Agglutination Reactions. Principles and Reactions

Agglutination Reactions Principles and Practice Sampler: Fixed and Adjustable Mouth pipetting is forbidden in lab Use digital or mechanical pipetting Agglutination tests 3 Pipetting method Agglutination tests 4 Serologic tests are based on Ag-Ab reactions • Previously we discussed that four items play role in Antigen-Antibody interaction: – Antigen and Antibody characteristics, their concentration and ratio and finally the environmental condition Agglutination tests 6 Antigen properties • Based on size, Ags could be: – Particulate: Whole Bacteria or RBC, other particles – Soluble: Protein antigens – Colloidal: Lipid antigens • Based on cellular location, Ags could be: – Intercellular – Extracellular Agglutination tests 7 Antibody • Abs could be from different class and subclasses that their Ag binding capacities varies. • IgM is produced in acute phase of infection and has 10 Fabs, so has more binding sites and is a better Ag agglutinating agent. IgM was more sensitive to reducing agents (2ME) • IgG is produced in chronic phase of infection and has 2 Fabs, but usually have higher binding affinity and is produced in higher amounts. So it can be a good Ag agglutinating molecule, too. Agglutination tests 8 Environmental factors • In the laboratory (in vitro) we try to mimic the similar physiologic pH (7.0 - 7.4) and thermal (37℃) conditions found in vivo. • Shaking and agitating can help us to obtain better and rapid reactions. • The optimal binding may take time, thus we need some incubation time. Agglutination tests 9 Classification of Ag-Ab reactions 1- Primary assays: Labeled immunoassays (Lattice formation is not necessary) Used for evaluation of low amounts of Ag or Ab 2- Secondary assays: Visible results (Lattice formation is the basics) Used for evaluation of high amounts of Ag or Ab 3- Tertiary assays: Functional assays Used for evaluation of the consequences following Ag-Ab reactions Secondary serologic reactions • Based on Ag type and size three types of reactions are possible: – Agglutination of particulate Ags – Flocculation (microscopic agglutination) of colloidal Ags – Precipitation of soluble Ags Agglutination tests 11 Agglutination • Agglutination is based on aggregation of particulate Ags (Agglutinogens) by Abs (agglutinins) or other agents such as lectins. • Agglutination is a simple and sensitive test with different applications, specially in diagnosis of febrile diseases such as Brucellosis and Salmonellosis. Agglutination tests 12 Classification of Agglutination tests based on method  Slide Agglutination Test (Rapid)  Tube Agglutination Test (Standard)  Microtiter plate Agglutination Test (Micro) Agglutination tests 13 Slid or Rapid Agglutination Test • Screening assays report format • Qualitative (Pos / Neg), (Pos: 1+, 2+, 3+, 4+): mostly used in passive agglutination tests (Reactive/ Non-Reactive): Blood grouping • Semi-quantitative (Titer): Brucellosis (Wright), Sallemonellosis (Widal) 14 Serodiagnosis of Brucellosis  Brucellosis is the most common global zoonosis  Serological diagnosis is based on agglutination of B. abortus bacterial particles with patients serum  Other species of the Brucella genus bacteria have similar antigens to B. melitensis and B. suis.  Thus inactivated B. abortus is used in the test as the available bacteria. Agglutination tests 15 Agglutination steps Agglutination takes place in two steps: 1. Sensitization or initial binding of molecules Rapid and reversible attachment of Abs to particulate Ags 2. Lattice formation or aggregation or stabilization Cross linking of particles and formation of visible enlarged aggregates Agglutination tests 16 Agglutination advantages • • • • • • Rapid Low cost Sensitive May be done on slide, tube or miroplate Easily monitored by eye Small amount of serum and reagent needed Agglutination tests 17 Classification of Agglutination tests based on Ag properties • Active agglutination: Ag is a native part or structure of the particle – Heamagglutination: Abs bind to RBC surface Ags • Blood grouping: Cell type, Back type – Microbial agglutination: Abs bind to bacteria surface Ags • Wright, Widal • Passive agglutination: A soluble Ag was bound on an inert particle to make a particulate Ag – Latex agglutination: ASO (Streptolysin O is bound to latex) – Heamagglutination: Viruses are bound to RBC and antiviral Abs studied Agglutination tests 18 Active Agglutination • Screening of surface Ags via specific Abs. • Serotyping of E. coli, Salmonella, Vibrio cholera, … with specific antisera • Blood groups (cell type) • Screening of produced Abs to surface Ags. • Wright and Widal tests • Blood grouping (Back type) Agglutination tests 19 Screening of Brucellosis • Bacterial suspension of Brucella abortus agglutinated with specific Abs in the serum • attenuated strain of B. abortus were inactivated and stained with rose Bengal or other stains for better visibility of the results Agglutination tests 20 Serologic tests for Brucellosis • • • • Rapid Wright test (screening) Standard Wright test in tube 2ME-Wright test Coombs’ Wright test Agglutination tests 21 Wright Tube Agglutination test 10 9 8 7 6 5 4 3 2 1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.9 Saline 0.1 Serum - Tube - 0.5 out 0.5 ← 0.5 ← 0.5 ← 0.5 ← 0.5 ← 0.5 ← 0.5 ← 0.5 ← 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Ag Control 1:5120 1:2560 1:1280 1:640 1:320 1:160 1:80 1:40 1:20 Titer Agglutination tests 22 Wright Tube Agglutination test In this case, the titre is 1/80 1/20 1/40 1/80 Agglutination 1/160 1/320 1/640 1/1280 Neg. ctrl No agglutination Agglutination tests 23 Interpretation of the results • Clinical history • Rapid tests • Standard tests • Follow up tests Agglutination tests 24 Passive Agglutination • Agglutination reaction of inert particles coated with soluble Ags that are not normally found on their surfaces • Inert Ag carriers: – Polystyrene latex – Red blood cells (O neg) – Charcoal Agglutination tests 25 Common Passive Agglutination tests • ASO (Anti-Streptolysin O) • RF (Rheumatoid factor) • CRP (C-reactive protein) • Gravindex (HCG) Agglutination tests 26 Passive Agglutination Agglutination tests 27 Tube Agglutination Test • Also known as the standard or Quantitative agglutination test, e.g. Brucellosis (Wright) • Serum diluted in 2-fold manner • Constant amount of Ag is added • Incubation at RT or 37°C (overnight or hours) • Visible agglutination at the bottom of tubes Agglutination tests 28 Microtiter plate agglutination Agglutination tests 29 Microscopic agglutination or Flocculation • VDRL (Venereal Disease Research Laboratory) • Nowadays RPR (Rapid Plasma Reagin) is substituted for RPR which is a agglutination test. • The positive results are checked by FTA-ABS Agglutination tests 30 CRP, RF, ASO, Gravindex • Sample + Reagent  Mix and Rotate for 3 minutes, Read the results within 3 minutes • • • • Use positive and negative controls Could be qualitative or semiquantitative Application of the tests Interpretation of the results Agglutination tests 31 CRP Agglutination tests 32