Antigen - Antibody Reaction (M) Flashcards
What are the diff general features of Ag - Ab rxn?
1) Physiochemical Properties
2) Affinity
3) Avidity
4) Specificity
5) Cross - Reactivity
6) Binding sites of Ag - Ab rxn
What are the characteristics of Physiochemical Properties?
1) Non-covalent
2) Reversible
How are Physiochemical Properties formed?
These are formed by a combination of hydrogen bonds, hydrophobic interactions, electrostatic, and van der Waals forces
What does “good fit” mean (or what is its purpose | in terms of Physiochemical Properties)?
The “good fit” bet an antigenic determinant and an Ab-combining site determines the stability of the Ag - Ab rxn
Multiple bonding of Ag and Ab ensures what (in terms of Physiochemical Properties)?
It ensures that the Ag will be bound tightly to the Abs
What is affinity?
It is the intensity of attraction bet Ag and Ab
What are the actions of affinity?
1) It measures the strength of interaction bet an epitope and an Ab’s Ag binding site
2) It describes how much Ab - Ag complex exists at the point when equilibrium is reached
What are the 2 types of affinity Abs?
1) Low-affinity Abs
2) High-affinity Abs
What are the actions of low-affinity Abs?
These bind Ag weakly and tend to dissociate readily
What are the actions of high-affinity Abs?
1) These bind Ag more tightly and remain bound longer
2) Bind a greater amt of Ag in a shorter period of time than low-affinity Abs
In high-affinity Abs, there is a very close fit bet what?
There is a very close fit bet the Ag-binding sites and the corresponding antigenic determinants
In the case of monoclonal Abs, how can the affinity be measured?
The affinity can be measured accurately because they are homogenous and selective for a single epitope
What are the characteristics of polyclonal Abs?
1) They are heterogenous
2) They are heterogenous, hence, they contain a mixture of Abs of diff affinities recognizing several epitopes
True or False.
Only an average affinity can be determined (in polyclonal Abs)
True, because Abs having diff affinities recognize several epitopes
What are the factors that influence affinity?
1) pH
2) Temperature
3) Buffer consumption
What is avidity?
1) It is the measure of the overall strength binding bet multivalent Ags and Abs
2) It is a better indicator of the strength of interactions in real biological systems than affinity
The avidity of an Ag - Ab rxn is dependent on what?
1) Affinity of the Ab for the epitope
2) Valency (# of binding site) of both the Ab and Ag
3) Structural arrangement of the parts that interact - stability
What is valency?
It is the # of binding site
What is specificity?
It refers to the ability of an individual Ab combining site to react w/ only 1 antigenic determinant or the ability of a population of Ab molecules to react w/ only 1 Ag
True or False.
Ag - Ab rxns does not usually show a high degree of specificity
False, because Ag - Ab rxns usually show a high degree of specificity
Abs can specifically recognize differences in what?
1) Primary structure of an Ag
2) Isomeric forms of an Ag
3) Secondary and tertiary structure of an Ag
When does cross-reactivity occur?
It occurs if 2 diff Ags share an identical or very similar epitope configuration
True or False.
The Ab’s affinity for the cross-reacting epitope is usually less than for the original epitope
True
Does antisera containing polyclonal Abs often found to cross-react w/ immunogens?
Yes, antisera containing polyclonal Abs can often be found to cross-react w/ immunogens partially related to those used for immunization, due to the existence of epitopes w/ similar configurations
What is the effect of cross-reactivity to the result in diagnostic tests?
It causes false result
What are the diff binding sites of Ag - Ab rxn?
1) Epitope
2) Paratope
What is an epitope?
It is a part of Ag w/c combines w/ Ab
What is antigenic determinant?
It specifically recognized by Abs and T cells
What is a paratope?
It is the part of the Ab that recognizes an epitope
Are most Abs bivalent?
Yes
What is bivalent?
2 binding sites
Is IgM a multivalent or polyvalent?
Either, IgM is multivalent or polyvalent
How many are the binding sites of IgM?
5 - 10 binding sites
What are the stages of Ag - Ab rxns?
1) Primary stage
2) Secondary stage
3) Tertiary stage
What is primary stage?
It is the initial interaction bet Ag and Ab (sensitization)
What is sensitization?
It is the interaction (initial?) bet Ag and Ab
What are the characteristics of primary stage?
1) It is rapid and reversible, but w/out any visible effects
2) It occurs at low temp
3) Ag and Ab is bound to each other by weak ionic bonds, hydrogen bonds, van der Waals forces
4) Covalent binding does not occur
What are the serologic tests used in the primary phase (or stage)?
1) Physical method
2) Chemical method
What are used by diff methods of serologic test (of primary stage)?
Uses markers
What are the markers utilized by methods of serologic tests (of primary stage)?
1) Radioisotope
2) Fluorescent dyes
What are the diff serologic tests for primary stage?
1) Enzyme linked Immuno-sorbent assay (ELISA)
2) Immuno-fluorescent antibody technique (IFAT)
3) Radio-immune assay (RIA)
What is the meaning of ELISA?
Enzyme linked Immuno-sorbent assay
What is the meaning of IFAT?
Immuno-fluorescent antibody technique
What is the meaning of RIA?
Radio-immune assay
What are the characteristics of secondary stage?
1) Irreversible interaction bet Ag and Ab
2) W/ visible effects, such as agglutination, precipitation, neutralization, complement fixation, and immobilization of motile organisms
3) Ag and Ab is bound by covalent bounds
4) A single Ab is capable of causing diff types of Ag - Ab rxns
5) A single Ag is capable of inducing production of diff classes of immunoglobulins
6) Has presence of lattice formation
What are the serologic effects of secondary stage?
1) Agglutination
2) Precipitation
3) Complement fixation (CFT)
4) Serum neutralization (NT)
5) Toxin-Antitoxin
6) Immobilization of motile organism
7) Enhancement of phagocytosis
What is the meaning of CFT?
Complement fixation
What is the meaning of NT?
Serum neutralization
What is a titer?
1) It is the expression of results (from serologic tests?)
2) It is the highest dilution of serum that gives a (+) rxn
What are the stages (or process) of Ag and Ab interactions?
1) Neutralisation (Exotoxin, toxoid | or neutralization)
2) Agglutination (Particulate Ag)
3) Precipitation (Soluble Ag)
4) Complement system
5) Phagocytosis
6) Inflammation
7) Cell lysis
What enhances phagocytosis?
Via the action of neutralisation, agglutination, and precipitation
How does Ag and Ab interaction lead to phagocytosis, inflammation, and cell lysis?
Via the complement system
What components induce? neutralization?
1) Exotoxin
2) Toxoid
What component induces? agglutination?
Particulate Ag
What component induces? precipitation?
Soluble Ag
What is tertiary stage?
It is the complete destruction and neutralization of Ags or tissue damage
What are included in the tertiary stage?
1) Opsonization
2) Chemotaxis and phagocytosis
3) Immune adherence
4) Humoral immunity
5) Clinical allergy
What are the purposes of serologic tests (in terms of Ag - Ab rxns)?
1) Used for detection of either serum Abs or Ags
2) For diagnosis of a wide variety of infectious diseases
3) For diagnosis of autoimmune diseases
4) For typing of tissues before transplantation
5) For blood typing
Provide an ex of serologic test (in terms of Ag - Ab rxns)
Precipitation test
What are the events that happen in precipitation test?
1) Ag occurs in a soluble form
2) Ab interacts w/ the soluble Ag in the presence of electrolyte at a specified pH and temp to produce a ppt
3) Lattice is formed bet the Ag and Ab forming a visible insoluble ppt
What are precipitins?
These are Abs that aggregate soluble Ags
Ags and Abs must be what (in precipitation test)?
These must be bivalent or polyvalent
What is the reason why Ags and Abs must be bivalent or polyvalent?
To form a ppt
What are the 2 types of media where precipitation occurs?
1) Liquid
2) Gel
What are the tests that can be done if the medium used for precipitation test is liquid?
1) Ring test
2) Slide test
3) Tube test
What are the tests that can be done if the medium used for precipitation test is gel?
1) Immunodiffusion
2) Electroimmunodiffusion
Are Ags soluble or insoluble in precipitation test?
Soluble
What is present in zone of equivalence (in precipitation test)?
Visible ppt
What are the characteristics of precipitation in liquid medium?
1) It is one of the easiest of the serological test
2) Occurs best when Ag and Ab are present in optimal proportions
What event occur in precipitation in liquid medium?
Soluble Ags react w/ Abs to form a lattice that eventually develops into a visible ppt
What test is performed in a small tube?
Precipitin ring test
What is the principle of lattice theory (Marrack)?
The interaction of multivalent Ag w/ multivalent Ab will, at optimum proportions of each (zone of equivalence), result in the formation of a lattice and a ppt
What is the indication? (or result?) if there is excess Ag (in lattice theory of Marrack?)
Early infection
What is the indication? (or result?) if there is excess Ab (in lattice theory of Marrack?)
Late in infection
In precipitation rxns, what is the result? if polyclonal antiserum is used?
Lattices or large aggregates
In precipitation rxns, what is the result? if monoclonal Ab is used?
No ppt is formed if an Ag contains only a single copy of each epitope
What is prozone phenomenon?
It is the zone of Ab excess
What is prozone phenomenon?
It is the zone of Ab excess
What are the events that occur in prozone phenomenon?
1) There is too much Ab for efficient lattice formation
2) Precipitation is inhibited and Ag combines w/ only few Abs and no cross-linkage is formed
Can prozone phenomenon be detected in supernatant?
Yes
What is postzone phenomenon?
It is the zone of Ag excess
What is the event that occur in postzone phenomenon?
Small aggregates are surrounded by excess Ag and again no lattice network is formed
What are the events that occur in zone of equivalence?
1) Ag and Ab rxn occurs optimally
2) Ag and Ab form large insoluble complexes
What is zone of equivalence?
It is the proportion of the Ag and Ab in the rxn mixture is equivalent
What should be done to detect the precipitation rxns?
Precipitation rxns must be run in the zone of equivalence
What are the types of precipitation rxns?
1) Precipitation in solution
2) Precipitation in agar
3) Precipitation in agar w/ an electric field
What are the tests that can be done in precipitation in solution (liquid)?
1) Ring test
2) Flocculation test (Slide Flocculation test and Tube flocculation test)
What is done in ring test?
The Ag solution is layered over antiserum in a test tube
What is the event that occur in ring test?
Precipitation bet Ag and Abs in antiserum solution
What is the appearance of ring test?
Marked by the appearance of a ring of precipitation at the junction of 2 liquid layers
What can be used? for ring test?
1) C-reactive protein (CRP)
2) Lancefield Streptococcal grouping
3) Ascoli’s thermoprecipitin test (anthrax)
What is used? for anthrax (for ring test)?
Ascoli’s thermoprecipitin test
Where can flocculation test be performed?
In a:
1) Slide
2) Tube (or test tube)
What is the process of slide flocculation test?
Place drop of Ag and antiserum on a slide, mixed by shaking
What is the (+) test or result of slide flocculation test?
Appearance of floccules
Provide an ex of slide flocculation test
VDRL test
What is the purpose of VDRL test?
For detection of reaginic Abs in syphilis is a slide flocculation test
What are the components that are present in tube flocculation test?
1) Ag
2) Antiserum
What is the (+) result of tube flocculation test?
Floccules appear
Provide an ex of tube flocculation test
Kahn Flocculation test for syphilis
What test can be done in precipitation in agar gel?
Immunodiffusion test
What is immunodiffusion test?
Precipitation test in a gel
What are the events that occur in precipitation in agar gel?
1) The reactants are added to the gel
2) The line of precipitation is visible as a band
3) Diff Ags observed (each Ag form a diff band)
Ag - Ab combination occurs by means of what?
Diffusion
What is the agar concentration in precipitation in agar gel?
0.3 % - 1.5 %
What is the purpose of immunodiffusion test?
It can detect identity, cross-rxn, and nonidentity bet diff Ags in a reacting mixture
What is the characteristic of Oudin Immunodiffusion?
Single diffusion (1 dimension)
What are the events that occur in Oudin Immunodiffusion?
1) Ab in an agar gel in the test tube
2) Ag solution is poured over it (test tube?)
3) Ag solution diffuses downward Ab-agar gel
4) Line of precipitation is formed
What is the action in Oudin Immunodiffusion?
The # of ppt bands shows the # of diff Ags present in the solution
What are the components in single diffusion?
1) Ag
2) Precipitin band
3) Ab in agar gel
What is the other term for radial immunodiffusion?
Mancini method
What are the events that occur in radial immunodiffusion?
1) Single diffusion in 2 dimensions
2) Antiserum solution (Ab) in agar gel - slide / petri dish
3) Ag is added to a well cut into the gel
4) Ag diffuses radially out of the well
5) Ab in the gel reacts w/ the Ag
6) A ring of precipitation is formed around the wells
Is the diameter of the ring directly proportional to the concentration of Ag (in radial immunodiffusion)?
Yes
What are the purposes of radial immunodiffusion?
1) It is used for the quantitative estimation of Abs and Ags in the serum
2) It is used to measure:
a. IgG, IgM, IgA in serum
b. complement components in the serum
c. Abs to influenza virus in sera
d. serum transferrin and alpha-fetoprotein
What is the procedure for double diffusion in one dimension?
Oakley-Fulthorpe procedure
What are the events that occur in Oakley-Fulthorpe procedure?
1) Ab is incorporated in agar gel in a test tube, above w/c layer of plain agar is placed
2) Ag is then layered on top of this plain agar
3) Ag and Ab move toward each other through the layer of plain agar
4) Ag and Ab react w/ each other to form a band of precipitation at their optimum concentration
What is the procedure for double diffusion in 2 dimensions?
Ouchterlony procedure
What are the events that occur in Ouchterlony procedure?
1) Both the Ag and Ab diffuse independently through agar gel in 2 dimensions, horizontally and vertically
2) Wells are cut in the agar gel (glass slide or in a Petri dish)
3) The antiserum (Abs) is placed in the central well
4) Diff Ags are added to the wells surrounding the center well
5) Incubation of 12 - 48 hrs in a moist chamber
6) Lines of precipitins are formed at the sites of combination of Ags and Abs
What are the 3 types of rxn (in Ouchterlony procedure?)?
1) Line of precipitation at their junction forming an arc represents serologic identity or the presence of a common epitope in Ags
2) A pattern of crossed lines demonstrates 2 separate rxns and indicates that the compared Ags are unrelated and share no common epitopes
3) Fusion of 2 lines w/ a spur indicates cross-rxn or partial identity
What is the event that happens in fusion of 2 lines w/ a spur indicates cross-rxn or partial identity (3rd type of rxn)?
The 2 Ags share a common epitope, but some Ab molecules are not captured by the Ag and traverse through the initial precipitin line to combine w/ the additional epitopes found in the more complex Ag
Double diffusion in 2 dimension has been used for what?
1) Demonstration of Abs in serodiagnosis of small pox
2) Identification of fungal Ags
3) Detection of Abs to extractable nuclear Ags
4) Elek’s precipitation test in gel is a special test used for demonstration of toxigenicity of Corynebacterium diphtheriae
What is agglutination?
It is an Ag - Ab rxn in w/c a particulate Ag combines w/ its Ab in the presence of electrolytes at a specified temp and pH resulting in formation of visible clumping of particles
When does agglutination rxn occur?
Occurs optimally when Ags and Abs react in equivalent proportion
What are the characteristics of agglutination rxn?
1) It is easier to perform and interpret
2) It is more sensitive than precipitation rxns for detection of Abs
3) This rxn is analogous to the precipitation rxn in that Abs act as a bridge to form a lattice network of Abs and the cells that carry the Ag on their surface
4) Because cells are so much larger than a soluble Ag, the result is more visible when the cells aggregate into clumps
5) Agglutination differs from precipitation rxn in that since the former rxn takes place at the surface of the particle involved, the Ag must be exposed and be able to bind w/ the Ab to produce visible clumps
What are the types of agglutination rxns?
1) Direct agglutination
2) Passive agglutination
3) Tube agglutination test
What is the characteristic of direct agglutination?
The Ags are found naturally on a particle known
What are the other types of direct agglutination?
1) Slide agglutination
2) Tube agglutination
3) Heterophile agglutination
4) Antiglobulin (Coombs’) test
What is passive agglutination?
It employs carrier particles that are coated w/ soluble Ags
Passive agglutination is usually done to what?
To convert precipitation rxns into agglutination rxns
What is reverse passive agglutination?
When the Ab instead of Ags is absorbed on the carrier particle for detection of Ags
What is slide agglutination test?
It is a basic type of agglutination rxn that is performed on a slide
What is the purpose of slide agglutination test?
It is used for identification of bacterial types (direct slide agglutination) that is still used today
What is the process in slide agglutination test?
A suspension of bacteria is prepared and is added to a drop of standardized serum
What is the (+) rxn or result of slide agglutination test?
It is indicated by clumping of bacteria and clearing of the bg solution
When does clumping (in slide agglutination test) occur?
It occurs instantly or within seconds in a (+) test
What are the uses of slide agglutination test?
1) As a routine procedure to identify bacterial strains, such as Salmonella, Shigella, Vibrio, etc., isolated from clinical sxs
2) For blood grouping and cross-matching
Where is tube agglutination test performed?
In glass tubes (test tubes)
What is the process in tube agglutination test?
The pt’s serum is diluted in a series of tubes and bacterial Ags specific for the suspected disease are added to it
How are Ag - Ab rxns demonstrated in tube agglutination test?
By demonstration of visible clumps of agglutination
Tube agglutination test is considered as what?
It is a std method used for quantitative estimation of Abs in the serum
What is the use of tube agglutination test?
It is routinely used for demonstration of Abs in the serum for serodiagnosis of enteric fever and brucellosis
What happens in Direct Coombs’ test?
The sensitization of RBCs w/ incomplete Abs take place in vivo
What is the purpose of Direct Coombs’ test?
To detect the cell-bound Abs
What is used to agglutinate pt’s RCs?
Antiserum against human Ig
What happens in Indirect Coombs’ test?
The sensitization of RBCs w/ INC Abs takes place in vitro
What is latex agglutination test?
It is the test that employs latex particles as carrier of Ag or Abs
What are the characteristics of latex particles?
1) These are inexpensive
2) These are relatively stable
3) These are not subject to cross-reactivity w/ other Abs
4) These Abs can be coated w/ Abs to detect Ag in the serum and other body fluids
The large particle size of the latex facilitates what?
It facilitates better visualization of Ag - Ab rxns by the naked eye observation
Where is latex agglutination test usually performed?
It is usually performed on cardboard cards or glass slides
How are (+) rxns for latex agglutination test graded?
Rxns are graded on a scale of 1 to 4
What are the uses of latex agglutination tests?
1) These tests are used for rapid identification of Ags of grp B Streptococcus, Staph aureus, Neisseria meningitidis, Cryptococcus neoformans, etc.
2) Useful for detection of soluble microbial Ags in urine, spinal fluid, and serum for dx of a variety of infectious diseases
3) Used to detect RA factor, ASLO, CRP, etc., in serum sxs
What is used as carrier particle in hemagglutination test?
RBCs
What are commonly used in hemagglutination test?
RBCs of sheep, human, chick, etc.
What is the event that occur in indirect hemagglutination?
When RBCs are coated w/ Ag to detect Abs in the serum
What is IHA?
It is the most commonly used test for serodiagnosis of many parasitic diseases including amoebiasis, hyatid disease, and toxoplasmosis
What is the meaning of RPHA?
Reverse passive hemagglutination
What is the event that occur in RPHA?
When Abs are attached to the RBCs to detect microbial Ag
What is the purpose of RPHA?
It has been used extensively in the past to detect viral Ags, such as in HBsAg in the serum for dx of hepa B infection
