Immunology & Serology (Midterms) - Lesson 1

prozone phenomenon

false negative result in serological tests caused by excess antibodies that interfere w/ formation of Ag-Ab complexes

prozone phenomenon

happens in very high antibody titers such as secondary syphilis or HIV co-infection

prozone phenomenon

address w/ repeated testing w/ serial dilutions

prozone phenomenon

can lead to delayed diagnosis and treatment; potential risk to fetus in pregnant women

prozone phenomenon

also hook effect or antibody excess

postzone phenomenon

also antigen excess

postzone phenomenon

false negative in IA like agglutination or precip tests

postzone phenomenon

false negative in cryptococcal antigen in CSF samples

postzone phenomenon

addressed w/ reducing concentration of antigen and allowing antibodies to bind more effectively

equivalence zone

point where optimal ratio of antigen and antibody concen leads to max amount of precipitation forming visible lattice structure

precipitation reaction

when soluble antigens and antibodies interact forming insoluble complex leading to precipitation and can be observed as visible precipitate

equivalence zone

specific point where antigen and antibody concen are in right proportions to maximize formation of complexes

lattice formation

at zone of equivalence, antigen and antibody molecules bind and forms cross-linked network or lattice which is insoluble and precipitates out of sol’n

precipitin curve

graph that shows relationship b/w amount of precipitation and concentration of antigen or antibody

equivalence zone

peak of precipitin curve

optimal binding

zone of equivalence represents point in titration or reaction where antigen and antibody molecules bind together in most efficient and stable manner

maximum precipitation/agglutination

in precipitation or agglutination assays, zone of equivalence is characterized by max amount of visible precipitation or clumping (agglutination)

antigen excess

before reaching equivalence point; some antigens are unbound to antibodies even though all antibody binding sites are occupied

antibody excess

after equivalence point; formation of immune complexes becomes less efficient and precipitation/agglutination may decrease

qualitative methods/precipitation tests

such as double immunodiffusion (ouchterlony assay); can be used to visualize zone of equivalence where visible ring or band of precipitation indicates optimal antigen-antibody interaction

equivalence zone

region in immunoprecipitation where concentration of antigen and antibody are optimal, leading to maximal formation of immune complexes

equivalence zone

zone crucial for accurate lab analysis and detection of antigen-antibody interaction

precipitation

occurs when soluble antigen combines w/ specific antibody forming insoluble antigen-antibody complex that precipitates out of sol’n

1. liquid precipitation

2. gel precipitation

types of precipitation:

liquid precipitation

adding increasing amounts of antigen to tubes containing constant amount of antibody

1. single diffusion

2. double diffusion

types of gel precipitation:

gel precipitation

uses semi-solid medium (agar or agarose) where both antigen and antibody diffuse towards each other, forming precipitation lines/bands

single diffusion

antigen or antibody diffuses from single well into gel medium

double diffusion

both antigen and antibody diffuse from separate wells into gel medium

precipitation

to detect presence and identify specific antigens or antibodies; in ring test and immunodiffusion

ring test

quick qualitative test for presence of antigen-antibody complex; ring of precipitate forms at interface of 2 liquid layers

immunodiffusion

antigen and antibody diffuse towards each other w/in semi-solid medium, forming precip line or bands

1. optimal proportions

2. valency

3. pH & temperature

4. electrolyte

factors affecting precipitation

valency

antibody must be bivalent and antigen must be polyvalent to form lattice

bivalent

at least 2 binding sites

polyvalent

multiple epitopes

agglutination

visible clumping of particulate antigens (bacteria, RBC, latex particles) when they react w/ specific antibodies forming lattice-like structure

agglutinins

antibodies that cause agglutinations

agglutination

antibodies have multiple binding sites allowing cross-linking w/ antigens on different particles forming visible aggregates

1. pH

2. temperature

3. ionic strength

4. antibody-antigen ratio

factors affecting agglutination:

1. direct agglutination

2. passive agglutination

types of agglutination:

direct agglutination

when antibodies recognize antigens naturally expressed by target cell

passive agglutination

when antibodies bind to antigens not naturally expressed by target cell but are artificially attached to carrier

agglutination tests

to detect presence of specific antibodies or antigens in sample

1. blood typing

2. identifying pathogens

3. latex agglutination

examples of agglutination tests:

blood typing

identifying blood groups by detecting presence of specific antigens on RBC

identifying pathogens

detecting antibodies against specific bacteria or virus

latex agglutination

using latex particles coated w/ antibodies to detect specific antigens

6.5-7.5

optimal pH for most antigen-antibody reaction in agglutination

IgM

antibodies that react best at colder temp

IgG

antibodies that react best at warmer temperature

flocculation

antibodies combine w/ soluble antigens forming visible precipitate or clumping of fine particles

flocculation

where suspended particles clump together forming larger aggregates or “flocs”; happens when antibodies bind to antigens leading to formation of visible precipitate

1. VDRL test

2. serum proteins

3. lipids

examples of flocculation test:

neutralization

ability of antibodies to render pathogen (virus or toxin) harmless by binding to it preventing infecting cells or causing harm

neutralization

antibodies respond to specific antigen and inactivates it

1. known amount of virus or toxin is mixed w/ px serum

2. mixture is inoculated into cell culture or animal model

3. if neutralizing antibodies are present, it will bind to virus/toxin

4. absence of disease or damage indicates antibodies are neutralizing antigen

how does neutralization test happen

1. viral neutralization test (VNT)

2. plaque reduction neutralization test (PRNT)

types of neutralization test:

viral neutralization test (VNT)

to detect neutralizing antibodies against virus

plaque reduction neutralization test (PRNT)

specific type of VNT; measures ability of antibodies to reduce number of plaque in cell culture

plaque

visible signs of viral infection

neutralization test

to diagnose viral infections and assess effectiveness of vaccines

neutralization test

provide info about person’s immune status and ability to fight off specific infections

neutralization test

to assess efficacy of vaccines by measuring level of neutralizing antibodies produced in response to vaccination