Ag ab reactions
- 1. Presenting by : Y. Santhoshi Lavanya 2020611005 Agricultural Microbiology
- 2. Antigens & antibodies combine specifically with each other. This interaction between them is called ‘Antigen- Antibody reaction’. Abbreviated as Ag – Ab reaction. They form the basis for humoral/antibody mediated immunity. They are used for detection of disease causing agents & some non-specific Ag’s like enzymes. When Ag-Ab reaction occurs in-vitro they are known as ‘serological reactions’.
- 3. The reactions b/w Ag & Ab occurs in 3 stages: Formation of Ag-Ab complex Leads to visible events like precipitation, agglutination etc. Destruction of Ag or its neutralization.
- 5. Specificity Immune complex Binding site Binding force Affinity Avidivity Cross reaction Sensivity
- 6. Refers to the ability of an individual antibody combining site to react with only one antigenic determinant (epitope). Each antibody binds to a specific antigen; an interaction similar to a lock and key.
- 7. When antigen & antibody are brought together , the antibody binds with the antigen to form a complex molecules called immune complex orAg- Ab complex. Ag +Ab Ag –Ab COMPLEX
- 8. The part of antigen which combines with antibody is called ‘Epitope’, recognized by the immune system, specifically by antibodies, B cells, or T cells. Part of an antibody that recognizes an epitope is called a ‘paratope’.
- 9. The binding b/w Ag & Ab in Ag – Ab reaction is due to three factors namely: Closeness b/w Ag & Ab -> more close = good strength of binding. Non – covalent bonds or Intermolecular forces -> hydrogen bonds, vander walls forces, hydrophobic bonds. Affinity of antibody -> strength of reaction b/w a single epitope & single paratope.
- 10. It refers to the intensity of the attraction between antigen and antibody molecule
- 11. It is the strength of the bond after the formation of antigen antibody complex
- 12. An antiserum raised against a given antigen may sometimes react with another closely related antigen. This reaction is called cross reaction. & the antigen which produce the cross reaction is called cross reactive antigen. The cross reaction is due to the presence one or more identical antigenic determinants on the related antigen.
- 13. Sensitivity refers to the ability of the test to detect even very minute antigen or antibody
- 14. PRECIPITATION TESTS AGGLUTINATION TESTS COMPLEMENT FIXATION TESTS ELISA IMMUNOFLUORESCENCE RADIO IMMUNOASSAY CHEMILLUMINESCENCE ASSAY IMMUNOELECTROBLOT ASSAY
- 15. When a soluble antigen combines with its antibody in the presence of electrolytes at a suitable temperature and pH, the antigen antibody complex forms an insoluble precipitate. Precipitation can take place in liquid media or gel Instead of sedimenting, if the precipitate remains suspended as floccules, the reaction is known as flocculation. The precipitate which is formed is influenced by the proportion of antigens and antibodies If increasing quantities of an antigens are added to the same amount of an antibody in different tubes, the precipitation will be found to occur in one of the middle tubes. The sensitivity of the precipitation reaction is comparatively less than the other immunoassay reactions.
- 16. Types of Precipitation tests Ring test Slide test Tube test Immunodiffusion Immunoelectrophoresis
- 17. Simplest form of precipitation test. Consisting of layering the antigen solution over a column of antibody in a test tube Precipitate forms at the junction of the two liquids. CLINICAL APPLICATIONS: Grouping of Streptococci Ascolis thermoprecipitin test for the diagnosis of anthrax Detection of adulteration of food stuffs.
- 18. When a drop of antigen and antibody are placed on the slide and mixed by shaking, floccules will appear between antigen and antibody. CLINICAL APPLICATIONS: VDRL test for syphilis
- 19. Serial dilutions of antigen made in the test tube to which fixed amount of serum is added. CLINICAL APPLICATIONS: Standardization of toxoids and toxins
- 20. In this method the specific antigens and antibodies are allowed to combine in a semi solid or gel media Immunodiffusion is performed by using agar or agarose gel The preferred concentration of agar is between 0.3-1.5 percent for the effective diffusion of the reactants to form a lattice. Immunodiffusion takes place through the intervening agar media whose rate is influenced or affected by the following factors like: The particle size of the reactants Temperature Gel viscosity Interaction between gel matrix and reactants
- 21. In agar, diffusion takes place by either of the two ways: 1. Single diffusion 2. Double diffusion Single and double diffusion can occur both in one dimension and double dimension. One dimension diffusion involves the diffusion of either antigen or antibody on the agar gel. Double dimension diffusion consists of the diffusion of both antigen and antibody on the solid agar media.
- 22. It is a type of single immunodiffusion method, which occurs in one dimension. In Oudin immunodiffusion, add antibodies in the molten agar media and pour it into the petri dish. After solidification of the gel matrix, add a layer of soluble antigen. As a result, the incorporated antibodies in the gel matrix will not diffuse, but the diffusion of antigen occurs. The antigen will diffuse towards the antibodies and form a line of a precipitate. OUDIN IMMUNODIFFUSION
- 23. It is a type of double immunodiffusion method, which occurs in one dimension. In Oakley Fulthorpe immunodiffusion first, add antibodies in the molten agar media. Over the layer of antibodies incorporated within the agar media, add a layer of plain agar media. Then, on the top of plain agar media, add a layer of soluble antigen. On incubation, both antigen and antibody will move towards each other to form a precipitin line. OAKLEY IMMUNODIFFUSION
- 24. It is a type of single diffusion method, which occurs in two directions. In radial immunodiffusion first, the antibodies in the serum are added in the molten agar media and poured into the glass slide. After the solidification of the gel matrix, create wells and add test antigen into it. Upon incubation, the antibodies incorporated within the agar gel will react with the specific antigens. A radial diffusion occurs exterior to the well’s surface in the form of precipitin ring. The diameter of the precipitin ring is directly proportional to the concentration of the antigen. RADIAL IMMUNODIFFUSION
- 25. This type of precipitation method involves diffusion (either in one or two directions) plus the technique of electrophoresis. The immunoelectrophoresis includes the following two methods that are given below:
- 26. It is performed on a glass slide, in which the reaction occurs in a single dimension. In Rocket immunoelectrophoresis, the agar is poured that is homogenized with the antiserum. Then, a well is created into which the antigenic suspension is added. Upon electrophoresis, antigens being negatively charged will migrate towards the positively charged antibody. This migration of antigen on interaction with an antibody will form a complex as a visible precipitin line. We could see the precipitin line in the form of a rocket. The height covered by the precipitin line is directly proportional to the amount of antigen-loaded into the well. ROCKET IMMUNOELECTROPHORESIS
- 27. It is a method of immunoelectrophoresis, in which the reactants move in two dimensions. In counter immunoelectrophoresis, pour the agar gel over the glass slide. Then, create wells on both the edges of the glass slide. On one end, add antigens and on the other, add antiserum. Upon electrophoresis, the antigen will migrate towards the anode, and the antibody will migrate towards the cathode. On the interaction between antigen and antibody, a precipitin line will form. COUNTER IMMUNOELECTROPHORESIS
- 28. To identify microbes (bacterial cells) To detect antigen in serum or other sample To detect antibody in serum or other sample To detect toxin or anti-toxin To detect food adulteration It can be used to measure concentration of antigen or antibody. Eg by radial immuno- diffusion
- 29. Agglutination reaction is a serological assay, which results in the clumping of reactants (antigens and antibodies) to form a large visible aggregated mass. It involves the mixing of large or particulate antigens with the antiserum containing antibodies over the solid matrices like glass side, microtitre plate or test tubes. The agglutination between antigen and antibody occurs at the optimal temperature, pH and ionic strength of the solution. The combination of both the reactants, i.e. antigen and antibody result in the formation of antigen-antibody network or lattice as “visible clumps”. The most common example of the agglutination reaction is “Blood typing”.
- 30. It involves direct interaction of antibodies present in the serum with the particulate antigens carrying epitopes of interest. Active agglutination is used to quantify antibodies against the particulate antigens like RBCs, pathogenic microorganisms like bacteria, fungi etc. In this method, add the particulate antigens into the wells of a microtitre plate. Then, serially dilute the antibodies and pour the suspension into the wells coated with an antigen. By increasing the concentration of antibody, the rate of agglutination will also increase.
- 31. 1. Positive result: Antibodies present in the serum will bind with the particulate antigen and results in the formation of the antigen-antibody mat at the bottom of the well. 2. Negative result: It occurs due to an insufficient amount of antibodies to agglutinate the antigens. Therefore, the agglutination reaction will not occur. In this case, the antigens would appear as pellets at the bottom of the well, instead of aggregates. Example: Slide agglutination test(Blood-grouping), Widal test for the diagnosis of typhoid fever, Coomb’s test to detect anti-Rh antibodies etc
- 36. It involves indirect interaction of the antibodies with the soluble antigens via carrier particles. Passive agglutination primarily involves the binding of soluble antigen with the carrier matrices like latex bead, polystyrene etc. In this type, the antigens do not carry epitopes of interest, which have to be agglutinated. Example: Latex bead agglutination for both antigen and antibody.
- 38. Reverse passive agglutination uses known antibodies in place of antigen, which attache with the carrier matrix. After the antibody’s attachment with the carrier molecule, the antigens attach to the Fab sites of the antibody to agglutinate it.
- 39. 1.Blood typing of recipient and donor at the time of blood transfusion. 2.Helps in the detection of antibody presence and to quantify the amount of antibody present in the patient’s blood. 3.Active agglutination helps in serodiagnosis of toxoplasmosis, brucellosis etc. 4.Passive agglutination helps in the determination of the Rh-factor. 5.It is extensively used in techniques like latex and haemagglutination. 6.It helps to detect the type of antigen and quantify the amount of antigen present in the patient’s blood.
- 40. The ability of antigen antibody complexes to fix complement is made use of in the complement fixation test Capable of detecting as little as 0.04 mg of antibody and 0.1 mg of antigen. CFT is a complex procedure consisting of two steps and five reagents
- 42. Flourescence is the property of absorbing light rays of one wavelength and emitting rays with a different wavelength. Flourescent dyes are conjugated to antibodies and are used to locate and identify antigens in tissues. Immunoflourescence can be direct or indirect Commonly used dyes are fluorescein isothiocynate and lissamine rhodamine CLINICAL APPLICATIONS: Sensitive method of diagnosing rabies
- 43. The term binder-ligand-assay has been used for these reactions Ligand: the substance whose concentration is to be determined is termed as ligand Binder: it is a protein which binds to the ligand. RIA permits the measurement of analytes up to picogram quantities In RIA fixed amounts of antibody and radiolabeled antigen react in the presence of unlabeled antigen The labeled and unlabeled antigens compete for the binding sites on the antibody After the reaction the antigen is separated as free and bound fractions The concentration of antigen can be calculated from the ration of the bound and total antigen, using a standard dose response curve.
- 45. The test can be used to determine very small quantities of antigens and antibodies in the serum. The test is used for quantitation of hormones, drugs, HBsAg, and other viral antigens. Analyze nanomolar and picomolar concentrations of hormones in biological fluids.
- 46. Term Was Coined By Engvall and Pearlmann in 1971. ELISA also known as an Enzyme Linked Immunosorbent Assay is a biochemical Techniques used mainly in immunology to detected the presence of an antibody or an antigen in a sample. ELISA used in the detection and quantization of several antigen as well as antibodies. ELISA can also be used in toxicology as a rapid presumptive screen for certain classes of drug.
- 48. The presence of antibodies and antigens in a sample can be determined. It is used in the food industry to detect any food allergens present. To determine the concentration of serum antibody in a virus test. During a disease outbreak, to evaluate the spread of the disease, e.g. during recent COVID- 19 outbreak, rapid testing kits are being used to determine presence of antibodies in the blood sample.
- 49. CILA refers to a chemical reaction emitting energy in the form of light Chemiluminescent compounds such as luminol or acridinium esters are used in CILA The signal during the antigen-antibody reaction can be amplified and measured and the concentration of the analyte are calculated.
- 51. Immunoelectroblot technique combines the sensitivity of enzyme immunoassay with much greater specificity It involves a combination of procedures: Separation of ligand-antigen components by polyacramide gel through electrophoresis Blotting the electrophoresed ligand fraction on nitrocellulose membrane strips Immunoblot: proteins separated by molecular weight and represented by a dark band on a blot.
- 53. The coronavirus antigen rapid test kit is a lateral flow assay that qualitatively detects the presence of nucleocapsid (N) protein in upper respiratory samples (nasal swabs). This lateral flow assay is designed with the sandwich immunoassay format. When the specimen is added onto the sample pad of a test cassette, coronavirus N protein binds with colloidal gold-labeled SARS-CoV-2 N protein antibody to form an antibody-antigen (Ab- Ag) complex. The Ab-Ag complex is captured by SARS-CoV-2 N protein antibody (Rabbit monoclonal antibody) when migrating to the test line under capillary action. A red-colored band will appear on the test line, which indicates the specimen is COVID-19 nucleocapsid protein positive. No color band will appear on the test line if the specimen does not contain any coronavirus antigen (N protein), or the antigen level is below detection limit.
- 56. Antigen-antibody reactions are very important for serological testing of human beings, as they give you a complete picture of all the immune responses occurring the body & helps determining the immunological disorders by the antigen (either self or non-self).