3. Precipitation and Agglutination Reactions Flashcards
How do antigens and antibodies interact?
Lock and key fit
The INITIAL FORCE of attraction the exist between a single FAB site on an antibody molecule and single epitope
Affinity
The SUM of all attractive forces between Ab and An
Avidity
Attach to antigen where it fits to the FAB for to shows the affinity between An and Ab
Epitope
What is the optimal pH for An-antigen reaction?
6.5 - 7.5
Ionic strength is base on?
Shielding
Zeta potential
What is the best temp reaction for IgM?
Room temp or colder
What is the best temp reaction for IgG?
36-38 C
An-Ab reaction are influenced by?
Ionic strength
pH
Reaction time
Temp
Dosage
Zone of antibody excess? causes false neg
Pro-zone
What do you call the where the antigen and antibody have the same amount?
Zone of equivalence
Zone of antigen excess? causes false neg
Post zone
Precipitation based method?
Nephelometry
Passive immunodiffusion techniques
Electrophoretic techniques
Involves combining soluble antigen with soluble antibody to produce insoluble complexes that are visible
Precipitation
Antibody reagent is combined with patient sample, this detects through Light refraction (Light scatter) from light source which detected by the light detector
Nephelometry
Uses agarose gel to detect An-Ab reaction which measures the rate of An-Ab reaction by the area being created around the agarose gel
Radial immunodiffusion
How many hours before reading radial immunodiffusion
24-72 hrs
What is being read in radial immunodiffusion?
Rate of diffusion
The rate of diffusion is affected by?
Size of the particle
Temp
Gel viscosity
Amount of hydration
What is the positive indicator for radial immunodiffusion (Being measured)
Area of the ring
NOTE: The diameter of the diffusion of the An-Ab reaction is proportional to the area/diameter of the ring
Where is the positive control located in Radial immunodiffusion?
middle
Performed in agarose gel in which both and antibody diffuses independently in two dimensions (Horizontally and vertically)
Ouchterlony Double immunodiffusion
What is the incubation period of Ouchterlony double immunodiffusion in a moist chamber?
12 to 48 hrs
What is the known sample in Ouchterlony double immunodiffusion?
Known Antibody
What is the sample used in Ouchterlony double immunodiffusion??
Patient Antigen
Presence of common epitope, curve shape
Pattern of identity
Curve pattern with spur, some antibodies are not capture
Pattern of Partial identity
Crossline pattern shows no commonalities
pattern of Non identity
An adaption of radial immunodiffusion but instead of allowing diffusion to take place at its own rate, electrophoresis is used to facilitate migration of antigen into the agar
Electrophoretic techniques: Rocket Immunoelectrophoresis
Double-diffusion technique that incorporates electrophoresis current to enhance results
Used for semi-quantification of a wide range of antigens
Immunoelectrophoresis
After electrophoresis takes place, anti-serum is applied to the gel’s surface rather than placed in a trough
Shorter time and higher resolution
Immunofixation electrophoresis
Used to test immunoglobulins, complement, C-reactive protein, other serum
Nephelometry
Used for immunoglobulins, and complement only
Radial immunodiffusion
Used for complex antigens such as fungal antigens
Ouchterlony double diffusion
Used for immunoglobulins, complement, and alpha-fetoproteins
Pocket electrophoresis
Used for Differentiation of serum protein
Immunoelectrophoresis
Used to test HIV, Lyme disease, and syphilis
Immunofixation electrophoresis
The interaction between antibody and a particulate antigen which results to clumping
Agglutination
The antibodies that produces such reaction are called?
Agglutinins
The particulate antigens aggregated are called
agglutinogens
Steps in agglutination
- Sensitization
- Lattice Formation
Antigen and Ab unite through antigen determinant site, this process is called?
Sensitization
Formation that rearrange of antigen and antibody bonds to cause agglutination
Lattice formation
Occurs when antigens are gound naturally on a particle
Can be used to detect antigen (Used known antibody as a reagent)
Direct agglutination
Grade for One solid clump?
4+
Grade for several large clump?
3+
Grade for numerous smaller clumps?
2+
Grade for BArely discernable clumps
1+
Grade for smooth suspension?
Negative
Antigens on a carrier molecule such as latex combine with the patient’s sample for antibody detection
Passive agglutination
Antibody is bound to the carrier molecule, which is then mixed with patient’s sample to detect antigen
Uses for passive agglutination include ID of bacteria measuring hormone and drug levels, and measuring levels of some proteins
Reverse passive agglutination
Antibody reagent is combined with patient’s specimen
If patient’s specimen contains that antigen, the antigens will react with antibodies
Agglutination inhibition
What is the positive reaction for agglutination inhibition?
No agglutination
What is the Negative reaction for agglutination inhibition?
Agglutination
What is the positive reaction to agglutination inhibition?
No agglutination
This use to detect virl antibodies and detects hormone
Test that uses bacteria as the inert particles to which the antibody is attached
Coagglutination
Bacteria that is the most frequently used in coagglutination
Staphylococcus aureus
Why S. aureus is used in coagglutination
Protein A
