Lecture 5 - Multiplex Bead Array

Question 0

Flow Cytometry

• principle
• Properties of a cell that can be measured by a flow cytometer.

Answer 0

Technology that measures and analyze physical characteristics of a single cell as they flow in a fluid stream through a beam of light.

Properties of a cell that can be measured by a flow cytometer.

1. Cell
2. Granularity
3. Fluorescence intensity

Question 1

Enzymes-linked Immunosorbent Assay

Answer 1

Technique use to detect and quantitate either antigens or antibody in which enzyme-labeled antibody or antigen is bound to a solid support.

Complex are detected by color change indicating the presence of the product of an enzyme-substrate reaction.

Question 2

Applications of Flow Cytometry

Answer 2

1. Immunophenotyping
2. DNA ploidy/Cell cycle analysis
3. Panel reactive antibody
4. Detection of HLA-B27 for antigen expression
5. Enumerations of;
   - T cell in pheripheral blood
   - Lymphocytes subsets
   - Reticulocytes

Question 3

Definition and Principle of Multiplex Bead Array

Answer 3

Permits simultaneous cytometric quantitation of multiple analytes in solutions by capturing these to spectrally distinct beads.

• Microbeads are covalently coupled with capture antibody
• Proteins captured on an antibody conjugated microbeads are detected by tagged detection Abs
• After incubation with the sample, the beads form an Ab-Ag complex
• Incubation with Streptavidin-linked fluorescent reporter, R Phycoerythin (SAPE) completes the “antigen sandwich”
• Beads are interrogated one at a time. Intensity of fluorescence is proportional to the amount of analyte bound to the surface of each microbead