L8 Flashcards
- Agglutination: mechanism, forms:
• bacterial agglutination
• Hemagglutination
–Agglutination Reaction Precipitation and agglutination are the visible expression of the aggregation of antigens and antibodies through the formation of a framework in which antigen particles or molecules alternate with antibody molecules
type 1 - direct bacterial Agglutination
Direct whole pathogens can be used to detect antibodies directed against pathogens. The most basic tests are those that measure the antibody produced by the host to determinants on the surface of a bacterial agent in response to infection with that bacterium , This type agglutination Because tube testing allows more time for antigen-antibody reaction
type 2 - Indirect or passive hemagglutination Hemagglutination
is agglutination of red blood cells, and tests for antibody detection. In the indirect or passive hemagglutination technique, erythrocytes are coated with substances such as extracts of bacterial cells, protozoa or purified polysaccharides or proteins. Erythrocyte of animals such as sheep or rabbits, or from group “0” humans, function as carrier for detecting and titrating the corresponding antibodies by agglutination. This technique is called indirect or passive hemagglutination testing because it is not the antigen of the erythrocytes themselves but the passively attached antigens that are bound by antibody , the result agglutination occurs
- Precipitation: mechanism, forms:
• Radial immunodiffusion
–Precipitation Reaction Precipitins can be produced against most proteins and some carbohydrates and carbohydrates- lipid complexes. Various system are available in which precipitation tests are performed in semisolid media such as agar or agarose, or nongel support medium such as cellulose acetate.
type 1 - 1. Radial double diffusion This technique also referred to as the Ouchterlony method, may be used to determine the relationship between antigen and antibodies
type 2 - Single radial immunodiffusion This is a simple and specific method for identification and quantitation of a number of proteins found in human serum and other body fluids.
—Principle: Radial immunodiffusion is based on a technique using reaction in which specific antibody is added to a buffered agarose medium, serum containing the test antigen is placed in a well centered in the agarose. The diameter of the resulting precipitin zone is related to the concentration of antigen placed in a well
• turbidimetry
– Turbidimetry (the name being derived from turbidity) is the process of measuring the loss of intensity of transmitted light due to the scattering effect of particles suspended in it. Light is passed through a filter creating a light of known wavelength which is then passed through a cuvette containing a solution. A photoelectric cell collects the light which passes through the cuvette. A measurement is then given for the amount of absorbed light. Turbidimetry can be used in biology to find the number of cells in a solution.
- Immunoturbidimetry is an important tool in the broad diagnostic field of clinical chemistry. It is used to determine serum proteins not detectable with classical clinical chemistry methods. Immunoturbidimetry uses the classical antigen antibody reaction. The antigen-antibody complexes aggregate to form particles that can be optically detected by a photometer
• nephelometry
– Nephelometry is a technique used in immunology to determine the levels of Nephelometry several blood plasma proteins. For example the total levels of antibodies isotypes or classes: Immunoglobulin M, Immunoglobulin G, and Immunoglobulin A. It is performed by measuring the scattered light at an angle from the sample being measured.
—This technique is widely used in clinical laboratories because it is relatively easily automated. It is based on the principle that a dilute suspension of small particles will scatter light (usually a laser) passed through it rather than simply absorbing it. The amount of scatter is determined by collecting the light at an angle (usually at 30 and 90 degrees). Antibody and the antigen are mixed in concentrations such that only small aggregates are formed that do not quickly settle to the bottom
—Nephelometry can be used to detect either antigen or antibody, but it is usually run with antibody as the reagent and the patient antigen as the unknown
• electroimmunodiffusion
—Eletroimmunodiffusion (EID) EID is a variation of the double immunodiffusion reaction in a support medium such as cellulose acetate or agarose through the use of an electric current that enhances the mobility of reactants and increase their movement towards each other. Antibody is placed in the well favoring its migration in the direction of the cathode; antigens that tend to be more negatively charged and placed in the well that favors migration of the anode. Precipitin bands form at a point of equivalence in a shorter periods of time. Electro immunodiffusion method, like immunodiffusion procedures, are classified into one-or-two-dimensional, singles, or double diffusion when a voltage is applied across the gels to move the antigens and antibodies together, immuno-double diffusion becomes counter current immuno electrophoresis (CIE) radial immunodiffusion (RID) becomes electro immunoassay (EIA)
• immunoelectrophoresis
- Immunoelectrophoresis is a combination of protein electrophoresis and immunoprecipitation. The test and reference samples are first separated by electrophoresis. The antiserum diffuses perpendicularly to the direction of
separation. Due to the formation of precipitating immune complexes, sharply defined lines of precipitation form in the range of equivalence. Proteins can be identified based on the intensity, shape, and position of the precipitation lines
