options o Prohibitive capital cost Fabrication Fabrication FDA Approval FDA Approval Financial Risk Risk Financial

Market o Rural Hospital Infection Rates Purpose Purpose o 300,000 total infections annually in Immunoassay Immunoassay rural hospitals Market Market Market Market o S. aureus responsible for Characteristics Characteristics approximately 76,000 infections Design Options Design Options o 300,000 assays required to test all Fabrication Fabrication patients presenting symptoms FDA Approval FDA Approval Financial Risk Risk Financial

Market o Rural Hospital Trends o Recent growth in rural population Purpose Purpose o 6% increase in hospital discharges between 1995 & Immunoassay Immunoassay 2000 o 11% growth in rural population between 1990 & Market Market Market Market 2000 Characteristics Characteristics Design Options Design Options o Rural hospital discharges to increase o Discharges expected to increase 10% between Fabrication Fabrication 2005 & 2014 FDA Approval FDA Approval Financial Risk Risk Financial

Market o Competition o Larger biotechnology companies Purpose Purpose o Remel, Sure-Vue, Murel Immunoassay Immunoassay Market Market Market Market o Our Advantages Characteristics Characteristics Design Options Design Options o Quantitative Detection Fabrication Fabrication o Shorter waiting time FDA Approval FDA Approval o Does not require sample culturing Financial Risk Risk Financial

Immunoassay Characteristics

Reagents & Components o What’s in the box? o Two 96 - well plates o HRP-IgG conjugate solution o Wash buffer Purpose Purpose o TMB Substrate solution Immunoassay Immunoassay o Stop Solution Market o Standard Antigen Solution Market o Standard Dose Response Characteristics Characteristics Characteristics Characteristics Curve Design Options Design Options Fabrication Fabrication FDA Approval FDA Approval Financial Risk Risk Financial

Reagents & Components o 96 well plate o Standard for use with ELISA o Allows for multiple sample testing Purpose Purpose o Polystyrene Immunoassay Immunoassay Market Market Characteristics Characteristics Characteristics Characteristics Design Options Design Options Fabrication Fabrication o IgG-HRP conjugate solution FDA Approval FDA Approval o Signal generation component of assay Financial Risk Risk Financial

Reagents & Components o Wash buffer o TRIS buffered saline with 0.1% Tween o Hydrogen peroxide Purpose Purpose Immunoassay Immunoassay o Oxidizing agent Market Market o TMB Substrate solution Characteristics Characteristics Characteristics Characteristics o 3,3’,5,5’-tetramethylbenzidine Design Options Design Options o Produces strong signal Fabrication Fabrication o Lacks mutagenic qualities FDA Approval FDA Approval Financial Risk Risk Financial