Antibodies and In Vitro Research (16 C)

Cytometry

measures physical and chemical characteristics of cells

Flow Cytometry

measurements are made as cells/particles FLOW through instrument in a stream

Flow Cytometry Cell Sorting (also called FACS)

fluorescence microscope analyzes moving particles in suspension

In flow cytometry, antibodies are bound to _____________ to detect cell-surface markers

fluorochromes

After being marked, cells are subjected (ONE AT A TIME) to ____________

lasers

Fluorochromes are _______ at a given wavelength and emit fluorescence

excited

The _________ detects fluorescence

filter

When a cell is hit with a laser, the __________ scatter is proportional to the size of the cell

forward
(more forward scatter = bigger)

The __________ scatter is proportional to granularity

side
(more side scatter = more granules)

Clinical uses of flow cytometry:

- diagnose inherited and acquired immunodeficiencies
- differentiate between different cancers
- evaluate bone marrow transplantation
- determine HLA expression
- detect pathogens in body fluids

Pregnancy test assay

immunochromatographic

Enzyme Linked Immunosorbent Assay (ELISA):
protein of interest is bound to a _______

plate

ELISA:
specific antibody is then conjugated to an ________ that converts a colorless substrate to colored

enzyme

ELISA:
the colored substrate is detected by a ______________

spectrophotometer

In ELISA, a standard _________ is generated to quantify samples

curve

ELISA can be used to measure _______ concentration in a serum

antibody

Sandwich ELISA measures

conc. of particular antigen

Western Blot steps

-It is performed by gel electrophoresis of the protein of interest, followed by transfer to a protein-binding membrane (eg, nitrocellulose) and incubation with antibodies specific to the protein of interest.

-The primary antibody or a labeled secondary antibody is then detected by fluorescence or chemiluminescence

Western Blot for HIV

used if patient tests positive on ELISA test
confirms presence of HIV
uses elctrophoresis