precipitation final
TRANSCRIPT
Zone in which the optimum precipitation occurs
Number of multivalent sites of antigen and antibody are approximately equal
*For a precipitation reaction to be detectable, the reaction must occur in this zone
Each antibody or antigen binds to more than one antigen or antibody, forming a stable network or lattice
Formulated by Marrack Each antibody must have at least two
binding sites and antigen must be multivalent
False negative reaction may occur due to the presence of high concentration of antibodies
If the reaction is suspected to be false negative: dilute out antibody and perform the test again may produce a positive result
Excess antigen may obscure the presence of small amount of antibody
To correct postzone phenomenon: test is repeated with an additional patient specimen taken about a week later
Modification of single-diffusion
Used to measure IgG, IgM, IgA, and complement components
Already replaced by more sensitive and automated methods
Standard Curve
Precipitin Rings A B C
a b c
Standards Samples
Diameter = log of concentration -log of diameter
One reagent: Either antibody or antigen is fixed
Other reagent moves in only one directionThe antibody is immobilized in a gelThin layer of Antigen Diffuses downward into the gel
Antgien Solution
Precipitation of Antigen/Antibody
Complexes
Antibody is distributed in the gel, and antigen is placed in wells cut in the gel.
Used to facilitatemigration of the antigen to the agar.
Antigen diffuses out of the well, and precipitation begins.
Precipitin line that is conical in shape,
resembling a rocket
Height of the rocket - Measure from the
well to the apex
Directly in proportion to the
amount of antigen in the sample.
Much more rapid than RID
Results can be obtained in a few
hours
It is essential, however, to
determine the net charge of the
molecules at the pH used for the test because this determines the
direction of migration within
the gel.
Using a buffer of pH 8.6
Other applications Too low to be detected
by nephelometry
Too high for RID
Agar or agarose gel mediumEquivalence ratioPrecipitation of immune complexParallel wellsPrecipitin lineNot widely use
•An immunochemical technique wherein both antigen and antibody diffuse independently through a semisolid medium in two dimensions, horizontally and vertically.
•Principle: Double Diffusion
Agar gel- usual transport medium
Incubation period- 12 to 48 hours
Zone of equivalence-Point of maximal precipitation
Ouchterlony plates
Different patterns possible:1.) Serologic identity
Fusion of the lines at their junction to form an arc represents serologic identity or the presence of a common epitope.
2.) NonidentityPattern of crossed lines demonstrates two separate reactions and indicates that the compared antigens share no common epitopes.
3.) Partial identityFusion of two lines with a spur.
Uses of this technique
- Identification of fungal antigens- Detection of antibodies to extractable nuclear antigens.
Irregular patterns maybe due to:
- Overfilling the wells- Irregular hole punching- Nonlevel incubation
Other factors affecting the accuracy of results:
- Drying out of the gel- Inadequate time for diffusion- Resulting in weakness of band intensity- Fungal or bacterial contamination of the gel
•Introduced by Grabar and Williams in 1953
•Double diffusion technique that utilizes an electric current to enhance results
• Performed as a two-step process and can be used for semiquantitation of a wide range of antigens
• Source of antigen is usually the serum, which is electrophoresed to separate out the main protein fraction
•Also used as a screening tool for the differentiation of more than 30 serum proteins including the major classes of immunoglobulins
Immunoelectrophoresis is a combination of electrophoresis and immunodiffusion
•Used in the clinical laboratory for the detection of myelomas, Waldenstrom’s macroglobulinemia, malignant lymphomas, and other lymphoproliferative disorders
• Also detects immunodeficiencies and deficiencies of complement components
• Replaced by immunofixation electrophoresis which gives quicker results and is easier to interpret
• Diagnosis of monoclonal gammopathy and in the urine is the demonstration of Bence Jones Proteins
Fig. 1 Urine immunoelectrophoresis
If BJ protein is present in a urine specimen, precipitin lines will form with either κ (kappa) or λ (lambda) anti-L chain antisera because BJ protein is composed of homogenous L chains of a single antigen type, either κ or λ.
SOURCES OF ERROR IN ELECTROPHORESIS
Turbidity
Ability of particles in suspension to refract and deflect light rays passing through the suspension such that the light is reflected back into the eyes of the observer.
measured through optical density (OD)spectrophotometer or colorimeter
Turbidimetry
A method for determining the concentration of a substance in a solution by measuring the loss in intensity of a light beam through a solution that contains suspended particulate matter.
Antigen and antibody are mixed
The initial turbidity will form a precipitate which is then quantified.
Device used to measure the light passing through a solution
Placed at an angle of 180 degrees from incident light
If light absorbance is insignificant: turbidity can be expressed as absorbance which is related to the concentration of suspended particles and path length
Measures low-level antigen-antibody reactions
Detect if there is a presence of contaminationQuantification of serum proteins,
complement components Detect the presence of either an antigen or
antibody
All reagents or sera to be used must be free of particles that could scatter the light
Polyethylene glycol
Measures the light that is scattered at a particular angle from the incident beam as it passes through a suspension
Amount of light scattered is an index of the concentration of the solution
Nephelometers Measure light scatter at angles ranging from 10°
to about 90°Preferred method for measurement of low-level-
antigen-antibody reactionsEnd point nephelometry
Reaction is allowed to run essentially to completion
Large particles tend to fall out of solution and decrease the amount of scatter
Kinetic or Rate nephelometryRate of increase of scattering is measured
immediately after the reagent is addedRate change is directly related to antigen
concentration if the antibody concentration is kept constant
APPLICATIONS OF NEPHELOMETRY
Quantification of serum proteins
IgG, IgA, IgM and IgEComplement components C3, C4 and C1
inhibitorHaptoglobin, C-reactive protein,
transferrin, albumin, alpha1-antitrypsinApha2-macroglobulin, fibrinogen,
ceruloplasmin and rheumatoid factor
COMPARISON OFPRECIPITATION TECHNIQUES
IMMUNODIFFUSION TECHNIQUES