Single Radial Immunodiffusion
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This document describes single radial immunodiffusion (RID), a quantitative technique used to determine the concentration of an antigen in a sample. RID involves incorporating specific antibody into an agarose medium with a central well for the antigen sample. The antigen diffuses radially and reacts with the antibody, forming a visible precipitate ring whose diameter relates to the antigen concentration. The technique is simple, cost-effective, and can quantify immunoglobulins, complement components, or other antigens from biological fluids by measuring ring diameters against a standard curve.
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Immunodiffusion principles and application
Immunodiffusion techniques such as radial immunodiffusion, Ouchterlony double diffusion, and immunoelectrophoresis can be used to detect and quantify antigens and antibodies through the formation of precipitin lines. These techniques utilize the diffusion of antigens and antibodies through a semi-solid medium like agar to form visible precipitin lines where the antigens and antibodies combine. They can be used to diagnose diseases, detect immunodeficiencies, and assess the purity and concentration of antigens and antibodies.
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Haptens
Haptens are small molecules that are antigenic but not immunogenic on their own. They are unable to induce an immune response because they cannot activate helper T cells due to their inability to bind MHC proteins or activate B cells directly as they are univalent. However, when haptens are covalently bound to a carrier protein, they form immunogenic conjugates that can induce an immune response by activating helper T cells and B cells. Pioneering work by Karl Landsteiner demonstrated that antibodies produced against hapten-carrier conjugates were specific for the hapten and carrier epitopes. Common examples of haptens include drug molecules, peptides, and steroids. Hapten-protein conjugates can cause drug
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Immunoelectrophoresis
Immunoelectrophoresis is a technique that combines electrophoresis and immunodiffusion to separate and characterize proteins based on their charge and reaction with antibodies. It involves electrophoresing an antigen mixture to separate components by charge, cutting troughs in the gel for antiserum, and detecting lines of precipitation where antibodies and antigens meet. Immunoelectrophoresis is used qualitatively in clinical laboratories to detect the presence or absence of proteins in serum and identify normal and abnormal proteins. It can detect immunodeficiencies or overproduction of proteins but is limited for quantitative analysis.
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[IMMUNOLOGY LAB REPORT] Antigen - Antibody Precipitation Reaction
[IMMUNOLOGY LAB REPORT] Antigen - Antibody Precipitation Reaction
Usage of Immunological reactions in diagnostics of infectious diseases.
Usage of Immunological reactions in diagnostics of infectious diseases.
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Spectrophotometric Instruments (Detector) and Application of UV – VIS spectro...
serological techniques for detection of plant virus.pptx
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Haematology Myloma.
Multiple myeloma is a malignant plasma cell disorder characterized by a monoclonal paraprotein, bone lesions, and excess plasma cells in the bone marrow. It has an incidence of approximately 50 cases per million population and occurs more commonly in black individuals and males. The cause is unknown but involves abnormal plasma cells secreting an immunoglobulin. Symptoms include bone pain, bone marrow failure, infections, and renal failure in some cases. Laboratory tests show anemia, elevated sedimentation rate, paraprotein, increased or abnormal free light chains, and lytic bone lesions on imaging. Related disorders include monoclonal gammopathy of undetermined significance, solitary plasmacytoma, plasma cell leukemia, and amyloidosis.
Inherited disorders of coagulation
Inherited disorders of coagulation can cause excessive bleeding due to defects in coagulation proteins. The most common is hemophilia A, which is caused by a deficiency in clotting factor VIII due to its location on the X chromosome. Symptoms range from severe spontaneous bleeding to mild symptoms depending on factor VIII levels. Hemophilia B is less common but similar, involving a factor IX deficiency. Von Willebrand disease results from mutations of the von Willebrand factor gene and causes bleeding from mucous membranes or following trauma due to defective platelet function and reduced clotting factor VIII transport. Rarer deficiencies involve factors II, V, VII, X, XI, and XIII.
Platelet disorders Haematology
This document discusses disorders of haemostasis related to platelets. It describes thrombocytopenia, which can be congenital or acquired. Congenital forms include conditions like thrombocytopenia with absent radii syndrome. Acquired causes include deficient platelet production or accelerated platelet destruction. Autoimmune thrombocytopenia is described where autoantibodies coat platelets, causing their premature destruction. Alloimmune thrombocytopenia can occur from transplacental passage of antibodies or after blood transfusion. Other causes of thrombocytopenia discussed include disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, and disorders of platelet function.
Haematology Mature lymphoid neoplasms
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Haemostasis introduction
This document provides an overview of haemostasis (the process of stopping bleeding from damaged blood vessels). It discusses the key components involved - the vessel wall, platelets, and coagulation factors - and how they interact. The vessel wall normally prevents clotting but releases substances upon injury that activate platelets and the coagulation cascade. Platelets form a primary plug which is stabilized by fibrin formed from coagulation factors. Tests are used to identify defects in haemostasis involving vessels, platelets or coagulation.
Immuology HUMAN CHORIONIC GONADOTROPIN Test (HCG)
Human chorionic gonadotropin (hCG) is a hormone produced during pregnancy that can be detected in blood and urine. HCG testing can detect pregnancy as early as 5-7 days after conception and should become negative again within 3-4 days after delivery. Elevated hCG levels can also indicate certain tumors. The one-step HCG urine pregnancy test uses immunochromatographic technology to detect the presence of hCG and determine if a person is pregnant based on whether one or two colored lines appear on the test strip within 10 minutes.
Immuology Injection in Lab Animals
This document discusses various injection methods used to immunize laboratory animals. It describes how injections are used to immunize animals against diseases, produce polyclonal and monoclonal antibodies, and for vaccine research. Common laboratory animals like mice and rabbits are used due to their specific weights, ages, and genetic purity. The document outlines different injection routes including intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, inhalation, intranasal, and oral administration. For each route, it provides details on the injection site and procedures to correctly administer the injection and avoid injury to organs or nerves.
Immuology Phagocytosis activity
Phagocytosis is the process by which immune cells called phagocytes engulf and destroy microorganisms and foreign particles. The most important phagocytes are neutrophils, macrophages, and dendritic cells.
The nitroblue tetrazolium (NBT) assay is a microscopic method used to determine the phagocytic activity of cells by measuring their ability to reduce yellow-colored NBT to a blue insoluble form through the production of superoxide during phagocytosis. The assay involves incubating blood or cells with NBT, then examining stained smears under a microscope to count the percentage of cells containing blue deposits, indicating NBT reduction and normal phagocytic function.
The NBT assay can be used to
Assessment of Circulating WBC
This document discusses the assessment of circulating white blood cells (WBCs) through WBC counting and differential analysis. It describes the five main types of WBCs and the purposes and procedures for WBC counting and differentials. WBC counting involves diluting a blood sample, loading it into a hemocytometer chamber, and counting the cells in specific areas to calculate the total WBC count per microliter. Differentials determine the percentage of each WBC type by staining and manually counting 100 white blood cells on a blood smear slide. Together these tests can help diagnose conditions that affect WBC numbers or types.
Indirect-ELISA in immunology
The document summarizes the indirect enzyme-linked immunosorbent assay (indirect-ELISA) method. It describes the principle of indirect-ELISA which uses an unlabeled primary antibody and an enzyme-conjugated secondary antibody. The general procedure involves coating a plate with antigen, blocking remaining sites, incubating with primary antibody, secondary antibody, adding a substrate to produce a detectable signal, and interpreting the data either qualitatively or quantitatively. The indirect-ELISA method has advantages of high sensitivity and specificity but requires multiple incubation steps and potential for cross-reactivity.
Antigen and Antibody reactions
This document discusses antigen-antibody precipitation reactions and double immunodiffusion techniques. Precipitation occurs when soluble antigen and antibody combine to form insoluble complexes called precipitins. Double immunodiffusion, or the Ouchterlony technique, involves placing antigen and antibody samples in wells in an agar plate where they diffuse and form visible precipitation lines if they react. The pattern of lines can indicate if antigens are identical, partially identical, or unrelated. This technique is used to detect, identify, and compare antigens for applications like disease diagnosis.
Cell-mediated immunity (CMI)
This document discusses cell-mediated immunity (CMI), which is mediated by T lymphocytes and is responsible for detecting and destroying intracellular pathogens like viruses or infected cells, graft rejection, and destroying tumor cells. CMI results in a delayed-type hypersensitivity reaction. Upon first exposure to an antigen, naive T-cells are sensitized and proliferate. Secondary exposures result in a faster and more effective response from memory T-cells. Lymphokines released by activated T-cells mediate immune responses. A positive tuberculin skin test indicates past tuberculosis infection. CMI differs from humoral immunity in its mechanisms, antigens recognized, and hypersensitivity responses.
Primary and Secondary Immune Responses
The document discusses primary and secondary immune responses. The primary response occurs during first-time exposure to an antigen, when the immune system must learn to recognize and make antibodies against it. The secondary response occurs upon re-exposure, when immunological memory has been established and antibodies are produced more quickly. Key differences are that the secondary response has a shorter lag time, higher antibody levels, and antibodies with greater affinity compared to the primary response.
Immuology
IgM represents about 10-15% of total serum immunoglobulins. It is the first antibody produced during a primary immune response and is responsible for agglutination, viral neutralization, and complement activation. IgM exists as a pentamer with 10 antigen binding sites, making it very efficient at combating pathogens early in the immune response.
IgA represents 15-20% of total circulating immunoglobulins. It exists as a monomer or dimer and is found predominantly in mucosal secretions like tears, sweat, saliva and gastrointestinal tract secretions. Secretory IgA provides local immunity by protecting mucous membranes from invading microorganisms.
IgD represents about 0.25%
Structure Function Relationship of Antibodies
1) Antibodies possess both an antigen binding capacity and a biological activity. Digestion with enzymes like papain and pepsin can yield fragments that retain one or the other function.
2) Papain digestion yields two Fab fragments that retain antigen binding and an Fc fragment that retains biological activity. Pepsin digestion yields an F(ab')2 fragment with two antigen binding sites and degrades the Fc fragment.
3) The major classes of antibodies in mammals are IgG, IgM, IgA, IgD, and IgE, which differ in their heavy chain constant regions. IgG is the most abundant in serum and provides a major defense against bacteria, viruses, and parasites through mechanisms like
Immuology
The document summarizes key aspects of immunoglobulins (antibodies):
- Antibodies are large Y-shaped glycoproteins produced by plasma cells that bind specifically to antigens. There are five classes of human antibodies that share a basic structure but vary to perform specific functions.
- Antibody molecules contain two identical heavy chains and two identical light chains. Light chains can be kappa or lambda type. The variable regions of the heavy and light chains give antibodies their antigen specificity.
- Each antibody contains two antigen binding fragment (Fab) regions and one crystallizable fragment (Fc) region. The Fab binds antigens while the Fc mediates immune responses through binding to other immune molecules and cell receptors.
Immunoglobulin Structure
Immunoglobulin or antibody molecules have a basic Y-shaped structure composed of four polypeptide chains: two identical heavy chains and two identical light chains. Each chain contains a constant region and a variable region. The variable regions from the heavy and light chains fold together to form the antigen-binding site. There are five classes of heavy chains (gamma, alpha, mu, delta, epsilon) that determine the five immunoglobulin classes (IgG, IgA, IgM, IgD, IgE). Each immunoglobulin class has different properties, distribution, and roles in the immune system.
B Lymphocytes
B lymphocytes differentiate into plasma cells that secrete antibodies. The development of mature B cells from pre-B cells occurs independently of antigens. However, the activation of B cells into plasma cells that produce antibodies is dependent on antigens. Mature B cells have surface receptors that allow specific antigens to bind to them, which then causes the B cells to differentiate into plasma cells that secrete antibodies with the same specificity. T helper cells are involved in the process of antibody class switching by B cells. In addition to antibody production, B cells also function as antigen presenting cells.
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Single Radial Immunodiffusion
- 1. Practical Immunology And Serology Single Radial Immunodiffusion By Ahmed Riyadh Abdul Rahman Al-Noor University College 1
- 2. Single Radial Immunodiffusion Radial immunodiffusion (RID) or Mancini immunodiffusion (Mancini method) or single radial immunodiffusion assay. Is a type of precipitation reaction (immunodiffusion technique). RID is a sensitive quantitative technique used to determine the quantity or concentration of an antigen in a sample. The technique is very simple and cost-effective; however, it may require a long incubation time. 2
- 3. Radial immunodiffusion is based on a technique using a precipitin reaction in which specific antibody is incorporated into agarose medium; sample containing the test antigen is placed in a well centered in the agarose. Antigens sample then diffuse through the agar in all directions from the well and react with the antibody forming visible precipitate or a precipitin ring. Ring shaped bands of precipitates from concentrically around the well indicating Ab-Ag reaction. The diameter of the resulting precipitin zone is related to the concentration of antigen placed in a well, the greater the antigen concentration the larger the circle of precipitation. 3 PRINCIPLE
- 4. 4 Fig: Single Radial Immunodiffusion
- 5. Objective and Application To determine the quantity or concentration of an antigen/antibody in a sample. Quantitation of immunoglobulins, complement components, and, any antigenic protein, in any biological fluid. Estimation of the immunoglobulin classes in sera. To compare properties of two different antigens. To determine the relative purity of an antigen preparation For disease diagnosis Serological surveys 5
- 6. PROCEDURE 6 1. An agar containing an appropriate antiserum (antibody) is poured in plates. 2. Carefully circular wells are cut and removed from the plates. 3. A single or series of standards containing known concentration of antigen are placed in separate wells, while control and “unknown” samples are placed in other remaining wells. 4. As the antigen diffuses radially, a ring of precipitate will form in the area of optimal antigen – antibody concentration. 5. The ring diameters are measured and noted. 6. A standard curve is prepared using the ring diameters of the standards versus their concentrations. This curve is then used to determine the concentration of the control and unknown samples.
- 7. 7
- 8. 8 Result and observation 1. The presence of a precipitin ring around the antigen wells indicates specific antigen-antibody interaction. 2. Absence of precipitin ring suggests absence of reaction. 3. The greater the amount of antigen in the well, the farther the ring will form from the well.
- 9. INTERPRETATION OF RESULTS 9 • The precipitin rings will be visible in (24 to 48) hours. • Carefully hold a plate up so that the overhead room lights shine through it. • You should be able to see opaque circles around each well where antigen and antibody have precipitated. • With a ruler, measure the diameter (through the centers of the wells) of the precipitin ring in millimeters. • To plot the standard curve, square the diameter value and plot antigen concentration on the X-axis and the diameter squared on the Y-axis. • Draw the best fit line through these points. Calculate the value of the unknown antigen concentration from this graph.
- 10. 10
- 11. THANK YOU 11