Antigen Antibody Reactions - JUdoctors 12, 2013 · Antigen – Antibody Reactions ... -Far Ag excess (no ppt. formed; free Ag in supernatant) ... -Immunofluorescence (IF) -12)Published

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.