W7.2

Question 1

immunoelectrophoresis

Answer 1

“Electrophoresis separates molecules according to differences in the electric charge
- Used to identify and characterize serum proteins (including immunoglobulins”

Question 2

Serum Protein Electrophoresis

Answer 2

”- Used to identify the presence of abnormal proteins, to identify the absence of normal proteins, and to determine when different groups of proteins are present in unusually high or low amounts in blood or body fluids
- Electrophoresis provides 5 different fractions:
albumin, alpha 1, alpha 2, and beta and gamma globulins
“

Question 3

Results of SPE (High values)

Answer 3

”* High albumin: Dehydration
* High alpha-1 globulin: Acute or chronic inflammatory diseases
* High alpha-2 globulin: Acute or chronic inflammatory diseases; kidney disease
* High beta globulin: Very high cholesterol; low iron
* High gamma globulin: Various inflammatory diseases; acute or chronic infection; chronic liver disease; some forms of cancer
“

Question 4

Results of SPE (Low values)

Answer 4

”- Low albumin: Malnutrition; inflammatory conditions; liver disease; kidney disease; Pregnancy
- Low alpha-1 globulin: Severe liver disease
- Low alpha-2 globulin: Malnutrition; severe liver disease; hemolysis
- Low beta globulin: Malnutrition; cirrhosis
- Low gamma globulin: Problems with the immune system
“

Question 5

Western Blot Procedure

Answer 5

“Serum, cell lysate, or protein extract is separated on SDS-polyacrylamide gels (SDS-PAGE) or isoelectric focusing gels (IEF).
- Samples are treated with denaturant, such as mixing 1:1 with 0.04 M Tris HCl, pH 6.8, 0.1% SDS.
- 5%–20% polyacrylamide gels
- Proteins may be renatured before blotting to optimize antibody (probe)-
epitope binding. (mild buffer 20% glycerol + 50mM TRIS, pH 7.4)
- Proteins are blotted to membranes by capillary or electrophoretic transfer.
- Probes are specific binding proteins, polyclonal antibodies, or monoclonal
antibodies.”

Question 6

Precipitation and Agglutination

Answer 6

visible expression of aggregation of antigens and antibodies through the formation of a framework in which antigen particles or molecules alternate with antibody molecules

Question 7

Precipitation

Answer 7

aggregation of soluble test antigens and soluble antibody to produce insoluble complexes

Question 8

Agglutination

Answer 8

“aggregation of particulate test antigens into larger complexes when a specific antibody is present
“

Question 9

Affinity

Answer 9

initial force of attraction (factor associated with binding of antibodies to antigenic determinants)

Question 10

Avidity

Answer 10

strength of binding (factor associated with binding of antibodies to antigenic determinants)

Question 11

How factors can affect sensitivity/specificity of immunologic assays

Answer 11

” Antibody with high affinity and high avidity = more antigen-antibody
complexes = more sensitivity in the assay
- Precipitation also depends on relative proportions of antigen and antibody present
- Optimum precipitation occurs in the zone of equivalence”

Question 12

Zone of equivalence

Answer 12

zone between antibody-excess and antigen-excess

Question 13

Prozone

Answer 13

”- precipitation is prevented because of excess of antibody
* May result in false-negative reaction as antigen combines with only 1 or 2 antibody molecules and no cross-linkages are formed
* May be overcome by serially diluting the antibody-containing serum until optimum amounts of each are present in the test system
“

Question 14

Postzone

Answer 14

”- precipitation is prevented because of excess antigen
* Small aggregates (clumps) are surrounded by excess antigens so no cross-linkages are formed
* A blood specimen should be taken from the patient ~1 week later
~ Titer of antibody should increase
~ Negative upon repeat = unlikely the patient has the antibody
“

Question 15

Detection of precipitation in a fluid matrix

Answer 15

turbidimetry and nephelometry

Question 16

Turbidmetry

Answer 16

measure of turbidity or cloudiness of a solution (ex. spectrophotometer)

Question 17

nephelometry

Answer 17

“measures the light that is scattered at a particular angle from the incident beam as passed through a solution

Question 18

Basic Principle of Turbidimetry and Naphelometry

Answer 18

”- When particles are suspended in a solution in a cuvette, they make the solution unclear (turbid)
- Incident light entering the cuvette will be subjected to three reactions:
* some of the light will be absorbed (blocked) by the particles
* some will be transmitted through the cuvette
* some will be scattered in various directions
“

Question 19

Turbidimeter

Answer 19

”- measuring the amount of transmitted light (and calculating the absorbed light) by particles in suspension to determine the concentration of the substance in question.
Amount of absorbed light, and therefore, concentration is dependent on ;
a) number of particles, and
2) size of particles.
Measurements are made using light spectrophotometers”

Question 20

Nephelometer

Answer 20

”- components are the same as a light spectrophotometer except that the detector is placed at a specific angle from the incident light to measure the amount of scattered light; amount is dependent on the size and number of particles in suspension.
(more antigen/antibody complexes gives an increase in scattering)
“