LEC3 - REVIEW OF BASIC SEROLOGY Flashcards
involves combining soluble antigen with soluble antibody to produce insoluble
complexes that are visible
Precipitation
the most efficient Ig for precipitation
IgG
is the process by which particulate antigens aggregate to form larger complexes when a specific antibody
is present.
Agglutination
most efficient in agglutination
IgM
antigens mainly found on
the surface of a particles. These particles can be
naturally or synthetically attached to a carrier
particle.
particulate antigen
IgG can also perform this but it needs to be
incubated at ____* C to enhance the agglutination
reaction
37
it is the visible aggregation of particles caused by combination with specific antibody
agglutination
Antibodies that produce such agglutination are often
called
____.
agglutinins
Agglutination involves a two-step process:
___ and ___
Sensitization and then Lattice Formation
Types of particles participating in agglutination
include ___
erythrocytes, bacterial cells, and inert carriers
such as latex particles.
Agglutination reactions can be classified into several
distinct categories:
o Direct agglutination
o Passive Agglutination
o Reverse Passive agglutination
o Agglutination Inhibition
o Coagglutination
o AHG-Mediated Agglutination
the most important category among the Agglutination reactions categories is the
AHG mediated agglutination
the initial interaction between antigen and antibody
sensitization
will there be a complex formed in sensitization?
none
is there a stability formed between the antigen and antibody interaction in sensitization stage?
alaws pa po
The first reaction involves antigen–antibody
combination through SINGLE antigenic determinants on
the particle surface
sensitization
a step of agglutination wherein it’s about the Initial binding. No complex formed
sensitization
Sum of interactions between antibody and multiple
antigenic determinants on a particle
Lattice formation or formation of large aggregates
Antibody must be able to bridge the gap between cells
in such a way that one molecule can bind to a site on
each of two different cells.
Lattice formation
Erythrocytes and bacterial cells have a slight negative
surface charge, and because like charges tend to
repel one another, it is difficult to bring such cells
together into lattice formation.
true or false
true
where there is a distance
between RBCs and bacterial cells
Zeta potential
a normal distance of rbc from each other and other cells that keeps them away from each other – repelling
25 nm apart
IgM with a potential valence of 10 is over _______
times more efficient in agglutination than is IgG with a
valence of 2
700
Wingspan of IgG is
15nm
Since IgG is small, it is not
able to connect antigen from different particle. Visible
reactions with IgG often require the use of
enhancement
medium like
22% albumin,
polyethylene glycol,
Low Ionic Strength Solution
(LISS),
incubating at 37C, adding of AHG reagent
commonly used enhancement medium.
Also used in crossmatching
Low Ionic Strength Solution
(LISS)
an enhancement medium for IgG that removes water from the
system. Removing water will lead to a high
concentration of antibodies that will improve
RBC sensitization.
polyethylene glycol
When using PEG, ___after 37C
incubation is not required.
centrifugation
Most sensitive type of enhancement
medium
polyethylene glycol
PEG is not applicable in samples that have
elevated plasma protein (multiple myeloma
pxs) because _______
precipitation of protein will
occur instead of agglutination
TYPES OF AGGLUTINATION REACTION
Direct Agglutination
Passive Agglutination
Reverse Passive Agglutination
Agglutination Inhibition
Coagglutination
AHG-Mediated Agglution
a type of agglutination reaction that occurs when antigens are found naturally on a
particle.
Direct Agglutination
Direct Agglutination
Can be done on ___ or ____
glass slides or test tubes
If an agglutination reaction involves red blood cells, it
is called
Hemagglutination
Hemagglutination is routinely done at ___ without enhancement technique
room
temperature
example of Hemagglutination
ABO blood typing
serological test where we are using known bacterial antigens
widal test
weil felix test
used to test for the
presence of unknown antibodies in the patient’s serum
known bacterial antigen
a rapid screening test to help determine
the possibility of typhoid fever
widal test
antigen used in widal test
Salmonella O (somatic) and H (flagellar)
antigens
bacterial suspension used for weil-felix test
proteus spp as they have the same antigenic determinants
weil-felix test detects what infection
rickettsial infection
the abo testing we are conducting is usually at room temperature, please explain why?
because the antisera that we are using is mainly IgM in nature, which works at room temp or even colder temp
criteria of agglutination under slide method
weak agglutination
strong agglutination
in tube method, what does negative stands for
smooth suspension
in tube method, what does 1+ stands for
barely discernable clumps
in tube method, what does 2+ stands for
numerous smaller clumps
in tube method, what does 3+ stands for
several large clumps
in tube method, what does 4+ stands for
one solid clump
in tube method, what does partial or complete hemolysis means?
positive reaction
Also known as the indirect agglutination
Passive agglutination
Reagent used in indirect or passive agglutination
antibodies attached to a carrier particle
what are we detecting in indirect agglutination
antigens
back in direct agglutination, what is the reagent used and what are we detecting
reagent is the bacterial antigen and we are detecting antibody
Carrier particles used in indirect agglutination
erythrocytes
latex
gelatin
silicone
bentonite
charcoal
most commonly used carrier particles
latex
disadvantage of rbc in carrier particles
causes cross reaction with heterophile antibodies
in Reverse passive agglutination, we are detecting ___
antigens
Reverse passive agglutination reagent used is
Antibody attached to a carrier particle
Antibody is attached to a carrier particle to detect antigen
Reverse passive agglutination
criteria for Reverse passive agglutination, the carrier particle must be
Carrier particle should have the ability to absorb the Fc region of the antibody so that the Fab region are
exposed to the environment finding specific antigen
example of test for Reverse passive agglutination
CRP latex agglutination reaction
example of test that uses Passive agglutination
Antistreptolysin O
Rheumatoid factor
Anti-nuclear Antibody test for SLE
a lack of agglutination as an indicator for a positive reaction
Agglutination inhibition
are based on
competition between particulate (first reagent) and
soluble antigens (patient sample) for limited antibody
combining sites (second reagent),
Agglutination inhibition reactions
example of Agglutination inhibition reactions
classic pregnancy test
ABO typing using saliva
ABO typing using saliva principle
Hemagglutination Inhibition
inhibitor agglutination is one of the carrier particle or a principle of ___
detector of secretor status
specimen used for detector of secretor status
saliva
what are the components we used for detector of secretor status
antisera + px saliva + known cells
indicator cells used for detector of secretor status
rbc
Coagglutination detects
antigen
uses bacteria as carrier
particle for antibody
Coagglutination
it is basically reverse passive agglutination, but in ___it uses bacteria as the carrier particle
coagglutination
which bacteria us coaggulation uses as a carrier particle
(Staphylococcus aureus
Staphylococcus aureus in coagglutination has the structure called ____ which absorbs FC portion of the antibody
protein A
AHG-Mediated Agglutination is also known as
Coomb’s test
AHG means
anti human globulin
Detects nonagglutinating antibody by means of coupling with a second antibody
AHG-Mediated Agglutination
prinicple of AHG-mediated agglutination
sensitization of rbc
2 types of AHG-mediated agglutination
direct and indirect
direct ahg mediated agglutination is used in (in vivo, in vitro)
in-vivo
indirect ahg mediated agglutination is used in (in vivo, in vitro)
in vitro
what are we detecting in AHG mediated agglutination
incomplete, unknown, weakly reacting, and unidentified antibodies
involves combining soluble antigen
with soluble antibody to produce insoluble complexes
that are visible
Precipitation
s the process by which particulate
antigens aggregate to form larger complexes when a
specific antibody is present
Agglutination
Measurement of precipitation by light scattering
Turbidimetry
Nephelometry
Determination of Precipitation via Passive
immunodiffusion techniques
Radial Immunodiffusion
Ouchterlony Double Diffusion/Immunodiffusion
Determination of Precipitation via
Immunoelectrophoretic technique
Rocket Immunoelectrophoresis
Immunoelectrophoresis
Immunofixation Electrophoresis
Countercurrent Immunoelectrophoresis
are designed for antigens
and antibodies that may be small in size or present in
very low concentrations.
Labeled immunoassays
The presence of such antigens or antibodies is
determined indirectly by using a _____ to
detect whether or not specific binding has taken
place.
labeled reactant
in labelled immunoassay, The substance to be measured is known as the
_______
analyte
are bound by molecules that react
specifically with them
Analytes
_______ have made possible rapid
quantitative measurement of many important
entities
Labeled immunoassays
Labeled immunoassays have made possible rapid
quantitative measurement of many important
entities such as ____
viral antigens
Classification of Labelled Immunoassay
Radio Immunoassay (RIA)
Enzyme Immunoassay (EIA)
Fluorescent Immunoassay
Common labeled antibodies – _________
monoclonal antibodies
monoclonal antibodies are discovered by
Discovered by Georges Köhler and César
Milstein
Constant source of highly specific antibody
monoclonal antibodies
are unlabeled analytes that are made up in known
concentrations of the substance to be measured.
Standards / calibrators:
They are used to establish a relationship between the
labeled analyte measured and any unlabeled analyte
that might be present in patient specimens
Standards / calibrators
once the reaction between antigen and antibody has
taken place, there must be a partitioning step, or a
way of separating reacted from unreacted analyte.
Separation methods
Separation methods example
Decantation, centrifugation, filtration, washing ste
the most common Separation methods used in most immunoassay when it comes to separation is
solid phase vehicle
what are the antigen and antibody reaction we need to take note of the
–physical absorption
–specific binding takes place
–complexes
purpose of washing rbc
removing unbound antibodies
why can’t we use unwashed rbc
results to false negative result
how many times we need to wash rbc
3-4 times
percent of unwashed rbc
2-5%
The last step common to all immunoassays is
detection of the labeled analyte.
Most immunoassays used the change in absorbance
measured by ______.
spectrophotometry
spectrophotometry examples
Enzyme immunoassay
immunofluorescene
radioimmunoassay
in radio immunoassay, in detecting radioactivity analytes. What test is associated?
scintillation counter
operational activity we need to establish in every test we are conducting before we run difference specimens
quality control
