Counter Current Immunoelectrophoresis
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Immunodiffusion -Different Types,Principle,procedureand application. it is a diagnostic technique for the detection or measurements of antibodies and antigens by their precipitation which involves diffusion through a substances such as agar or gel agarose .common types -oudin procedure,oakley fulthorpe procedure ,mancini technique ,ouchterlony double immuno diffusion
Counter immunoelectrophoresis
Counter immunoelectrophoresis (CIEP) is a laboratory technique used to detect antigen-antibody binding by facilitating the rapid migration of antigens and antibodies towards each other using an electric current in an agar or polyacrylamide gel. CIEP is similar to Ouchterlony immunodiffusion but uses electrophoresis to speed up the process, forming a precipitin line within 30-60 minutes if binding occurs. CIEP is commonly used to detect antigens and antibodies in serum to diagnose infectious diseases like hepatitis B and detect antigens in body fluids like cerebrospinal fluid. The procedure involves placing antigen in one well and antiserum in another on an agarose gel slide, applying an electric current,
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This document provides information on Ouchterlony double diffusion and radial immunodiffusion techniques. Ouchterlony double diffusion involves placing antigen and antibody in wells in an agar gel plate and observing the interaction over 24-48 hours. Possible results are identity, non-identity, or partial identity based on fusion of lines. Radial immunodiffusion is similar but incorporates antibody into the gel and antigens diffuse outward, forming measurable circular precipitin rings. Both techniques are used to detect and quantify the presence of antigens and antibodies.
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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 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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Agglutination
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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.
Complement fixation tests
Complement fixation tests (CFT) detect antibodies that do not agglutinate or precipitate by measuring their ability to fix complement. CFT involves incubating patient serum with antigen and complement, then determining if complement is still available to lyse indicator cells. If complement is fixed in the antigen-antibody complex, it cannot lyse the indicator cells, indicating antibody presence. CFT can detect antibody levels below 1 microgram/mL, but it is time-consuming and not sensitive enough for immunity screening due to occasional nonspecific reactions. Interpretation involves whether indicator cell lysis occurs, indicating the absence or presence of antibodies in the patient serum.
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Immunoelectrophoresis is a technique that combines electrophoresis and immunodiffusion to separate and identify protein antigens in a sample. The sample is first separated into individual protein components via electrophoresis in an agar gel. Antibodies are then placed in troughs cut parallel to the electrophoretic migration. As the separated antigens and antibodies diffuse toward each other, visible precipitin lines or arcs form where antigens and antibodies interact, allowing identification of different proteins present in the original sample. Immunoelectrophoresis is useful for protein identification, quantification, and detection of abnormalities.
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This document discusses various precipitation reactions and immunological techniques used to detect antigens and antibodies, including: Ouchterlony double immunodiffusion, single radial immunodiffusion, immunoelectrophoresis, and rocket electrophoresis. It explains that precipitation reactions involve two soluble reactants forming an insoluble precipitate. These techniques use diffusion and electrophoresis of antigens and antibodies in semi-solid media like agar to form visible precipitin lines, rings, or rockets, allowing detection and sometimes quantification of proteins. The techniques have various applications in medicine and clinical laboratories.
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This document discusses antigens, haptens, and adjuvants. It defines antigens as molecules capable of inducing an immune response, and notes they are usually proteins or polysaccharides. Haptens are small molecules that are not antigenic alone but can become antigenic when bonded to a carrier molecule. Adjuvants are substances that can enhance the immune response to antigens when mixed with a vaccine. The document provides examples and mechanisms of action for each.
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To learn and contribute in the professionally challenging environment in a manner that certainly benefits for man kind and enhances my skills
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This document discusses key concepts in immunology including antigens, immunogens, epitopes, haptens, innate immunity, and adaptive immunity. It defines antigens as molecules recognized by the immune system and immunogens as antigens that elicit an immune response. Epitopes are the smallest part of an antigen recognized by B and T cell receptors. Haptens are small molecules that require a carrier to induce an immune response. Innate immunity provides the first line of defense using soluble proteins and cells like phagocytes. Adaptive immunity develops over time through T and B cell responses and produces immunological memory.
Agglutination
1) Agglutination tests detect antigens or antibodies by exploiting the ability of antibodies to cross-link antigen-coated particles, forming visible clumps or lattices.
2) There are several types of agglutination tests including direct, passive, and reverse passive agglutination as well as hemagglutination and hemagglutination inhibition.
3) Agglutination tests are useful, rapid techniques for detecting various infectious diseases and other analytes but can be limited by prozone effects at high antibody concentrations.
Immunological techniques
Immunological techniques use antigens and antibodies to detect pathogens or their components in patient specimens. Agglutination tests couple antigens or antibodies to particles and look for cross-linking and agglutination. Complement fixation tests measure complement-consuming antibodies by incubating specimens with complement and antigens. Enzyme immunoassays like ELISA use enzyme-linked antibodies to detect antigens and quantify antibodies. Precipitation tests look for visible precipitation of antigen-antibody complexes to detect antigens or antibodies.
Ouchterlony double diffusion
ODD is a immunodiffusion technique is used in detection, identification and quantification of antibodies and antigens. (Analyzing the antigen and antibody)
Immunofluorescence technique
Immunofluorescence is a technique that uses antibodies conjugated with fluorescent dyes to visualize specific antigens under a microscope. It works by binding fluorescent antibodies to target antigens, which then glow when exposed to ultraviolet light, allowing visualization of the antigen's distribution. There are two main types: direct immunofluorescence uses a single fluorescent antibody, while indirect uses an unlabeled primary antibody followed by a fluorescent secondary antibody for signal amplification. Immunofluorescence has various applications like identifying proteins in cells and tissues and is useful for diagnosing autoimmune disorders.
Agglutination
Agglutination is the clumping together of antigens and antibodies. It occurs when the antibodies bind to particulate antigens. This causes the antigens to crosslink and form visible aggregates. Common applications of agglutination tests include blood typing (ABO and Rh), diagnosis of typhoid (Widal test), and identification of antibodies against Rh antigens (Coombs test). The titer or end point of an agglutination test refers to the highest dilution at which antigen-antibody clumping is still visible.
Monoclonal and Polyclonal Antibodies
This document discusses monoclonal and polyclonal antibodies, including their production and uses. Monoclonal antibodies are produced from a single clone and recognize a single epitope, while polyclonal antibodies recognize multiple epitopes of an antigen. Monoclonal antibodies are produced via cell fusion and screening of hybridomas, while polyclonal antibodies are produced by injecting animals with antigens to elicit an immune response. Both have advantages and disadvantages for diagnostic and therapeutic applications.
Antigenic determinants
There are three major categories of antigenic determinants on immunoglobulin molecules: isotypic, allotypic, and idiotypic determinants. Isotypic determinants distinguish each antibody class and subclass within a species. Allotypic determinants are subtle amino acid differences encoded by different alleles of isotype genes. Idiotypic determinants are generated by the conformation of the amino acid sequences of the heavy- and light-chain variable regions specific for each antigen.
Complement fixation test (CFT)
The complement fixation test is a traditional test used to detect the presence of specific antigens or antibodies. It involves incubating a patient's serum sample with a known antigen, then checking if any complement was activated and "fixed" or bound by the formation of antigen-antibody complexes. If complexes formed, the complement is fixed and will not react with indicator cells, showing a positive result. If no complexes formed, free complement will react with the indicators, showing a negative result. While economical for screening multiple infections, it is not very sensitive and can produce non-specific results.
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- 3. • Immunoelectrophoresis is the electrophoresis of a determined antigen mixture in an agarose gel that allows the separation of different antigens along the gel slide, and then the lateral diffusion of an antibody in the gel
- 4. • Counter Current Immunoelectrophoresis is a modification of immunoelectrophoresis
- 5. • The principle of counter current immunoelectrophoresis is based on the movement of antigen towards the cathode and of the antibody towards the anode during the passage of electric current through agar. The meeting of the antigen and antibody is greatly accelerated by this method and this made visible in 30 to 60 minutes.
- 6. • This has been applied to the detection of bacterial and fungal polysaccharide antigens in cerebrospinal fluid.
- 7. • Agarose, Prepare 10 ml of 1.0% Agarose (0.1 g/10 ml) in Assay Buffer by heating slowly till agarose dissolves completely. • Antigen, • Test antiserum, • Positive antiserum, • Assay Buffer, • Electrophoresis apparatus, Glass slides.
- 8. 1.Mark the end of a glass slide as +ve and -ve, so that when placed the glass slide in electrophoresis apparatus, the +ve mark faced towards anode and the negative mark faced towards cathode.
- 9. 2. Place the glass plate or slide on a horizontal surface. Pipette and spread 5 ml of agarose onto the glass slide. Allow to solidify for 15 minutes. Take care that the slide is not disturbed and allow the gel to solidify. 3. Cut wells of according to the template using gel puncher. The distance between the two wells should not be more than 0.5 cm. 4. Place the slide in the electrophoresis tank and fill the tank with electrophoresis buffer till the buffer just covers the gel surface. Do not add excess of buffer.
- 10. Wells by using Gel puncher
- 13. • 5. Add 10μl of antigen in each of the two wells towards cathode (Negative electrode) and 10μl of positive control antiserum and test antibody in wells towards anode (Positive electrode) as shown. Cathode (-) Anode (+)
- 14. 6. Connect the power cord to the electrophoretic power supply according to the convention: red : anode and black : cathode. 7. Apply 50 V and allow the electrophoresis to continue for about 60 minutes. 8. Observe for precipitin line between the antigen and antibody wells.
- 15. • a) Precipitin line indicates the presence of antibody for the antigen in the test sera. • b) The absence of the precipitin line indicates the absence of any antibody for the antigen in the test sera.
- 18. Plasmodium Glutamate dehydrogenase (pGluDH) separated by Counterimmunoelectrophoresis
- 19. The presence of precipitin line between the antigen and antibody indicates its specificity while the absence of precipitin line indicates non-specificity.