FINALS - IMMUNOASSAY 2 Flashcards
agglutination means there will be a formation of
large complexes
What would be the particular antibody involved when
it comes to the agglutination reactions
IgM
Many of the immunoassays right now are based on those
principle of __ AND ___ reaction
agglutination and precipitation
Immunoassays have been developed to detect either __ or ___, and they vary from easily performed manual tests to highly complex automated assays.
antigen or antibody
is the initial force of attraction that exists
between a SINGLE FAB SITE on an antibody molecule and a SINGLE EPITOPE or determinant site on the corresponding
antigen
affinity
how antige and antibody binds? through ___
non covalent bond
Antibodies are capable of reacting with antigens that
are structurally similar to the original antigen that
induced antibody production.
This is known as ___
CROSS-REACTIVITY
The strength of attraction depends on the ___
of antibody for a particular antigen.
specificity
represents the sum of all the attractive forces
between an antigen and an antibody.
Avidity
This involves the strength with which a multivalent antibody binds a multivalent antigen, and it is a measure of the overall stability of an
antigen–antibody complex
avidity
it is the forces that keeps the molecules together. We need to stabilized the antigen-antibody
complex that would be the mechanisms of
the ___.
avidity
A high avidity can actually compensate for a __
low affinity
Stability of the antigen–antibody complex is essential to detecting the presence of _____, whether it
is antigen or antibody.
an unknown
antigen and antibody binding is
affinity
satbilization of antigen antibody binding
avidity
what is the requirement for the precipitation reaction?
equal or proportion strength of affinity and avidity
The precipitation reaction will be depending on the ___ of antigen and antibody
relative porportion
precipitation curve is also known as
precipitin curve
the optimum precipitation or the precipitin
curve that we have occurs in the ___in
which the number of the multivalence sites of the antigen-antibody are approximately equal
zone of
equivalence
in the zone of equivalence, the precipitation reaction may be declined on the
other side of that particular equivalence zone due to the excess of either antigen or antibody
rthat is what we called as
PROZONE and POST ZONE
excess antibody
prozone
his phenomenon of
Antigen combines with only one or two antibody molecules
and cross linkages are formed that would be the ___.
prozone
excess antigens
postzone
In this case every
available antibody site is bound in a single antigen and no
cross-links are formed
POSTZONE
his phenomenon is not
correlated with a lattice network, so basically there is no
lattice network formed on the ___.
postzone
Measurement of precipitation by light scattering.
o Turbidimetry
o Nephelometry
Determination of Precipitation via Passive
immunodiffusion techniques.
o Radial immunodiffusion
o Ouchterlony double diffusion or Ouchterlony
double immunodiffusion
Determination of Precipitation via
Immunoelectrophoretic technique
o Rocket Immunoelectrophoresis
o Immunoelectrophoresis
o Immunofixation electrophoresis
o Countercurrent Immunoelectrophoresis
is a measure of the turbidity or cloudiness of a
solution.
TURBIDIMETRY:
On that particular turbidimetry the light
scatter occurs in proportion to the __, __ and ____ of the
molecules present in the solution
size, shape and the concentration
So the measurement when it comes to
the turbidimetry can be made using a
___ or ___.
spectrophotometer or automated clinical
chemistry analyzer
In turbidimetry, A detection device is placed in ___ with the incident light
direct line
Collecting light after it has passed through the
solution.
turbidimetry
It thus measures the reduction in light intensity
due to reflection, absorption, or scatter
turbidimetry
It is recorded in absorbance units, a measure of
the ratio of incident light to that of transmitted
light.
turbidimetry
In turbidimetry
The unscattered light is measured at ___
degrees angle from the incident light.
180 degrees
If we are referring with the nephelometry the
intensity of ___ is measured.
scattered light
measures the light that is scattered at a particular
angle from the incident beam as it passes through a
suspension.
nephelometry
It is also the index of the solution
concentration
NEPHELOMETRY:
Nephelometers measure light scatter at angles
ranging from ___ degrees to about ___ degrees
10; 90
this would be the
reaction; we are allowing to run essential
completion but large particles tend to fall out of solution and decrease the amount of the scatter.
End-point nephelometry
the rate of
scattering increases and it is measured
immediately after the reagent is added
Kinetic or rate nephelometry
Many automated instruments utilize this principle
(_____) for the measurement of serum proteins.
nephelometry
provides accurate and precise
quantitation of serum proteins, and due to
automation, the cost per test is typically lower
than other methods.
Nephelometry
Nephelometry can provide ___
quantitation of complement components
quantitation of c reactive components and even clotting factors
quantification of immunoglobulins such
as the IgG, IgA, IgM and IgE and even the kappa and lambda light chain
among the precipitation reaction, it is less expesive
NEPHELOMETRY
When it comes to the nephelometry the
light detection device is at angle to the ___
incident light
If we are referring with the ___ again this would be the intensity of the light transmitted through a particular
medium wherein the unscattered light is measured at 180-degrees angle from the incident light beam.
turbidimetry
If we are referring with the ___ this would be the intensity of that scattered light that would be the factor that being measured on that particular nephelometry
can used an angle for about
10 to 90-degrees.
nephelometry
The precipitation of antigen–antibody complexes can also
be determined in a support medium such as a ___.
gel
In this particular GEL consists of a
high-molecular-weight complex polysaccharide derived from____, are used for this purpose.
seaweed, and agarose, a purified
agar
In passive
immunodiffusion technique, The reagent that we have here is an
___
antibody
In passive immunodiffusion technique, Yung pinakagamit na gamit natin dito ay
yung ___
agar at agarose
why agar at agarose is commonly used in passive immunodiffusion technique?
it helps to stabilized the
diffusion process and allow the
visualization of the precipitate bonds
The rate of diffusion in passive immunodiffusion is affected by the ___
size of the particles,
the temperature,
the gel viscosity,
and the amount of hydration.
RADIAL IMMUNODIFFUSION (RID): described by
JAMES OUDIN
was the first to use gels for precipitation
reactions, and he pioneered the technique known as
single diffusion
JAMES OUDIN
Antibody was incorporated into agarose in a test tube.
The antigen was layered on top, and as the
antigen moved down into the gel, precipitation
occurred and moved down the tube in proportion
to the amount of antigen present.
RADIAL IMMUNODIFFUSION (RID):
A modification of the single-diffusion technique
was the ___.
RADIAL IMMUNODIFFUSION
the positive result of RADIAL IMMUNODIFFUSION
formation of the precipitate bond.
two tchnique tomeasure RID
mancini method
fahey and mckelvey method
one of the method to measure RID that uses end point method
mancini
a method to measure RID in which the antigen is allowed to diffuse to completion
Mancini
in mancini method, the determination of IgM is completed for how many hrs?
50-72 hrs
in mancini method, the determination of IgG is completed for how many hrs?
24 hrs
the presence of rig formation in mancini method signifies,
antigena nd antibody formation
within the diameter is the ANTIGEN CONCENTRATION
a method to measure RID in which the method used is kinetic method, using measurements taken before the point of equivalence is reached
fahey and mckelvey method
the readings for fahey and mckelvey takeshow many hrs
reactions is measured after 18 hrs
sources of error RID
overfilling and underfilling of wells
nicking the side of the wells when filling
spilling sample outside the well
improper incbatio time and temp
incorrect measurement
RID has been used to measure ___
Ig M, G , A, and complement components
it is simple to perform and requires no instrumentation but it is fairly expensive to run
Radial immunodiffusion
I radial diffusion, we can as well use ELISA but not for __
not for low volume analytes such as Ig D or IgG
in this technique, both antigen and antibody diffuse independently through a semisolid medium in two dimensions, horizontally and vertically
ouchterlony double diffusion
wells are cut in a gel - reactants are added to the wells incubate for 12 and 48 hrs in a most chamber - precipitin lines form here the moving front of antigen meets that of antibody
which process is this one?
ouchterlony double diffusion
ouchterlony double diffusion is also called as
ouchterlony double immunodiffusion
In letter A in ouchterlony double diffusion
common epitope - resembles smooth curve
In letter B ouchterlony double diffusion
cross line pattern - demonstrate 2 separate reactions
no common epitope
no identity
category C ouchterlony double diffusion
diffusion of 2 lines - partial identity - with spur?
rocket immunoelectrophoresis is also called as
laurell technique
antibody is distributed i the fell and the antigen is added just like RID
switch ON the electric current
when antigen diffuses, precipitation begis
end result: conical shape precipitin line
rocket immunoelectrophoresis
why do we need to switch on th electric crrent in rocket imunoelectrophoresis?
to facilitate the migration of the antigen
end result of the rocket immunophoresis
conical shape precipitin line
double diffusion technique that incorporates electrophoresis crrent to enhance results
immunoelectrophoresis
immunoelectrophoresis is introduced by
grabar and williams in 1953
this is performed as a two major step process and can be used for semi quantitation of a wide range of antigens
immunoelectrophoresis
2 step in immunoelectrophoresis
electrophoresis - separation of protein components
diffusion of antigen and antibodies
immunoelectrophoresis takes how many hrs
18 - 24 hrs
what is the source of antigen of immunoelectrophoresis
serum
typically the source of antigen is serum, which is electrophoresed to separate out the main protein fractions
a trough is cut in the gell parallel to the line of separation
antiserum is places in the trough, and the gel is incubated for 18 - 24 hrs
these lines or arcs ca be compared in shape , intensity, and location to that of a normal serum to detect abnormalities
immunoelectrophoresis
used as a screening tool for many serum proteins including the major class of Ig and a qualitative and semi quantitative technique
for detection of myeloma
can detect Waldenstrom macroglobulinemia
immunoelectrophoresis
in immunoelectrophoresis, we are using goat antibodies and a serum with ___
immunodeficiency
___, either mataas
yung value nung isang antibody at yung isa naman ay
deficient.
B lymphocyte deficiency
deficiency that will cause increase bacterial deficiency
IgG deficiency
in immunodeficiency, what deficency is involved?
serum IgA
deficiency.
As first described by Alper and Johnson
IMMUNOFIXATION ELECTROPHORESIS
It is similar to electrophoresis except that it has no trough for the antibody reagent. The antibody is applied directly to the gel surface rather than phase
IMMUNOFIXATION ELECTROPHORESIS
IMMUNOFIXATION ELECTROPHORESIS reaction time
less than 1 hr
Modification of immunoelectrophoresis with only 30 - 60 mns of reaction time
COUNTERCURRENT IMMUNOELECTROPHORESIS
There is a side-to-side distribution of the antigen and
antibody
COUNTERCURRENT IMMUNOELECTROPHORESIS
negative charge for COUNTERCURRENT IMMUNOELECTROPHORESIS
anode
we have antigen 1 (Ag1), antigen 2 (Ag2),
and antigen 3 (Ag3).
in countercurrent immunoelectrophoresis,
Kapag sa ___ naman, we have the positive
charge. We have the antibodies there
cathode
in counter current mmunoelectrophoresis, a side to side means
concentration of that Ig or Ag
___ are designed for antigens and antibodies that may
be small in size or present in very low concentrations.
Labeled immunoassays
in labelled 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 immunoassays
The substance to be measured is known as the __.
analyte
in labelled immunoassays
__ are bound by molecules that react specifically with them.
analytes
Characteristics of Labelled Immunoassay
▪ Competitive Assay
▪ Non-competitive Assay
Classification of Labelled Immunoassay
▪ Radioimmunoassay (RIA) -
Competitive and Noncompetitive RIA
▪ Enzyme Immunoassay (EIA) -
Competitive: EIA -
Noncompetitive: Indirect ELISA
▪ Fluorescent Immunoassay (FIA)
All the reactants are mixed together simultaneously, and labeled antigen
competes with unlabeled patient antigen for a limited number of antibody
binding sites.
Competitive Immunoassay
The amount of bound label is Inversely Proportional to the concentration of
the patient antigen.
Competitive Immunoassay
Antibody, is first passively absorbed to a solid phase (which is also a medium
well).
Noncompetitive Immunoassay
Unknown patient antigen is then allowed to react with and be captured by the
antibody.
non competitive immunoassay
After washing to remove unbound antigen, a second antibody with label is
added to the reaction
non competitive immunoassay
The amount of label measured is Directly Proportional to the amount of
patient antigen
non competitive immunoassay
what type of antibodies used for labelled immuoassays
monoclonal antibodies
monoclonal antibodies which are
discovered by __
George Kohler and Cesar Milstein
. __ remains as a
constant source of high specific antibody.
Monoclonal antibodies
Aka Calibrators
standards
Are unlabeled analytes that are made up in known concentrations of the
substances to be measure
Standards
So the bound and unbound fractions are usually separated by physical means (such
as ___).
decantation, centrifugation, and filtration
The last step common to all immunoassays is the ___
detection of the labeled
analyte
Radioimmunoassay Used ___ as a label
radioactive substances
