precipitation Flashcards
electro-immunodiffusion
- When RID is applied with current, it becomes electroimmunoassay/ rocket electrophoresis
- Ag and Ab different charges at selected pH
- Band coverage finally meet at the middle, when all antigen is precipitated , the precipitin pattern resembles that of a shooting rocket.
ROCKET ELECTROPHORESIS/ LAURELL TECHNIQUE
(ONE DIMENSIONAL SINGLE ELECTROPHORESIS)
immunodiffusion
Ag and Ab diffuse radially
double dimension
passive immunodiffusion
- immunoglobulins, complement
- Ab in gel + Ag in well»_space; diffusion
- precipitin ring
radial immunodiffusion (RID)
patterns
crossed bands
non-identity
electro-immunodiffusion
- Antigen and antibody migrate toward each other by electrophoresis.
- Used only when Ag and Ab have opposite charges.
- Detection of the semi-quantitative antigen of the body fluid
- Faster and more sensitive than Ouchterlony Method
COUNTERCURRENT IMMUNOELETROPHORESIS (ONE DIMENSIONAL-DOUBLE ELECTROPHORESIS)
The strength with which a multivalent antibody binds a multivalent antigen.
avidity
- A semiquantitative gel precipitation technique in which proteins are first separated by electrophoresis and then subjected to double diffusion with antibodies directed against the individual proteins.
- Reliable and accurate method fro detecting structural abnormalities and concentration changes in proteins
- Useful in screening circulating immune complexes/ Myeloma proteins
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METHOD
1) Ag is separated by electrophoresis,
2) Ab is placed in trough cut in the agar,
3) Trough is filled with an anti-serum
4) Incubation: 18 to 24 hours -
APPLICATIONS
1) Serum: detection of Monoclonal Gammopathy
2) Urine: detection of Bence Jones Protein
immuno electrophoresis
- Immunodiffusion reaction in a support medium with the use of electric current to enhance mobility of reactants and to increase movement toward one another.
- PRINCIPLE:
1) Antibody: Positively Charged: Migrates toward the cathode (-)
2) Antigen: Negatively Charged: Migrates toward the anode (+)
electro-immunodiffusion
- ELECTRO-IMMUNODIFFUSION
- IMMUNO-ELECTROPHORESIS
- IMMUNO-FIXATION
electrophoretic method
types
- RADIAL IMMUNODIFFUSION
- OUCHTERLONY DOUBLE DIFFUSION
passive immunodiffusion method
- Soluble Antigen (Kraus, 1897).
- The combination of soluble antigen with soluble antibody to produce visible insoluble complexes.
- SOLUBLE ANTIGEN + SOLUBLE ANTIBODY → INSOLUBLE COMPLEX
precipitation
passive immunodiffusion
- 27-72 hrs
- IgG: 24 hrs
- IgM: 50-72 hrs
- SQUARE of diameter is directly proportional to concentration
- precipitin ring
mancini (endpoint)
Antibodies are capable of reacting with antigens that are structurally similar to the original antigen that induced antibody production
cross-reactivity
immunodiffusion
both Ag and Ab are moving
double diffusion
passive immunodiffusion
- precipitation (rabbit)
- flocculation (horse)
- Ab in agarose + Ag on top»_space; Ag diffuses downward
- precipitation occurs in proportion to the amount of Ag present
OUDIN
radial immunodiffusion
- Antigen is allowed to diffuse to completionand when equivalence is reached, there is no further change in the ring diameter
- longer reaction time (sensitive)
- occur 24 - 72 hours
- ring diameter = antigen conc.
- uses graphing paper
mancini (endpoint diffusion)
patterns
- fusion of two lines with a spur
- share a common epitope but some Ab are not captured by Ag
partial identity
immunodiffusion
pertains to the antigen or antibody
“immuno”
precipitation
- VDRL and other non-treponemal tests (except: RPR)
- standardization of toxins and toxoids
- a specific type of precipitation that occurs over a narrow range of antigen concentrations
- antigen consists of very fine particles that Ag-Ab complexes remain suspended instead of settling as sediments
- may require microscopy
flocculation
combination
double diffusion-single dimension
Oakley and Fulthrope
patterns
fused bands
serological identity
immunodiffusion
only one reactant is moving (usually Ag)
single diffusion
types
- TURBIDIMETRY
- NEPHELOMETRY
precipitation by light scatter
passive immunodiffusion
- complex antigens
- fungal antigens
- both Ag and Ab diffuse in two dimensions
- PARTIAL ID = diff. Ags but similar epitopes
- precipitin paterns: X (non identity), C (identity), K (partial ID)
ouchterlony
A law used to mathematically describe the equilibrium relationship between soluble reactants and insoluble products. It can be applied to antigen–antibody relationships
law of mass action
immunodiffusion
movement of the antigen or antibody
“diffusion”
combination
single-diffusion-double dimension
Radial Immunodiffusion/ Mancini
- A simple , specific method for identification and quantification of a number of proteins found in serum and other body fluids.
- Internal reactants like specific antibodies added to buffered agarose medium, and serum containing standard volume of CHON or Ag is placed in well, centered in agarose.
- Result: Precipitin Ring
- Interpretation: Diameter of the ring is proportional to the concentration of the Antigen.
- Quantitative: Ig Levels, Serum CHON’s, Serum Complement
radial immunodiffusion
combination
double-diffusion-double dimension
Ouchterlony and Elek
radial immunodiffusion
- Measurement taken before the point of equivalence is reached, Antigen isnot allowed to diffuse completely
- shorter reaction time
- read wihtin 18 hours (24 hours)
- ring diameter = log conc. (Ag)
- uses semi-log paper
fahey & Mckelvey (kinetic diffusion)
precipitation
- lancefield
- neufeld quellung
- carbohydrate cell wall Ag
- capsular swelling Ag
liquid precipitation
- A detection device is placed in direct line with the incident light, collecting light after it has passed through the solution.
- Measures the reduction in light intensity due to reflection, absorption, or scatter.
- Scattering of light occurs in proportion to the size, shape, and concentration of molecules present in solution.
turbidimetry
- Ag/Ab Complexes form and precipitate when the 2 reactants meet at the equivalence zone.
- Use to determine the relationship between Ag and Ab
- Both Ag and Ab diffuse
- Clinical application: Detection of antibody associated with autoimmune disease (RA, SLE Sjogren’s)
- Disadvantage: Semiquantitative only
ouchterlony double diffusion
combination
Single diffusion- single dimension
OUDIN
- Measures the light that is scattered at a particular angle from the incident beam as it passes through a suspension.
- measure light scatter at angles ranging from 10 degrees to about 90 degrees.
- Light scatter may be recorded in arbitrary units of “relative light scatter,” or it may be directly extrapolated by a computer to give actual concentrations in milligrams per deciliter (mg/dL) or international units per milliliter (IU/mL)
nephelometry
passive immunodiffusion
plain gel is placed between Ag and Ab to fascilitate double diffusion
Oakley And Fulthrope
Is the initial force of attraction that exists between a site on an antibody molecule and a single epitope single Fab or determinant site on the corresponding antigen.
affinity
immunodiffusion
the movement are either UP or DOWN
single dimension
passive immunodiffusion
- 18-19 hours (24hrs)
- diameter is proportional to the LOG of the concentration
- precipitin ring
fahey and Mckelvy (kinetic)
gel precipitation
- Both reactants (Ag and Ab) diffuse within a gel.
- Both reagents are addedafter gel has set.
DOUBLE IMMUNODIFFUSION TECHNIQUE
gel precipitation
- One reactant (Ag or Ab) remains fixed in gel.
- Other reactant is allowed to moved.
- Interaction with the reagent that is immobilized.
- Reagents become fixed in the gel if they are added to the gel medium while it is in liquid form.
SINGLE IMMUNODIFFUSION TECHNIQUE
- A semiquantitative gel precipitation technique similar to that of immunoelectrophoresis, except that antibody is added directly to the surface of the gel after electrophoresis has taken place.
- A cellulose acetate strip impregnated with antiserum is placed over the separate proteins after serum , urine or CSF are electrophoresed.
- Diffusion of antiserum into the gel occurs rapidly, resulting in precipitation
immuno-fixation electrophoresis
