CHAPTER 6.2: AGGLUTINATION Flashcards
(110 cards)
1
Q
Technique in which molecules with a net charge are separated when an electric field is applied
A
ELECTROPHORESIS
2
Q
Negative charged particles migrate to the
A
ANODE (+ Pole)
3
Q
Positive charged particles migrate to
A
CATHODE (- Pole)
4
Q
FACTORS THAT INFLUENCE RATE OF PROTEIN MIGRATION
A
5
Q
• The bigger and the larger the size, it will be hard to migrate
A
SIZE AND SHAPE OF PROTEIN
6
Q
• Used agarose gel and the amount of solvation has a great impact with regards to the rate of protein migration
A
AMOUNT OF SOLVATION
7
Q
• alkaline pH
A
PH OF BUFFER: >8
8
Q
• Room temperature
A
TEMPERATURE
9
Q
• Protein will denature once it is exposed to high temperature
A
TEMPERATURE
10
Q
• flow of ions goes toward the cathode and can impede movement of proteins toward the anode
A
ENDO-OSMOSIS
11
Q
DIFFERENT TESTS FOR ELECTROPHORESIS
A
12
Q
Laurell Technique (1960)
A
ROCKET IMMUNOELECTROPHORESIS
13
Q
Radial immunodiffusion (RID) + electrophoresis
A
ROCKET IMMUNOELECTROPHORESIS
14
Q
Single reactant moving in one dimension
A
ROCKET IMMUNOELECTROPHORESIS
15
Q
Electrophoresis is used to facilitate migration of the antigen into the agar
A
ROCKET IMMUNOELECTROPHORESIS
16
Q
End result: precipitin line that is conical in shape, resembling a rocket
A
ROCKET IMMUNOELECTROPHORESIS
17
Q
The height of the rocket, measured from the well to the apex, is directly in proportion to the amount of antigen in the sample.
A
ROCKET IMMUNOELECTROPHORESIS
18
Q
This technique has been used to quantitate immunoglobulins, using a buffer of pH 8.6
A
ROCKET IMMUNOELECTROPHORESIS
19
Q
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 (?)
A
applied electric field
cone/ rocket shape band
20
Q
• Ressler’s method
A
CROSSED IMMUNOELECTROPHORESIS
21
Q
• Single reactant moving in 2 dimensions
A
CROSSED IMMUNOELECTROPHORESIS
22
Q
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)
A
electrophoresis
Ab-containing agarose
23
Q
• Countercurrent electrophoresis
A
COUNTER IMMUNOELECTROPHORESIS
24
Q
• Voltage Facilitated double immunodiffusions
A
COUNTER IMMUNOELECTROPHORESIS
25
• Double reactants moving in one dimension
COUNTER IMMUNOELECTROPHORESIS
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COUNTER IMMUNOELECTROPHORESIS Use:
Identify bacterial, fungi or virus in fluids
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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
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• Grabar and Williams
CLASSIC IMMUNOELETROPHORESIS
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• Double reactants moving in 2 dimensions
CLASSIC IMMUNOELETROPHORESIS
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• Two-step process
CLASSIC IMMUNOELETROPHORESIS
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• Used as a screening tool for the differentiation of many serum proteins, including the major classes of immunoglobulins.
CLASSIC IMMUNOELETROPHORESIS
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• 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
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CLASSIC IMMUNOELETROPHORESIS Use:
Differentiate the Ig Class, identify abnormal proteins, myeloma proteins, Monitor purity of pharmaceutical products
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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
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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
36
CLASSIC IMMUNOELETROPHORESIS
Sequence:
• Cathode (+) to Anode (–)
1. Albumin
2. Alpha-1 globulin
3. Alpha-2 globulin
4. Beta globulin
5. Gamma globulin
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Immunoglobulin
Gamma globulin
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STEPS IN AGGLUTINATION
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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
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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
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Process by which (?) such as cell aggregate to form larger complexes when a (?) is present
particulate antigens (agglutinogen)
specific antibody (agglutinin)
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Antigen-Antibody reaction
SENSITIZATION
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Stabilization of agglutinogen + agglutinin
SENSITIZATION
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Stabilization of antigen–antibody complexes with the binding together of multiple antigenic determinants.
SENSITIZATION
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is affected by the nature of the antibody molecules themselves
SENSITIZATION
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Best antibody for agglutination is IgM
SENSITIZATION
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IgM
potential valence of 10 is over (?) more efficient in agglutination than is IgG with a valence of 2
700 times out of
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Cross linking
LATTICE FORMATION
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Representing the sum of interactions between antibody and multiple antigenic determinants on a particle (Avidity)
LATTICE FORMATION
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There is visible agglutination
LATTICE FORMATION
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FACTORS THAT AFFECT AGGLUTINATION
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1. Buffer pH Routine:
(close to physiological pH)
pH 7
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Affects the zoning phenomenon
2.Relative concentration of Ag and Ab
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• Abs will not detect determinants buried within the particle
3. Location and concentration of Antigenic determinants of the particle
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• More number of determinants, the higher the likelihood of cross bridging
3. Location and concentration of Antigenic determinants of the particle
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Non covalent interaction
4. Electrostatic interactions between particles
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• in the buffer plays an important role in agglutination
5. Electrolyte concentration (ionic strength)
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• Electrolytes reduce (?) that interfere with lattice formation
electrostatic charges
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6. Antibody isotope Best:
IgM
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7. Temperature
: Cold reacting (range 4-22oC)
: Warm reacting with optimum temperature at 37oC
IgM
IgG
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9. Time of incubation of coated particles with patient’s serum Incubation times ranges from
15-60 minutes
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0
No agglutinates
Dark, turbid, homogenous
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W+
Many tiny agglutinates, many free cells, may not be visible without microscope
Dark, turbid
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1+
Many small agglutinates, many free cells (25% are agglutinated)
Turbid
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2+
Many medium sized agglutinins, moderate number of free cells (50% are agglutinated)
Clear
66
3+
Several large agglutinates, few free cells (75% are agglutinated)
Clear
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4+
One large solid agglutination, no free (100% are agglutinated)
Clear
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MAJOR CATEGORIES OF AGGLUTINATION REACTIONS
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It will not use any carrier particle
DIRECT/ ACTIVE AGGLUTINATION
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Detecting the presence of antigen
DIRECT/ ACTIVE AGGLUTINATION
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Occurs when antigens are found naturally on a particle
DIRECT/ ACTIVE AGGLUTINATION
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Reaction is due to an Ag-Ab reaction where in the Ag is inherent native to the cell
Direct Immune
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Example: ABO grouping (hemagglutination), Widal Test
• ABO antigens are found in the RBC
• Reagent: Antisera
Direct Immune
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Aggregation of indicator rod blood cells are NOT due to AgAb reactions
Direct Non Immune
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Example: Viral Hemagglutination test
Direct Non Immune
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Direct non immune agglutination
VIRAL HEMAGGLUTINATION
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Virus can stick to agglutinate RBC in the process
VIRAL HEMAGGLUTINATION
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Rubella virus, dengue virus, influenza virus, mumps virus
VIRAL HEMAGGLUTINATION
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Viral receptor: Peplomers
VIRAL HEMAGGLUTINATION
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Competitive binding Assay Procedure:
1. Patient serum incubated with (?)
2. Viral particles will bind to the (?)
3. (?) added to reaction mixture
4.
Positive result: Negative result:
viral particles (Commercially available)
Fab region of Anti-viral Abs
Indicator RBCs
Inhibition or Absence of Agglutination; (Presence of Ab)
Agglutination
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Reactions where Ag has been fixed or absorbed to a carrier/ inert particle
INDIRECT/ PASSIVE AGGLUTINATION
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Detecting the presence of antibodies
INDIRECT/ PASSIVE AGGLUTINATION
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Example: Antistreptolysin-O (ASTO)
INDIRECT/ PASSIVE AGGLUTINATION
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Different passive carriers:
o Human RBCs
o Clay (Bentonite)
o Latex particles
o Colloidal gold
o Charcoal particles
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Antibody is bound to the carrier
REVERSE PASSIVE AGGLUTINATION
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The antibody must still be reactive and is joined in such a manner that the active sites are facing outward.
REVERSE PASSIVE AGGLUTINATION
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Fluid is detected for the presence of Ag
REVERSE PASSIVE AGGLUTINATION
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Example: CRP, Reverse agglutination test for Candida and Nisseria
REVERSE PASSIVE AGGLUTINATION
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Patient sample (Ag) incubated with Ab in test kit
LATEX PARTICLE AGGLUTINATION INHIBITION
90
Complex will form if the patient sample contains the corresponding Ag and the Fab sites are no longer available for the Ag-coated latex particles
LATEX PARTICLE AGGLUTINATION INHIBITION
91
Reactions are based on competition between particulate and soluble antigens for limited antibody-combining sites, and a lack of agglutination is an indicator of a positive reaction
LATEX PARTICLE AGGLUTINATION INHIBITION
92
If the patient sample has no free hapten, the reagent antibody is able to combine with the carrier particles and produce a visible agglutination. In this case, however, agglutination is a negative reaction
LATEX PARTICLE AGGLUTINATION INHIBITION
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Example: HCG/ pregnancy test
LATEX PARTICLE AGGLUTINATION INHIBITION
94
Agglutination inhibition.
(?) is added to the patient sample. If patient antigen is present, (?) results. When (?) are added, no agglutination occurs, which is a positive test. If no patient antigen is there, the (?), and agglutination results, which is a negative test.
Reagent antibody
antigen–antibody combination
antigen-coated latex particles
reagent antibody combines with latex particles
95
systems using bacteria as the inert particles to which antibody is attached
Coaglutination
96
Coaglutination
(?) is most frequently used, because it has a protein on its outer surface, called protein A, which naturally adsorbs the fragment crystallizable (FC) portion of antibody molecules.
Staphylococcus aureus
97
The active sites face outward and are capable of reacting with specific antigen
Coaglutination
98
particles nonspecifically bind the FC portion of immunoglobulin molecules. When reagent antibody is used, combination with patient antigen produces a visible agglutination reaction.
Staphylococcus aureus
99
detects non agglutinating antibody by means of coupling with a second antibody
ANTIGLOBULIN TEST
100
Detects IgG Ab bound to Ag on Red cells (in-vivo)
Direct Antiglobulin Test
101
Direct Antiglobulin Test Purpose:
• HDN investigation
• HTR investigation
• AIHA (Autoimmune Hemolytic Anemia)
• Drug induced Hemolytic Anemia
102
Direct Antiglobulin Test Example:
Direct Coomb’s test
103
• Detects presence of Abs in the serum that is still to be attached to an analyte
Indirect Antiglobulin Test
104
Indirect Antiglobulin Test Purpose:
o Crossmatching
o Ab determination
o Ab identification
o RBC Ag phenotyping
105
Example:
Indirect Coomb’s test
106
QUANTITATIVE AGGLUTINATION REACTION Best
107
Gold-inorganic colloidal particle
SPIA/ Sol Particle Immunoassay
108
Dye-organic colloidal particle
DIA/ Disperse Dye Immunoassay
109
Latex particle
IMPACT/ Immunoassay by Particle Counting
110
IMPACT/ Immunoassay by Particle Counting
