17-1 Immunological testing Monoclonal antibodies Serology Quantifying antigen – antibody reactions.
Antigen Antibody Reactions - JUdoctors 12, 2013 · Antigen – Antibody Reactions ... -Far Ag...
Transcript of Antigen Antibody Reactions - JUdoctors 12, 2013 · Antigen – Antibody Reactions ... -Far Ag...
Antigen – Antibody Reactions
8 الشيت سوف تكون حاوية كل شيء ال داع للساليدات بدءا من ساليد رقم سالم
سوف تكون ابسط ما يمكن صياغة ضمن مصطلحات الدكتور لذا من يرجع للتسجيل سيجد اختالف لكن نحو األسهل و األكثر ترابطا محافظة على ما طرح إن شاء هللا
نصف الفهم = االشكال التوضيحية
Quick revision
Ag + Ab ↔ AgAb
** antibody reactions can be called serological reactions because most of antibodies are produced and
are mainly present in serum.
- Non-covalent interaction , driven to the right but it is reversible .
- Very specific interaction , this is the most important characteristic of the adaptive immune response
- forces that hold the reactants together :
* Vander waal force
* Electrostatic force
* Hydrophobic force
- Strength of reaction
how far it is driven to the right ( affinity refers to a single binding site ) >> IgM and IgD>> low affinity ; the secreted IgM >> more affinity.
the affinity of the secondary immune response (production of IgG ) is more
than that of the primary immune response (production of IgM)
- Avidity : the collective affinity of multiple binding sites on an antibody molecule . measures the True
strength of the Ab -Ag interaction within biological systems so Avidity is a better measure of antigen-antibody
interactions ; it measures the total number of reactants involved.
cross reaction can exist in related Ag*
Occurs if two different Ags share identical or very similar epitope >> the amino acid sequences in the Fab region are usually very specific for an antigen but the possibility of antigenic
determinants to be similar is also present in nature; this leads to cross-reactivity.
Therefore, cross-reactivity refers to the possibility of an antibody directed against a certain antigen to bind to a
similar/related antigen.
Non-covalent bonds that hold the
antigen to the antibody in Ag-Ab
Reactions.
* the interaction at one site will increase the possibility of reaction at 2nd site . so if the affinity is high, it will result in increased binding and therefore influence the avidity of the interaction.
* High avidity can compensate for low affinity (secreted pentameric IgM has a
higher avidity than IgG) >> the affinity of IgG is higher than that of IgM when it comes to comparing a
single binding site BUT if the whole antibody is taken, the strength of the reaction is more for IgM because of the
presence of five binding sites; this is actually a measure of avidity.
Stages OF Ag – Ab reaction
>> antigen- antibody reactions go through phases .
>> The formation of complexes in the early phases takes place between an antigenic determinant (single epitope)
and Fab of Ab (( forming few invisible primary complexes : Small Ag - Ab complexes )) which facilitates the
binding of other epitopes , so as the time elapses the complexes that be formed become larger and larger (large
complex : secondary reaction which is explained by the lattice hypothesis ) , this allows the formation and
development of macroscopic manifestation reactions ( forming visible Ag - Ab aggregates or precipitate .),
this reaction continues until the largest possible complexes are formed .
>> primary complex may be the final reaction in the cases where we have Ag with limited binding sites (2-3 ), they
don't form largest complex because Ag can accommodate in maximum of 1 or 2 Ab and in this case the complexes
that result are small , e.g : hormones
while large proteins usually have multi-binding sites ( large no. of antigenic
determents ) , allows the formation of macroscopic reactions .
>> Ag-Ab reactions can take place in vitro ( lab ) or vivo ( body ) , those that
take place in vivo are either intravascular or both intra & extra vascular .
** IgM are confined to circulation and cannot leave it because of the high
molecular weight = intravascular , while those of lower molecular weight (IgG and IgE) can leave the
vasculature and enter tissues = ( both intravascular and extravascular ) , because 50% of IgG is present outside
the vasculature , IgE is usually present fixed on the mast cells and enter basophilic tissue . Time required for the
formation of such immune-complexes (precipitin formation ) is hours to days leading to irreversible
immunoprecipitates .
Lattice Theory
24-72 hrs are needed to form
the end reaction between Ag
and Ab that depends on the
present of multivalent Ag
primary
complexes
Antigen-antibody reactions pass
through initial phases that are
associated with the production of
small Ag-Ab complexes ( primary
complex ) and as the reaction is
allowed to proceed, the complexes
become larger and visible ( precipitate
or agglutinate ) .
>> the formation of large complexes (visible Ag - Ab aggregates) is explained by the Lattice Theory .
just imagine that we have Ag.s with multivalent (more than one antigenic determents ) & we have weather
IgG ( 5 binding sites ) or IgE ( 2 binding sites ) ,,
IgG molecule will bind to 2 determents of 2 different antigens since it has 2 binding sites , each anti-
gene can be linked with another IgG molecule , and that why the formation of complexes proceed in a way of
network ( a mesh of Ag-Ab ) , and that's called Lattice hypothesis.
** complexes are formed better when IgM involved because of the 5 binding
sites *2
So the Lattice Theory explains the formation of large complexes with time
and this required multivalent Ag (contains more than 2 identical epitopes)
& Cross-linking of Ags by specific Abs (2 or more antigen-binding sites).
all antibodies have at least two antigen binding sites like IgG unless we’re
talking about a single Fab which is mono-valent and does not allow the
formation of visible complexes.
>> the complexes are formed at ratio that called the zone of equivalence (Molar ratios of epitopes and
antigen-binding sites are optimal )
so Lattice formation (visible Ag - Ab aggregates) occurs when:
- Ag is multivalent (contains more than 2 identical epitopes)
- Cross-linking of Ags by specific Abs (2 or more antigen-binding sites)
- Molar ratios of epitopes and antigen-binding sites are optimal (zone of equivalence)
If antigens and antibodies are mixed and allowed to interact then measurements of either the antigens or
antibodies concentrations are to be made then the result will curve like this one ( check the picture below )
Ab
Ag
1) [Ab] > [Ag] , so Ab compete for few numbers of
Ag & there is no chance for the formation of the
large complexes >>> no precipitate as the reaction
processed [Ag ]& [Ab] increases leading to (2) the
formation of large complexes at the zone of
equivalence , usually the ratio at this zone is 1:1 (
e.g 100 Ab : 100 Ag ) this ratio at which optimal
complexes are made & the largest complexes are
formed >>> visible precipitate ,,, the formation of
these complexes required incubation of time that
varies depending on the reactant , IgM for example
required more time than IgG .
3) [Ag] is extreme , more than that of Ab so more
Ag compete for few number of Ab & that why there
is no large complexes formed >>> no precipitate .
1 2 3
so,, zones of lattice formation
-Far Ag excess (no ppt. formed; free Ag in supernatant) -- “postzone” ,, no complexes
-Ag excess (sub-optimal ppt.; free Ag in spnt.),, There might be very small complexes that cannot
be seen by the naked eye.
-Zone of equivalence (maximum ppt.; no Ag or Ab in spnt.),, Complexes are so large that they can be
seen by the naked eye.
-Ab excess (sub-optimal ppt; Ab in spnt.) Small complexes are formed that cannot be seen
-Far Ab excess (no ppt; Ab in spnt.) -- “prozone” ,, no formation of complexes
For the sake of simplicity of discussions, it is enough to classify them into pro-zone, post-zone, antigen-excess,
antibody-excess and the zone of equivalence between Ag & Ab excess/production.
large complexes
Small complexes
>> If we consider the beginning of the curve , is actually
extreme [Ab] ( far [Ab] excess ) , (no ppt. formed; free Ab in
supernatant) -- “prozone” >> no complexes formed because
there is no enough Ag ,,
as the reaction processed , small invisible complexes are
formed , at the zone of Ab excess (sub-optimal ppt; Ab in
supernatant) , many Ab aren't involved in the reaction .
as the ratio of Ab:Ag reaches 1:1 ,the largest possible
complexes are formed = maximum ppt >> no Ag or Ab in
supernatant that's called the Zone of equivalence .
then as the [Ag] increases above the [Ab] = Ag excess , no
large complexes are formed , just few small complexes>>
(sub-optimal ppt.; free Ag in supernatant .) ,,
finally as the Ag increases extremely (Far Ag excess) >> (no
ppt. formed; free Ag in supernatant) -- “postzone”
It's important to mention that some times when we run an Ag-Ab reaction , the [Ab] for example is extremely
large & that result in –ve reaction ( this happens when we expect individual with certain disease that produce
certain amounts of Ab , all manifestations says that he suffers of that disease although the reaction is –ve ) so the
concentrations in this case required dilution .
>> If antigens and antibodies are mixed and allowed to interact then measurements of either the antigens or antibodies are
to be made to detect the amount of what we looked for ,,, just a drop of serum + a drop of Ab & that lead to the detection of
the presence of the reactant weather we look for Ag or Ab .
always we have a known & look for unknown( e.g we have Ag so we look for Ab ,, we have Ab so we look for Ag ).
serial dilutions of one of the reactants should be made depending on whether it is the antigen or the antibody that is being
looked for.
If the antibody is the one being looked for, a known antigen is used and diluted. If an antigen is the one being looked for, a
known antibody should be used and diluted.
>>Test tubes containing variable amounts of the known or unknown (whether antigen or antibody) reactant are prepared.
For example, if antibodies are to be detected in serum, 0.9 ml of saline and 0.1 ml of antibodies in serum should be added;
this will make the ratio 1:10 in a constant volume of 1 ml , if we mix this well and transfer .1 ml from this tube to the 2nd
one
and another .1 ml were transferred to the 3rd
,,,, the first tube will have a ratio of 1:10, the second tube (since it’s been
diluted twice) will have a ratio of 1:20, the ratio of the third tube will be 1:40 & after all the mixing and transferring the
fourth tube’s ratio will be 1:80….1:160 & so on... This is called serial dilutions.
This dilution can be expressed as one tenth, one hundredth…etc. these are simple calculations made according to the
concentrations of the antibody.
>>So a serial dilution is prepared of one of the reactant either antibodies or antigens are looked for.
Then a constant/ same amount of the second reactant is added, (It was demonstrated that reactions take place at
concentrations that are optimal, in which the ratio of the reactants is optimal at 1.1 to 1; almost 1.1 antigens to 1
antibody.) >>the reaction will be in the form of PPt or agglutinates .
>>the reaction were +ve in the 1st
4 tubes & -ve in the rest .
>> the serum was diluted 160 times and stills give +ve reaction that is of course a measure of the[ Ab] in serum , which called
titer , so titer represent the highest dilution that gives a +ve reaction >> so the titer is 160
Therefore, the titer is either the highest
dilution that gives a positive reaction or
the reciprocal of the dilution that gives a
positive reaction.
Methods that detect Ag-Ab reactions
Primary reactions may be in the early phases of reaction or it may be the end result of the reaction
because the Ag are small & they will never make large complexes , here we cannot make calculations &
serum dilution ..etc.
the reaction is invisible so we need to label them to detect the small complexes .
** the substances that used to label the reactions :
-Immunofluorescence (IF)
substances that have a characteristic of absorbing UV light and emitting it in a visible light so the use of
UV light (280 ) and exposing it to substance , the substance will absorbed UV light become excited and
emit the light to a visible range ,,, special microscopes are used to detect the immunofluoresoence that
labels the primary immune response ,,, the color called be green like in the case of Florisene or it could
be red for example like rodamain , so the color that’s visible depending on the wave length and the use
of the substance is just to make the reaction visible
-Radioimmunoassay (RIA)
Substances that emit gamma or beta rays and we can use hydrogen, C14 , I125 , phosphorus 132 ,,,, etc
many radioisotopes can be used and here the labeling of the Ag or the Ab , we can labeled either and
look for the 2nd ,,,,so complexes can be detected by counting radioactivity in the reaction and
radioactivity can be counted using special scanter gamma or beta counter depending on the isotopes
that’s used and this method is so sensitive that can detect picograms and nanograms of substances
-Enzyme immunoassay (EIA)
use enzymes to label Ag or Ab instead of IFL or RID ,,, like peroxides and the substrates is hydrogen
peroxide ,,, different enzymes can be used ,, and we detect the enzyme by the using of substrate but
substrates alone will not make the reaction visible so we add pH indicator ; a substance that changes in
the color when pH changes ,, so +ve reaction can give a color change ,,, so we can judge whether it is +ve
or –ve reaction but if we want a quantitative a mount we measure the intensity of the color and in all of
these we should use standard of known concentrations to construct a curve and then from the curve we
can determine the concentration of a substance .
-Immunonephelometry
is a method that depends on the detection of light scattered ,,, primary immune complexes that formed
in the medium don’t precipitate because these are small in size so to detect these complexes which
present in the spnt we expose the medium to light and light will pass through this medium and will face
in counter these primary complexes , when it is in counter primary complexes , light will scattered and it
will scattered in all direction so we can measure light scattered at a certain angle for example at an
angle of 90 ° and was found that the amount of light scattered at a certain angle is proportional to the
concentration of the complexes so we can make complexes with standard with known concentrations
and then we apply the reaction on unknown , and from the amount that scattered detect the
concentration of these substances so primary immune complexes which cannot be visible , cannot be
seen by naked eye can be detected by the use of labels
(measures picogram to nanogram quantities of analyte) this is for very small complexes (10 -9 to 10-12)
Secondary Reactions that form when we have large multivalent Ag with IgG or IgM Ab , these
complexes will form ppt or agglutinate depending on the solubility of the reactant .
if both reactants (antigen & antibody) are soluble, the product will be >> precipitate so the reaction is
called precipitation.
But if one of the reactants is insoluble (usually the antigen is insoluble however the antibody can be
made insoluble by attaching it to a particle like RBC, latex, charcoal ) . In such case, the reaction will be
in the form of >> clumps or agglutination .
so the secondary reactions are measured by agglutination and precipitation.
latex and charcoal are used to increase the sensitivity of reaction , if we have a soluble Ag it will form
ppt , ppt as a reaction is less sensitive than the agglutination so if we make the substance insoluble by
attaching to latex and charcoal >> the reaction will be more sensitive , may be agglutinate ,,,, if we use
RBC to react with Ab the reaction is called hem-agglutination, this can be used in typing of blood or we
can attach an Ag to RBC , using them as a carrier of an Ag >> determine the positivity of a reaction ,,,,
large immune complexes that formed will be either agglutinate or ppt .
we can measure the amount of this reactant by doing serial dilution of the serum , this will detect the
titer of Ab.
o Agglutination Techniques
o Precipitation Techniques ± Electrophoresis
Let’s start with precipitation as the first form of Ag-Ab
reactions.
It takes place when soluble antigens are mixed with soluble
antibodies.
Precipitation can take place in capillary tubes, test tubes, and
in gel
Capillary tubes have one application for ppt ; that measures .
antibodies produced against bacillus anthracis, the causative
agent of anthrax الجمرة الخبيثة. if serum of individual of anthrax
is mixed with anthrax Ag a ring of ppt will formed at the zone of equivalence ( visible large
complexes ) ,,,, this test is called the syphilis Test to detect the anthrax Ab and this take place at
capillary tube
The most important applications of precipitations are performed in gel as it has more than one form.
Precipitation in gel
o Double diffusion
o Single (radial) diffusion
o Combination of diffusion in gel and electrophoresis
double diffusion or the ouchterlony technique
Gel is made from agar which is from the cell walls of sea plants , has no intrinsic reactivity and there
are no reactants present in it.
it is only used as a medium in which 2 wells are cut in the gel, one is used to place the antigen inside
& the other is used to place the antibody .
agarous usually used as a medium to perform precipitations because it allows the diffusion of Ab or
Ag in agar .
now if we take agar plate we can cut wells in the agar and put Ab in one and the Ag in the other ,
they will move in all direction & toward each others that will meet at the zone of equivalence and
that allows the formation of immune complexes that can be seen in the agar , agar will allow the
visibility of these reactions , . This is known as double diffusion or the ouchterlony technique.
double diffusion
Gel is inert (contains no reactants)
Both Ag and Ab travel through the
medium
The gel contains nothing! One well is used
to add antibodies whereas the other is used
to add the antigen; both migrate to meet
each other. In this case a line of
precipitation is formed between the antigen
and the antibody at the optimal zone of
equivalence.
Ouchterlony Plates Precipitin Patterns
Ag & Ab placed in wells cut into an agarose gel (both reactants diffuse)
Precipitin line (or arc) indicates Ab has specificity for Ag
Position of precipitin between wells depends on MW and concentration of reactants
3 possible patterns of reaction: identity, non-
identity, partial identity
If the antibody is known and it’s required to
determine whether the antigen is present in a
certain fluid, the fluid is just added so that double
diffusion is allowed to proceed. If a line of
precipitation is found then the antigen really is
present. This is the first application; to determine
whether an antigen is present in biological fluid via
using a known antibody.
The second application is to determine the number
of antigens than can be used. Biological fluids can contain more than one antigen; their numbers
can be determined.
Also, the relationship between antigens can be determined.
If a certain antigen is known to be present and its
respective antibody is known whereas the
relationship between this antigen and another
unknown antigen is to be established/studied,
both the antigen and its antibody are placed;
- if lines of precipitates form and cross >> non-
identity meaning that the antigens are not related/
non-identical.
- sometime the lines fuse together with the
presence of an extra spur for an antigen>> partial
identity; the two antigens share some antigenic determinants but one of them has extra antigenic
determinants that present in the form of a “spur”.
- If the lines fuse completely >> identical; this is referred to as a line of identity.
Therefore, by double diffusion, the presence of an antigen and its relationship with other antigens
can be studied.
OUCHTERLONY DOUBLE DIFFUSION
This is a practical case in which
multiple/ six antigens are compared
to each other.
Here, some antigens are
identical(1+2 ),
(3+4 ) have one identical antigene
(5+6) have no related ag .
Single (radial) diffusion
is when one of the reactants is fixed in the gel (usually the antibody is fixed in gel).
. It has the characteristic of being fluid at high temperatures and solidifying at 45⁰.
45⁰ does not influence antibodies so the gel is heated until it becomes liquid then the antibodies are
added and mixed very well then it is allowed to cool down so that it becomes solid at a temperature
of 45⁰.
In such a case, the antibody is evenly mixed in the gel; the concentration of the antibody is the same
or in other words, it is homogenous within the gel. If a well is cut in the gel and the antigen is added,
it will diffuse in all directions and it will make a ring of precipitation at the optimal zone. Precipitates
are formed as the antigen migrates in the gel. Lines of precipitates form but they disappear/dissolve
because the reaction is reversible until they reach the optimal zone of equivalence and make large
complexes in the form of a ring with certain diameter , was found that the diameter is proportional
in some way to the concentration of the antigen
both techniques, whether single or double diffusion, can be combined with electrophoresis to
enhance the migration of the antigen. Therefore, electrophoresis is utilized to speed up the reaction
only.
The reaction requires 48-72 hrs so instead of waiting , we can have the result in 2-3 hrs by performing of
the electrophoresis .
Single diffusion
Ab are mixed and fixed in the agar ,,, wells for the Ag , Ag will diffused in all direction forming a ring of ppt
o Supporting medium (gel) contains one reactant at a uniform concentration
o Only the unknowns move through the medium ( we can measuring the concentration of that
reactant by measuring the diameter of the ring
>> Antibodies are mixed with agar gel; a well is used to add the antigen. The antigen will migrate in
all directions and it usually forms a ring of precipitation at the optimal zone where the reactants are
present in optimal concentrations.
>> the concentrations of different reactant can be made and there are 2 methods if we allow the
reaction to complete ,,,, the diameter square of the ring is proportional l to the looked Ag concentration
that’s why use similar paper to determine the amount of substance rather than graph paper in gel
>> If it’s the single radial immune-diffusion, the antibody is mixed with the gel and the antigen is added
in serum. In this case, the gel has anti-IgG (if it is the IgG that is to be measured). IgG will spread and
wherever it meets the anti-IgG it will form a ring of precipitation which dissolves until the concentration
of the reactants reach their optimum concentrations in the zone of equivalence. Here, the diameter of
the ring is measured. If the reaction is allowed to be completed, one can infer that the antigen is directly
proportional to the diameter (of the ring of precipitation) squared. By this way, the concentration of the
antigen can be measured.
Ab uniformly distributed in gel; Ag diffuses outward from a well (single diffusion)
Ag-Ab complexes form as concentric rings around the well at zone of equivalence
At a set time, ring diameters are measured
[Ag] is directly proportional to the ring d2
Unknown value is determined by comparing to a 3-standard curve
The gel usually has the capacity to hold 12 wells if not more. So in addition to the standard wells, 9
additional wells can be made and are used to add the unknowns; or the antibodies that are to be
measured. So it is 3 wells for the standards (known concentration of Ag , 9 wells for measure
concentration of unknowns.
Three standards are used with 3 different concentrations; low, medium, & high.
Then the diameter is measure, squared and plotted so that the concentration can be measured by
constructing a standard curve. So a standard curve is constructed first from which the
concentration of the unknown reactant can be determined.
After measuring the diameter of the precipitation ring, the diameter is squared and plotted versus
the concentration (which is known already as these are standards). The result is a straight line that
does not pass through the origin (point (0, 0)) because of the diameter of the well itself.
>> assume that the diameter is doubles so it\s more than the largest standard in this case we should
do dilution ( as the dilution increase the diameter will decrease ) & then multiple by the factor of
the dilution
Standard Curve
Precipitin RingsA B C a b c
Standards Samples
With respect to the standards,
-The diameter is measured
-The concentration is already
known for the standards.
RADIAL IMMUNODIFFUSION can be performed in two forms:
1- Fahey method (kinetic). It does not allow the reaction to be completed; it is ended at 18 hrs.
Completion of the reaction may require 48-80 hours. For IgM is 80 hours. For IgG it is 48 hours.
Read at 18 hours
Plot [std] vs. ring diameter on semi-log paper
the ratio will not be related to the diameter squared. The diameter is proportional with the log of the
concentration so semi-log paper is used since the relationship is between the diameter and log of the
concentration.
2-Mancini method (endpoint). This is the one shown in previous diagram. In this case, the reaction is
allowed to proceed to completion. The relationship is between the diameter squared and the
concentration of the antigen.
Read at 48 or 72 hours
Plot [std] vs. ring diameter squared on graph paper
>>> can be used to measure all immunoglobulin except IgE used for many reactions as in the
measurement of antibody concentrations, complement components, transferrin, ceruplasmin, &
different proteins can be measured by this technique.
Results reliable only if the ring size is within the range of the standards; if greater than highest std,
dilute and repeat test. If the diameter of an unknown is larger than that of the largest standard, the
sample should be diluted and the test repeated as the results will be unreliable.
Used to measure IgM, IgG, C4, C3, transferrin, CRP, other proteins.
Fahey Method
Electroimmunoassay (rocket)
Electrophoresis hastens movement of Ag (placed in wells) through Ab -imbedded gel
(single diffusion) :
When the single immnodiffusion is combined with electrophoresis, the technique is called
Electroimmunoassay , or the rocket technique because precipitation lines that are produced
assume the shape of rockets
So, similar to radial immnodiffusion , agar is mixed with an antibody , so the antibody is
incorporated in the agar , wells are cut and then antigens are added , that are allowed to
migrate , migration here is speeded up by electrophoresis and instead of forming circle / radial
of preciptations ,they form rocket techniques .
Selected pH (8.6) keeps Abs at their isoelectric point; they will not move :
It is very important to mention that proteins are negatively changed and they have an isoelectric
point which is 8.6 , at which their net charge is zero which allows them to move with
electrophoresis .
Rocket-shaped precipitin bands will form at zone of equivalence (changes as reactants
move:
And the shapes that are produced are shown here in the slide, so the antigen migrates in the
agar where antibody is homogenously evenly mixed , and the results are rockets of
electrophoresis .
These rocket can be stained by a stain called coomassie blue to visualize the rocket.
[Ag] proportional to length of rocket:
It was found “roughly speaking” that the concentration of the antigen is proportional to the height
of the rocket but more accurately , it is proportional to the area under the rocket,
– So the antigen concentration is proportional either to the height of rocket or the area under
the rocket. And by constructing a standard curve , using known concentration of the antigens
, we can determine the concentration of unknown reactant by just blotting the height of the
rocket against the standard curve.
May be used to quantitate plasma proteins such as coagulation factors, alpha-fetoprotein, C3,
C4, CRP, haptoglobin
This technique is more sensitive , faster than the radial immunodiffusion and can be
used to measure the same reactants like abs, complement components .etc, so sensitivity is
similar . compared with RID , it requires electrophoretic equipment and more technological
finesse
this technique has been Largely replaced by immunonephelometry.