Horseradish Peroxidase Immunoassay-Powerpoint presentation

Horseradish Peroxidase Immunoassay-Powerpoint presentation...

Info iconThis preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: Horseradish Peroxidase Immunoassays Patrick A. Williams Ian M. Klink Brandon D. Shaw Chemical Engineering Senior Design Project Overview Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk I. II. III. IV. V. VI. VII. VIII. Purpose Immunoassay Market Characteristics of Our Immunoassay Design Options Fabrication FDA Approval Financial Risk Objectives & Goals Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk o Produce a quality diagnostic test o Provide leading immunoassays for bacterial detection o Target rural hospital market initially o Make Money Product Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk Immunoassay Kit o Analytical test o Targets specific molecule o Results obtained by signal generation Why S. aureus immunoassay? Staphylococcus aureus Purpose Purpose Immunoassay Market Characteristics Design Options o o o o Major cause of hospital infections 8 common infections Can cause deadly complications Hospital Infections 4th leading cause of US deaths Fabrication FDA Approval Financial Risk www.ulb.ac.be/sciences/biodic/ ImBacterie2.html Why S. aureus immunoassay? Common Conditions Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk Infection Description Folliculitis inflammation of the follicles Boils painful, pus-filled inflammation of the skin and subcutaneous tissue Sties Inflammation of one or more sebaceous glands of an eyelid Impetigo a contagious skin infection Abscesses A localized collection of pus in part of the body, formed by tissue disintegration and surrounded by an inflamed area Staphylococcal pneumonia inflammation of the lungs Osteomyelitis onset after surgery Toxic shock syndrome acute infection associated with tampon use during menstruation S. aureus immunoassay Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk o Early detection yields better treatment o Sanitation control o Can be modeled and adapted to other immunoassays Immunoassay Components Antibody o Immune system vesicle Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk Antigen o Molecule being detected Enzyme o Oxidizing agent using in signal generation Substrate o Signal generator in conjunction with enzyme Background Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk o Antibody o Chains o Regions o Types o Polyclonal o Monoclonal Polyclonal •Same form of antibody with Variable Region varying binding specificities and Lightaffinities for the targeted antigen Chain Monoclonal Constant Region •Arises from a single cell and have Heavy Chain Background o Which antibody for our assay? o Immunoglobulin G (IgG) Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication o Why IgG? o Widely available o Higher antigen affinity compared to other antibodies FDA Approval Financial Risk o Structure of IgG Background o Antigen — Protein A o Source - Staphylococcus aureus Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk Background o Immunoassays Purpose Purpose Immunoassay o Immunometric o Enzyme linked immunosorbent assay Market E Characteristics E E Design Options E Fabrication E E E E FDA Approval Financial Risk Solid phase Antigen coated with antibody Enzyme labeled antibody Wash to remove unbound labeled antibody Background o Immunoassays Purpose Purpose Immunoassay Market S E Characteristics S S E E Design Options Fabrication FDA Approval S E S E S E S S Financial Risk Enzyme Catalyzed Reaction to Produce Signal Immunoassay Operation Purpose Purpose Immunoassay o Equilibrium test o Approximately 3 hours Market Characteristics Design Options Fabrication FDA Approval Financial Risk o Working Range o Pico-molar o Laboratory operation Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk Sample Wash 96 Well Plate Conjugated HRP Wash Chromogenic Reagent Stop Spectrophometer λ=403 nm Market Market o Target market segment o Rural hospitals Purpose Purpose Immunoassay o Why rural hospitals? o Untapped market Market Characteristics Design Options Fabrication FDA Approval Financial Risk o Technology — Cost barrier o Automated throughput vs. manual o Prohibitive capital cost Market o Rural Hospital Infection Rates Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk o 300,000 total infections annually in rural hospitals o S. aureus responsible for approximately 76,000 infections o 300,000 assays required to test all patients presenting symptoms Market o Rural Hospital Trends Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk o Recent growth in rural population o 6% increase in hospital discharges between 1995 & 2000 o 11% growth in rural population between 1990 & 2000 o Rural hospital discharges to increase o Discharges expected to increase 10% between 2005 & 2014 Market o Competition Purpose Purpose Immunoassay o Larger biotechnology companies o Remel, Sure-Vue, Murel Market Characteristics Design Options Fabrication FDA Approval Financial Risk o Our Advantages o Quantitative Detection o Shorter waiting time o Does not require sample culturing Immunoassay Characteristics Reagents & Components o What’s in the box? Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk o o o o o o o Two 96 - well plates HRP-IgG conjugate solution Wash buffer TMB Substrate solution Stop Solution Standard Antigen Solution Standard Dose Response Curve Reagents & Components o 96 well plate Purpose Purpose Immunoassay o Standard for use with ELISA o Allows for multiple sample testing o Polystyrene Market Characteristics Design Options Fabrication FDA Approval Financial Risk o IgG-HRP conjugate solution o Signal generation component of assay Reagents & Components o Wash buffer o TRIS buffered saline with 0.1% Tween Purpose Purpose Immunoassay Market Characteristics Characteristics Design Options Fabrication FDA Approval Financial Risk o Hydrogen peroxide o Oxidizing agent o TMB Substrate solution o 3,3’,5,5’-tetramethylbenzidine o Produces strong signal o Lacks mutagenic qualities Reagents & Components o Assay buffer Purpose Purpose o TRIS buffered saline with 1% BSA (Bovine Serum Albumin) Immunoassay Market Characteristics Characteristics Design Options Fabrication FDA Approval Financial Risk o Standard antigen solution o Protein A antigen solution o 4 concs. that span working range Reagents & Components o Stop solution o 1M hydrochloric acid Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk o Standard dose response curve o Signal vs. antigen conc. o Conc. as low as 800 pg/mL HRP Kinetics Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk Enzyme Reaction o HRP undergoes three reactions to generate signal kα HRP + H 2 O2 → HRP − 1 + H 2 O kβ HRP − 1 + AH 2 → HRP − 2 + AH • kγ HRP − 2 + AH 2 → HRP + AH • + H 2 O Color is generated from oxidized substrate radical HRP Kinetics o Stop solution will be added to the mix after 10 minutes. Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication o Spectrophotometer is used to measure the concentration of colored substrate in solution FDA Approval Financial Risk o λ=403 nm Kinetic Analysis o Antibody-antigen binding o Surface adsorption kinetics Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk Ab + Ag k1 → ← k−1 Ab − Ag o Three binding reactions of interest o Antibody to surface o Antigen to antibody o Labeled antibody to antigenantibody surface complex Kinetic Analysis o Evaluation of rate constants o How? Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk o BIAcore s51 Kinetic Analysis Plate Antibody Binding o Binding reaction of antibody to surface Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk S + Ab k3 → ← k−3 SAb o First order surface adsorption kinetics o Adsorption and Desorption reactions considered Adsorption = rAD = k3C AbCv , S Desorption = rDES = k−3CSAb k3 Equilibrium = K 3 = k−3 Netrate = rAD − rDES = k3C AbCv , S − k−3CSAb Kinetic Analysis o Binding of antigen to antibody Purpose Purpose SAb + Ag k 2 → ← k−2 SAb − Ag Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk Adsorption = rAD = k2Cv , SAbC Ag Desorption = rDES = k−2CSAb − Ag k2 Equilibrium = K 2 = k−2 Net rate = rAD − rDES = k2Cv , SAbC Ag − k−2CSAb − Ag Kinetic Analysis o Surface complex binding Purpose Purpose SAb − Ag + HRP − Ab k5 → ← k −5 SAb − Ag − Ab − HRP Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk Adsorption = k 5 C HRP − Ab C v , SAb − Ag Desorption = k −5 C SAb − Ag − Ab − HRP k5 Equilibrium = = K5 k −5 C SAb − Ag = Ab − HRP Netrate = k 5 C HRP − Ab C v , SAb = Ag − K5 Kinetic Analysis Equilibrium interaction model Purpose Purpose Immunoassay rSAb − Ag C SAb − Ag = k C C − Ag v , SAb 2 K2 Market Characteristics Design Options Fabrication FDA Approval Financial Risk r = k1CHRP − AbCv , SAb − Ag − CSAb − Ag − Ab − HRP K1 CSAb − Ag − Ab − HRP rbind . = k1 CSAbCHRP − Ab − Ag − K1 Kinetic Analysis Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk o Assume Adsorption & Desorption rates are equal to zero o Solve for unknown concentrations C SAb− Ag = K1 ( Ag Cv , SAb ) C C Ag = C Ag , 0 − C SAb− Ag − C SAb− Ag − Ab− HRP − C HRP − Ab− Ag [ C SAb− Ag = K1 (C Ag ,0 − C SAb− Ag − C SAb− Ag − Ab− HRP − C HRP − Ab− Ag )Cv , SAb o Initial antigen concentration is known o Initial surface antibody conc. is known Kinetic Analysis o System of 3 equations o Solved using Polymath™ Purpose Purpose Immunoassay Market Characteristics o Conc. of surface antibodyantigen complex critical variable Design Options Fabrication FDA Approval Financial Risk o Strength of signal is defined by this concentration Kinetic Analysis o Dose response curve Purpose Purpose o Indicates antigen concentration o Curve asymptotes as surface antibody concentration is approached Immunoassay Dose Response Curve-varying CSAb(mol/cm2) Market 2.00E-12 1.80E-12 Characteristics 1.60E-12 Design Options FDA Approval Financial Risk CSAbAgAbHRP (mol/cm^2 Fabrication 1.40E-12 1.20E-12 2.5e-10 CSAb (mol/cm^2) 5e-10 CSAb (mol/cm^2) 7.5e-10 CSAb (mol/cm^2) 1e-9 CSAb (mol/cm^2) 1.25e-9 CSAb (mol/cm^2) 1.00E-12 8.00E-13 6.00E-13 4.00E-13 2.00E-13 0.00E+00 0 550000 1100000 1650000 2200000 2750000 3300000 CAg, 0 (pg/mL) 3850000 4400000 4950000 5500000 Sensitivity o Assay sensitivity o How low can we go? Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk o Determined by surface complex concentration o Minimum complex conc.=1.8x10-15 mol/cm2 Sensitivity Purpose Purpose o Signal strength increases with surface antibody concentration Immunoassay Market Characteristics Design Options Fabrication o Optimize cost of surface antibody using this plot FDA Approval Financial Risk o Use minimum amount of surface antibody that can generate signal Sensitivity Dose Response Curve-varying CSAb (mol/cm2) 1.00E-14 9.00E-15 Purpose Purpose 8.00E-15 Immunoassay Characteristics Design Options 7.00E-15 CSAbAgAbHRP (mol/cm^2) Market 2.5e-10 CSAb (mol/cm^2) 5e-10 CSAb (mol/cm^2) 7.5e-10 CSAb (mol/cm^2) 1e-9 CSAb (mol/cm^2) 1.25e-9 CSAb (mol/cm^2) 6.00E-15 5.00E-15 4.00E-15 Fabrication 3.00E-15 FDA Approval 2.00E-15 Financial Risk 1.00E-15 0.00E+00 0 400 800 1200 1600 2000 CAg, 0 (pg/mL) 2400 2800 3200 3600 4000 Sensitivity Purpose Purpose Immunoassay o Dose response curve is only used in linear range Market Characteristics Design Options Fabrication FDA Approval Financial Risk o Linear range= 800 pg/mL – 4000 pg/mL Design Options Evaluated Processes Purpose Purpose Immunoassay Market Characteristics Horseradish Peroxidase (HRP) Extraction o 2 Differential Extractors o Biphasic Salt Solution o Polyethylene Glycol (PEG) & KPO4 o Reagent Cost≈$20,000 o FCI≈$150,000 Design Options Design Options Amount: 1.24kg Activity: 117U/mL KPO4 PEG RECYCLE Fabrication FDA Approval Crude Extract Extract 1 P-2 / DX-102 PRODUCT Differential Extraction Financial Risk HRP/BUFFER P-3 / V-101 PEG/ water P-1 / DX-101 Storage Salt Phase Raff Differential Extraction Modeled using SuperPro Designer 4.7 Evaluated Processes o Cost Comparison of Produced and Purchased HRP Purpose Purpose Immunoassay Market Characteristics Design Options Design Options Fabrication FDA Approval Financial Risk o Purchase HRP o Annual requirement: 5 g/yr o Cost: $15,000/yr o Produce HRP o Approximate Labor cost: $35,000/yr Evaluated Processes Purpose Purpose Immunoassay Market Characteristics Design Options Design Options Fabrication FDA Approval Financial Risk HRP Extraction continued o Decided not to produce o Purchase HRP o Lyophilized o Small amounts needed o Efficient for small company o $266 per 100 mg (Sigma) Fabrication Plate Preparation Polystyrene Plate Steps Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk 1. 2. 3. 4. 5. 6. Fill wells with IgG coating solution Incubate for 18 hours Remove coating solution. Wash. Fill wells with 2nd coating solution Incubate for 30 minutes. Remove coating solution. Wash, and dry for 24 hours. Manufacturing Purpose Purpose Immunoassay Market Characteristics Equipment o 3 major pieces o Sterilization o Bottling o Labeling Design Options Fabrication FDA Approval Financial Risk STS Series KFVG x212 HEA 105 Manufacturing Gantt Chart: Components Purpose Purpose Immunoassay Market Characteristics ID 1 2 3 4 5 6 8 Duration 10 11 Financial Risk 0.5h 0.5h 0.5h 0.5h 1.0h 0.5h 1.0h 15 16 17 18 11 12 1 2 3 4 5 0.5h Packaging 13 10 0.5h 0.5h 12 9 0.5h 14 FDA Approval Dail y O per at ion 8 0.5h Operating Labor 9 Fabrication Assay Solution - Sterlization Standard Solution - Sterilization Wash Buffer - Sterilization Conjugated HRP - Sterilization Substrate (TMB) - Sterilzation Stop Solution - Sterilization Assay Solution - Filling Standard Solution - Filling Wash Buffer - Filling Conjugated HRP - Filling Substrate (TMB) - Filling Stop Solution - Filling Assay Solution - Labeling Standard Solution - Labeling Wash Buffer - Labeling Conjugated HRP - Labeling Substrate (TMB) - Labeling Stop Solution - Labeling Raw Materials 7 Design Options Task Name 0.5h 1.0h 1.0h 1.0h 1.0h 1.0h $7,000,000 $490,169 $24,370 Packaging Purpose Purpose Shipping Box o 6”x6”x4” Indestructo Mailing Immunoassay Market Characteristics Design Options Gray Convoluted Foam o 8”x6”x2” Fabrication FDA Approval Financial Risk Yearly Packaging Costs $24,370 Quality Control o Random quality audits o 2.5% of each assay batch Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk o Verification of valid working range o Error greater than 5% fails audit o 2.5% of tested assays fail - discard batch Quality Control o Apparatus Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk o Xiril™ X100 combination liquid handler and plate reader o Higher test throughput o Less chance of human error FDA Approval Why FDA Approval Food & Drug Administration o Government organization Purpose Purpose Immunoassay Market Characteristics Design Options o Protects Consumers o Promotes Public Health o Regulates Medical Devices Fabrication FDA Approval Financial Risk o Approval o Require for US sales o Quality product endorsement FDA Testing Phases Purpose Purpose Scrap Phase 1 Accuracy and Reproducibility Test Scrap Phase 2 Human Serum Testing Scrap Phase 3 Scrap Phase 4 Immunoassay Market Characteristics Design Options Fabrication Chemical Inhibition Testing FDA Approval Financial Risk Quality Control FDA Testing Decisions o How much pre FDA testing should we do? Purpose Purpose Immunoassay Market Characteristics Design Options o Should we perform many tests in the same phase, or just a few? o After what phase should we enter the FDA approval process? Fabrication FDA Approval Financial Risk o To answer these questions we developed a novel model of the FDA approval process. Pertinent Steps Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk Phase 1: Accuracy and Reproducibility Phase Chemical Serum Testing Phase 3: 2: Human Inhibition Testing Tests: 500 Time: 4 weeks Phase 4:Cost: $28,000 Quality Control FDA Approval Outline Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk Business Establishment & Registration FDA Testing Phases Pre-Clinical Testing Financial Risk Stochastic Modeling o Evaluated Design Variables Purpose Purpose Immunoassay Market Characteristics Option Number of Tests FDA 1 Phase 1 & 2 Phase 3 & 4 1000 500 Phase 4 2 Phase 1 & 2 Phase 3 & 4 1000 500 Phase 3 3 Phase 1 & 2 Phase 3 & 4 500 250 Phase 4 4 Phase 1 & 2 Phase 3 & 4 500 250 Phase 3 Design Options Fabrication FDA Approval Financial Risk Stochastic Modeling Purpose Purpose Immunoassay Market o Evaluate all possible pathways in the FDA model for each design variable to determine: o Cost Characteristics Design Options o Time Fabrication FDA Approval Financial Risk o NPW Stochastic Modeling 4000 3500 Immunoassay 3000 Market 2500 Characteristics Design Options Frequency Purpose Purpose 2000 1500 Fabrication 1000 FDA Approval Financial Risk 500 0 (6000) (5000) (4000) (3000) (2000) NPW ($1000) (1000) 0 1000 Stochastic Modeling o Risk Assessment 1 Purpose Purpose 0.9 Immunoassay 0.8 0.7 Characteristics Option 2 Option 3 0.6 Design Options Probability Market Option 1 Option 4 0.5 0.4 Fabrication 0.3 FDA Approval 0.2 0.1 Financial Risk 0 (7,000) (6,000) (5,000) (4,000) (3,000) NPW ($1000) (2,000) (1,000) 0 1,000 Stochastic Modeling o Option 1 gave the best results Purpose Purpose Immunoassay Market o Thorough testing through all phases reduces the uncertainty in the FDA approval process Characteristics Design Options Fabrication FDA Approval o Estimated testing cost: $1.8 Million o Pre FDA Testing: $1 Million o FDA Testing: $800,000 Financial Risk o Estimated time: 1.7 years Breakdown o Estimated Annual Sales Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk o 250,000 Immunoassay Kits o $125 per kit o Total Sales: $31 Million o Total Product Cost o $30 million o Total Capital Investment o $5.8 Million o Estimated NPW o -$2 Million Questions Purpose Purpose Immunoassay Market Characteristics Design Options Fabrication FDA Approval Financial Risk ??? ...
View Full Document

{[ snackBarMessage ]}

Ask a homework question - tutors are online