MAJOR CATEGORIES OF AGGLUTINATION

1. DIRECT/ ACTIVE AGGLUTINATION

- Antigens are found naturally on a particle

- Known bacterial antigens: used: test for presence of unknown antibodies in patient

- Patient serum: diluted into a series of tubes or wells: reacted with bacterial antigens specific for the

suspected disease

- Used in diagnosis: bacterial agents: extremely difficult to cultivate

- WIDAL TEST:

 Rapid screening test: determine possibility of typhoid fever

- Hemagglutination: agglutination involving RBCs

 Hemagglutination: syphilis

 Passive agglutination: Rubella

 Latex particle agglutination inhibition: HCG

2. PASSIVE AGGLUTINATION

- Particles are coated with antigens not normally found on their surfaces

- Use of synthetic beads or particles: provide: advantages of consistency and uniformity

- (+) AGGLUTINATION: patient antibody is present

3. REVERSE PASSIVE AGGLUTINATION

- Antibody rather than antigen is attached to a carrier protein

- Must still be active: joined in a manner that the active sites are facing outward

- Adsorption:

 Spontaneous

 Require some of the manipulation as is used for antigen attachment

- Detect microbial antigen

- Rapid identification of:

 Group B Streptococcus

 Staphylococcus aureus

 Streptococcal group A and B

 Rotavirus
 Cryptococcus neoformans

- Latex particles coated with antibody: reacted with patient sample containing the suspected antigen

- (+) AGGLUTINATION: patient antigen is present

4. AGGLUTINATION INHIBITION

- Based on competition between particulate and soluble antigens for limited antibody-combining sites.

- Positive reaction: Lack of agglutination/ No agglutination

- Reagent antibody: added to patient sample

- If patient antibody is present antigen-antibody combination

- Antigen-coated latex particle: added: no agglutination  POSITIVE

- No patient antigen: reagent antibody combine with latex particle: agglutination  NEGATIVE

5. COAGGLUTINATION

- Uses bacteria as the inert particle to which the antibody is attached

- Staphylococcus aureus

 Most frequently used

 Protein A: protein on the outer surface; naturally absorbs the Fragment crystallizable (FC) portion of

an antibody molecules

 S. aureus particles nonspecifically bind to the FC portion of immunoglobulin molecules

 When reagent antibody is used, combination with patient antigen produces a visible agglutination

reaction

- Coagglutination reagents: used in identification of:

 Streptococci

 Neisseria meningitis

 Nesseria gonorrhoeae

 Vibrio cholera 0139

 Haemophilus influenza

PRECIPITATION

- Aggregation of soluble test antigen; combination of soluble antigen and soluble antibody to produce a

visible insoluble complex

TERMS:
 Precipitin: Antibody

 Precipitinogen: Soluble antigens

 Precipitates: Insoluble complexes formed from the union of the two

 Flocculation: Natural clumping

o Observed: ―fleecy mass‖: suspension of Ab-Ag is agitated

 White or cloudy appearance

IMMUNOGLOBIN INVOLVED

 IgG: much better precipitating antibody

 IgM: much better agglutinating antibody

 Precipitation: IgG>IgM>IgA

 IgE: Non-precipitating

PREREQUISITES TO REACTION

 Antigen

o Multivalent

o Soluble

 Antibody

o At least 2 binding site

 Both present in correct proportions that allow lattice formation

 Optimum Temperature

 pH

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PRECIPITATION CURVE

A. PROZONE:

- Zone of antibody excess

- Insufficient antigen= no cross linking

B. ZONE OF EQUIVALENCE:

- Maximal amount of precipitation that can occur

C. POST ZONE

- Zone of antigen excess

- Decreased cross linking

- Result: insoluble immune complex

RING TEST/ INTERFACIAL TEST/ FLUID PPT TEST

- Place undiluted antiserum: Bottom

- Antigen solution: layered over

- Incubate

- RESULT: hazy/cloudy/milky

- APPLICATIONS:

 ASCOLI TEST: detect capsular antigen of Anthrax bacillus

 Lancefield Serological Typing: detect streptococci: Non-specific test

 C-REACTIVE PROTEIN: Increased: MI and RF

PRECIPITATION IN GEL MEDIUM

1. Single Diffusion

- Only one reactant (antigen) is moving

- Ag and Ab diffuse through the pores of the gel until the concentration reaches optimum ratio & form a

stable immunoprecipitate

2. Double Diffusion

- Both Ag and Ab are moving through medium

3. Single Dimension

- Reactions in tubes: Ag & Ab migrate up and down

4. Double Dimension

- Petri dish- Ag & Ab diffuse radially

1. Single Diffusion- Single Dimension (Oudin)

- Antibody/ Antiserum is incorporated in melted agar

- Mixture is poured into a tube and allowed to solidify

- Antigen solution placed above the agar

- Precipitin bond: appears in the agar

2. Single diffusion- Double Dimension (Radial Immunodiffusion/Mancini Test)

- USES:
 Popular method for quantitating a variety of proteins normally found in serum

 Quantitating Ab titer (concentration)

- Antibody: incorporated into an agar poured into a glass plate to form a uniform layer

- With Circular wells: antigen is introduced

- Ring-shaped bonds: appear concentrically around the well

- Used to quantitate the amount of antigen present

- Read by measuring the size of area: precipitin ring

- RADIAL IMMUNODIFFUSION BY MANCINI

 Diameter of the disk is proportional to the logarithm of the concentration of antigen

- TECHNICAL ERROS:

 Over and underfilling of wells

 Spilling sample outside of the well

 Nicking the well

 Improper incubation/Temperature

3. Double Diffusion-Single Dimension (Oakley and Fulthrope)

- Antibody/Antiserum: incorporated: Agar

- Poured into a tube to harden

- Second layer: agar without antibody

- Precipitation bond: place in the plain agar column: ppt ring

4. Double Diffusion-Double Dimension (Outerlony and Elek)

- Other names:

 Double immunodiffusion

 Double angular Immnunodiffusion

- Outerlony:

 Two or more wells are cast into the agar

 Antibody is added on one well

 Antigen on the other well

 Lines of precipitate forms where Ag-Ab diffuse meet in optimal concentration

- Use: find relationship between antigen

- 3 possible pattern:

i. Serological Identity

 Fuse pattern of precipitate around the antibody

 Anti-1

ii. Serological Non-identity

 Lines of precipitation cross one another

 Antigens: serologically distinct

 Anti-1,2,4

iii. Serological Partial Identity

 Spur formation

 Antigen: not identical but posses a common determinant

ELECTROPHORESIS

PRINCIPLE:

- Technique in which molecules with a net charge are separated when electric field is applied to the

system

- FACTORS:

 Size and shape of protein

o Bigger CHON: less travel/migration

 Viscosity of the buffer: thick/thin

 pH: 8.0 or higher

 Temperature

o Room Temperature: ideal

o Higher temperature: Higher mobility: can denature CHON

 Endo-osmosis: Flow of ions toward the cathode

o (-) charge electron, anion- (+) Electrode: anode

o (+) charge electron, cation - (-) Electrode: cathode

SUPPORT MEDIA

- Filter paper: rarely used

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- Cellulose Acetate: favored: relatively inert (immobile)

- Polyacrylamide gel: use: Research laboratories: HIV confirmatory test

- Agarose gel: separation of large molecules:

 DNA

 IgM

CATEGORIES OF IMMUNOELECTROPHORESIS

 Single Reactant moving in one dimension  Rocket technique of Laurell

 Single Reactant moving in two dimension Crossed immunoelectrophoresis

 Double Reactant moving in one dimension  Counter immunoelectrophoresis

 Double Reactant moving in two dimension  Classic immunoelectrophoresis

A. Rocket Electrophoresis: Voltage Facilitated Single Immunodiffusion

- Shape formed: Rocket-like

- Antigen is pushed through the antibody containing gel under the influence of applied electric field

 Quantitative Electrophoresis Method

o Used with a calibration curve

o The length of the rocket: proportional: logarithm of the antigen concentration

B. Crossed Immunoelectrophoresis/ Double Crossed Immunoelectrophoresis

- Two dimensional

- First Step: Electrophoretic separation of proteins in a biological sample

- Second Step

 Separated CHON: subjected to second electrophoresis at angle 90o

from the first electrophoresis

 Move through agarose gel containing antibodies leading to the formation of arc

- Formation of arc: Ag and Ab reach equivalence

C. Counter current immunoelectrophoresis

- AKA:

 VOLATAGE FACILITATED IMMUNODIFFUSION

 COUNTERCURRENT ELECTROPHORESIS

 DOUBLE ELECTROIMUNODIFFUSION
- Antigen and antibody are added to separate parallel well cut out of an agar gel

- Electric field: applied

 Migrate at opposite direction

 Antibody: migrate to the cathode

 Antigen: migrate to the anode

D. Classic Immunoelectrophoresis

1. Biological sample (antigen): introduced into a well

- Electrical field is applied resulting in separation of proteins

2. An antibody is applied in a trough located parallel to the separated CHON

- Both the CHON & trough antibodies diffuse in the gel

- Result: characteristics arc when Antigen-Antibody complex forms