Precipitation Methods

Question 1

Principle: Soluble antigen combines with soluble antibody to produce visible insoluble complexes

Answer 1

Precipitation

Question 2

Clumping together of particles to form visible masses over a narrow range of antigen concentration

Answer 2

Flocculation

Question 3

Principle: Soluble antigens react with specific antibodies to form a precipitate of fine particles

Answer 3

Flocculation

Question 4

Applications of Flocculation

Answer 4

Venereal Disease Research Laboratory (VDRL) tests

Rapid Plasma Reagin (RPR)

Question 5

Principle: Light scattering by immune complexes is measured
● scattering of light is proportional to the size and amount of immune complexes formed

Answer 5

Nephelometry

Question 6

Applications of Nephelometry

Answer 6

Immunoglobulins

Complement

C-reactive protein

Question 7

Measures the decrease in light intensity in a solution containing immune complexes

Answer 7

Turbidimetry

Question 8

Measures the reduction of light transmitted at 180 ° angle.

Answer 8

Turbidimetry

Question 9

Measures transmitted light at 90 ° angle

Answer 9

Nephelometry

Question 10

if only one reactant (usually antigen) is moving

Answer 10

Single Diffusion

Question 11

if both antigen and antibody are moving through the medium

Answer 11

Double diffusion

Question 12

if the reaction in a medium have only one effective dimension for antigen and antibody migration (i.e., up and down)

Answer 12

Single dimension

Question 13

if the reaction is in circular holes (i.e., wells) cut in a gel on a flat surface, diffuses from the wells radially

Answer 13

Double dimension

Question 14

Principle: Known Antibody fixed in agar + Unknown Antigen (overlaid) → Precipitin lines

Answer 14

Single Linear Diffusion (SLD) or Oudin Technique

Question 15

Applications of Single Linear Diffusion (SLD) or Oudin Technique

Answer 15

Detects multiple antigen- antibody reactions

Question 16

Principle: Known Antibody fixed in agar + Unknown Antigen (well cut in agar plate) → Precipitin ring

Answer 16

Single Radial diffusion

Question 17

2 types of Single Radial diffusion

Answer 17

Fahey Method & Mancini Method

Question 18

diameter of precipitin ring at 24 hours (Read before it reaches the maximum at 6-12 hours)

Answer 18

Fahey Method

Question 19

area of precipitin ring formed at 48 hours.

Answer 19

Mancini Method

Question 20

Uses antibody not in excess.

Answer 20

Fahey method

Question 21

● Diffusion time for result reading is critical.
● Diffusion endpoints may not be reached,
affecting accuracy.

Answer 21

Fahey method

Question 22

Uses excess antibody in the gel for higher
sensitivity and accuracy.

Answer 22

Mancini method

Question 23

More commonly used in commercial
immunodiffusion plates.

Answer 23

Mancini method

Question 24

Principle: Antigen diffuses out of well in a gel containing antibody → Precipitin ring forms → Diameter proportional to the concentration of antigen

Answer 24

Radial Immunodiffusion (RID)

Question 25

technique used to measure antigen concentration based on the formation of a lattice network between ag and ab in a gel.

Answer 25

Radial Immunodiffusion (RID)

Question 26

2 types of Radial Immunodiffusion (RID)

Answer 26

Endpoint Method (Mancini Method) Kinetic Method (Fahey Method)

Question 27

● Ring diameter readings are taken at about 19 hours before equivalence is reached. ● The diameter is proportional to the log of the concentration.

Answer 27

Kinetic Method (Fahey Method)

Question 28

● Antigen is allowed to diffuse to completion until equivalence is reached, indicated by no further change in ring diameter. ● The square of the diameter is directly proportional to the antigen concentration.

Answer 28

Endpoint Method (Mancini Method)

Question 29

Applications of Radial Immunodiffusion (RID)

Answer 29

Immunoglobulins, complement

Question 30

No longer commonly performed except for low-volume testing of IgD and IgG

Answer 30

Radial Immunodiffusion (RID)

Question 31

Principle: Antigens and antibodies diffuse out from wells cut in gel and precipitin lines where they meet

Answer 31

Ouchterlony technique / double immunodiffusion

Question 32

Ouchterlony technique / double immunodiffusion Three basic reaction patterns:

Answer 32

Identity Non-identity Partial identity

Question 33

a single smooth arc of precipitation forms between the antigens and antibodies

Answer 33

Identity

Question 34

two separate lines of precipitation cross each other

Answer 34

Non-identity

Question 35

two precipitating lines meet, forming a spur

Answer 35

Partial identity

Question 36

Applications of Ouchterlony technique / double immunodiffusion

Answer 36

Fungal antigens Extractable nuclear antigens

Question 37

Principle: Antigens and antibodies are placed in wells that are directly opposite one another in a gel → an electrophoretic charge is applied to drive the reactants toward each other → precipitin band forms where they meet

Answer 37

Countercurrent immunoelectroph oresis (CIE)

Question 38

● To rapidly check any antisera for the presence and specificity of antibodies for a particular antigen. ● To detect antigens and/or antibodies in serum for diagnosis of a particular disease

Answer 38

Countercurrent immunoelectroph oresis (CIE)

Question 39

Applications of Countercurrent immunoelectroph oresis (CIE)

Answer 39

Bacterial antigens

Question 40

Principle: Proteins are separated by electrophoresis, then subjected to double diffusion with reagent antibodies placed in a trough cut in the agar → shape, intensity, and location of the precipitin arcs developed are compared with those of a normal control

Answer 40

Immunoelectrophoresis (IEP)

Question 41

Applications of Immunoelectrop horesis (IEP)

Answer 41

Serum proteins, including immunoglobulins

Question 42

Principle: Proteins are separated by electrophoresis → cellulose acetate strip impregnated with antiserum is placed on the separated proteins → the antiserum diffuses into the gel, and antigen-antibody complexes precipitate

Answer 42

Immunofixation electrophoresis (IFE)

Question 43

Gel diffusion + Electrophoresis

Answer 43

Immunoelectrop horesis (IEP)

Question 44

Protein electrophoresis + immunoprecipitation

Answer 44

Immunofixation electrophoresis (IFE)

Question 45

Applications of Immunofixation electrophoresis (IFE)

Answer 45

Identification of immunoglobulin s in monoclonal gammopathies, Bence-Jones proteins

Question 46

Principle: An electrical charge is applied to a RID assay → the height of the rocket-shaped precipitin band is proportional to the concentration of antigen

Answer 46

Rocket Electrophoresis

Question 47

Applications of Rocket Electrophoresis

Answer 47

Immunoglobulins, complement, alpha-fetoprotein