13. Labeled Immunoassays I

Question 1

Outline the procedure of a direct IFA that can be used to quantitate T lymphocytes in a sample of patient blood.

Answer 1

Patient cells + Fluorescent-labelled Ab –Incubate, wash–> Look for fluorescence of cells with a fluorescent microscope or flow cytometry

Question 2

Outline the procedure of an indirect IFA that could be used to detect antibody to Treponema pallidum in a sample of patient serum.

Answer 2

Cells known to be positive for Ag fixed onto microscope slide + Pt. serum Ab –incubate,wash–> add anti-human Ig-FITC conjugate —> incubate, wash —-> look for fluorescence under fluorescent microscope

Question 3

Outline the procedure of a fluorescent microbead immunoassay that could be used to detect patient antibody to a nuclear antigen called “Sm”.

Answer 3

1. A specific set of beads is labelled with a different antigen
2. Add patient serum antibody in the test sample binds to the Ag
3. Fluorescent (PE) labelled anti-human Ig binds to the patient’s antibody

Question 4

Outline the procedure of a fluorescent microbead immunoassay that could be used to detect the cytokine, IL-2, in fluid from a cell culture.

Answer 4

1. Each bead is labelled with a capture antibody specific for an analyte (Ag)
2. Capture antibody binds analyte in the test sample
3. Fluorescent labelled reporter antibody (PE - labeled anti-Ag) binds to capture analyte

Question 5

What are two disadvantages of RIA methods?

Answer 5

1. Handling and disposal of radioactive material

2. instability of radioactive isotopes