Overview of the Immune System and Immunological Assays with Real World Connections Cassandra Bonvissuto, Poland Seminary High School, Poland, OH Mentor: Prosper Boyaka, Ph. D, The Ohio State University Supported by The American Association of Immunology 1 Table of Contents: A. Teacher Guide………………………………………………………………………3 I. Background…………………………………………………………………………………3 II. Student Outcomes…………………………………………………………………….13 i. Science concepts covered……………………..………………………….13 ii. Standards……………………………………..………………………………..…14 • NGSS……………………………………..…………………………….…..14 • Introduction to Biotechnology: Georgia Standards…..16 iii. Placement of instructional unit…………………………………………18 iv. Technical skills…………………………………………………………………..18 v. Relevance………………………………………………………………………….18 III. Student Learning Objectives………………………………………………………19 IV. Time Requirements……………………………………………………………………20 V. Advanced Preparations……………………………………………………………..21 VI. Materials and Equipment…………………………………………………………..25 VII. Student Prior Knowledge…………………………………………………………..28 VIII. Daily Unit Plans………………………………………………………………………….30 IX. Assessment Methods…………………………………………………………………32 B. Student Guide…………………………………………………………………….94 I. Rational……………………………………………………………………………………..94 II. Materials…………………………………………………………………………………107 III. Procedure………………………………………………………………………………..110 IV. Data Collection………………………………………………………………………..119 V. Discussion and Analysis……………………………………………………………121 2 A. Teacher Guide I. Background: i. Introduction to Immunology Part 1 and Cell Research Project What is our immune system? What is immunity? What are we trying to protect ourselves against? Those are the questions that many students will ask. This unit on immunology was created to answer these basic questions with videos, notes, labs, research projects, presentations, and collaborative work. Students will learn the difference between innate and adaptive immunity. Innate immunity is the fast acting, non-specific immune system that acts against a broad range of pathogens, but if that is not enough, it triggers the adaptive immune system to help fight the pathogens. The adaptive immune system takes longer to activate because it uses very specific antibodies and receptors against varying pathogens and antigens. The following link is to the National Institute of Allergy and Infectious Disease and is a great place to start to learn about the immune system: https://www.niaid.nih.gov/topics/immunesystem/Pages/default.aspx There are a variety of WBC’s (white blood cells) in your immune system that help protect you against pathogens that are constantly attacking your body. You can think of your immune system as the military for your body, and there are different branches that have different functions to protect us. The students will research and become experts on one assigned cell of their body’s immune system. They will then make a presentation that they will use to teach the class about their assigned cell. You can find more information about the specifics of each cell type at the following link: https://www.niaid.nih.gov/topics/immuneSystem/Pages/immuneCells.aspx ii. Introduction to Immunology Part 2 and Vaccines Article As the students start to learn about their immune system, you will introduce the topic of vaccines. You may ask students how do vaccines work? What causes allergies? Why can’t I take antibiotics to treat a viral infection? What is MRSA? This part of the unit will focus on answering these questions. You will answer some of these questions in this part of the lesson. A vaccine typically contains an agent that resembles a disease-causing micro- organism and is often made from weakened or killed forms of the microbe, its toxins, or one of its surface proteins. Improving vaccinations and creating new vaccinations is a hot topic in the research community right now. Medicine is 3 always changing and evolving due to new discoveries completed by researchers. In 1998, a study was published by Dr. Andrew Wakefield that tied autism to the MMR (measles, mumps, and rubella) vaccine. This was later retracted because of lack of replication and a conflict of interest that arose during the study. More information about this topic can be found at the following link: http://www.cnn.com/2011/HEALTH/01/05/autism.vaccines/index.html. But, researchers have been trying to find a way to create more powerful vaccines that will have longer protection with less harmful side effects for their recipients. Vaccines actually inject inactivated viruses, parts of the pathogens DNA, or a variety of other antigens specific to the pathogen of interest. Currently, many vaccines also need booster shots to enable your body to maintain appropriate levels of antibodies in your serum to provide a protective effect against the pathogen. If we could find a way to decrease the amount of injectable pathogen/antigen while increasing the initial immune response to provide a stronger, longer lasting immunity that would be a great leap in the medical field. They are also trying to develop vaccines that would treat rapidly mutating viruses like HIV, but we haven’t gotten there yet. The purpose of this lesson is to have students practice reading and dissecting research articles. They will also have to present their article findings to the class and lead a class discussion on the benefits and/or complications of the study. These articles are all tied to vaccinations so that it ties into the next activity (the ELISA lab on vaccination methods), but can be changed based on your needs. They will use these articles to jump start their project idea on comparing vaccination methods that will be discussed in the ELISA protocol. iii. Quantitative ELISA Lab ELISA stands for Enzyme Linked Immunosorbent Assay. ELISA’s are a common immunological assay with a variety of uses, such as to check antibody titers when giving someone a booster of tetanus, when checking for the presence of an antigen in serum (such as malaria or HIV), when checking the presence of a specific antibody in serum (like for allergens or against other specific antigens), and many other things. Your body’s immune cells make antibodies against the specific antigens. ELISA’s are run to detect either the antibody or the antigen in your serum. To detect these, ELISA’s use antibodies that are linked to enzymes to detect antigens in a patients’ serum. The enzyme that is attached to the antibody 4 produces a visible color change when a substrate is added in its presence. A substrate is something that binds to the enzyme that the enzyme then acts on to change it in some way. If the antigen is not in the sample (which could be another antibody, a protein, a virus, etc.), the antibody that is linked to the enzyme will not be able to bind to anything, so it will be washed away. When the substrate is added, there will be no color change. If the there is a color change, that means that your enzyme linked antibody was able to bind to its specific target antigen in the well. You can determine the qualitative concentration of the antigen present in the well by observing the intensity of color change. The darker the color change, the more you have. If you have standards that you serially dilute at 1:2 of a known concentration, you can determine the exact quantity of antigen present in your well. You can reference the attached ELISA PowerPoint to obtain more details about the assay. It has links to helpful videos as well. Knowing the difference between a qualitative and quantitative assay is crucial in this lesson because you will be combing both types on one plate. Qualitative assays give you a relative concentration of antibody or antigen present in your serum in comparison to other samples. Quantitative assays allow you to determine the exact concentration of antigen or antibody present in your sample. The Quantitative assay uses standards, which have known concentrations, to generate a graph against the obtained absorbance values. Absorbance is the ratio of incidence to transmission of radiant energy (light) through a material. These values are placed on a log scale graph (this is to present a wide range of data in a more compact area) to generate a standard curve like the one above. You can then use that curve as a reference to determine unknown sample concentrations when given the absorbance of your unknown sample by your plate reader. For example, if your unknown sample had an absorbance reading of 1.5 nm, your concentration would be about 10.5 ng/ml. 5 iv. Diagnosis of HIV/AIDS with the use of Flow Cytometry HIV stands for Human Immunodeficiency virus. It is a virus that attacks helper T- cells (Th cells), also known as CD4 T-lymphocytes, in your immune system. Th cells are also called CD4 T-cells because they have CD4 receptors on their plasma membrane surfaces. CD4 T-cells have roles in both the humoral and cell mediated immunity in the adaptive branch of the immune system as seen in figure 1 below. With lower levels of CD4 T-cells, the adaptive immune system will struggle to mount an appropriate antibody mediated response or cytotoxic T-cell response to new infections, leaving the infected individual more susceptible to opportunistic infections and cancer. https://www.intechopen.com/source/html/39824/media/image2.jpg Figure 1 The HIV virus is coated with a gp41 (glycoprotein-41) and gp120 (glycoprotein- 120) viral envelope complex. The looped domains on gp120 are recognized by the CD4 TCR (T-cell Receptor) and CCR5 (Chemokine co-receptor 5) or CXCR4 (chemokine co-receptor 4) (figure 2). When gp120 binds with CD4 and a co- receptor, conformational changes occur in the membrane proteins, which bring the virus toward the cell, allowing their membranes to fuse. Once membrane fusion occurs, the contents of the virus are released into the cell. Viral RNA is reverse-transcribed into DNA and gets integrated into the T-cells DNA. The T-cell then uses its own resources, such as ribosomes, nucleotides, amino acids, etc. to transcribe and translate viral DNA into necessary proteins, additional viral RNA, etc. to assemble more viruses.