CHAPTER 6.2: AGGLUTINATION Flashcards
Technique in which molecules with a net charge are separated when an electric field is applied
ELECTROPHORESIS
Negative charged particles migrate to the
ANODE (+ Pole)
Positive charged particles migrate to
CATHODE (- Pole)
FACTORS THAT INFLUENCE RATE OF PROTEIN MIGRATION
• The bigger and the larger the size, it will be hard to migrate
SIZE AND SHAPE OF PROTEIN
• Used agarose gel and the amount of solvation has a great impact with regards to the rate of protein migration
AMOUNT OF SOLVATION
• alkaline pH
PH OF BUFFER: >8
• Room temperature
TEMPERATURE
• Protein will denature once it is exposed to high temperature
TEMPERATURE
• flow of ions goes toward the cathode and can impede movement of proteins toward the anode
ENDO-OSMOSIS
DIFFERENT TESTS FOR ELECTROPHORESIS
Laurell Technique (1960)
ROCKET IMMUNOELECTROPHORESIS
Radial immunodiffusion (RID) + electrophoresis
ROCKET IMMUNOELECTROPHORESIS
Single reactant moving in one dimension
ROCKET IMMUNOELECTROPHORESIS
Electrophoresis is used to facilitate migration of the antigen into the agar
ROCKET IMMUNOELECTROPHORESIS
End result: precipitin line that is conical in shape, resembling a rocket
ROCKET IMMUNOELECTROPHORESIS
The height of the rocket, measured from the well to the apex, is directly in proportion to the amount of antigen in the sample.
ROCKET IMMUNOELECTROPHORESIS
This technique has been used to quantitate immunoglobulins, using a buffer of pH 8.6
ROCKET IMMUNOELECTROPHORESIS
ROCKET IMMUNOELECTROPHORESIS Procedure:
1. Antigen is pushed through antibody containing gel under influence of an (?)
2. When they are equivalence, precipitation will occur forming a (?)
applied electric field
cone/ rocket shape band
• Ressler’s method
CROSSED IMMUNOELECTROPHORESIS
• Single reactant moving in 2 dimensions
CROSSED IMMUNOELECTROPHORESIS
CROSSED IMMUNOELECTROPHORESIS Procedure
1. Proteins are separated by (?)
2. Proteins are subjected to a 2nd electrophoresis where they will move through a (?) until rocket is formed (Ag-Ab reach equivalence)
electrophoresis
Ab-containing agarose
• Countercurrent electrophoresis
COUNTER IMMUNOELECTROPHORESIS
• Voltage Facilitated double immunodiffusions
COUNTER IMMUNOELECTROPHORESIS
• Double reactants moving in one dimension
COUNTER IMMUNOELECTROPHORESIS
COUNTER IMMUNOELECTROPHORESIS Use:
Identify bacterial, fungi or virus in fluids
COUNTER IMMUNOELECTROPHORESIS Procedure:
1. Ag and Ab are added to separate parallel wells cut out in an (?)
2. When an electric field is applied, the Ag will migrate to the (?) and Ab to the (?)
3. Zone of equivalence will form a (?)
agar gel
Anode; cathode
precipitate
• Grabar and Williams
CLASSIC IMMUNOELETROPHORESIS
• Double reactants moving in 2 dimensions
CLASSIC IMMUNOELETROPHORESIS
• Two-step process
CLASSIC IMMUNOELETROPHORESIS
• Used as a screening tool for the differentiation of many serum proteins, including the major classes of immunoglobulins.
CLASSIC IMMUNOELETROPHORESIS
• It is both a qualitative and a semiquantitative technique and has been used in clinical laboratories for the detection of myelomas, Waldenström’s macroglobulinemia, malignant lymphomas, and other lymphoproliferative disorders.
CLASSIC IMMUNOELETROPHORESIS
CLASSIC IMMUNOELETROPHORESIS Use:
Differentiate the Ig Class, identify abnormal proteins, myeloma proteins, Monitor purity of pharmaceutical products
CLASSIC IMMUNOELETROPHORESIS Procedure:
1. Ag is introduced in a well and an electric field is applied resulting in separation of proteins
2. Ab is introduced in a trough parallel to the separated protein
3. Ag-Ab complex form
CLASSIC IMMUNOELETROPHORESIS Procedure:
1. Ag is introduced in a well and an (?) is applied resulting in separation of proteins
2. Ab is introduced in a (?) parallel to the separated protein
3. (?) form
electric field
trough
Ag-Ab complex
CLASSIC IMMUNOELETROPHORESIS
Sequence:
• Cathode (+) to Anode (–)
- Albumin
- Alpha-1 globulin
- Alpha-2 globulin
- Beta globulin
- Gamma globulin
Immunoglobulin
Gamma globulin
STEPS IN AGGLUTINATION
published the first report about the ability of antibody to clump cells, based on observations of agglutination of bacterial cells by serum.
Gruber and Durham
This finding gave rise to the use of serology as a tool in the diagnosis of disease, and it also led to the discovery of the ABO blood groups (1902)
Gruber and Durham
Process by which (?) such as cell aggregate to form larger complexes when a (?) is present
particulate antigens (agglutinogen)
specific antibody (agglutinin)
Antigen-Antibody reaction
SENSITIZATION
Stabilization of agglutinogen + agglutinin
SENSITIZATION
Stabilization of antigen–antibody complexes with the binding together of multiple antigenic determinants.
SENSITIZATION