Laboratory Immunological Techniques

Front 1

Define agglutination

Back 1

Particulate antigens react with specific antibody resulting in visible clumping by forming bridges between antigenic determinants on adjacent particles.

Front 2

What is the basis of agglutination?

Back 2

- Latex covered with Ab so each bead can bind with numerous Ag,
- Ag must bind to multiple beads
- Limited to large molecules limited with repetitive antigenic domains
- eg: microbial capsules, flagella or lipopolysaccarides
- repeating antigen molecules will clump pattern

Front 3

What was the specific agglutination tests used in the practical classes?

Back 3

- Staphytect (latex slide test)
- Syphilis agglutination test

Front 4

Which test was used for rapid detection of an antigen?

Back 4

Staphytect (latex slide test)
- blue beads coated with fibrinogen and IgG

Front 5

Which test was used for the detection of an antibody?

Back 5

Syphilis test --> rapid plasma reagin carbon antigen

Front 6

Describe the reaction of the Staphydect / Latex test:

Back 6

Blue latex particles are in the solution and are coated with human fibrinogen (clot forming glycoprotein) to detect clumping. Latex beads most importantly is coated with IgG, which will bind with Protein A and surface antigens found in MRSA and MSSA

Front 7

Describe the process for the syphilis test

Back 7

- Rapid Plasma Reagin carbon antigen
- Reagins and Abs are produced when exposed
- Reagins are Abs which mediate immediate hypersensitivity reactions and occur when lipoidal material is expelled from damaged host cells, reagins are called in this instance non treponemal antilipoidal Abs.
Carbon particles are the vehicle to host treponemal Ag to detect non treponemal reagins, which will form black clumps

Front 8

Define precipitation

Back 8

an Ab-Ag reaction resulting in visible precipitate, which can quantify the amount of Ab from the amount of precipitate obtained from a known amount of antigen
o Ag-Ab reaction which produces a precipitate – Ag and Ab soluble.
o Ag and Ab come together, reach equivalence point and precipitate.

Front 9

what is the zone of equivalence?

Back 9

o Ag-Ab reaction which produces a precipitate – Ag and Ab soluble.
o Ag and Ab come together, reach equivalence point and precipitate.

Front 10

List methods of precipitation performed in class

Back 10

1. Ouchterlony
2. Counter Immunoelectrophoresis (CIEP)
3. Serum Immunoglobulin Levels (SRID)

Front 11

Describe the process of Ouchterlony

Back 11

- An agarrose gel layer is punctured and impregnated alternately with antibody and antigen.
- They are allowed to diffuse which will develop a precipitin line when the ideal Ab-Ag ratio is formed
- numbers of lines represent the number of different antigen/Ab systems present
- A continuous line = line of identity
- non continuous line = non identity
- anti human serum --> Ab
- human serum --> Ag

Front 12

In Ouchterlony when does a line form>

Back 12

At zone of equivalence which is where there is either no Ab or Ag remaining (free).

Front 13

Describe an agglutination test to detect an antigen

Back 13

Ag - Staphytect
- coat particle in anti- Ag Ab
- this case it is IgG which is anti-proteinA and surface antigens found in sera

Front 14

Describe an agglutination test to detect an Ab

Back 14

Ab - Treponemal
- Cardiolipin antigen suspension containing added carbon particles react with positive serum containing reagins produce black clumps

Front 15

Define a line of identity

Back 15

A precipitin line which develops when an optimimum Ab:Ag. Each line represents a Ab:Ag system reaction.
The precipitin line develops only when the Ab:Ag system reaches the zone of equivalence, where the amount of Ab precipitated is 1:1 (or close) to Antigen present.

Front 16

What is the relationship between Ouchterlony test and the zone of equivalence?

Back 16

The ouchterlony test will not cross link if either Ag or Ab is in excess

Front 17

Describe a line of non identity

Back 17

Precipitin lines that cross

Front 18

Describe a line of partial identity

Back 18

- Ab in middle
- Ag in other wells
- spurs present due to unshared determinant

Front 19

Describe radial immunodiffusion or SRID

Back 19

http://www2.hawaii.edu/~johnb/micro/medmicro/medmicro.11.html