Immunology- Mic : First observed by Gruber and Durham when serum antibody was found to react with bacterial cells.

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1 Immunology- Mic 451 Lab -Notes 1. Agglutination is the aggregation of particulate matter caused by the combination with specific antibody 1896: First observed by Gruber and Durham when serum antibody was found to react with bacterial cells. Agglutination occurs if an antigen is mixed with its corresponding antibody called isoagglutinin. This term is commonly used in blood grouping. This occurs in biology in three main examples: The clumping of cells such as bacteria or red blood cells in the presence of an antibody or complement. The antibody or other molecule binds multiple particles and joins them, creating a large complex. This increases the efficacy of microbial elimination by phagocytosis as large clumps of bacteria can be eliminated in one pass, versus the elimination of single microbial antigens. Another example occurs when people are given blood transfusions of the wrong blood group. Types of particles that participate in such reactions: Erythrocytes Bacterial cells Inert carriers such as latex particles Primary phenomenon (SENSITIZATION)

2 First reaction involving Ag-Ab combination Initial reaction: rapid and reversible Cross link formation visible aggregates (stabilization) Secondary phenomenon: (LATTICE FORMATION) stable network (visible reaction) Governed by physiochemical factors: Ionic strength of milieu ph temperature 2. HEMAGGLUTINATION Hemagglutination is the process by which red blood cells agglutinate, meaning clump or clot. The agglutin involved in hemagglutination is called hemagglutinin. In cross-matching, donor red blood cells and recipient's serum or plasma are incubated together. If agglutination occurs, this indicates that the donor and recipient blood types are incompatible DIRECT AGGLUTINATION Test patient serum against large, cellular antigens to screen for the presence of antibodies. Antigen is naturally present on the surface of the cells. In this case, the Ag-Ab reaction forms an agglutination, which is directly visible. PASSIVE AGGLUTINATION An agglutination reaction that employs particles that are coated with antigens not normally found in the cell surfaces

3 Particle carriers include: Red blood cells Polystyrene latex Bentonite Charcoal The latex particles are coated with IgG and mixed with the patient's serum REVERSE PASSIVE AGGLUTINATION Antibody rather than antigen is attached to a carrier particle For the detection of microbial antigens such as: Group A and B streptococcus Staphylococcus aureus Neisseria meningitidis Haemophilus influenzae PRINCIPLE: latex particles coated with antibody are reacted with a patient sample containing suspected antigen.

4 Latex Agglutination Antibody molecules can be bound to each latex beads It will increase the potential number of exposed antigen-binding sites. When an antigen is present in test specimen, it may bind to the latex bead thus forming visible cross-linked aggregates. Latex particles can be coated with antigen (pregnancy testing, rubella antibody testing) 3. Blotting Techniques What is blotting? Blots are techniques for transferring DNA, RNA and proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis. The Southern blot is used for transferring DNA, the Northern blot for RNA and the western blot for PROTEIN.

5 TYPES OF BLOTTING TECHNIQUES Blotting technique Southern Blot It is used to detect DNA. Northern Blot It is used to detect RNA. Western blot It is used to detect protein. This method Involves separation, transfer and hybridization. It is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. The DNA detected can be a single gene, or it can be part of a larger piece of DNA such as a viral genome. Southern blotting combines agarose gel electrophoresis for size separation of DNA with methods to transfer the size separated DNA to a filter membrane for probe hybridization. The key to this method is Hybridization. Hybridization - Process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target patient DNA.

6 Western blotting Western blotting (1981) is an Immunoblotting technique which rely on the specificity of binding between a protein of interest and a probe (antibody raised against that particular protein) to allow detection of the protein of interest in a mixture of many other similar molecules. The SDS PAGE technique is a prerequisite for Western blotting. Dot blot A dot blot (or slot blot) is a technique in molecular biology used to detect biomolecules, and for detecting, analyzing, and identifying proteins. It represents a simplification of the northern blot, southern blot, or western blot methods. In a dot blot the biomolecules to be detected are not first separated by chromatography. Instead, a mixture containing the molecule to be detected is applied directly on a membrane as a dot, and then is spotted through circular templates directly onto the membrane or paper substrate. This differs from the western blot because protein samples are not separated electrophoretically. This is then followed by detection by either nucleotide probes (for a northern blot and southern blot) or antibodies (for a western blot).

7 4. What Is An ELISA? It uses a 96-well plate to measure a protein or substance based on an antigen/antibody Analyte The sample being analyzed and in immunoassays the analyte is either Antibody or Antigen ELISA technique Is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. An ELISA test uses components of the immune system and chemicals to detect immune responses in the body (for example, to infectious microbes). The technique is divided into 1- Competitive ELISA 2- Sandwich ELISA (also called direct ELISA) 3- Indirect ELISA 1- Competitive ELISA The labelled antigen competes for primary antibody binding sites with the sample antigen (unlabeled). The more antigen in the sample, the less labelled antigen is retained in the well and the weaker the signal). 2- Sandwich ELISA (also called direct ELISA) Method ) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen;

8 (4) enzyme-linked secondary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme to detectable form. 3- Indirect ELISA Method The protein antigen to be tested for is added to each well of ELISA plate, where it is given time to adhere to the plastic through charge interactions. A solution of non-reacting protein is added to block any plastic surface in the well Then the serum is added, which contains a mixture of the serum antibodies, of unknown concentration, some of which may bind specifically to the test antigen that is coating the well. Afterwards, a secondary antibody is added, which will bind to the antibody bound to the test antigen in the well. This secondary antibody often has an enzyme attached to it A substrate for this enzyme is then added. This substrate changes colour upon reaction with the enzyme.

9 The colour change shows that secondary antibody has bound to primary antibody, which strongly implies that the donor has had an immune reaction to the test antigen. The higher the concentration of the primary antibody that was present in the serum, the stronger the colour change. spectrometer is used to give quantitative values for colour strength Indirect ELISA

10 5. SEROLOGICAL DIAGNOSIS IN IMMUNOLOGY METHODS OF DETECTION OF ANTIBODIES 1. Immuno-precipitation Assays = detect antibodies in solution = qualitative indication of the presence of antibodies = end-point is visual flocculation of the antigen and antibody in suspension 2. Complement Fixation = based on the activation or fixation of complement following binding of complement factors to Ag-Ab immune complexes 3. Neutralization = the ineffectivity of an organism or the activity of toxin is neutralized by specific antibody = rarely used for diagnostic purposes = mainly used to detect antibody formation after vaccination 4. Particle Agglutination = relatively simple and fast = capable of detecting lower concentration of antibodies = designed to detect antibodies to viruses, subsequent to interaction or vaccination = utilize Ag coated latex particles, coal particles, bentonite particles or erythrocytes 5. Immunofluorescence = requires use of microscope equipped to provide ultraviolet illumination or an instrument capable of irradiating the assay with UV light and detecting the resultant fluorescence with a fluorometer

11 6. Enzyme Immunoassay = the most sensitive = usually indirect assay that depends on the use of an antihuman IgG or IgM antibody conjugate 7. Radioimmunoassay = high sensitivity 6. What is an Epitope? An epitope is the small site on the antigen which is recognized by the antibody. Usually between one and six sugars or amino acids on the surface of the antigen. Monoclonal Antibodies Much more complicated to produce than polyclonal antibodies. Process begins by immunizing an animal (most commonly a mouse) with an antigen. The animal s spleen is removed. Monoclonal antibodies recognize one epitope only. Polyclonal Antibodies Produced by immunizing an animal with the appropriate antigen. The immunized animal s serum is collected. Antibodies can then be purified from the serum. Since one antigen induces the production of many antibodies the result is a polyclonal mixture of antibodies. Fluorophore Fluorophore is the region of the molecule capable of exhibiting fluorescence. Fluorochrome is a dye or molecule capable of exhibiting fluorescence

12 How Does Fluorescence Microscopy Work? Excitiation: Energy in the form of a photon of light is absorbed by a fluorophore. While in the excited state, the fluorophore undergoes conformational change. Emission: a photon of energy is emitted and the fluorophore returns to its original state. A single fluorophore can be repeatedly excited and detected. 7. THIN LAYER IMMUNOASSAY Background In formation Many antigens adsorbs firmly to the hydrophobic surface of polystyrene. The exposed antigenic determinants on the monomolecular film are fully reactive with antibody. Antibody bound to the antigen may be detected by fluorescence in ultraviolet light after reaction with fluorescein labeled anti-immunoglobulin. Thin-layer immunoassay (TIA) is a technically simple and accurate method for the assay of antigen-antibody reaction. The technique basically uses the principle that macromolecules may be absorbed as a thin layer to a polystyrene surface. These macromolecules most often preserve their serological reactivity and hence capable of binding antibodies. The presence of antibodies on the antigen surface is visualized as a distinct hydrophilic condensation pattern when surface is exposed to water vapor. TIA has been used for the determination of antibodies against purified proteins, polysaccharides, and lipopolisaccharides also proved suitable for screening of serum samples.

13 8. PHAGOCYTOSIS Background In formation Inflammation is one of the ways our body reacts to an infectious agents or injury. During the inflammatory response, white blood cells with multilobed nucleus, PMNs (Polymorphonuclear neutrophils) migrate out of capillaries and into the infected tissue. PMN attracted to the site of infection through a process known as chemotaxis. In Chemotaxis, phagocyctic cells move migrate towards the infected cite where phagocytosis begin. The phagocytic cells are PMNs, monocytes, and macrophages. Monocytes are white blood cells with one large nucleus. Monocytes circulate in our pheripheral blood for only about 1 day. They may eventually move into tissue where they mature into a cell called a macrophages. Macrophage may life for several month. PMNs and macrophage are the major phagocytic cells of the immune system. Phagocytosis is the process when phagocytes ingest, kill, and digest infectious organisms and unwanted cellular debris such as old cells or particulate matter. The particulate susceptible to the phagocytosis can be intracellular, such as Mycobacterium tuberculosis, or extracellular such as Streptococcus pneumonia. The intracellular pathogen are ingested by macrophage when they are released from a dying cell. 9. PRECITPITIN: Ring Test Background Information The ring test is one of the oldest methods for detecting soluble antigen and antibody. It is simple, quick, and reasonably sensitive for the detection of trace quantities of antigen, as used forensically to detect adulteration of food meats with our meat proteins. It is less sensitive for the detection of antibody. A good antiserum usually gives a ring test visible in minutes at a dilution factor 1:5.

14 The ring test is based on the propensity of antibodies to form complexes with their corresponding antigens. When antibodies attach to antigen molecules in solution, the molecules become part of an insoluble antibody-antigen complex, and a visible precipitate forms. Ring Test 10. IMMUNODIFFUSION : Radial Immunodifussion Background Information In Immunodiffusion tests, antibodies and or soluble antigens are loaded into separate wells of a gel and are allowed to difuse, each reagent moving radially into the gell. An immobile precipitate, visible as a band (precipitin line) in the gel, develops if specific antibody- antigen binding takes place, and if antibody-antigen components are present at optimal proportions. In radial immunodifussion, the antibodies are equally distribute, loaded into a well, diffuses. When antigens molecules move through the gel, they bind to and carry antibodies, until the ratio of antibodies are optimal for complex formation.

15 Procedure (Immunodiffusion) 1. Obtain 5 µl of human serum, and pipette it into designated wells of the RID assay gel. 2. Reserve the three vails for standard concentrations. High standards, low standard, and a serum control (Provided in the radial immune diffusion kit) 3. Place the gel into moist box, and incubate for 24 hours at room temperature to allow diffusion on and banding (The gel can be stored in refrigerator for several weeks, if the box is keep moist) 4. Read the result of your assay: Measure the diameter of the circle of precipitate (in centimeters) 11. Antigen & Antibody Reactions Affinity vs. Avidity Affinity Avidity Measure of the binding strength between an antigenic determinant (epitope) and an antibody combining site. It is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site. The cumulative binding strength of all antibody-epitope pairs which results from multivalent antigen and antibody.

16 Precipitation Serological Tests One of the easiest of serological tests Soluble AG & Ab interact and form a lattice that develops into a visible precipitate. Occur best when antigen and antibody are present in optimal proportions (Equivelance). Antibodies that aggregate soluble antigens are called precipitins. Polyclonal antibodies can form lattices, or large aggregates. However, monoclonal antibody can link only two molecules of antigen and no precipitate is formed. Simple Immunodiffusion Reactions The region of equivalence Diagrammatic representation of radial & double immunodiffusion. (Precipitation reactions in gels yield visible precipitin lines; no visible precipitate forms in regions of Ab or Ag excess.)

17 III- Electro-Immnodiffusion In electro-immunodiffusion, diffusion is combined with electrophoresis Electrophoresis separates antigen molecules according to differences in their electrical charges and molecular weight then specific antibodies diffuse and react with separated antigen forming precipitin bands.

18 a) Immunoelectrophoresis Method + Ags are separated by electrophoresis Ab is placed in trough cut in the agar Ag - Ag Ab Ag Ab Interpretation- Precipitin arc represent individual antigens Qualitative - Relative concentration c) Rocket Electroimmunodiffusion Antigen is electrophoresed into gel containing antibody. The precipitin reaction results in a rocket-shaped precipitin formation. The distance from the starting well to the front of the rocket shaped arc is related to antigen concentration.

19 IV-Measurement of Precipitation by Light Principle: Antigen-antibody complexes, when formed, will precipitate in a solution resulting in a turbid or cloudy appearance that can be measured by: Turbidimetry Passing light through a cloudy solution. (Net decrease in light intensity) Nephelometry Measuring light scattered at a particular angle after being passed through a solution i.e. indirect measure. Amount of light scattered correlates to the concentration of the solution Evaluation: Ag concentration is proportional to the turbidity of complexes (end point)