immuoagglutination test yasser m. alatawi pharm.d college of pharmacy umm al-qura university
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Immuoagglutination test
Yasser M. alatawiPharm.D
College of PharmacyUmm Al-Qura University
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Key point
Definition Factor affecting agglutinationClassification of agglutination reactionMethodology
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Definition
Agglutination occurs due to the cross-linking of antibodies with particulate antigens to form clumping of insoluble particles .
when antigen an erythrocyte the term hemagglutinin is often used .
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Cont.
General priciple of agglutination :Tow phase reaction :
1-Binding of antigen an antibody
2- Lattice formation ( clumping)
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Cont.
Application of agglutination tests : Determination of blood type . To assess and monitoring bacterial
infection .
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factor affecting agglutination
Class of antibody : IgM class are larger molecule containing 10 binding
site , therefore are more effective than other immunoglobulin's class ( e.g. IgG (
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Cont.
Charge of the carrier particle : In slain , RBCs will attract +ve charge and an ionic
cloud will form around cell ,thus the cells will be repelled and stay in distance ( zeta potential).
Can be reduce by addition of Albumin and low ionic strength solution .
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cont .
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Cont.
Number of Antigen Sites : The number of antigen sites is an important since the
more antigen sites result in more antibodies being attached and forming cross-linkages. These cross-linkages result in agglutination
• Concentration of reaction : Ab-Ag reaction is more rabid in higher conc.
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Cont.
Enviromental factor : Mixing , centrifugation and appropriate
incubation time and tem >>> Enhance agglutination reaction .
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Classification of agglutination reaction
A. Direct agglutination :
+ ↔
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Cont.
RBC antigen ABO blood groups and Rh . I antigens- cold agglutinin test for mycoplasma
pneumonia . Paul-bunnel antigen –associated with infection
mononucleosis .
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Cont.
Bacterial antigen Use for detect recent infection . Febrile agglutinin: Ab produce during bacterial
infection with fever . Widal test: salmonella Bactria as antigen for detection
of Ab to typhoid paratyphoid organism .
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Cont.
B. Passive agglutination ( latex agglutination)
Antigen is attached to a particulate carrier , then react with antibody .
Advantage: rabidity Carriers include : charcoal , gelatin , RBCs
+ ↔
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cont
Used for : Rheumatoid factor : ASO: antistreptolysin O in serum associated
with streptococcal infectioin SLE :detection of antinuclear antibodies in
human sr. associated systemic lupus erythematousus .
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Cont.
C. Reverse passive agglutination : Ab is attached to carrier . Use to detect C-reactive protein is an
acute phase protein increase druing infection and inflammation, so it an inflammatory marker .
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Cont.
D. Agglutination inhibition reaction Use to measure soluble antigen …. e.g. pregnancy test in urine A tow step process :
+ ↔Prior to Test
+ ↔+Test
Patient’s sample
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METHODOLOGY
Agglutination either a qualitative or quantitative :
Method
Slide test Tube tests Microwel assay
•Rabid•Semiquntitative
•Room temp. incubation
•Longer incubation•Different incubation
Condition•Semiquntitative
•Modification tube assay
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Slide test.
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Tube tests
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Microwel assay
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Qualitative agglutination test:
Negative control sample positive control
Negative result : no agglutination Positive result: agglutination
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Quantitative agglutination test:
agglutination test can also be used to quantities the level of antibodies particulate antigens
1/2
1/4
1/8
1/16
1/32
1/64
1/12
8
1/25
6
1/51
2
1/10
24
Pos
.
Neg
.
Titer
648
512<232
128324
Patient
12345678
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Cont.
Interpretation : mg\dl of serum = conc. Of positive control x last
dilution showing a positive result Titer 1:4 and positive control .8 mg\dl So , conc. Of Ab .8 x 4 =302 mg\dl
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Cont.
Interpretation of result for otherTube method : Strength can be graded in flowing manner: 4 + large clumping 3 + moderate clumping 2 + small clumping 1 + very small clumping
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Quality control
Postive and negative control sholud include in each test Reaction time critical .( more than 3 min >>> drying of sample
false positive result) Freezing the latex reagent lead to spontaneous agglutination …
so always bring reagent to room temp. before starting the test . Do not mix component form different kit or manfacturers . Always use new pipette when transferring sample . Do not use reagent after expiration date Sample showing turbidity or hemolysis may yield incorrect result
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Cold agglutinins
Cold agglutinin disease is an autoimmune disease characterized by the presence of high concentrations of circulating antibodies, usually IgM, directed against red blood cells. It is a form of autoimmune hemolytic anemia, specifically one in which antibodies only bind red blood cells at low body temperatures, typically 28-31°C.
The primary form is idiopathic ,and secondary Occur with infection especially mycoplasma pneumonia and some cancer
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