Avram ECPrep
Alexandru Aaron Globerman Blake Sowerby September 27, 2005
Presentation Overview
Technical Background Client Background Problem Statement Client Requirements Proposed Designs
Slush Model Sandwich Model Spot Model
Design Evaluations
HIV - retrovirus
Infects T-lymphocyte cells by binding to CD4 receptors Viral RNA released in cell undergoes reverse transcription -> DNA DNA integrated into genetic material of host cell and replicated New proteins and viral RNA assemble at cell membrane into new virus Studies of CD4gp120 CD4 interaction lead to antiretroviral treatment (HAART)
Monitoring HIV/AIDS
To assess efficacy of treatment regimen, must examine viral effect on patients Viral Load
Examines concentration of virusproduced peptide fragments in blood serum Requires extensive preparation; allows for concrete measurements to be prepared
CD4 Cell Concentration
HIV destroys T-Helper cells (CD4); measuring cell concentration an indirect method of ascertaining virulence. Far lower cost if done with ELISA vs. Flow Cytometry Of questionable clinical utility
Flow Cytometry Overview
Uses Antibody-conjugated Fluorescent beads to mark certain cell populations Monochromatic Light induces fluorescence in beads, can be detected at many wavelengths Computer gating allows for separation of subsets of populations Cell behavior can be tested through examination of metabolic products
ELISA Overview
Enzyme Linked ImmunoSorbent Assay Sandwich Elisa Most Common Method
Capture Antibody attaches Antigen to plate Display Antibody conjugated to Enzyme attaches to Antigen Substrate added to effect a color change reaction Spectrophotometry conducted to measure the extent of color change
Flow Cytometry vs. ELISA
Flow cytometry (hematology) method
System: $80.000 - $120.000 Monoclonal antibodies: $100 - $200 per test Commercially available methods:
EasyCD4/CD8 (Guava Technologies) CyFlow (Partec) (Partec)
ELISA method (Enzyme-Linked Immunosorbent Assay) :
System: $8.000 - $1.200 Assay reagent: $15 - $20
ELISA methods were commercially available:
Coulter Manual CD4 Count kit: storage period only 6 hrs Dynabeads uses magnetic beads TRAx CD4 kit:
Labor intensive Disintegrate cell membrane Requires careful calibration Storage period 3 days (at room temp)
Client Information World Wide Laboratories
Non-profit, faith-based corporation Provides medical instrumentation to developing world Trains technical staff in use of equipment and procedures Overriding premise is minimal cost of goods to the end-user
Problem Statement
ECPrep is a method of preparation of cells and apparatus that will facilitate the use of the method of sandwich ELISA to determine the level of CD4+ cells in whole blood. The purpose of this is to develop a low-cost method of conducting test this for application in the developing world so that antiretroviral treatments can be adjusted in patients diagnosed with HIV/AIDS.
Client Requirements
Low Cost (less than current standard cost - $5 per test) Short Processing Time 8-12 hours maximum Can differentiate cell count in a critical region 50<cell load<200 cells per microliter Must function in cooperation with WorldWide Labs StatFAX ELISA instrument Calibration of plate preparation methods to current instruments output Adaptable to either 8-well strips or 96-well plates
Proposed Design 1: Slush Model
Degrade cell membranes with detergent Provides more antigen groups for recognition More total enzyme for signal amplification
Design 1 cont.
Addition of permeablizing solution adds cost, complexity, and time Competitive loss of antigen groups will increase cost of primary and secondary Ab Loss of accuracy due to variable size of antigen groups
Proposed Design 2: Sandwich Model
Uses whole cells Fewer Binding groups, but more antigen per group Less competitive binding Less Cost/Time of preparation without perm Fewer Ab necessary b/c fewer total groups
Design 2 cont.
Nearly uniform cells allow for predictability in performance Density of surface antigen makes competitive binding unlikely Less complex due to minimization of steps, lowers cost and complexity
Proposed Design 3: Spot Sandwich Model
Uses Sandwich concept with separable regions of Ab coating on plate Allows for exact titer of Cells in sample Higher accuracy and precision Prohibitive labor/cost in preparation More Primary Ab needed
Design 3 cont.
Cost of preparation of plates would be astronomical, would require aut...