CHAPTER 6 - AGGLUTINATION Flashcards
Process by which particulate antigens (agglutinogen) such as cell aggregate to form larger complexes when a specific antibody (agglutinin) is present
AGGLUTINATION
particulate antigens
agglutinogen
specific antibody
agglutinin
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
2 STAGES OF AGGLUTINATION
Antigen-Antibody reaction
SENSITIZATION
Cross linking
LATTICE FORMATION
Visible agglutination
LATTICE FORMATION
No agglutination yet
SENSITIZATION
Forms bridges between Ab and Ag
LATTICE FORMATION
Represents binding of Ag and Ab
SENSITIZATION
Stabilization of antigen–antibody complexes with the binding together of multiple antigenic determinants.
SENSITIZATION
FACTORS THAT AFFECT AGGLUTINATION
Routine Buffer pH
pH 7
(physiological pH)
7.35 – 7.45
Affects the zoning phenomenon
Relative concentration of Ag and Ab
Abs will not detect determinants buried within the particle
Location and concentration of Antigenic determinants of the particle
More number of determinants, the higher the likelihood of cross bridging
Location and concentration of Antigenic determinants of the particle
Electrostatic interactions between particles
Non covalent interaction
(Zeta potential)
Electrolyte concentration
in the buffer plays an important role in agglutination
Electrolyte concentration (ionic strength)
: charge bet RBC and the elctrolyte/ECF
Zeta potential
Zeta potential:
in RBC
outside the body
↑
↓
reduce electrostatic charges that interfere with lattice formation
Electrolytes
Antibody isotope Best: l
IgM
(dealing w/ particulate Ag)
IgM
IgM: Cold reacting with optimum temperature at
4-22oC
IgG: Warm reacting with optimum temperature at
37oC
Incubation times ranges from
15-60 minutes
No agglutinates
0
Dark, turbid, homogenous
0
Many tiny agglutinates, many free cells, may not be visible without microscope
W+
Dark, turbid
W+
Many small agglutinates, many free cells
1+
(25% are agglutinated)
1+
Turbid
1+
Many medium sized agglutinins, moderate number of free cells
2+
(50% are agglutinated)
2+
Clear
2+
3+
4+
Several large agglutinates, few free cells
3+
(75% are agglutinated)
3+
One large solid agglutination, no free
4+
(100% are agglutinated)
4+
MAJOR CATEGORIES OF AGGLUTINATION REACTIONS
Reaction is due to an Ag-Ab reaction where in the Ag is inherent (inside) native to the cell
Direct Immune
Direct Immune Example:
ABO grouping (hemagglutination), Widal Test
Aggregation of indicator rod blood cells are NOT due to Ag-Ab reaction
Direct Non Immune
Direct Non Immune Example:
Viral Hemagglutination test
Virus can stick to agglutinate RBC in the process
VIRAL HEMAGGLUTINATION
VIRAL HEMAGGLUTINATION viruses
Rubella virus, dengue virus, influenza virus, mumps virus
Viral receptor: (binds RBC)
Peplomers
Competitive binding Assay
VIRAL HEMAGGLUTINATION INHIBITION (HAI)
Procedure:
1. Patient serum w/ Ab incubated with viral particles (Commercially available)
2. Viral particles will bind to the Fab region of Anti-viral Abs
3. Indicator RBCs added to reaction mixture
VIRAL HEMAGGLUTINATION INHIBITION (HAI)
VIRAL HEMAGGLUTINATION INHIBITION (HAI)
Positive result:
Negative result:
Inhibition or Absence of Agglutination (Presence of Ab)
Agglutination
Reactions where Ag has been fixed or absorbed to a carrier/ inert particle
INDIRECT/ PASSIVE AGGLUTINATION
INDIRECT/ PASSIVE AGGLUTINATION Example:
Antistreptolysin-O (ASTO)
Different passive carriers:
Human RBCs
Clay (Bentonite)
Latex particles
Colloidal gold
Charcoal particles
Antibody is bound to the carrier
REVERSE PASSIVE AGGLUTINATION
Fluid is detected for the presence of Ag
REVERSE PASSIVE AGGLUTINATION
REVERSE PASSIVE AGGLUTINATION Example:
CRP, Reverse agglutination test for Candida and Nisseria
Patient sample (Ag) incubated with Ab in test kit
LATEX PARTICLE AGGLUTINATION INHIBITION
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
LATEX PARTICLE AGGLUTINATION INHIBITION Example:
HCG/ pregnancy test, Screening test for Drug
Systems using bacteria as the inert particles to which antibody is attached
COAGULATION
- most frequently used bacteria in COAGULATION
Staphylococcus aureus
– responsible for attaching Ab to the bacteria; naturally adsorbs the fragment crystallizable (FC) portion of antibody molecules
Protein A
The active sites face outward and are capable of reacting with specific antigen
COAGULATION
Detects IgG Ab bound to Ag on Red cells (in-vivo) Purpose:
Direct Antiglobulin Test
Direct Antiglobulin Test Purpose:
Hemolytic Disease of the Newborn investigation
Hemolytic Transfusion Reaction investigation
Autoimmune Hemolytic Anemia
Direct Antiglobulin Test Example:
Direct Coomb’s test
– maternal Ab attacking the fetal RBC
Hemolytic Disease of the Newborn investigation
– recipient Ab attacking the donor RBC
Hemolytic Transfusion Reaction investigation
– selfAb attacking own RBC
AIHA (Autoimmune Hemolytic Anemia)
Detects presence of Abs in the serum that is still to be attached to an analyte (invitro)
ANTIGLOBULIN TEST
ANTIGLOBULIN TEST Purpose:
Crossmatching
Ab determination
Ab identification
RBC Ag phenotyping
ANTIGLOBULIN TEST Example:
Indirect Coomb’s test
– metabolite of drug attached to RBC
Drug induced Hemolytic Anemia
In concentration or in exact value
QUANTITATIVE AGGLUTINATION REACTION
SPIA/ Sol Particle Immunoassay
Gold-inorganic colloidal particle
DIA/ Disperse Dye Immunoassay
Dye-organic colloidal particle
IMPACT/ Immunoassay by Particle Counting
Latex particle
Best QUANTITATIVE AGGLUTINATION REACTION
IMPACT/ Immunoassay by Particle Counting
VIRAL HEMAGGLUTINAT ION INHIBITION (HAI)
Reagent/s:
Unknown:
1) Viral particles 2) Indicator RBC
Antibody
INDIRECT/ PASSIVE AGGLUTINATION
Reagent/s:
Unknown:
Antigen
Antibody
REVERSE PASSIVE AGGLUTINATION
Reagent/s:
Unknown:
Antibody
Antigen
LATEX PARTICLE AGGLUTINATION INHIBITION
Reagent/s:
Unknown:
1) Antibody 2) Ag-coated latex particle
Antigen
VIRAL HEMAGGLUTINAT ION INHIBITION (HAI)
(+) Result
INDIRECT/ PASSIVE AGGLUTINATION
(+) Result
LATEX PARTICLE AGGLUTINATION INHIBITION
(+) Result
COAGULATION
Unknown
(+) Result
Ag
Agglutination
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 (Karl Landsteiner)
AGGLUTINATION
Antigen and antibody unite through antigenic determinant sites.
Sensitization
Sensitization or Lattice formation
Particulate Ags: w/ multiple determinants
Sensitization
Sensitization or Lattice formation
Cross-linking or bridging
Lattice formation
Rearrangement of antigen and antibody bonds to form stable lattice.
Lattice formation
Blood type determines the Ag present in RBC
ABO grouping (hemagglutination)
Ex.
A = AAg
B = Bag
AB = AB Ag
O = None (w/o A or B Ag)
ABO grouping (hemagglutination)
ABO grouping (hemagglutination) Types:
- Forward Typing
- Reverse Typing
– employs use of antisera containing Abs
Forward Typing
Types of anti-sera:
Anti-A and Anti-B
Competition between the Viral particle (VP) and Indicator RBC (I. RBC) for the binding sites of the Ab
[Px serum w/ Ab + Viral particle w/ Ag = Ag-Ab binding] + [Indicator RBC (to indicate agglutination)]
(+) Result:
No Agglutination
[Px serum w/o Ab + Viral particle w/ Ag = Ag-Ab binding] + [Indicator RBC (to indicate agglutination)]
VP binds w/ I. RBC due to peplomer to show agglutination
Result:
Agglutination
Antigen is attached to the carrier particle, and agglutination occurs if patient antibody is present.
INDIRECT/ PASSIVE AGGLUTINATION
Antibody is attached to the carrier particle, and agglutination occurs if patient antigen is present.
REVERSE PASSIVE AGGLUTINATION
REVERSE PASSIVE AGGLUTINATION
Has a fab site facing outwards, which is important to detach Ag
Ab
REVERSE PASSIVE AGGLUTINATION
– portion of Ab attached to the coated particle
Fc portion of the Ab
Reagent antibody is added to the patient sample.
LATEX PARTICLE AGGLUTINATION INHIBITION
If patient antigen is present, antigen–antibody combination results.
LATEX PARTICLE AGGLUTINATION INHIBITION
When antigen-coated latex particles are added, no agglutination occurs, which is a positive test.
LATEX PARTICLE AGGLUTINATION INHIBITION
If no patient antigen is there, the reagent antibody combines with latex particles, and agglutination results, which is a negative test.
LATEX PARTICLE AGGLUTINATION INHIBITION
Screening test for Drug
THC -
MET -
CONTROL – for
THC - marijuana
MET - methamphetamine (shabu)
CONTROL – for the validity of the test
Screening test for Drug Result
1 line for CONTROL -
1 line for MET –
positive for both
positive for THC
Same as reverse passive agglutination but bacteria is used instead of carrier particles to w/c Ab is attached
COAGULATION