immuno . lec 4

7/31/2019 Immuno . Lec 4

http://slidepdf.com/reader/full/immuno-lec-4 1/14

Wednesda 6/ /2011

Ziad Al-Nasser

Emad Al-Gazo

Antigen antibody reactions -

Immunoglobulin

2

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Immunoglobulins

*Represents 20 % of our plasma proteins that prevent us against infectious agents

(pathogens).

*They come from B cells when they change to plasma cells.

*They are detected by analytic techniques such as electrophoresis in the (gamma

globulin) fraction of serum ((from book)) see figure 4.3.

*The specificity of Igs depends on B-cell receptor structure and the protein

composition of the Ig itself ((the gamma wave of the protein electrophoresis and

within this gamma wave we see sub-waves of different Igs)).

IgG:

*It is the most common Ig in our body that is produced in most of infections related to

secondary immune response.

*Has 4 subtypes IgG1 IgG2 IgG3 IgG4.

*Has the longest half life and the only antibody that crosses the placenta.

*It is the main neutralizing antibody and antitoxin.

IgM:

*It is first antibody to be produced; it is the one that presents on B-cell surface as a

monomer. Now when it comes out it comes as a pentamer.

*Has a violencey of 10 in order to bind 10 antigenic determinants, we need that in the

area of infections and this is so important in newborns to protect them against

pathogens .

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*IgM can’t pass placenta so the baby develop them by himself.

IgA:

*it is the Ig of the mucous membranes so we see it in the respiratory tract and GIT and

urogenital tract so it acts as a 1st

line of defense.

*it is a dimer 2 monomers adherent to each other and we have 2 types (IgA1) (IgA2).

IgD:

*it is essential component of B-cell receptor so it is important to IgM function on the

surface and it is an indicator of maturity of B-cells and has a role in the stability of B-

cells and rarely we see it free ,usually on the surface .

IgE:

*it is the antibody of inflammation it is the one that responsible for type 1

hypersensitivity reactions and what we call it anaphylactic shock.

*we see it in response of parasitic type of infections.

*IgE and eosinophils are highly related to each other.

*hapten: simple chemical compounds can’t -by itself- induce antibody response so it has

be to coupled to a macromolecule (eg: protein carrier) and this protein carrier has a

role in activating the T-helper cells and the cytokines that are needed to stimulate the

B-cells then antibodies can be generated .

*immunogen: that a substance that can (always) produce antibodies by itself and cause

immune response so it has to be: 1.forgien body to host.

2. Fairly large, MW must be more than 6000.

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3. Chemically complex.

Now in Ig model in the pic above we have 4 chains 2 heavy and 2 light chains ,,the 2

heavy in which the substance of it are the determinant of the type of Ig so when I say (

IgG ANTIBODY) then the heavy chain here we call it of the gamma type ,,we have 4

types of IgG (1,2,3,4) and we have 4 types of heavy chains( gamma 1)(2)(3)(4),,,, and the

substance of heavy chains we call them the isotypes that form the classes and

subclasses of Igs. and the same for IgA we have IgA1 and IgA2 SO the heavy chains

are either alpha1 or alpha2,,,, and IgM the heavy chain is MUO ,,, IgE the heavy chain

is epsilon and for IgD it is delta .and all these heavy chains are coded by chromosome

num 14 .

Now if we look at the heavy chain we see what we call it the variable region and the

constant region,, here the variable means the amino acids variation so they differ from

Ig to the other of the same type while the constant region all are the same in same

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species. so isotypes of human they are the same but they differ from isotypes of mice

,,,so if u inject Igs of mice in human immune response will happen but nothing will

happen from human to human .

Now in the variable region of both heavy and light chains of Igs we have the hyper -

variable region and this is the most specific part of Ig so u can imagine it as tees of key

that is going to enter the lock which we call it the complementary region.

On the other side we have the light chain (2 types kappa and lambda) ,,now in the Ig

ethier u have kappa or lambda so u can’t have kappa & lambda okay.

Now the f igure in page 3 is an animation to the structure of Ig doesn’t represent the

real shape ,,which looks global in ( 3D), And have a domains which they areinterconnected to each other by disulfide bonds so these bonds link the domains

between heavy and heavy and light and heavy chains .

Now the region that we talked about it so far is called fab region which is the region

where the antigen binds ,, and in the middle we have the hinge region which allows

flexibility ,,and in the lower we have the fc region that binds cellular receptors and the

complement ,,play a major role in the biological function of Ig.

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(The pic above shows (3D) shape of Ig )

In the heavy chain of Igs the constant part is divided to 3 parts in most Igs and 4 in IgM

and IgE constant 1 ,2,3 from upper to lower in the figure in page 4 of the lecture. Now

the CH2 (CONSTANT HEAVY 2) Is the area where complement is going to bind and will

be fixed so it plays a role in complement fixation in the heavy chain and also u can see in

the CH2 the carbohydrates are there ,,,so Igs they are glycoproteins =carbohydrates

+proteins,,, so if u inject those into animals, they are going to develop antibodies against

them,,, and if u inject them into human then antibodies could developed against the

variable part of heavy chain protein ,,, Now u have to know we call the heavy chain

proteins isotypes and the variable region proteins allotypes and the hyper-variable

region proteins adiotypes .

Now our body will develop antibodies against the adiotypes we call them anti-adiotypes(Igs against Igs), those anti-adiotypic molecules, our body use them as messenger for

interaction between cells, exactly our body use them as cytokines for activation or

suppression of cells.

Back to fc portion,, u have to know that on the lymphocytes we have receptors for fc

portion of Ig so fc portion will bind on these receptors of the B-cells and T-cells or even

the NK cells ,,, this important to NK cells function ,,so tumor cells or infected cells bind

to fab region and the NK cells will come and bind to fc region by fc receptors,, those fc

receptors are not specific ,,so NK cells also not specific they are servalent cells ,,so they

will bind and become activated and produce the cytolysins and interferons that will kill

the virally infected cells and the tumor cells we call that APCC (ANTIBODY DEPENDANT

CELLULAR CYTOTOXISTY ).

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Now the Dr. showed this slide and started to talk:

You can see here the Igs (M,D,G,E,A) the heavy chain symbol for IgM is ( mu) this is the

isotype the mean serum concentration = 0.4-2.5,,, we can compare it with the highestconcentration of IgG which is from 7 to 18,,, but the IgE concentration is less than

0.0005 this is the lowest concentration (important) ,,and u have to know the heaviest

one is IgM and it is the most efficient in activating the complement by opsenization then

phagocytosis take place ,,,now the IgE: the mast cells and basophils have a receptors for

IgE and this is important for its function and anaphylactic. About IgA: that is important

in mucosal immunity, and about IgG is the only one that can pass the placenta and

longest half life so it is the Ig that allow maternal protection of newborns.

Then the doc showed the pic that in the next page which is about the effect of enzymes

on Igs for example: IgG the most stable one.

If we add pepsin enzyme then the fab region will appear as f(ab’)2 because still there is

a disulfide bond but the pepsin enzyme will degrade the fc region(the biological region )

into fragments . Now if we add the papain enzyme then it will degrade the Ig into 3

parts; 2 parts of fab and one fc ,,the fab one can bind antigen (fragment antigen –binding

), the fc doesn’t bind the antigen but activate the complement and do the biologicaleffector function .

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Now in the figure (4.9 in the book) u can see the light chain has 1 constant domain and

one variable, and the heavy chain has 1 variable domain and 3 or more constant

domains.

Now in the figure (the next page) u can see a summary of the functions of Igs:

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IgM: pentamer 5 monomers they are connected by j (joining) chains so it will have the

higher MW 900,000.

FUNCTION: elevated in the primary response, and activating the complement because it

has 10 potential antigen binding sites.

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Now IgD: it is chiefly found on the surface of B-cell receptor molecule and involved in

cell activation and has a role in maturity of B-cells.

Now IgG: is the main neutralizing antibody, look here for example at IgG in the 3rd

box u

can see the virus each angle of this virus act as ligand (protein binding ) which binds to

the receptor on cell surface so our body will produce IgG that block the function of

ligand (neutralize the virus ) ,,so u have to remember IgG it has the longest half life (3

weeks) so it is most prevalent in the serum ,,, and remember that it is the one that cross

the placenta ,, we need 2 IgG to fix the complement while 1 of IgM is enough because it

is a pentamer.

Now IgE: it is responsible for inflammation and anaphylactic shock ,,it binds to the mast

cells and basophils by fc region not fab,, so when fab bind to the allergen then the mast

cell will be granulated and produce mediators like histamine ,,, so u have to know thatIgE is responsible for hypersensitivity reaction ,,,it is important in clinic to avoid these

hypersensitivity reactions by taking a good history from the patient ,,, but sometimes

we can’t predict the allergy example: a patient sensitive to epinephrine ,,,or

hydrocortisone and these are rare cases u cant predicts it.

Now IgA : 2 subtypes A1 AND A2 ,,, it is a dimer 2 monomers that are connected by

(J CHAINS) ,, and this Ig normally in the serum ,,now when infection happen in the

mucous membranes like in the gut ,,then the Ig transferred to the gut by a process

resembles the endocytosis by binding to the S protein receptors, then it will pass to the

site of infection, now the S protein will protect the IgA from microbial enzymes.

Now the Dr. showed slide 23 and said :

U have to know what( fab ,fc ,f(ab’)2 ,VL,VH ,CL,CH ) MEAN ,,,,,

The domains: they are segments of Igs.

The Ig supergene family: similar folded polypeptide domain.

Now slide 24:

The opsonization : is the process that facilitate the phagocytosis by binding Ig with

microbe ,,we call this microbe opsonic ,,and the complement activation also help in

phagocytosis ,,and remember the complement is fixed on (CH2) .

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,,,,,SO FAR WE END CHAPTER 4 ,,,,

((NOW LET’S GO TO GHAPTER 5: ANTIBODY ANTIGEN INTERACTION))

Antibody antigen interactions they are similar to those between enzyme and its

substrate, u have to know there are 4 forces contribute in this interaction:

1. Electrostatic forces.

2. Hydrogen bonds.

3. Van der walls forces.

4. Hydrophobic interaction.

All of these forces are non covalent bonds (weak bonds) but as a sum it will act strongly.

Now we have different types of antigens, the variety is infinite and we have different

binding sites at the Ig. Now the strength of anti-body antigen interaction we call it the

affinity, and the accumulative antigen antibody interactions we call it additive, these are

important two terms as the Dr. said.

Look at figure in the next page:

You have to know that we have different structures and shapes of antigens:

1. Simple antigen.

2. Larger epitope.

3. Complex antigen.

And we have different binding sites of Igs to bind these antigens. So the simple antigen

(like: Vitamin k) will bind to binding site which is crevice (pocket), and the larger epitope

(like: linear peptide epitope) this will bind with a groove binding site, and complex

antigen like: folded protein that present in lysosome this will bind to extended surface.

Antibodies importance:

1. protection our cells from the antigen by neutrolizing them and preventing them

from binding to their receptors.

2. for diagnostic tests: meuserment of the antibodies concentration indicate the

infection by the pathogen for exampl: if u infected with hepatitis B virus then the

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antihepatitis antibodies concentration will high (high titer) so this indicat the

infection, u have to know that the concentration must be more than normal by 4

foldes at least.

** The Cross Reactivity**Or we call it multi –specificity, this means that antibody binds to more than one antigen,

here the antibody will bind to antegin that isn’t responsible for its production.

Now plz refer to the box 5.1 in the book page 27, it is important.

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Now if u remember (group A Beta Hemolytic Strep) that cause tonsillitis then it will

cause rheumatic fever or rheumatic heart disease and that happened by cross reactivity

of protein M that present in the bacteria and our tissue and this will cause autoimmune

disease. quantity

Now refer to the diagnostic tests, in the medical microbiology lab there is a section we

call it immunology or serology department, in this department simply they do antibody

antigen interactions, so if the antigen is known then I will know the antigen and vice

versa because they are matching, the problem sometimes is the cross reactivity then the

test will be false positive :/.

In these tests u have to know some terms like specificity and sensitivity, 100% specificity

then no false positive results, sensitivity: this means that any small amounts of Igs

should be detected .

Now in the serology tests u can determine quantity (how much positive results there)

(the titer) and quality (positive or negative) of the tests.

The 1st

tests done in the past was the agglutination and precipitation tests, u are familiar

with agglutination in experiments of blood grouping, now the precipitation: this happen

when the antigen and the antibody are part of a solution so they will bind together and

form what we call it latex (network-like ) and if u draw this as curve it will looks like a

bell shape ,,this means that the maximum amount of immune complexes in the middle

and these immune complexes will precipitate and this happen better in the optimal

concentration, this optimal means that the proportion of antibody to antigen nearly 1:1

so no excess antibodies or antigens .

***ELISA test:

Enzyme linked Immunosorbent Assay:

The most common test in the lab through the measure of antibody titer. Here in the

experiment we know the antigen but the antibody is unknown.

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We add the antigen to the plate then antigen will bind to plastic plate but the excess is

washed out then we add the serum patient (antibody) then the plate is washed out for

excess antibodies, if the antibody in the serum then it will bind to antigen then we add

the secondary Igs (anti human Igs) we get it from the horse or the goat then we tag

them by enzyme (horseradish peroxidase) so this will be like the enzyme and substrate

complex so this complex is colored then the result of the test is positive.

To Make It Clear Look To The Diagram

“”Add the antigen→ Add the patient’s serum and washed→ If they matched they will

stay→ Add the secondary antibody with the enzyme→ It will consider the primary

antibody as antigen an interact with→ Washed them out they will stay if they

matched→ Add the substrate→ Interact with the enzyme→ Color product→ Positive

test “” Correction Team.

THE END

Finally I would to thank my friend and my cusion (alaa al kofahi ) for helping me in

writing this Lec ma gasart abn 5alti ,, and sorry for mistakes ,,and gd luck all ,,,,,,,,,,,,

))

))

DONE BY: EMAD AL GHAZO

immuno . lec 4

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