Introduction to Antibodies

My name is Denise Woolley and before I took on this role as implementation consultant, I used to be a senior scientist in antibody research and development at the Leica Biosystems site in Newcastle at the United Kingdom. Today I'm going to talk to you about introduction to antibodies.

Viruses, bacteria, toxins, anything that doesn't belong in the body is generally considered foreign matter. Therefore, it's potentially pathogenic, which means it has the potential to cause disease in the body that it's infected. Antibodies are specialized proteins called immunoglobulins, and these are generated by the body's B cells in response to this foreign matter, to the viruses, the bacteria, the toxins. Now our cells have protein structures attached to them, which identify our cells as belonging to us, and these are recognized by our immune cells. Sometimes this recognition system can malfunction, which causes autoimmune diseases. But generally, anything unlabeled in our bodies is targeted for destruction by our immune cells, but our cells are not. 

Antibodies, immunoglobulins, are roughly Y-shaped and are formed by 4 polypeptide chains. Two of the four chains are heavy chains and two of the four chains are light chains. The difference between the heavy and the light chain is the amino acid sequence and, as the name suggests, heavy chains weigh more than the light chains. An average heavy chain is about 50 kilodaltons, which is double the weight of an average light chain at 25 kilodaltons. A single heavy chain will combine with a single light chain to form a heterodimer structure. Two heterodimers join to form a complete immunoglobulin. The join between the heterodimers is a disulfide bridge and this acts as a hinge which gives flexibility to the Y-shaped structure that you can see on the screen.

There are four, sorry, there are two types of light chain, not four. You've got kappa and lambda. And generally, there's no functional difference between the two. Now, antibodies will only ever have kappa chains or light chains or lambda chains. They'll not have one of each. And in humans, kappa chains are usually more abundant than the lambda chains with a ratio of two to one.

Structurally, antibodies have two significantly different areas. The first can be called the constant domain. This forms the lower part of the Y shape and it only includes the heavy chains. The variable domain is the second area, and this includes the heavy and light chains at the top of the Y shape, the arms of the Y. The variable domain is so-called due to the variations or differences in the sequence of the first 110 amino acids in the heavy and light chains. This is the area of the antibody that can detect and bind to its specific antigen. The variability of amino acids allow the antibody to be generated against the specific protein.

The heavy chain sequences form the constant domain. This gives rise to the antibody isotype, which we'll discuss in a couple of slides. The constant domain is the same for every antibody within that specific isotype, so it's easily recognized by the host immune cells. The variable domain also contains the FAB region. FAB stands for fragment antigen binding, and as it sounds, it's the area of antigen binding. The constant domain contains the FC region, or fragment crystallizable. Now there's no antigen binding capabilities in this region. This fragment is responsible for interacting with the host immune cells, so it's easily identified by the immune cells. The flexibility from the disulfide bridge allows the two FAB fragments, so the two arms of the Y, to move independently of each other, which allows them to interact with more antigens. The Y-shaped structure allows for variability in the FAB regions whilst maintaining the similarity in the FC region. It allows the antibody to detect a range of antigens, but it still allows the host immune cells to be able to recognize itself, recognize the FC region, the constant domain, and generate an immune response.

As mentioned earlier, the isotypes are determined by the constant domain, the FC regions of the heavy chains. There's 5 different isotypes of mammalian antibodies and they're categorized by the sequence and number of amino acids in the heavy chains. The 5 sequences are labelled with lowercase letters from the Greek alphabet. You've got IgG, which is gamma, IgM, mu, IgA, alpha, IgD, delta and IgE, epsilon. We're going to go through one at a time.

IgG containing the gamma chain. This is the most abundant immunoglobulin in the human body. It's very small. It exists as a single Y-shaped structure with a molecular weight of about 150 kilodaltons. Now, most pathogenic infections are confronted by IgG antibodies, and this is part of the humoral or cell mediated immune response, which basically means it's an immune response driven by antibodies. The Fab fragment, it binds to a specific piece of the pathogen, which then triggers the immune response. The FC region, recognized by the specific immune cells. Now, these cells are phagocytes. The phagocytes such as macrophages engulf and digest the pathogen or an infected human cell, destroying the potential pathogen and saving the host from infections. Multiple IgG antibodies tend to bind to a single pathogen or an infected cell, and it produces a coating of IgG antibodies all around it. and this is called opsonization. IgG is further subdivided into four more classes and they're numbered one to four with IgG one is the most abundant and IgG four is the least abundant. The subclasses generally are very similar, but some differences are seen in the disulfide bridge or the hinge area. IgG3 has around four times as many amino acids in the gamma chain, in the heavy gamma chain, as the other subtypes do. And it's this subtype that's involved with the immune complement cascade. This is the cascade which triggers the phagocytic response to opsonization. Another interesting fact about IgG is it can cross the placenta. and it can confer a degree of immunity to a developing fetus.

IgM has the mu heavy chain. This is the first antibody generated in response to the body detecting a pathogen. The pentameric structure makes it the largest antibody found in mammals, and that's five individual IgM units that have joined together. Sometimes it can exist as a hexamer, which is 6 units, but it's most commonly found as a pentamer. These individual units are formed into the pentamer and held in place by the disulfide bonds and adjoining chain, which is also called a J chain. It's the presence of the J chain that's thought to trigger the formation of more pentamers when there's loads of single IgM antibodies. in the area, in the vicinity. The pentamer structure has 10 separate Fab fragments, so technically it can bind up to 10 individual antigens, which makes it extremely effective. And just like IgG3, IgM also triggers the complement cascade and phagocytosis. Now, due to its larger size, it's generally found in lymphatic and larger blood vessels around the body.

IgA, so this is formed with the alpha heavy chain. It's found in monomeric, in dimeric formats, so one or two units, with the dimeric structure connected by another J chain and a secretory component. So we have IgA1 and IgA2. IgA1 is the monomeric version, the single unit, usually found in blood serum. IgA2 is called secretory IgA or SIGA and is most commonly present in the dimeric form with the secretory component. SIGA or IgA2 is found in abundance at mucosal surfaces, such as those that are in prostatic ducts, endometrium, and especially the respiratory and the GI tract. Anything with mucus, basically. The secretory component protects the antibody from undergoing digestion by gastric pepsin, which is what makes it capable of survival in the harsh acidic stomach environment. The secretory component also allows the Dima format to be secreted in mucus and body fluids, and it's found in high concentrations in human colostrum and breast milk, which goes towards conferring some degree of immunity to a breastfed newborn baby. The primary rule of IgA is to protect the host from ingested or inhaled pathogens. So unlike IgM and IgG, it doesn't trigger the complement cascade in the blood. IgA works by forming a physical barrier between the pathogen and the epithelial cells. It stops the pathogen from getting into the host. It creates a barrier and it protects the mucosal linings. Once it's stopped the invasion, the infection of the pathogen, in the GI tract you've got the peristaltic motion of the bowel, of the muscle around the bowel organs, which push the pathogen out of the body.

Now, as of this recording, immunoglobulin D is the least understood antibody. It's formed by the delta heavy chain and exists in a monomeric form, which is located primarily on B-cell membranes, and it's usually in conjunction with an IgM. It's not present in very high levels and it has a very short half-life of two to three days. It's thought to make up about 1% of all immunoglobulins in the body. And currently it's thought that it's involved with tamping down autoimmune reactions, but so much more work is needed before we can save a definite.

IgE uses the Epsilon heavy chain, and usually it's produced in response to parasitic infections. However, we tend to associate it more with allergic reactions. It's present in small levels in a monomeric form, estimated at about 0.0002% of total immunoglobulin in the body. It's usually found in mucosal surfaces, such as those in the respiratory and the GI tract. Now, allergic reactions are basically an overreaction of the immune system in response to something that it shouldn't be allergic to. It's termed, this item, this, it's not a pathogen, we'll call it the allergen. The first time an allergen enters the body, and this could be something like pollen entering the respiratory system, it's presented to the B cells, who differentiate into a specialized type of B cell called plasma cells. Now these cells are short-lived and all they do is exist to produce multiple copies of IgE which will detect the specific antigen that triggered its formation. It'll detect the specific allergen. The constant FC region of the IgE has a very high affinity for certain white blood cells, and these are basophils and mast cells. And this allows the antibody to bind to these cells. The next time the allergen enters the host body, they're ready, waiting, and will detect it. There's no delay in an immune response. So on re-exposure to the allergen, the B cells don't need to have the allergen presented to it to form plasma cells and to produce IgE. The antibodies are already there and they're attached to the mast cells and the basophils. Every time the allergen is present in the host body, the IgE antibodies bind to it and trigger the adjoined mast cell to release histamine from its granules. And it's histamine that causes allergic symptoms. it initiates the inflammatory response, and that can manifest as a runny nose, watery eyes, coughing, itching, throat problems, breathing, and in extreme cases, it can result in anaphylactic shock.

We've covered the structure of the five different isotypes, and now we're going to discuss specificity and in vitro manufacture of antibodies over the next few slides. For some reason, I struggle to say specificity, so I have to say it very slowly. Antibody specificity is the ability of the Fab regions to bind to one or more antigens. So the difference in the amino acid residues of the antigen binding site is what's responsible for the specificity. Antibodies detect unique areas on antigens called epitopes, and every antigen has multiple epitopes.

Monoclonal antibodies will only detect a single epitope on the antigen, and that's the one that triggered their formation. Polyclonal antibodies are more generalized, and they can recognize more than one epitope on an antigen. Therefore, monoclonal antibodies are more specific than polyclonal antibodies, which makes them usually more expensive. They're more expensive to develop and they take longer to manufacture.

Polyclonal antibodies are generally cheaper and quicker to manufacture as they produce from several different B cell clones, but they don't have the same degree of specificity as a monoclonal antibody. Now, both types of antibody have the benefits and also have the disadvantages. It depends on what you want to use them for. Why are we talking about antibodies? Why are they so important? Antibodies are a crucial part of cancer research around the globe. Normal mammalian cells express a large range of different proteins, that can be on the cell membrane, in the cell cytoplasm, and on the cell nucleus. These proteins are altered when a cell undergoes malignant changes. The normally expressed proteins, they can change in structure, they can stop it, the cells can stop expressing the usual proteins, they can, the changes can cause the cells to start expressing different proteins, they can start over expressing the usual proteins, they can express different proteins that can stop expression altogether.

It's these changes in the expressed proteins that are detected using a technique called immunohistochemistry, which is abbreviated to IHC, or immunocytochemistry, which is abbreviated to ICC. IHC is when the technique's performed on tissue, on pieces of tissue, and ICC is when it's performed on individual cells, groups of individual cells, but generally people say one or the other to cover both.

Generating high quality, specific, robust antibodies for research is quite a rigorous process. Against human cancers, what we need to do is isolate the tumor cells and purify them. You pick the tumor that you want to raise antibodies against, the tumor that's growing in the human, you take it out, you harvest the tumor cells, you purify it, you make sure you've just got tumor cells. The relevant proteins that are the cells that are expressing the proteins, these proteins are harvested, and the tiny protein fragments are used to inoculate specialized lab animals, and this triggers the immune reaction within the lab animals. The animal's immune response recognizes it as not being labelled as self, it's foreign, so it has an immune reaction. The antibodies it produces are designed to attack the proteins that have been inoculated into it. Once there's a sufficient degree of these antibodies, the animal's splenic cells are isolated. These splenic cells are introduced to a specialized malignant HGPRT negative cell line.

I'm going to say this and it's quite hard. Biology can be compared to learning a new language, so please bear with me. But HGPRT stands for hypoxanthine-guanine-phosphor-ribosyl-transferase, which is basically an enzyme that's found in the human body. And the cell line that we use is negative for it. All of that just to be negative. The myeloma cell line is immortal. And once it's fused with the splenic B cells, it generates immortalized B cells which produce the specific antibody against the inoculated human proteins. This fusion is called a hybridoma. Now, any unfused cells are removed using hypoxanthine-aminopterin-thymidine, or HAT medium, which is easier to say than the other one. It prevents the unfused cells from undergoing cell division, so they'll die off and not form part of the hybridoma.

Initially, Within the hybridoma, there will be multiple B cell clones and they will produce a range of antibodies against various epitopes on the protein antigens. To produce a monoclonal antibody, you need to isolate a single immortalized B cell clone. To do this, you have several different dilutions of HAP medium over multi-well plates. You keep diluting out and out until eventually you end up with a single cell and a single well in the plate. And it's this single cell that will be cultured, that will grow and will eventually produce a supply of monoclonal antibodies. It will be replicated, it will produce the specific monoclonal antibody that it was first raised against. It's a very complicated process and I've shortened it down to like 6 very, very simplified steps, but you can appreciate it's not simple, but it is a very important process.

Now we've made the antibodies, what are we going to do with them? Generally, there are 6 classifications of mammalian cancers and they're all, they're named from the cell, the type of cell from which they originate. Carcinoma is the name given to malignancies that start in epithelial cells. These carcinomas derive from cells from the skin, from specific organs. You've got basal cells in the skin, the thyroid is epithelium, lungs, liver, colorectal cancers are all examples of carcinomas. And most cancers are carcinomas.

Sarcomas occur in the tissues that are between the organs. Connective tissues such as muscle, tendons and bones are classified as sarcomas. Leukemia and lymphoma are both blood cancers. Leukemia originates in the white blood cells within the bone marrow. These malignant white blood cells migrate out of the bone marrow and go into the bloodstream. They circulate around the body, therefore it's not a solid tumor. It can be detected throughout the whole body. Lymphoma also develops from white blood cells, but unlike leukemia, it's a solid tumor. It forms within the lymph nodes, within the lymphatic system, so it doesn't circulate around the body.

Malignant melanoma develops from specialized skin cells called melanocytes. Now, these specialized skin cells produce melanin pigment, which is a brown insoluble deposit, and it's responsible for the different shades of skin that we see. We'll discuss malignant melanoma in a bit more detail in a few slides' time. And finally, the last one is central nervous system tumors. These arise from nerves, from the spinal cord and from the brain. Due to medical developments over the last 200 years, we humans are living much longer. This increased lifespan, combined with various lifestyle choices that we make, mean we are more susceptible to cancer.

In a very simplified description, cancer is uncontrolled cell growth. When cells grow, what they do is they divide, they make a copy of the DNA for each new cell that produces. One cell will divide into two. The first cell makes a copy of its DNA and that copy will go into cell two. The second cell will make a copy and that will go into cell three, a third copy of the DNA. This third copy will make a fourth copy and so on and so on. With the DNA being duplicated each time, it's at a risk of damage. Every time DNA is replicated, you run the risk of a mistake in the sequence. Think of it as a very, very broad comparison with photocopying. When you have your single copy, you have an original document, sorry, you make a single copy and it's very clear, it's sharp, it's crisp. Now, if you take that copy and make another copy and keep doing that and making copies of each copy, by the time you get to the 10th, the 20th, the 30th, you can't see the clarity that you had in the first one. It's not sharp, it's not crisp, it's not clean, it's blurred. Linking it back to the cells, every time your DNA is copied, it's getting blurry, which would mean it's a potential mistake in the DNA sequence. We've got a piece of DNA or a mistake in the DNA, which can trigger malignant changes. This change in the DNA sequence can result in the overexpression of protein, the under expression of protein, different proteins, which is what we will detect with antibodies.

Malignant melanoma is one of the most prevalent cancers in males and females. It starts when the sun's rays damage the DNA and the melanocytes. And this damage causes the switch that usually stops the cells from dividing. It turns it off. and the cells just keep dividing and dividing and dividing. The top image on the screen is a typical clinical presentation of malignant melanoma. The outline of the darkened area, it's irregular, the color’s uneven, and they are both very, very common symptoms for malignant melanoma. But we need to make sure that the changes are due to these damaged melanocytes, so a biopsy of the lesion will be taken. That biopsy will be processed in a pathology lab and from this an H&E slide will be generated and that's what's in the middle image.

H&E is hematoxylin and eosin. Hematoxylin is a dye which stains cell nuclei blue so you can see the DNA or you can see the nucleolus where the DNA is contained. An eosin is a dye which stains the rest of the cellular components, various shades of pink. The brown area, now this is the melanin pigment, the naturally occurring insoluble melanin pigment. While the H&E is used to visualize the tissue structure, we need to use immunohistochemistry, and that's what's seen in the lower image.

This shows the tissue which has been stained with an antibody that was generated against a specific protein called HMB45. Now HMB45 protein is only expressed by malignant melanocytes, not normal melanocytes, just malignant melanocytes. An antibody created against HMB45 will then identify lesions which are malignant melanomas. But the antibodies are microscopic. They're invisible to the naked eye and even at a very high magnification, you can't see them. We tag them with specific enzymes. These enzymes, horseradish peroxidase, react with another reagent called DAB to produce a rich brown insoluble precipitate at the location where the antibody is bound to the antigen structure in the tissue. It's a much stronger, richer, deeper brown than what you see with the normal melanin pigment. You can differentiate between the two.

Hematoxylin is commonly used as a counter stain and this allows you to be able to identify the tissue structure. You can see if the layer of skin where the malignant melanocytes are, you can see if they've moved out of that layer and into underlying tissues. Now, immunohistochemistry is the technique that we use to use the antibodies for diagnosing disease and training materials for immunohistochemistry are also available on the Life Science portal of the Leica Biosystems website.

We've discussed malignant melanoma, but all through the body, every single cell, or nearly every single cell, has the potential to become malignant. Each different tumor that arises from these malignant cells will have a different protein expression profile. Now, having said that, some proteins are expressed by a wide range of normal and malignant tissues. It could be that using a single antibody, is not the most reliable method. It is with HMB45, but generally with other cancers, it's not. Several different tumor types can express the same protein. They can lose the same protein. They can express the same modified protein.

It's common to use a variety of antibodies during testing, and some of these will you will expect to be negative and some of these you will expect to be positive. Negative would be no antibody staining and positive would-be detection of the antibody. It's this staining profile that gives you an answer. On this slide, we have several examples of abnormalities in epithelial cells and antibodies that are commonly used to identify them. Image one shows positive staining for the antibody raised against high molecular weight cytokeratin, CKAE1 and AE3. The antibody is bound to cells expressing these specific cytokeratins. It's a very close up image of a ductal breast cancer. The breast, the ductal cancer cells are the ones expressing the CKAE1 and AE3.

Image 2 demonstrates bowel carcinoma and it's displaying positivity for CDX2 protein. The bowel has a mucosal lining and the mucosal cells have become malignant and they've spread through the underlying tissue where they shouldn't normally be. Using antibodies raised against the CDX2 protein allows a clearer identification of the malignant cells among the normal cells. The brown cells, the malignant cells and the blue cells around it are normal cells.

Image 3 is an example of prostatic ductal hyperplasia. Prostatic ducts express prostatic specific antigen or PSA. Using this antibody allows clear visualization of the thickening ducts, so they shouldn't be that thick and normal prostate. Now, hyperplasia is not a true malignancy, but it's still an abnormal change that arises from a specific epithelial cell type.

The last one, image four, is a normal human thyroid, and it's been stained with antibodies raised against thyroid transcription factor one. which we also call TTF-1. Whilst the name and the tissue suggests TTF-1 is used in diagnosing thyroid tumors, it is, but it's also used in identifying lung tumors. TTF-1 protein is commonly expressed in type 2 pneumocytes in the lung. When the cells undergo malignant changes, they continue to express TTF-1, which allows the identification of lung adenocarcinoma.

In this webinar, we've looked at the structure of antibodies. We've learned the nomenclature for each region, how the structure supports the function. We've looked at the five different isotypes and the function in the human body. Briefly, we've discussed the manufacture of antibodies and how they're used to determine different types of cancer. We've touched on why a longer lifespan means a greater risk of cancer developing later in life. And it's this increased risk that makes the production of high-quality antibodies for use in reproducible immunohistochemistry techniques extremely valuable to cancer researchers around the world. Thank you for listening. I hope you enjoyed it and I'm happy to answer any questions. Thank you.