Labeled Immunoassays

Question 1

What is the purpose of labeled immunoassays?

Answer 1

Testing that is used for antigens or antibodies that may be small in size or present in very low concentrations.

Question 2

What are the two major formats of immunoassays?

Answer 2

Heterogenous and homogenous.

Question 3

Describe heterogenous immunoassays.

Answer 3

The separation of bound and free components. A wash process is needed (otherwise false positives occur).

Question 4

Describe homogenous immunoassays.

Answer 4

The lack of separation or wash phase.

Question 5

Which type of immunoassay (homogenous/heterogenous) is less prone to analytical errors?

Answer 5

Homogenous.

Question 6

What are some labeling techniques that can be used in immunoassays?

Answer 6

Radioactivity, enzymes, chemiluminescence, and fluorescence.

Question 7

Describe the process of noncompetitive immunoassays.

Answer 7

Antibody or antigen is passively absorbed to a solid phase; unknown patient Ab/Ag can react with and be captured by antibodies. After washing, a second antibody with a label is added.

Question 8

In noncompetitive immunoassays, the amount of labeled measured is ___ proportional to the amount of patient antigen.

Answer 8

Directly.

Question 9

Describe the process of competitive immunoassays.

Answer 9

All the reactants are mixed together at the same time. Labeled antigens then compete with unlabeled patient antigens for antibody binding sites.

The patient sample “competes” with labeled antigen.

Question 10

In competitive immunoassays, the amount of bound label is ___ proportional to the concentration of labeled antigen.

Answer 10

Inversely.

Question 11

The radioimmunoassay method (RIA) can measure trace amounts of analytes such as ___ and ___ that are small in size.

Answer 11

hormones; vitamins.

Question 12

In radioimmunoassay method (RIA) labeled in bound phase is ___ proportional to the amount of patient antigen present.

Answer 12

Indirectly.

Question 13

Enzyme immunoassays use ___ as labels.

Answer 13

Enzymes.

Question 14

In enzyme immunoassays, what is the purpose of the substrate?

Answer 14

What enzymes use to produce breakdown products that are chromogenic.

Question 15

Describe the principle of competitive ELISAs.

Answer 15

Sample antigen competes with enzyme-conjugated antigen for a limited number of binding sites on antibody molecules attached to a solid phase.

Question 16

In competitive ELISAs, the product signal is ___ proportional to the concentration of the test substance.

Answer 16

Inversely.

Question 17

In competitive ELISA, the more antigen in the sample, the ___ reference antigen detected, indicating a ___ signal.

Answer 17

less; weaker.

Question 18

Describe the principle of noncompetitive ELISA.

Answer 18

Excess solid-phase antigen binds patient antibody, and a second labeled antibody is added.

Question 19

In noncompetitive ELISAs, the results are ___ to the amount of enzymatic activity detected.

Answer 19

Proportional.

Question 20

Capture (Sandwich) ELISA, is best for antigens that have what?

Answer 20

Multiple determinants.

Question 21

What is the principle behind the Capture (Sandwich) ELISA method?

Answer 21

Excess solid-phase antibody binds patient antigen, and a second labeled antibody (to the antigen) is added after a wash step. Antigen is ‘sandwiched’ between two antibodies.

Question 22

With Homogenous ELISA, if reagent antigen binds to purchased antibody, what happens to the active site and enzyme?

Answer 22

The active site is closed and the enzyme is no longer active.

Question 23

With homogenous ELISA, if a patient binds to the purchased antibody, what happens with the enzyme?

Answer 23

The reagent is still active.

Question 24

Homogenous ELISA contains ___ proportional results.

Answer 24

directly.

Question 25

What are advantages and disadvantages of ELISA testing?

Answer 25

Advantages: automated, inexpensive, safe, sensitive, stable chemicals.
Disadvantages: not as sensitive as RIA.

Question 26

Rapid immunoassays are ___ tests that are easy to perform and give reproducible results.

Answer 26

membrane-based.

Question 27

Discuss the principle of immunochromatographic assay (noncompetitive).

Answer 27

Patient sample is added to a test strip and migrates through the strip. Patient antigen complexes to antibody-labeled particles or competes with antigen-labeled particles across individual test lanes at unique detection zones.

Question 28

Define fluorochromes.

Answer 28

Fluorescent compounds that absorb energy from a light source and converts it to a wavelength.

Question 29

Discuss the principle of direct immunofluorescent assays.

Answer 29

Uses tissue sections or live cell suspensions. Antibody conjugated with fluorescent tag is added to an UNKNOWN antigen that is fixed to a slide, incubated, and washed. Read using fluorescence microscopy where a bright apple green is a positive result.

Question 30

Discuss the principle of indirect immunofluorescent assays.

Answer 30

Patient serum is incubated with KNOWN antigen attached to solid phase. The slide is then washed with AHG where fluorescent tag is added. Sandwich forms with first antibody which fluoresces.

Question 31

Discuss chemiluminescent immunoassay (CLIA).

Answer 31

The emission of light caused by a chemical reaction (oxidation) which produces an excited molecules that releases light during return to its ground state.

Question 32

Chemiluminescent immunoassays can be ___ or ___ because labels can attached to either antigen or antibody.

Answer 32

heterogenous; homogenous.

Question 33

What kind of interferences can occur with labeled immunoassays?

Answer 33

Antigen interference (postzone effect: false decrease), antibody interference, biotin, and cross-reactivity.