IS LAB MID

• Precipitation involves combining soluble antigen with soluble antibody to produce insoluble complexes that are visible.
• Precipitation involves the interaction of antigen with antibody in correct proportion resulting in a visible precipitate. The antibodies can either be polyclonal or monoclonal. The simplest form of precipitin reaction would be layering of antigen in solution over a small volume of anti-serum. Precipitation occurs at the interface of the two reagent, forming a ring
• Factors Affecting Visible Reaction:

  1. Antibody concentration
  2. Antigen concentration
• In zone of equivalence, the number of multivalent sites of antigen and antibody are approximately equal
• In the case of antibody excess, the prozone phenomenon occurs, in which antigen combines with only one or two antibody molecules and no cross-linkages are formed.
• Zone of equivalence - when both antigen and antibody concentrations are equivalent, they will combine to form large aggregates (precipitate).
• In the zone of equivalence, precipitation is the result of random, reversible reactions whereby each antibody binds to more than one antigen and vice versa, forming a stable network or lattice
• where there is antigen excess, the postzone phenomenon occurs in which small aggregates are surrounded by excess antigen. Again, no lattice network is formed.
• precipitation of antigen–antibody complexes can also be determined in a support medium such as a gel. Agarose, a purified high-molecular-weight complex polysaccharide derived from seaweed, is used for this purpose
• When antigen and antibody diffuse toward one another in a gel matrix, visible lines of precipitation will form. Agarose helps stabilize the diffusion process and allow visualization of the precipitin bands
• Antigen and antibody are added to wells in the gel and antigen–antibody combination occurs by means of diffusion. When no electrical current is used to speed up this process, it is known as passive immunodiffusion. The rate of diffusion is affected by the size of the particles, the temperature, the gel viscosity, and the amount of hydration. Immunodiffusion reactions can be classified according to the number of reactants diffusing and the direction of diffusion.
• Immunodiffusion

  • RID
  • Rocket Electrophoresis
  • Double Immunodiffusion
• A single-diffusion technique, called radial immunodiffusion (RID), has been used in the clinical laboratory. In this technique, antibody is uniformly distributed in the support gel and antigen is applied to a well cut into the gel. As the antigen diffuses out from the well, antigen– antibody combination occurs in changing proportions until the zone of equivalence is reached and a stable lattice network is formed in the gel. The area of the ring obtained is a measure of antigen concentration that can be compared with a standard curve obtained by using antigens of known concentration.
• One technique for the measurement of radial immunodiffusion was developed by Mancini and is known as the endpoint method. In this technique, antigen is allowed to diffuse to completion; when equivalence is reached, there is no further change in the ring diameter. Equivalence occurs between 24 and 72 hours. The square of the diameter is then directly proportional to the concentration of the antigen.
• Radial immunodiffusion has been used to measure IgG and IgA subclasses as well as complement components. Immunodiffusion is simple to perform and requires no instrumentation, but it has largely been replaced by more sensitive methods such as nephelometry and enzyme-linked immunoassays.
• Rocket Electrophoresis - APPLY ELECTRICITY, Electricity INDUCED UNTIL ZONE OF EQUIVALNCE IS REACHED

  3 -4 HRS MAXIMUM
• One of the older, classic immunochemical techniques is Ouchterlony double diffusion. In this technique, both antigen and antibody diffuse independently through a semisolid medium in two dimensions, horizontally and vertically.
• The position of the precipitin bands between wells allows for the antigens to be compared with one another. Several patterns are possible: (1) Fusion of the lines at their junction to form an arc represents serological identity or the presence of a common epitope, (2) a pattern of crossed lines demonstrates two separate reactions and indicates that the compared antiens share no common epitopes, and (3) fusion of two lines with a spur indicates partial identity.
• Ouchterlony double diffusion is still used to identify fungal antigens such as Aspergillus, Blastomyces, Coccidioides, and Candida.
• Precipitation is one of the simplest methods of detecting antigen–antibody reactions because most antigens are multivalent and thus capable of forming aggregates in the presence of the corresponding antibody.
• Precipitates in fluids can be measured by means of turbidimetry or nephelometry.
• Turbidimetry is a measure of the turbidity or cloudiness of a solution. A detection device is placed in direct line with an incident light, collecting the light after it has passed through the solution. This device measures the reduction in light intensity caused by reflection, absorption, or scatter. The amount of scatter is proportional to the size, shape, and concentration of molecules present in solution.
• Nephelometry measures the light that is scattered at a particular angle from the incident beam as it passes through a suspension. The amount of light scattered is an index of the solution’s concentration.
• Turbidimetry


  A measure of the turbidity or cloudiness of a solution
• Turbidimetry

  1. Detection device placed in direct line with incident light

  2. Collecting light after it has passed through the solution

  3. Measuring reduction in light intensity caused by reflection, absorption, or scatter
• Turbidimetry


  • Amount of scatter is proportional to the size, shape, and concentration of molecules present in solution
• Turbidimetry is a measure of the turbidity or cloudiness of a solution
• A detection device is placed in direct line with an incident light, collecting the light after it has passed through the solution
• This device measures the reduction in light intensity caused by reflection, absorption, or scatter
• The amount of scatter is proportional to the size, shape, and concentration of molecules present in solution
• Nephelometers typically measure light scatter at angles ranging from 10 degrees to about 90 degrees.
• Although the sensitivity of turbidity has increased, nephelometry is more sensitive, with a lower limit of detection of 1 to 10 mg/L for serum proteins.
• Quantification of immunoglobulins such as IgG, IgA, IgM, and IgE, as well as kappa and lambda light chains, is mainly done by rate nephelometry because other methods are more labor intensive.
• 1. Collect blood to anti-coagulated tube.
  2. Prepare 2-3% RCS.
  3. Prepare 2 ml plasma. See to it the RCS and plasma are of different blood types.
  4. Add 2 ml of 2-3% RCS over 2 ml of plasma.
  5. Stand for 15-30 minutes.
  6. Observed for the reaction.
• Nephelometry


  Light that is scattered at an angle is measured, indicating the amount of antigen or antibody present
• Radial immunodiffusion


  Antigen diffuses out into gel that is infused with antibody. Measurement of the radius indicates concentration of antigen
• Ouchterlony double diffusion


  Both antigen and antibody diffuse out from wells in a gel. Lines of precipitate formed indicate the relationship of antigens
• Antigen-antibody complexes


  • The first step is rapid and reversible. The second step, or lattice formation, is the formation of cross-links that form the viable aggregates. This represents the stabilization of antigen-antibody complexes with the binding together of multiple antigenic determinants
• TECHNIQUE

  • Nephelometry
  • Radial immunodiffusion
  • Ouchteriohy double diffusion
  • Immunoelectrophores
  • Immunoactivpe stophoresis
• APPLICATION

  • Immunoglobulins, complement, C-reactive protein other serum proteins
  • Immunoglobulins complement
  • Complex antigens such fungal antigens
  • Differentiation of senum proteins
  • Oven or underproduction of antidy