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Toolbox assignment for Immunology lab ...

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BIOL 4300 11. 21. 2016 Immunologist Toolbox

1. Haptens Small molecules of the immune system response to even though they are missing some of the other essential characteristics that define immunogens are called haptens. These molecules create antibodies because larger carriers bind to it and either a combined state or in the free state. This fools the immune system into believing that these molecules need to be addressed. A hapten will not elicit this response on its own. Without having these larger carriers, it would not have the same kind of effect (Anderson).

(Antigen Properties)

2. Routes of Immunization and Effects of Antigen Dose Routes of administration for immunization are typically oral, intramuscular, subcutaneous, or intradermal. The definition of a route of administration is “the path by which a vaccine is brought into contact with the body” (Route of Administration). An antigen is usually a protein, but sometimes it is a polysaccharide. These substances will create a specific immune response that forms into antibodies that are specialized, or a sensitized T cell which will affect the way in which the body responds to an intruder (Antigen Properties). The

route of administration of an immunization is critical because it activates a mechanism that will take the antigen dose to the right part of the body. Therefore, the right dose must be accompanied by the right type of administration in order for immunization to take place.

(Route of Administration)

3. Adjuvants Adjuvants are defined as material that is non-immunogenic by itself, but will enhance the immunogenic properties of any immunogen that is added. The theory is that adjuvants are able to act either in combination or alone to signals zero, one, or two in order to initiate 𝑇! cell responses. Bruno Guy states that type A adjuvants act on signal zero, but also indirectly on signal two. These are called toll – like receptor agonists. Type B act on signal one because they have an effect that is mediated by the enhancement to AG presentation into T cells. Some emotions are in in this category as can be liposomes and microspheres

(Guy)

4. Affinity chromatography Affinity chromatography is the process of separating biochemical mixtures based on the specific interaction between antigens and antibody, receptors and lagoons, or enzymes and substrate. Previous knowledge must be understood about the way these materials will react. In the example of a ligand, materials are isolated and must be able to bind in reversible manner to a specific ligand. The reason for using Affinity chromatography is that it offers a high level of selectivity and capacity for protein purification schemes. This technique is the only one that provides an opportunity to utilize the biological structure of a protein or the way in which it functions for the purpose of purification. Without this technique, purification would be both time-consuming and overly complicated.

(Affinity Chromatography)

5. Equilibrium dialysis: measurement of antibody affinity and avidity Affinity can be defined as the strength and antibody has been binding a monovalent ligand to a site with a single antigen-binding. Equilibrium dialysis can be defined as determining the ability of an antibody to bind small antigens, for example haptens, so that they may freely defuse across the membrane of dialysis. Affinity is a measure of that an antigenic determinant will have in terms of its binding to the single antigen-binding site. However, the avidity is a measurement of strength of the binding of an antibody to an intact antigen. The use of equilibrium dialysis help determine how much strength exists within antibody affinity and avidity (Janeway and Travers).

(Janeway and Travers)

6. Coombs tests and the detection of Rhesus incompatibility A Coombs test and working toward the detection of Rhesus incompatibility is conducted through using anti-immunoglobulin antibodies the purpose of finding antibodies that will create hemolytic disease in newborns. Robin Coombs was the first to develop anti-immunoglobulin antibodies and design the test that is still referred to as the Coombs test. Hemolytic disease of the newborn happens when a mother’s body is creating IgG antibodies for the Rh blood group antigen that is then transferred to the red blood cells in her fetus. Mothers who are Rh negative make antibodies when they are exposed to Rh positive red blood cells from the fetus. This comes from a paternally inherited antigen for Rh. Fetal red blood cells are destroyed when the anti-Rh antibodies and become destroyed within the

phagocytic cells within the liver, which causes hemolytic anemia in the fetus and newborn infant (Janeway and Travers).

(Janeway and Travers)

7. Monoclonal Antibodies and Phage display libraries for antibody V-region production Monoclonal antibodies from identical immune cells that are cloned from a unique parent cell. The process was created by Georges Köhler and Cesar Milstein who created a technique that they used for taking mouse cells in order to

generate a population of homogenous antibodies. This was accomplished by fusing spleen cells from a mouse who had been immunized to cells from mouse myeloma for the purpose of creating hybrid cells that could proliferate indefinitely and secrete the specific antibody that was used as an antigen to immunize the donor of the spleen cells. Phage display libraries for antibody V – region production is a technique used in producing molecules that are like antibodies. Gene segments are fused to gene encoding that is on the coat protein of the bacteria phage and are then used to infect bacteria, which creates coats on the phage particles that are antibody like fusion proteins. A collection of recombinant phage which show varieties of antigen-binding domains on the surface are known as the phage display library. This provides an opportunity for isolating selected phage for binding to the specific antigen (Janeway and Travers).

(Adenosine)

8. Immunofluorescence microscopy Immunofluorescence microscopy is a method that is used to determine localization and end of genus expression levels where researchers have taken interest in certain proteins. This is an example of immunostaining and a form of

immunohistochemistry which uses fluorophores for the purpose of being able to see the location of bound antibodies. There are several things that must be considered before using this method. This includes the “nature of the antigen, specificity and sensitivity of the primary antibody, properties of the fluorescent label, permeabilization and fixation technique of the sample, and fluorescence imaging of the cell” (Immunofluorescence Microscopy). This technique can be used on individual cells, cultured cells, or tissue sections as long as they are fixed in some method. These antibodies are used as a way of analyzing the distribution proteins and antigen targets, as well as glycoproteins (Immunofluorescence of Microscopy).

Example of Monoclonal Antibody as seen through Immunofluorescence Microscopy technique (Immunofluorescence Microscopy)

9. Immunoprecipitation and Immunoblotting Immunoprecipitation is used for small-scale affinity purification of antigens for a specific antibody so that it can be immobized onto a solid support. This may be magnetic particles or the use of an agarose resin. This is one of the most commonly used methods for the purpose of isolating proteins so they can be detected by Western blotting or other techniques for assay. The technique was originally developed from traditional column affinity chromatography. However, a much smaller amount of resin is put into microcentrifuge tube which is then incubated with beads to create a slurry that will eventually be removed after centrifugation through the use of a pipet (Overview).

Immunoblotting is an immunoenzymatic technique in which a nitrocellulose filter is used for antigenic support with the intention of creating a reaction with viral proteins that had been previously transferred. An A peroxidase protein is use to detect to the specific antibodies. This allows for a researcher to find reactivity in antibodies within a serum that can be compared against different proteins. The technique is effective in serological diagnoses and can confirm a variety different illnesses (Sanchez-Vizcaino, Mur and Arias)

(Sanchez-Vizcaino, Mur and Arias)

10. Use of antibodies in the isolation and identification of genes and their products In this process, the use of genes that are encoded for a particular protein are isolated. This allows for a purified protein cell to be observed for the antibodies that are specific to that protein through affinity chromatography. Amino acid sequencing takes place that is gathered from the amino terminal end of the protein or from the peptide fragments that come from the proteolysis. The amino acid sequences provides for the construction of synthetic oligo-nucleotides which can give information that suggests DNA sequences. These are used as a method of isolate the encoding to produce an answer to the problem (Janeway and Travers).

(Janeway and Travers)

11. Flow cytometry and FACS analysis Flow cytometry is a powerful tool for numbering and defining lymphocytes. This procedure finds and counts individual cells as they pass through stream that comes from a laser beam. The flow cytometer separate and identify cells through a tool that is called a fluorescence activated cell sorter, or FACS. This provides a method of identifying properties of cell subsets in monoclonal antibodies that are used by cell surface proteins. Specific monoclonal antibodies tag individual cells in a mixed population through labels from fluorescent dyes or through antibodies that are labeled with anti-immunoglobulin antibodies and then forced through a large volume of saline which creates a stream of liquid that has cells and at that are spaced singly at intervals. The dyed molecules excite and fluoresce, giving information to the researcher (Janeway and Travers).

(Janeway and Travers)

12. Biosensor assays for measuring the rates of association and disassociation of antigen receptors for their ligands Biosensor assays measure the amount of association and disassociation of ligands to matching antigen receptors. A gold-plated surface is used on which to rest the bounding ligand so that the T cell receptor will contact the ligand and allow for binding, which will later be observed disassociating. The time between Association and disassociation are measured through the use of the biosensor. Because of the challenging nature of T cells, studying membrane-bound T cell receptors can be very difficult. However, the biosensor assays provide for an opportunity to study these activities (Reina).

(Yakimchuk)

13. Stimulation of lymphocyte proliferation by treatment with polyclonal mitogens Polyclonal mitogens incite lymphocytes to proliferate. They create mitosis in lymphocytes from a variety of different specificities and clonal origins. Polyclonal mitogens can create the same results by affecting growth response mechanisms as antigen. The treatment of lymphocytes for proliferation by polyclonal mitogens as the stimulants can assess how lymphocytes are behaving in patients who have immune-deficiencies when given nonspecific stimuli.

Finding the antigen – specific T cell proliferation in a culture means optimizing the response of lymphocytes to polyclonal mitogens.

(Janeway and Travers)

14. Measurements or apoptosis by the TUNEL assay TUNEL staining or apoptosis by TUNEL assay is a process through which DNA fragments are dyed at the enzyme terminal deoxynucleotidyl transferase (TdT). The streptavidin, which is enzyme tagged, then binds to the biotin which creates a detectable biotin label. A reaction between a substrate of the enzyme, which is colorless, and the tagged streptavidin will indicate whether or not cells have gone through the process of apoptosis (APO-BrdU TUNEL Assay).

(APO-BrdU TUNEL Assay)

15. Assays for cytotoxic T cells and CD4 T cells

Cytotoxic T cells are usually detected through a Cr-release assay. Live cells will not usually spontaneously release radioactive labeled sodium chromate, but they will usually take them up. The radioactive chromate is released when the cells are killed and will be observable in supernatant mixtures of both cytotoxic T cells and target cells which can then be measured. When DNA is attacked by a cytotoxic T cell, the target cells will fragment rapidly and can be then retained on a filtrate while on fragmented DNA is collected on the filter. Measurement can be taken of either the fragments that are released are those that are retained of the H – thymidine in chromosomal DNA. This creates a method of quickly measuring activity of the cytotoxic T cells.

(Janeway and Travers) Assays for CD4 T cells include cytokines which are released when the T cell recognizes antigen. CD4 T cell function can be studied when it is possible to measure the kind and amount of release proteins that are the site again. Therefore, learning about the effector potential of a T cell can be done through measuring the amount of protein it produces. Biological assays provide for a method of measuring the activity of cytokines through modification of ELISA called a capture or sandwich ELISA. This type of assay provides an ability to create a bridge between two monoclonal antibodies so that they react in relationship to epitopes that can be found on the site it can molecule. The ELISPOT can detect cytokine secreting cells. The variance to both ELISA and ELISPOT is that it avoids the problem of different side of cytokines responding to different stimuli in the same way which can often be found in a bioassay.

(Cytokine ELISA Plate Arrays)

16. DNA microarrays DNA microarrays were developed in the 1990s. They allowed for studying transcriptome, which means studying cells as they are active in real time. The technology exploited a principal that is very similar to the reasons that nucleic acid is essential to information storage, to the hybridization in terms of complementary sequences. The basis of the development of the DNA microarray is that hybridization that occurs between a cDNA that is taken from a biological sample and applied to a predesigned complementary DNA probe and then arranged on a slide or array. Therefore, a pre-designed library can be achieved through the development of synthetic nucleic acid probes which have been immobilized and then spatially placed into an array that exists on a solid matrix. Micro arrays come from Southern blotting, which is a technique where DNA fragments are placed into a substrate and then examined with a known gene sequence (Grigoryev).

(Grigoryev)

17. Assessment and transfer of protective immunity A population can be assessed for the number of new infections and the severity of those infections in order to examine how immunization has spread. The efficiency with which a vaccine has eradicated an illness can be assessed through studies that evaluate quantitative information that indicates how immunity has been transferred. Transfer of protective immunity occurs as body fluids transfer the immunity of one host to another host. Immunity only takes place as long as antibodies are active in the body into which they were transferred. However, there are consequences to transfer of immunity including a temporary immunity or even a problem of being allergic to the serum (Janeway and Travers).

(Janeway and Travers)

18. Testing for Allergic Responses Testing for allergy or allergic responses takes place as allergens are introduced to the skin or blood in order to see if an allergic response is triggered. The most rapid and reliable form of test is a skin test, which is actually less expensive than blood tests although either are effective. A skin prick test takes place as scratches or needle pricks are used in order to allow solutions that have possible allergens to penetrate the skin. Wheals are the reactions that the skin may have becomes red, raised and itchy. This means that person is usually allergic to that allergen. This be considered a positive reaction. An intradermal

test takes place when the allergen is actually injected into the skin. This occurs when an allergen did not create a reaction to the skin prick test, but it is still suspected to be causing allergic reactions. In addition, a skin patch test takes place when a solution with an allergen is put on a pad and taped to the person for between 24 and 72 hours. The intention is to seek out whether or not there is contact dermatitis. In blood tests, antibodies or search for to see if the blood is trying to fight off the allergen. The most common type of blood test for allergies is an immune assortment assay, which is either a ELISA or EIA (Allergy Tests).

(Rare Diseases)

19. Hematopoietic cell transfers Lymphocytes can be used to replace an entire hematopoietic system through the transfusion of other bone marrow or stem cells that come from another source. Clinical models have begun to show that the repopulation of stem cells can be done across different types of species. As an example, in a study evaluated by Vollweiler, Zielske, Reese and Gerson, it appeared that murine oncoretroviruses and lentiviruses to be controlled where immunodeficiency is caused issues and patient were T cell chimerism was shown to be dramatic. Chimerism occurs as when the DNA of two separate entities are present in one body.

(Encyclopedia Britannica)

20. In vivo depletion of T cells and B cells In vivo depletion of T cells is a serious problem because it means that there are no T cells in order to increase immunity. Studies in mice have provided information about the importance of T cells in the way that they can be reproduced through a variety of different treatments. By depleting the T cells, the study of their function has been enhanced. However, mice do not have a single site of B cell development. Therefore, birds have been used in order to study depletion of B cells. The absence of gamma globulin’s appear to be most affected by the viruses that require the presence of B cells (Janeway and Travers).

(Schloot).

21. Transgenic and Knockout Animals Transgenesis takes place when a gene is cloned and introduced into the species through micro-injection into a male pro-nucleus within a fertilized egg. This egg is then implanted into the uterus of a species for the purpose of creating an animal that has an extra genetic element. This extra genetic element is called the transgene. This is most often done with mice. When an animal has mutated, the functions of the gene are better understood when the gene is not expressed. Therefore, a gene knockout takes place when a normal gene is identified and isolated and then replaced in vivo with a copy that is defective. This provides a method of evaluating and assessing a gene that is inactive (Janeway and Travers)

(Janeway and Travers)

Works Cited “Affinity Chromatography”. Biotechniques Den, n.d. Web. 10 November 1016. “Allergy Tests”. WebMD, n.d. Web. 12 November 2016. Anderson, William L. Immunology. Madison Conn: Fence Creek Publishing,

1999. Print.

“Antigen Properties, Types, and Determinants of Antigenicity”. Microbiology Info.,

2016.

Web. 11 November 2016. “APO-BrdU TUNEL Assay”. Phoenix Flow Systems, 2016. Web. 12 November

2016.

“Cytokine ELISA Plate Arrays”. Signosis, 2016. Web. 12 November 2016. Encyclopedia Britannica, 2014. Web. 12 November 2016. Guy, Bruno. “Where do Adjuvants Act?” Nature Reviews, 5 (July 2007): 505-517. Grigoryev, Yevgeniy. “Introduction to DNA Microarrays”. Bite Size Biology, 12

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