unfortunately I need it rather quickly as it is due tomorrow. I didn't realize how long these take to get done (I actually completed 3 other sections). The length is specified: no less than 10 sentences for each answer, although I think there's some flexibility of +/- 3 sentences. How long do you think this will take you to complete because I'm working on them as well. I've already completed number 2, so no need to work on that number. Thanks.
Please do not work on number 3 either
So I just got home and can start working on the questions you need. Please tell me the numbers I need to do. And each question needs to be answered in 10 sentences, is that correct? For example, here is what I have done so far. If this is what you are looking for, let me know, and will continue. However, I will need help with the last part of the last question, since I don't know your book?? Thank you!
Thank you for getting back to me. I like your example response so far.
Q1: I'm happy with the response- we're done with that one.
Q2: I've done this one but would like you to review/correct it (below).
Q3: I've only done part of it and can send it to you. I am not sure if I'm correct so please do this question as well (sorry to add it back in!).
Q4a: In response to your last sentence in the question, why would the vaccination only produce T helper and B cell responses?
Q4b: I'm happy with the response- we're done with that one.
Q5i and Q5ii: I'm happy with the response- we're done with those.
Q5iii: requires some input from the book/me. I've attached the molecular tests from my book/professor. Hopefully this will help to answer the question. If not, I can do this one - just let me know.
Q2: can you review this and make changes/corrections?
Here is my response:
2. Describe five principle categories of antibody effector functions. For each category, explain the roles of antibody Fab and Fc domains and cellular Fc receptors.
The five antibody effector functions are neutralization, opsonization, antibody dependent cell mediated cytotoxicity, sensitization, and complement activation.
-In neutralization, antibodies bind to the pathogen or foreign substance and block the pathogen’s ability to interact with their intended target cell. For example, antibodies can bind to and neutralize a bacterial toxin, preventing it from interacting with host cells and causing pathology. The complex of toxin and antibodies binds to macrophage receptors through the antibody’s constant region.
-An antibody binding to a pathogen can opsonize the material and induce phagocytosis (neutrophils and macrophages). The Fc region of the IgG antibody interacts with Fc receptors on phagocytic cells causing the pathogen to be more readily phagocytosed.
-In antibody dependent cell mediated cytotoxicity, IgG antibodies coat the surface of the target cell and act to bring the natural killer (NK) cells and the target cell together to allow destruction of the target cell. NK cells have on their surface an Fc receptor for IgG antibody Fc domains and thus they can recognize, bind, and kill target cells coated with antibody.
-Sensitization: Mast cells have high-affinity Fc receptors on their cell surface that are bound to IgE antibodies. Antigen cross-linking triggers the mast cell to release granules containing inflammatory mediators like histamine and serotonin.
-Complement activation: Activation of the complement cascade by antibodies can result in pathogen lysis. Also, some components of the cascade opsonize pathogens and induce phagocytosis. Antibodies that bind with their Fc region to the surface of antigens activate the classical complement cascade. The constant region of IgG and the combination of phagocyte receptors for complement enhance phagocytosis.
For Q3, I'm not sure if it's correct at all. Please correct, modify, or completely re-do the answer. Here's what I've got so far:
3. Monoclonal antibody technology was first developed in the 1970’s and was accompanied by hopes that they would serve as “magic bullets”.
a) What was meant by this concept of “magic bullet”?
The “magic bullet” was a concept that became known throughout the early 19th century that an ideal therapeutic agent can be created to kill a target pathogen. This, of course, came after the development of monoclonal antibodies that showed specific binding affinity.
b) Why didn’t it work right away and how was this problem overcome?
Cesar Milstein, an Argentinean biochemist, wanted to analyze the rate and nature of mutations of antibody-producing cells to see how the mutations affected an antibody’s ability to bind to antigen. His experiments used Potter’s mouse myeloma tumors to make cell cultures but they ultimately failed. The cells in cultures used in Milstein’s experiments weren’t able to show high maturation rates in the variable regions of the antibodies. This was due to the fact that his myeloma cell cultures, which were usable for lab studies, produced abnormal antibodies that were weak when it came to binding with an antigen in vivo. In Switzerland, Kohler was trying to validate the role mutation plays in antibody structure. Kohler was using cultured B cells that had high antigen-binding ability but quickly died out. After hearing Milstein give a talk about his research, Kohler came to work in his lab. The two fused antibody-producing B cells from mouse spleen to mouse myeloma cells. The mice were previously injected with antigen and a culture medium was used to select fused cells producing the antibody to the antigen. After years of failed experiments, the cell fusion worked and they could produce pure, monoclonal antibodies that only bound specific antigen.
c) Explain the key differences between chimeric, humanized, and fully humanized antibodies; and briefly explain the process by which each type is created.
Q5iii: Here's what my professor provided for us:
In addition to the many protective effector functions that antibodies provide in vivo, antibodies have long served as incredibly versatile and powerful tools for biological and medical studies. In this section, you are introduced to some widely used experimental techniques that involve the use of antibodies. Specifically, western blotting, ELISA, immunoaffinity chromatography, immunofluorescent microscopy, and FACS techniques are introduced.
Western Blot This technique is useful for a qualitative (semi-quantitative) identification of a particular protein, from within a mixture of proteins. The proteins are subjected to separation by electrophoresis through a polyacrylamide denaturing gel (SDS-PAGE). The proteins are then transferred in place onto a filter paper that is exposed to a specific antibody which is allowed to bind specifically to the protein of interest. The excess antibody is washed off and the bound antibody is revealed by mixing with a second antibody that is specific for the Fc region of the primary antibody. Typically this secondary antibody has been conjugated to an enzyme such as alkaline phosphatase or beta-galatosidase which can react with a substrate to form a color and thus reveal the location of the protein antigen.
Here is a link to a tutorial about the western blotting technique: http://www.biology.arizona.edu/immunology/activities/western_blot/w_main.html
ELISA ELISA stands for enzyme linked immunoassay. This is a very sensitive quantitative technique for specifically detecting antigens or antibodies. It is widely used in research labs around the world and is also used in clinical laboratories for diagnosing diseases. The technique involves the use of 96 well plastic microtiter plates. Each well is coated with a protein sample, and then a primary antibody is added to each well. If the specific antigen is present in the well, the antibody will bind. Excess antibody is removed and then a secondary antibody specific for the primary antibody is added, as described for the Western Blot process. Again, the secondary antibody has been conjugated to an enzyme that will create a color when the substrate is added. In this case the amount of color can be measured by using a spectrophotometer.
Here is a link to a tutorial about the ELISA technique: http://www.biology.arizona.edu/immunology/activities/elisa/main.html
Here is another very good ELISA tutorial: http://www.sumanasinc.com/webcontent/animations/content/ELISA.html
Immunoaffinity Chromatography This technique is used for purification of large quantities of a particular macromolecule, usually a protein. An antibody specific for the protein of interest is conjugated to chromatography beads which are loaded into a column. A mixture of macromolecules, typically a mixture derived from a cellular lysate (broken up cells) or tissues, is passed over the column. The macromolecule of interest should specifically bind to the antibody while all other substances pass through. The bound molecule can subsequently be "eluted" from the column by altering the salt of pH of the washing buffer that is passed over the column. This is a technique commonly used in research for large scale purifications and in the biotechnology industry for purifying large quantities of biological molecules that may be used as therapeutic agents.
Here is a link to an animated tutorial for affinity chromatography: http://www1.gelifesciences.com/APTRIX/upp00919.nsf/Content/AD018C9E293F99BEC1256E92003E865A?OpenDocument
Immunofluorescent Microscopy Central to this technique is the use of antibodies that are conjugated to a fluorescent molecule that gives off a distinct color which can be detected in a fluorescent microscope. This technique has been incredibly useful in the field of cellular biology for identifying and studying the distributions and functions of cellular structures.
Here is a link to a short explanation of immunofluroescence microscopy that includes some beautiful microscope images of cells that have been "stained" with fluorescent antibodies: http://www.andor.com/learning/applications/Immunofluorescence/
FACS Fluorescence Activated Cell Sorting, FACS, also involves the use of antibodies that are conjugated to a fluorescent molecule. However, instead of merely looking at the cells in a microscope, the FACS technique enables the labeled cells to be physically separated and counted from those that are not labeled with antibody. See figure 4.14 in the textbook. A mixture of cells is allowed to combine with the labeled antibody and then passed through a flow cytometer nozzle that allows the passage of only one cell at a time. A laser activates the fluorescent antibody which is then detected and recorded on a computer. This technique has been particularly useful for identification of different types of leukocytes based on differential expression of CD molecules described in module 1.
Wonderful! Thank you! Enjoy dinner :)
I updated 3b. Is this better or should I scrap it?
Cesar Milstein, an Argentinean biochemist, wanted to analyze the rate and nature of mutations of antibody-producing cells to see how the mutations affected an antibody’s ability to bind to antigen. His experiments used Potter’s mouse myeloma tumors to make cell cultures but they ultimately failed. The cells in cultures used in Milstein’s experiments weren’t able to show high maturation rates in the variable regions of the antibodies. This was due to the fact that his myeloma cell cultures, which were usable for lab studies because they grew uncontrolled in vitro culture but produced abnormal antibodies that were weak when it came to binding with an antigen in vivo. In Switzerland, Kohler was trying to validate the role mutation plays in antibody structure. Kohler was using cultured normal B cells that had high antigen-binding ability but quickly died out as they had a finite life. After hearing Milstein give a talk about his research, Kohler came to work in his lab. They decided to combine the strengths of both their approaches and fused antibody-producing B cells from mouse spleen to the immortal mouse myeloma cells. The mice were previously injected with antigen and a culture medium was used to select mouse B cells producing the high-affinity antibody fused to the immortal myeloma cells. After years of failed experiments, the cell fusion experiment worked and they could produce immortal B cells that made pure, monoclonal antibodies that only bound a specific antigen.
Sounds good- I kept my updated 3b. thanks!
Explain the key differences between chimeric, humanized, and fully humanized antibodies; and briefly explain the process by which each type is created.
shoot- sorry, I think we forgot the process part...
Last bit of help! Thanks
Thanks for your help!
Are you available to work again?
Here is the catch- when I post the questions, I will only have 20 minutes to answer the questions so we would have to coordinate when we can do this so that there is no delay between posting and answers. Is this something you'd be interested in? Thanks! Also, I have feedback from my professor on our previous work. Would you be interested in reviewing the comments? I can send the document over so you can get an idea of what the professor is looking for the next time around- let me know. Take care...
Hello- I made a mistake -we have 60 minutes total and it's only 10 questions. However, those were previous chapters and it may change (maybe more questions). This next set of questions will be on immunology and specifically B-cell and T-cell development. These questions are not like the mass set of questions I sent you previously - they're easier! yey...
I am available this weekend. I am on the west coast and will have time between 3 and 7pm on Saturday and all day Sunday. What works for you?
Also, the file with the comments is pretty large. Is there an export feature on this website I can use? Thanks!
Well the timed part is multiple choice and I would be working on it too. I just feel like I need someone else to verify my answers. I understand and appreciate your honesty. Thanks!
Hi BMW! I'm hoping you will reconsider working with me! You were great! The timed, 10 question quizzes aren't too bad (I get between 80 and 100% usually) but sometimes just need a second opinion. Please help!!!
I have received comments from my professor in order to improve my midterm. Can you help update the questions that need modification?
I would be happy to work on this project for you, but please confirm that you'd like to do so.