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Antibody-Antigen Interaction

Sara Al-Zu'bi

Ziad Al-NasserThursday, 7/7/2011

5

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The Dr. started the lecture by talking about the attendance and seat numbers, I think all of us knowevery thing regarding that :D

Dont look how many pages this lecture is, you will think its long and boring before reading it!! Just lookat the info in it and get the maximum benefit and good luck

we started talking about antigens, antibodies and antigen-antibody

interaction, we talked in details about Immunoglobulins and the structure of the antibody;

remember the specific parts, the Fab region (binds to the antigen), Fc portion (binds to the cells or

complement), Hypervariable region, we talked also about antigens and how they bind to the

antibodies, remember the forces that keeps antigens and antibodies together (these forces are

electrical or non-covalent).

we talked about the benefit of the antigen-antibody interaction to clear out infections; neutralize

viruses, binding to ligands; attachment proteins preventing those from binding to receptors &

also we said that we can use this interaction for diagnostic purposes; if we see antibodies, this is

an indication that we have been invaded by antigens so if you determine the antibody type, then

you can by default determine the antigen type that has induced the production of this antibody.

This -the substance that induce the production of antibodies by itself- is called immunogen.

We also talked about cross-reacting antibodies: antibodies that react with antigens that are not

responsible for their production, remember the significance of that in explaining many

autoimmune diseases, drug allergy and giving you false positive results in serology.

And dont forget the significance of the serological tests; to have 100% specificity which means

zero false positive, 100% sensitivity means zero false negative and not to have any problem.

We talked also about quantity and quality >> quality: the test is either positive or negative,

quantity: measure the titer

Routinely, we do a lot of diagnostic tests in the lab in the section of serology (or clinical

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We also have a test called adsorption inhibition test as well lots of applications related to that. (The Dr said

this sentence without any explanation just like that)

one of the most common tests that we do in our serology lab and over the world of course where

simply we are using tagged secondary immunoglobulins, tagged here is an enzyme which is

horseradish peroxidase enzyme), and you detect the enzyme activity;

Enzyme + substrate enzyme + products;

** If the products are colored then its a positive test.

** The more the intense the color the more antibodies or reaction you are going to have.

look at the picture and follow:

The antigen is known and adherent on the

bottom of the plastic plate.

We add the patients serum; if it has the

antibody that we are looking for; it is going to

bind to the antigen.

Incubate and wash; incubate: collect the

antibodies bind to the antigens and wash any

excess of the antibody.

Add secondary Immunoglobulins that are tagged with horseradish peroxidase enzyme.

Incubate and wash

add the substrate so it will produce enzyme-substrate complex, it will gives you back the

enzyme & products, products are colored and sometimes you could add something to color the

products so,

- the color that develops means positive test that means the patient has an antibodies against

the antigens,

- When no color is appeared, no enzyme activity, the antibodies dont match the antigens so

they will be washed out so it will be negative.

So, to sum up:

** Agglutination inhibition test >> agglutination = negative test, inhibition of agglutination = positive test.

** In case of pregnancy (positive test):

HCGH in urine antibodies interact with it no free antibodies no agglutination when latex (HCG) added positive

** In case of NO pregnancy (negative test):

NO HCGH in urineNO antigen-antibody interaction free antibodies agglutination when latex (HCG) added negative

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** we do positive control as well at the same time here to be sure, so ELISA has much higher specificity and

sensitivity compared to agglutination, precipitation or even the complement fixation test, sensitive means that you

can detect very small amount of the antigen, so the chance of giving false negative will be very very low.

Here, the substance that could be tagged is an immunofluorescent material and we use asubstance called fluorscein isothiocyanate (FITC),,, if you hit this substance with the source of UV

light, it will go into a higher state of energy and then it will lose this energy in the form of light, so

it emits light. So,

- If you see light (means that it fluoresces) = positive test.

- Iftheres no light = negative test.

In this test, we could have 2 variants: direct & indirect.

Where we detect the antigen and the antibody is known and we label the antibody with an

immunofluorescent material so you detect the antigen; we detect tissue antigens

We have a slide and it contains an antigen that is unknown, I want to know what the nature of

the antigen is, if Im suspecting, for example, x antigen so I buy an antibody specific for that

antigen (x antigen).

So, to sum up:

** ELISA test >> color = positive test, no color = negative test.

** In case of positive test:

Antigen is known antibodies interact with it when wash, the antibodies still present secondary antibodies added

theyll interact with the primary onewhen wash, the complex wont be washed add substrate enzyme activity

colored products.

** In case of negative test:

Antigen is known

no antigen-antibody interaction

when wash, the antibody will be washed out

secondaryantibodies added no interaction with the primary onewhen wash, secondary antibodies also will be washed out

add substrate no enzyme activity no products or color.

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Add the antibodies that are fluorescenated with the fluorescein isothiocyanate and these

antibodies are known.

Incubate and wash.

Put it under the fluorescent microscope.

- if the antigen matches the antibodies, so it want to bind & when you put it under the UV light

of the fluorescent microscope, the fluorescent material will fluoresces and emits light so this is

the positive test.

- Ifthe antigen doesnt match the antibody, it wont bind so it will be washed out so when we

put it under the UV light, no fluorescent material and no light development,,, negative test.

-

- Used to detect the antibodies so the antigens have to be known and its exactly like the ELISA test,but the difference is: instead of using an enzyme on the secondary antibodies, we use the

fluorescein isothiocyanate.

-

-

The difference between the light and fluorescent

microscopes is the light that is used, UV in the

fluorescent and regular light in the light)

So, to sum up:

** Direct immunofluorescent test >> light = positive test, no light = negative test.

** In case of positive test:

Antigen unknownmatching between antibody & antigen fluorescenated antibodies interact with it exposure to UV

light emission of light positive

** In case of negative test:

Antigen unknown no matching between antibody & antigen no antigen-antibody interaction exposure to UV light

no emission of light negative

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- We have the antigen is known on the slide.

- Add the patients serum that contains antibodies which are unknown.

- Incubate and wash so if it has attached it will stay.

- Then we add a secondary antibody with the fluorescenated material on the top.

- incubate and wash- Put it under the fluorescent microscope.

-

- - If the antigen matches the antibodies, then they will bind and when we wash, they wont be

washed so the immunofluorescent material will fluoresces. This is a positive test, that means that

the antibodies that you are looking for is matching that of the antigen.

- - if the antigen doesnt match the antibodies so there will be no binding and the antibodies will be

washed out and theres no immunofluorescent material so no light,,, this is a negative test.

*** for example, if Im looking for anti-rubella antibodies, here on the slide put rubella antigen

then anti-rubella antibodies are positive.

*** The indirect immunofluorescence is more sensitive (means that its ability to detect smaller

amounts of the antigens or antibodies is better), because what you need here is just one antibody

to bind to one antigenic determinant. Also, the intensity of light is stronger and you could have so

many other antigens that the fluorescenated antibody could bind to, they could bind to the

isotypes, allotypes and so on.

So, to sum up:

** Indirect immunofluorescent test >> light = positive test, no light = negative test.

** In case of positive test:

Antigen is known antigen & antibodies match each other primary antibodies interact with it when wash, the

antibodies still present secondary antibodies addedtheyll interact with the primary onewhen wash, the complex

wont be washed exposure to UV light emission of light positive test.

** In case of negative test:

Antigen is known no matching between the antigen and antibody no antigen-antibody interaction when wash,the antibody will be washed out secondary antibodies added no interaction with the primary onewhen wash,

secondary antibodies also will be washed out exposure to UV light no light.

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We can use the immunofluorescence for the confirmation of syphilis; the causative

agent is: treponema pallidum, the test called: fluorescent treponema antibody

absorption test.

In the routine screening for syphilis, we use an antigen called cardiolipin and the

antibodies react with cardiolipin [what cardiolipin has to do with T.pallidum is like a

cross reaction in those patients, when they are infected with syphilis organisms,

they developed immunoglobulins that react with cardiolipin]

BUT,,, this test is not that specific so we need to confirm the test BY fluorescent

treponema antibody absorption test.

SO, we get the patients serum, we add some treponemes other than T.pallidumto

absorb other antibodies that are not against T.pallidum, so the patients serum will

be left only with antibodies supposed to react with the T.pallidum, so we get

T.pallidumorganisms fixed into a slide and then we put the patients serum on the

top then incubate & wash then add a secondary antibodies with the fluorescenated

material then incubate & wash then we put it under the fluorescent microscope so,

- If the patient is positive (have syphilis), then the primary antibodies (in the serum)

are going to bind to the T.pallidum organisms(its shape like the spring ( )) and

the fluorescenated antibodies bind to the primary antibodies so it will fluoresces.

This is a positive test for syphilis that means that the patient is having syphilis.

So, this is a confirmatory test

** We can do other tests here; like that >> you have syphilis antigen of T.pallidum

adherent to RBCs for example and you can do microheme agglutination and so on.

- Student: does the secondary antibody consider the primary antibody

as an antigen?

- dr.: yes, the secondary antibody considers the primary antibody as an

antigen and it will bind into those antigenic determinants that we have

talked about; isotypes or allotypes or the carbohydrate antigen and so

on. And many sites thats why its so sensitive.

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Firstly, we willdiscuss flow cytometry then western immunoblot technique

also, we can use immunofluorescence to determine the type of cells, we can use a machine that

we have in the lab we call it: flow cytometer and the process is called flow cytometry and you caneven sort these cells or separate cells from each other through a process we call it FACS

(Fluorescenated Active Cell Sorter), you can separate the cell that you want, you use that if you

want to determine the number of the CD4 cells, like in the AIDS patients.

If I want for example to see how many CD4 cells I have, I want even to separate those CD4 cells, we

identify different types of cells that we have in our body by a specific marker present on their

surface; marker means specific antigen that is not shared by others if that marker is there then we

call that CD4 cells or CD8 and so on

What do we do?!

We get the patients serum and we get the Buffy coat, which has the WBCs, they contain T helper

cells plus other cells like CD8 (T cytotoxic), for example; the CD4 has the CD4 antigen on the top

and the CD8 has the CD8 antigen on the top.

we start thinking of ELISA to detect antibodies against the antigens of the HIV virus (indirect

process), BUT we could get 1-2% false positive SO we dont say directly that this patient has AIDS

so we need to confirm that with another test (because if the patient is diagnosed having AIDS then

hes died until proving otherwise so we have to be sure in making diagnosis)..

** the test is called: and after we confirm that, the patient is having

AIDS (HIV positive) then we start treatment and we start monitoring that patient whether she or he

is improving or not we do that by counting the number of T helper cells (CD4) because those

are the target cells of the HIV virus; since it binds to the CD4+ cell antigen (this is the specifity)..

we have in our body around 1200 /l CD4 cells, usually if they go below 200, the patient is going

into severe opportunistic type of infections, maybe 500 or less start to have a trouble and so on.

We see how the patient is responding by calculating how many CD4 cells by a technique called

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We add a fluorescenated antibodies against the CD4 (if I

want to detect CD8 cells, I will add a fluorescenated

antibodies against the CD8 antigens). So anti-CD4

fluorescenated antibodies bind to the CD4 cells.

In the flow cytometer; it has a very narrow tube that fitsthe size of only one cell at a time, very small so these cells

start to pass; the CD8 or the CD4.

we have laser, it will hit the fluorescenated antibodies

(bound to cells) and every time it hits them, it will give

you a click -dot- (if theres no fluorescenated cells pass

then it will not click), So it will give you how many of

those cells have passed, also you could separate them

either to the right or to the left, so you can see how manydots here from CD4 you have and how many dots of CD8

you do have.

>> For example, if you look into this patient (panel B), the number of the dots of the CD4 is much

much less than here (panel A), that means this patient have AIDS.

As you know, in making diagnosis of leukemias and lymphomas, they detect certain markers on

lymphocytes (the abnormal one) or abnormal leukemic cells that are not present on normal cells

and they do that exactly with the same mechanism to detect the abnormal markers and herethese markers often are immature markers; they supposed to be present in immature cells not in

the mature cells in the blood and so on.

We get the HIV virus and we do an electrophoresis to this virus so it is going to be separated and all

the proteins of HIV virus are going to be separated in the gel (the polyacrylamide gel) based on the

So, to sum up:

** Flow cytometry used to count number of cells and know their types and separate them.

** We detect a specific cell marker like CD4, CD8, CD10.

** If you want to detect CD4:

take the serum add fluorescenated antibodies (anti-CD4) they will bind to T helper cells use flow cytometer machine

has laser

when fluorescenated antibodies hit it, gives click

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molecular weight or charges; the lower the molecular weight will be fast as well the heavy will be

slow so now the proteins will be separated on the gel, then,

you get a nitrocellulose filter paper and you stick it in the gel, then you do a transverse

electrophoresis from the gel into the nitrocellulose filter paper so all the bands in the gel will betransferred into the nitrocellulose filter paper so we get those filter papers and just we cut them

into streps put them in blot and then we sell them to labs!!!!!!!!!!

So nitrocellulose filter paper has all the HIV proteins adherent into it, we dont see

them so now we want to see them or we want to see which antibody is going to bind

to which protein on the nitrocellulose filter paper so We do an ELISA for example.

Now, we take the patients serum and we put it in the blot so if the patient has specific antibody,

its going to bind to these bands, then incubate for half an hour and then we wash it out then we

add a secondary antibody with an enzyme like horseradish peroxidase, incubate and then wash..

- the primary antibody is bound to the secondary antibody

Then you add the substrate and it will give a color so in the nitrocellulose filter paper, you will see

colored bands,

- to make diagnosis of AIDS, we should show a band from every group of antigens of the HIV

means that we have antigens on the surface of the virus, we have the antigens for the reverse

transcriptase enzyme, we have antigens from the core of the virus, you should have antigens

from each of those groups, if you see that then the patient is going to be labeled as HIV

positive.

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So this is how its going to look like

this is nitrocellulose, this is real test, you can see the different proteins of

the HIV virus and the colors that develops so it shows all the proteins, for

example if you look here into this patient -whos having just one band-

and the negative control so one band here will not be enough to make

the diagnosis so we tell the patient that we cant confirm, you have to

come may be after one or 2 weeks or one month and we keep monitoring

the patient tell we confirm either positive or negative.

this is a new technology where you can use a combination of ELISA with immunofluorescence

together, if you want to detect more than one antigen at the same time and we use this test if the

patient have hepatitis and we want to know which type of hepatitis the patient has; A, B or C.

So we get a microbeads; bead is a glass sphere, very small

glass sphere and they are colored and each one of those can

give you a spectrum of color when you get it with the source

of UV light and you can bind an antigen on these spheres,

put antigens of hepatitis A, B or C and so on.,,

Add the patients serum on the top, if you have an infection

its going to bind to one of the beads

Extremely rare to have patients with hepatitis A or B or C together,

we have never seen that but usually the infectious disease is caused

by one organism; this is the rule of the thumb

So, to sum up:

** Western immunoblot technique used to detect antibodies!! To confirm AIDS.** get patients serum (contain primary antibodies) add it to nitrocellulose filter paper (have HIV antigens & proteins) if

antibodies matches the antigens, they will interact add secondary enzyme-tagged antibodiesDO ELISA TEST!!

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So if the patient has one of them, A, B or C then its going to bind the antibody

Add a secondary antibody that is fluorescenated

Hit it with the source of UV light so as you see here (the picture)

After looking at the picture, the dr. said >> the color of the 1st

,2nd

and the 3rd

and the differentcolors here for that of 1 so it means that if that color for hepatitis A then the patient is going to

have only hepatitis A so the is designed to look for antibodies thats feeling that theres so many

antigens at the same time and its very sensitive as you can see, its an immunofluorescent

technique.

Now, we use hybridization techniques used to detect specific sequence, the microarray test as

well. We now have the sequence of so many microorganisms and then you can add antibodies into

that chip and determine the type of the infectious agent that we are looking for.

Complement: complementary to antibody, it binds to see H2 part of the heavy chain so we say

that the complement is fixed, this means that there will be no more of them present.

** If I want look for the concentration of one of the complement component of a patient lets say

C3 and its so low in that patient >> that means It could be used up by antigen-antibody reaction or

genetically it could be deficient in that patient >> this is the principle used up by the complement

fixation test.

The dr. invited us to visit the clinical

microbiology lab or the serology

section in the king Abdullah hospital

in order to get more details about

the tests used there.

So, to sum up:

** immunofluorescence microbeadbased immunoassay used to detect more than an antigen at the same time.

** get the different antigens we want to detect put them in different microbeadsadd pateints serum the antibody

that matches any of the antigens will bind to it add fluorescenated antibodies expose to UV light each bead

fluoresces with different colors

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Now, the dr. started with chapter 6. The pictures hes explaining are in the book, I didnt put

them since the slides arent available yet!!

Going back to the antibody structure and the antibody diversity which we talked about, do you

remember how many immunoglobulin specifities we -the humans- have?! Around 1011 different

specifities and different shapes of antigens that can bind to! From where do we get that number?

Also, we said around 1017-1018 different specifities of B-cells we born with! How did you get into

that number? We said, if we are genetically diverse then we have a higher chance to have both

diversities (in phenotype & genotype)

So, remember we said that antibodies come from B-cells and in order to form an antibody, you

need genes that code for the polypeptide proteins of this immunoglobulin. And also, we havegenes that will code to the area that is so specific; the hypervariable region, the complementary

determining region! Also, We need genes to code for the constant region,,,

You will see that genes that will code for the variable regions and the genes that will code for the

constant regions come from different areas, and the constant regions, in the heavy chain and in

the light chain also they come from different regions!

How all of those come together to form the immunoglobulins and how we calculated how many

different specifities we do have?

We will answer those questions in what we call

So, you have to know that proteins come out from genes; those genes when they are expressed,

we call them EXONS and we have intervening sequences we call them Introns, SO, the gene if it

was expressed, it go into transcription and then translation as you know to end up with a protein.

So, the proteins that will develop are simply a reflection that we have genes, for that, the different

polypeptides we get are related to the different genes that we have.

So it starts happening in the Germline, at the DNA level

Variable (V), diversity (D), joining (J; the last 100 amino acids in the variable region of the light

and heavy chains), leader (L), constant (C),, all will be rearranged.

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So, in the light and Heavy we have the variable area; V genes in the light and V genes in the heavy!

And we have D genes in the heavy but not in the light.

What codes for the variable region of the light chain are V and J segments and in the heavy

chain: V, D and J because the specific part of the Ig (the variable region) comes from part of the

light and part of the heavy; Two polypeptide chains!

Now, we want to determine that specifity which comes from the variable genes for the light

whether its kappa () or lambda () and the variable genes of the heavy; the different isotypes

that you know; Mu (), Delta (), Gamma (), Epsilon () and Alpha ()!

And remember, we have diversity genes for the heavy and J genes (joining) for both the light and

the heavy.

>> all Those will be rearranged and they will be selective; 1V 1D 1J, you will have gene

rearrangement and then gene splicing at the level of the mRNA, they come together from

different locations, they go into transcription, translation and produce the polypeptide. You can

see it depends on how many different V genes I have, how many different J genes I have or D

genes I have.

So V (D) J then recombination occurs at the level of the DNA, transcription at the level of mRNA

and then translation and the polypeptide that will form the Ig.

So, as an outcome of the proteins that will develop here will form the heavy and light chain

polypeptides and those will come together to form the Ig; Ig will assemble.>> This is all what we are going to talk about for today and tomorrow. The chart below is summery

of whats explained before:

The introduction to understand this, one should know the structure of Immunoglobulin, We have

heavy and light chains of the Immunoglobulin, we have variable and constant regions, remember

on the light and the heavy, they are coded by different gene segments; the more genes we have

the more the chance to have diversity.

germline heavy

and light chain

gene segments

V(D)J

recombination

transcription

translation

heavy and light

chain polypeptides

immunoglobulins

assembly

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Genes will be rearranged during development, on B-cell development and T-cell development for

the T-cell receptor, its almost the same. We have an L sequence to start the process, then we have

J segment, D segment in the heavy in addition to V and C genes, constant for the isotype classes

and subclasses that we have talked about before.

They will be next to each other by gene rearrangement and we need enzymes to break and unite.

If you have those enzymes then the gene rearrangement is going to take place and the

Immunoglobulins are going to develop, if genetically you are missing these enzymes, then you are

going to be immunocompromised,, at the level of DNA; we have RAG-1 and RAG-2 enzymes, and

they are so important for the production of those antibodies, if they are missing you become

immuncompromised.

What really will add up to the development diversity is not only the number of genes, the

rearrangement and rematching, but also we can see other things that will add up into the

diversity, like for example; the somatic recombination that will take place, possibilities, products

and then they assemble, V for binding (the variable region binds to the antigen), C (the constant

part for the biological function; complement binding, T-cell receptor binding or macrophages). So

you can understand that the most important part in the diversity regarding the antigen binding is

the VARIABLE region not the constant.

So this is what I was talking about, here for an example; for an Immunoglobulin, you must have a

light chain, which is either or never BOTH. And you require a heavy chain class or subclass (1,

2, , 1, 2, 3, 4, or ). So what are the genes that are present in those chains?! we start with

the DNA at the germline where we start with 5 and the L sequence and then you have the variable

genes, we have 35 variable genes for (V1, V2 or V3); you need just one gene of them and each

one gives you specifity as simple as that for the . And the same thing for in which we have 30

different V genes, you dont have to remember the number of genes in those just have an idea!

Then, we have J genes for joining; we said that the variable region is formed from the products of

V and J, where the products of the J form the last 100 amino acids.

So the specifity now for the will be determined by those genes as you can see here, the V and theJ.

We have the C gene for the and we have one type for that biological function. For the , we have

around 5 or 6 J genes, and also you can have more than 1 constant region (C1, C2, C3 and C4), so

we have more than 4 types for and those will add up to the diversity as you know regarding to

the antigen binding, what really adds up to the antigen binding as you know is the V and J only.

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And then we have the heavy chain, the heavy here is the same thing, we have around 50 V genes &

we have D genes, also will add up and those are around 25 genes and then we have around 6 J

genes.

>>>> So the variable region, you must have one of the V, one of the D and one of the J. you need

to have the constant regions of the heavy, which is one of the (1, 2, , 1, 2, 3, 4, or ).

So the process starts here by the gene rearrangement,

this is a summary of what I was talking about (figure 6-1), you have exons; expressed genes and

Introns; intervening in between and they are spliced out by enzymes, so this gene is going to be

selected and then the Introns will be spliced out, so the J will come very close, 1V and 1J, no more

than that, they will come together, V-J joining as you can see.

For the heavy chain, they start with the V and the J, and then the D will be added as you can see,

and you end up with V (D) J.both now go through the transcription, so you have mRNA, and then into translation that will code

for proteins of the V and J to form the variable region of the light and then the variable region of

the heavy V (D) J.

This is more detailed of what I was talking (figures 6-2, 6-3) (if you dont understand any thing, I

think that the dr. will repeat what hes said in the next lecture so dont worry!!).

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