Precipitation Reactions

Question 1

What is a precipitation reaction?

Answer 1

involves combining soluble antigen with soluble antibody to produce an insoluble complex that becomes visible

Question 2

What conditions are required for a successful precipitation reaction?

Answer 2

antigen and antibody must have multiple binding sites for one another, and their relative concentrations must be equal

Question 3

What is the difference between an immunodiffusion and a passive immunodiffusion?

Answer 3

passive immunodiffusion does not use an electrical current to accelerate the reaction

Question 4

What factors affect the rate of diffusion?

Answer 4

particle size, temperature, gel viscosity, hydration level, and the interactions between the matrix and reactants

Question 5

Describe the Oudin single diffusion.

Answer 5

quantitative gel diffusion in which antibody is added to agarose and poured into a test tube; antigen is layered over this agarose and diffuses down into it, forming a precipitin band at the zone of equivalence

Question 6

Why is the Oudin technique called “single” diffusion?

Answer 6

the antigen is only travelling in one direction

Question 7

What is radial immunodiffusion?

Answer 7

modification of the single diffusion technique, where the antigen is applied to a well cut into the support gel; instead of a precipitin band, the zone of equivalence is represented by a ring of precipitation, the area of which is a measure of the antigen concentration

Question 8

What is the end-point method of radial immunodiffusion?

Answer 8

antigen is allowed to diffuse to completion (takes anywhere from 24-72 hours), after which the square of the diameter of the precipitin ring is directly proportional to the antigen concentration

Question 9

What is the kinetic method of radial immunodiffusion?

Answer 9

uses measurements taken before the zone of equivalence is reached; diameter of precipitin ring is proportional to the log of the concentration

Question 10

What is the Ouchterlony double diffusion technique?

Answer 10

qualitative gel diffusion in which both antigen and antibody diffuse independently through the medium in two dimensions (horizontally and vertically); in most cases, multispecific antibody is placed in the central well, and different antigens are placed in the surrounding wells; the position of the resulting precipitin bands allows for comparison of the antigens to one another

Question 11

What is the Ouchterlony reaction of identity?

Answer 11

fusion of the precipitin lines at their junction to form an arc, which represents serological identity or the presence of a common determinant

Question 12

What is the Ouchterlony reaction of non-identity?

Answer 12

a pattern of crossed precipitin lines, which demonstrates two separate reactions and indicates that the compared antigens share no common determinants

Question 13

What is the Ouchterlony reaction of partial identity or cross-reactivity?

Answer 13

fusion of the two precipitin lines with a spur, which indicates that while the two antigens share a common determinant, some of the antibody molecules are not captured by antigen and travel through the initial precipitin line to combine with additional determinants found in more complex antigen

Question 14

Which antigen does the spur in a partial identity reaction point to?

Answer 14

the simpler antigen

Question 15

What are enzyme immunoassays (EIA)?

Answer 15

techniques where enzymes are used to label antigen-antibody reactions

Question 16

What is the difference between heterogeneous and homogeneous EIA?

Answer 16

heterogeneous EIA requires a step to physically separate free ligand (antigen) from bound ligand; homogeneous does not require this step

Question 17

What are the five typical enzyme labels, and which are most commonly used (*)?

Answer 17

horseradish peroxidase* (readily available and inexpensive), glucose oxidase, glucose-6-phosphate dehydrogenase (fluorometric), alkaline phosphatase* (expensive), and beta-D-galactosidase

Question 18

What are the advantages of EIA?

Answer 18

no need for expensive equipment (reactions can be read with a spectrophotometer or through observing color changes), no health hazards or disposal problems

Question 19

What are the disadvantages of EIA?

Answer 19

some specimens may contain natural inhibitors, nonspecific protein binding, sensitivity of enzymes to temperature

Question 20

Describe competitive heterogeneous EIA.

Answer 20

enzyme-labeled antigen competes with unlabeled patient antigen for a limited number of binding sites on antibody molecules attached to a solid phase; after carefully washing to remove any nonspecifically bound antigen, enzyme activity is determined

Question 21

How is enzyme activity related to the concentration of the test substance?

Answer 21

inversely proportional - the more patient antigen is bound, the less enzyme-labeled antigen can attach

Question 22

Sensitivity of what degree can be achieved?

Answer 22

nanograms/mL

Question 23

What is another name for noncompetitive heterogeneous EIA and what does it mean?

Answer 23

ELISA - enzyme-linked immunosorbent assay

Question 24

Why is ELISA considered indirect?

Answer 24

the enzyme-labeled reagent does not participate in the initial antigen-antibody binding reaction

Question 25

Describe ELISA.

Answer 25

antigen is bound to a solid phase, and unknown patient antibody is added and given time to react and incubate; after a wash, enzyme-labeled antiglobulin is added, which reacts with the Fc portion of any patient antibody that is bound to the antigen on the solid phase; second wash is performed, and an enzyme substrate is added to detect the reaction

Question 26

How is ELISA interpreted?

Answer 26

the amount of enzyme label detected is directly proportional to the amount of patient antibody

Question 27

What is an immunoenzymetric assay?

Answer 27

another form of ELISA that detects unknown antigen by means of excess labeled antibody

Question 28

Describe the immunoenzymetric assay process.

Answer 28

labeled antibody and patient antigen are allowed to react, and then excess solid phase antigen is added (usually attached to glass beads); solid-phase antigen combines with any unreacted labeled antibody, and these complexes separate out from the supernatant upon centrifugation

Question 29

How are immunoenzymetic assays interpreted?

Answer 29

the activity of the supernatant, which contains the complexes of patient antigen and labeled antibody, is in direct proportion to the concentration of patient antigen

Question 30

Describe sandwich or capture assays.

Answer 30

form of ELISA that uses antigens with multiple epitopes; excess solid-phase antibody is combined with the test sample to capture any antigen present; after incubation, enzyme-labeled antibody is added which may recognize the same or different epitope that solid-phase antibody did, and therefore complete the “sandwich”; used to detect antigens that are present in very low concentrations

Question 31

How is a sandwish/capture assay interpreted?

Answer 31

enzymatic activity is directly proportional to the amount of antigen in the patient sample

Question 32

What antigens are best suited to detection by sandwich/capture assay?

Answer 32

those with multiple epitopes, such as polypeptide hormones, proteins, tumor markers, and microorganisms (especially viruses)

Question 33

How do most membrane-based cassette assays work?

Answer 33

also known as immunochromatography; analyte is applied at one end of a reaction strip and migrates towards the other end via the capillary action of an absorbant pad; labeling and detection zones are set between these two ends, and as the sample is loaded, it reconstitutes the labeled antigen or antibody, forming a complex that migrates toward the detection zone; antigen or antibody in the detection zone captures these complexes and produces a color reaction if the assay is positive

Question 34

What is another name for competitive homogeneous EIA, and what does it mean?

Answer 34

EMIT - enzyme multiplied immunoassay technique

Question 35

What level of sensitivity is EMIT testing capable of?

Answer 35

ug/mL (less sensitive than ELISA)

Question 36

What are the chief uses of EMIT testing?

Answer 36

detecting hormones, therapeutic drugs, and drugs of abuse in serum and urine which cannot be easily measured by other means

Question 37

Describe the EMIT process.

Answer 37

reagent antigen is labeled with an enzyme tag; when antibody binds to specific determinant sites on the antigen, steric hindrance to the enzyme occurs, resulting in a measurable loss of activity

Question 38

How is EMIT testing interpreted?

Answer 38

enzyme activity is directly proportional to the concentration of patient antigen

Question 39

Which enzymes are inhibited by EMIT?

Answer 39

lysozyme, neuramidase, trypsin, papain, bromelain, glucose-6-phosphate dehydrogenase, and beta-D-galactosidase

Question 40

What four factors affect the sensitivity of EMIT testing?

Answer 40

detectability of enzymatic activity; change in enzymatic activity; strength of the antibody bindings; and assay susceptibility to interference from endogenous enzyme activity, cross-reacting antigens, or enzyme inhibitors