A Highly Versatile Microscope Imaging Technology Platform for the Multiplex Real-Time Detection of Biomolecules and Autoimmune Antibodies - Molecular Diagnostics - page 36

small reaction volumes, are presented together with a high-content detection of
autoimmune antibodies using a HEp-2 cell assay. Its adaptiveness and versatility
gives VideoScan a competitive edge over other bioanalytical technologies.

Keywords Autoimmune diagnostic Hybridization kinetics Immunofluorescence
patterns Microbeads Microscope imaging technology Multiplex Real-time

Contents

1 Introduction.......................................................................................................................... 36

2 The VideoScan Platform ..................................................................................................... 38

2.1 Hardware and Software Components ........................................................................ 38

2.2 Data Acquisition ......................................................................................................... 39

3 Design Principles of VideoScan Microbead Assays .......................................................... 42

3.1 Immobilization of Microbeads ................................................................................... 42

3.2 Immobilization of Biomolecules on the Surface of Microbeads.............................. 44

3.3 Determination of Minimum Number of Microbeads Per Assay .............................. 46

3.4 Determination of Functional Capture Probe Density ................................................ 47

4 VideoScan Technology Applications.................................................................................. 47

AKLIDES Application for the Automated Detection of Autoantibodies
by Cell-Based Assays .................................................................................................

4.1

47

4.2 Semiquantitative Autoimmune Diagnostics of Antinuclear Antibodies ................... 49

4.3 Immobilization and Detection of Biotinylated Antigens from Crude

Bacterial Lysates......................................................................................................... 54

5 VideoScan Assays for the Detection of Nucleic Acids ..................................................... 56

5.1 The VideoScan Multiplex Hybridization Assay........................................................ 56

Homogeneous Microliter-Volume Hybridization Using Advalytix Slides ............ 5.2 59

6 VideoScan PCR: Analysis and Application ....................................................................... 60

6.1 PCR in Solution .......................................................................................................... 61

6.2 Melting Curve Analysis in Solution .......................................................................... 64

6.3 Multiplex Melting Curve Analysis on the Surface of Microbeads .......................... 65

6.4 Multiplex Hybridization Temperature Optimization on a Microbead Surface ........ 68

7 Conclusions.......................................................................................................................... 69

References.................................................................................................................................. 70

1 Introduction

The stunning achievements in the life sciences are based mainly on the develop-
ment of innovative bioanalytical technologies. Many of these technological plat-
forms are of high technical complexity but are suitable for just one application.
Most of them are capable of detecting either proteins/peptides or nucleic acids
at different multiplex levels ranging from low-density (\100 features) to high-
density ([1,000 features) assays. Although highly sophisticated, many technolo-
gies like the planar microarray are unable to measure desirable kinetic data but
provide endpoint measurements instead.