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Antibody-Antigen Interaction
Sara Al-Zu'bi Ziad Al-Nasser Thursday, 7/7/2011

The Dr. started the lecture by talking about the attendance and seat numbers, I think all of us know every thing regarding that :D Dont look how many pages this lecture is, you will think its long and boring before reading it!! Just look at 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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immunology; clinical means hospital based ( ))so in serology or clinical immunology, we do lots of antigen-antibody reactions, either to determine the presence of the antigen (so the antibody has to be known) or vice versa; to determine the presence of the antibody (so the antigen has to be known). The tests that we are going to talk about are: agglutination inhibition, ELISA, immunofluorescence, complement fixation, flow cytometry, western immunoblot technique, fluorescent microsphere based immunoassay & immobilization.. So lets start with the first one:

The application of this test is: pregnancy determination, the pregnancy test that present in the markets is an agglutination inhibition test. In pregnancy, they look for HCG hormone (human chorionic gonadotropin hormone). If the lady is pregnant, she is going to have HCG hormone in her urine and this test can be positive after 4 to 5 weeks of pregnancy and may be less in the blood (I think that we can test pregnancy earlier than 4 or 5 weeks if we use blood, of course by another test different from this one).

The steps of the test and interpretations:

By the KIT, you will see that it has antibody against the HCGH, so: Put one drop of urine of the suspected pregnant and then you put one drop of anti-HCGH on the top, - If shes pregnant, then the antibodies are going to react with the antigens so there will no more free antibodies present. - If shes not pregnant, then the antibodies wont react with HCGH (cuz its not present), so there will be free antibodies. Then we put another drop of latex particles that have HCGH attached into them so, - If the lady is pregnant, nothing will happen because theres no antibodies (because they are already utilized by the HCGH antigen thats present in the urine), so this is a positive test,,, theres NO agglutination; agglutination is inhibited. - If the lady is not pregnant, then there will be agglutination; agglutination is NOT inhibited because there are free antibodies against HCGH and when you put another drop of HCG coated latex particles on the top, then the free antibodies are going to bind to the HCGH and will give you agglutination.

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 urine NO antigen-antibody interaction free antibodies agglutination when latex (HCG) added negative

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.
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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 one when 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 secondary antibodies added no interaction with the primary one when wash, secondary antibodies also will be washed out add substrate no enzyme activity no products or color.

** 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 a substance 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. - If theres 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).
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 Add the antibodies that are fluorescenated with the fluorescein isothiocyanate and these antibodies are known. The difference between the light and fluorescent Incubate and wash. microscopes is the light that is used, UV in the Put it under the fluorescent microscope. fluorescent and regular light in the light) - 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. - If the 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.
So, to sum up: ** Direct immunofluorescent test >> light = positive test, no light = negative test. ** In case of positive test: Antigen unknown matching 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

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.

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 added theyll interact with the primary one when 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 one when wash, secondary antibodies also will be washed out exposure to UV light no light.

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.pallidum to 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.pallidum organisms 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.

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: western immunoblot technique 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 flow cytometry.

Firstly, we will discuss 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 can even 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.
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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 fits the 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 many dots 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 here these markers often are immature markers; they supposed to be present in immature cells not in the mature cells in the blood and so on.
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

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
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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 be transferred 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 bandand 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.

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 antibodies DO ELISA TEST!!

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

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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 different colors 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.
So, to sum up: ** immunofluorescence microbead based immunoassay used to detect more than an antigen at the same time. ** get the different antigens we want to detect put them in different microbeads add 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

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.
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.

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.

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So if I want to look for specific antibody, the antigen has to be known so I put here the antigen and the antibody together in a well and then I add the complement,,, If you have the antigen that matches the antibody >> the complement will be fixed that means that you will not have any free complement in the well,,,

You are going to be sure by adding up what we call testing system and we will get what we call sensitized sheep RBCs; we get sheep RBCs injected into a rabbit, get the serum of the rabbit and then you put it in sheep RBCs so the antibodies are going to bind to the surface of sheep RBCs so sensitized sheep RBCs means sheep RBCs that have antibodies on the surface, we take those and then we put them in the testing tube, If the patient has an antibody and antigens that are bound to each other so there will be no complement free in that particular well so when you add the testing system, nothing will happen, so this is a positive test. If neither the antigen nor the antibody is matched that means that the antibody is free and not reacted with the antigen so when you add the testing system then the complement is not going to be fixed So its going bind to the testing system and final outcome of complement activation will be lysis of RBCs so when I say the RBC is lysed this is a negative test >> so positive: no lysis and negative: lysis [This test is rarely done nowadays however still some labs do it]
So, to sum up: ** Complement fixation test >> fixed complement, no lysis = positive test, non fixed complement, lysis = negative test. ** In case of positive test: Antigen is known antibodies interact with it complement bind to the antibody (fixed) low concentration of the complement, NO free one add testing system (sensitized sheep RBCs) no complemrnt to bind no lysis ** In case of negative test: Antigen is known antibodies dont interact with it complement not fixed normal concentration of the complement, free add testing system (sensitized sheep RBCs) complement binds it lysis

. Here you have the bacteria or any organism moving then you put antibody on the top, if the antibody is specific to those bacteria, its going to bind to the complement and so the bacteria will stop -immobilize-, this is a positive test.

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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 have genes 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:
V(D)J

germline heavy and light chain gene segments

recombination transcription translation

heavy and light chain polypeptides

immunoglobulins assembly

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.
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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 the J. 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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