1. Immuoagglutination test Yasser M. alatawi Pharm.D College of Pharmacy Umm Al-Qura University

2. Key point Definition Factor affecting agglutination Classification of agglutination reaction Methodology

3. 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.

4. Cont. General priciple of agglutination : Tow phase reaction : 1-Binding of antigen an antibody 2- Lattice formation ( clumping)

5. Cont. Application of agglutination tests :  Determination of blood type.  To assess and monitoring bacterial infection.

6. 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 (

7. 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.

8. cont.

9. 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.

10. Cont. Enviromental factor :  Mixing, centrifugation and appropriate incubation time and tem >>> Enhance agglutination reaction.

11. Classification of agglutination reaction A. Direct agglutination : + ↔

12. Cont. RBC antigen  ABO blood groups and Rh.  I antigens- cold agglutinin test for mycoplasma pneumonia.  Paul-bunnel antigen –associated with infection mononucleosis.

13. 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.

14. Cont. B. Passive agglutination ( latex agglutination) Antigen is attached to a particulate carrier, then react with antibody. Advantage: rabidity Carriers include : charcoal, gelatin, RBCs + ↔

15. 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.

16. 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.

17. Cont. D. Agglutination inhibition reaction Use to measure soluble antigen …. e.g. pregnancy test in urine A tow step process : + ↔ Prior to Test + ↔ + Tes t Patient’s sample

18. METHODOLOGY Agglutination either a qualitative or quantitative : Method Slide test Rabid Semiquntitative Room temp. incubation Tube tests Longer incubation Different incubation Condition Semiquntitative Microwel assay Modification tube assay

19. Slide test.

20. Tube tests

21. Microwel assay

22. Qualitative agglutination test: Negative control sample positive control Negative result : no agglutination Positive result: agglutination

23. 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 1/128 1/256 1/512 1/1024 Pos. Neg. Titer 64 8 512 <2 32 128 32 4 Patient 1 2 3 4 5 6 7 8

24. 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

25. Cont. Interpretation of result for other Tube method : Strength can be graded in flowing manner: 4 + large clumping 3 + moderate clumping 2 + small clumping 1 + very small clumping

26. 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

27. 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.autoimmune diseaseantibodiesIgM red blood cellsautoimmune hemolytic anemia The primary form is idiopathic,and secondary Occur with infection especially mycoplasma pneumonia and some canceridiopathic