Testing: Agglutination/Precipitation

Question 1

Ouchterlony formation patterns

Answer 1

Identity, non-identity, partial-identity

Question 2

Immunodiffusion Techniques

Answer 2

• Radial Immunodiffusion (RID)
• Immunoelectrophoresis (IEP)
• Immunofixation electrophoresis (IFE)

Question 3

RID

Answer 3

• Agar with Ab incorporated
• Dilute the Pt’s sample and inoculate into well.
• Pt’s IgG diffuses across the agar to zone of equivalence and precipitin ring is formed and measured.

1. Mancini method: incubated 48 hrs, read at endpoint, plot d2 vs conc on linear graph paper
2. Fahey method: plot the diameter vs conc on semilog paper, read at 18hrs.

Question 4

Flocculation Tests

Answer 4

Precipitate of fine particles that is microscopic or macroscopic. VDRL or RPR

Question 5

Complement Fixation Tests

Answer 5

• Complement is “fixed’within Ag/Ab formation
• Uptake of complement is an indicator of the Ag/Ab formation
• Lack of hemolysis indicates that complement has reacted with the test Ag/Ab complex
• Lysis indicates that complement is not fixed by an Ag/AB reaction and is available to react with the hemolysin

Question 6

Heterophile Antibodies

Answer 6

Antibodies which have the ability to react with antigens that are not responsible for their production.

Question 7

IFE

Answer 7

• Separation of proteins into discrete bands, Ab applied directly onto the surface after electrophoresis; Ab contacts the Ag immediately
• More sensitive
• Easier interpretation yielding distinct Ag/Ab complex

Question 8

IPE

Answer 8

• Separation of proteins as Ab is placed in a trough running parallel to electrophoresis.
• Diffusion of Ag & Ab and precipitin arc is formed
• Interpretation dependent on comparison of control arc vs. pt arc

Question 9

Immunoelectrophoresis

Answer 9

A semiquantitative gel precipitation technique in which proteins are first separated by electrophoresis and then subjected to double diffusion with antibodies directed against the individual proteins

Question 10

Immunofixation electrophoresis

Answer 10

A semiquantitative gel precipitation technique similar to that of immunoelectrophoresis, except that antibody is added directly to the surface of the gel after electrophoresis has taken place

Question 11

VDRL

Answer 11

• Uses Cardiolipin as an Ag to find Reagin Ab
• Produces Flocculation that is read under a microscope; good for screening tests, treatment monitoring
• Used for CSF testing

Question 12

RPR

Answer 12

• Cardiolipin Ag to find Reagin Ab to lipoproteins
• Modified VDRL with charcoal particles that causes macroscopic flocculation
• More sensitive than VDRL in primary syphilis, recommened for patients who have negative Dark Field results but are suspected of having Syphilis
• Not used in CSF testing

Question 13

Hemagglutination inhibition (HAI)

Answer 13

• Rubella virus can agglutinate chick erythrocytes
• When Pt serum has Rubella antibody and is incubated with Rubella antigen, binding occurs. Unbound antigen is present to aggutinate chick erythrocytes.
• Serum is diluted in phosphate buffer in titration plates and Ag-RBC; After 2hrs at room temperature, the plates are read for the presence or absence of agglutination

Question 14

Passive hemagglutination (PHA)

Answer 14

Human RBC are coated with soluble rubella virus Ag. These RBC will agglutinate In the presence of rubella Ag

Serum is diluted in phosphate buffer in titration plates and Ag-RBC; After 2hrs at room temperature, the plates are read for the presence or absence of agglutination

Question 15

Reagin

Answer 15

An Ab-like substance present in those infected with syphilis or occasionally in other chronic conditions

Question 16

RPR Antigen

Answer 16

Cholesterol lipin particles coated in cardiolipin and lecithin in fluid containing choline chloride and charcoal among other things

Choline chloride acts as a chemical inactivator of inhibitors prsent in fresh serum or plasma that can prevent agglutination; nomally these were inactivated by heating

Question 17

FTA-ABS

Answer 17

• Patienst serum is absorbed with non-T. pallidum Ag to reduce cross-reactivity
• Serum is added to a slide that has fixed treponemes on it
• Flourescent antihuman Ab is added, and will bind to any anti-Treponemal Ab present from the Pts serum

Question 18

MHA

Answer 18

• Microhemagglutination assay
• RBCs with T. pallidum Ag mixed with Pt serum, if Ab are present agglutination will form

Question 19

TPPA

Answer 19

• Treponema Pallidum Particle Agglutination
• Works like MHA, but uses colored gelatin particles coated with T. pallidum instead of RBCs