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Antigen antibody 
reactions
plasma 
• Albumin 
• Fibrinogen 
• Gobulins 
– Alpha 
– Beta 
– Gammaglobulis
Gammaglobulis 
• Five types 
– IgG 
– IgA 
– IgM 
– IgE 
– IgD
functions of antibodies 
• to neutralize 
– toxins 
– viruses, 
• to opsonize microbes 
– so they are more easily phagocytosed, 
• to activate complement 
• to prevent the attachment of microbes to 
mucosal surfaces. 
• antibodies have a catalytic (enzymatic) 
capability
• Strength of the reaction between an antigenic 
determinant (antigen) and an antigen combining 
site (antibody) 
High Affinity 
Ab 
Ag 
Affinity 
Low Affinity 
Ab 
Ag 
Affinity =  attractive and repulsive forces
Avidity 
• The overall strength of binding between an Ag 
with many determinants and multivalent Abs 
Keq = 104 
Affinity 
106 
Avidity 
1010 
Avidity
Specificity 
• The ability of an antibody combining site to react 
with only an antigenic determinant. 
• The ability of antibody to combine with an antigen
Cross Reactivity 
• The ability of an individual Ab combining site to 
react with more than one type of antigenic 
determinant. 
• The ability of a Ab molecules to react with more 
than one Ag 
Anti-A 
Ab 
Ag A 
Cross reaction 
Anti-A 
Ab 
Ag C 
Similar A.D
Factors Affecting Happening of 
Ag/Ab Reactions 
• Affinity 
• Avidity 
• Ag:Ab ratio 
• Physical form of Ag 
Ab excess Ag excess 
Equivalence – Lattice formation
Tests Based on Ag/Ab Reactions 
• All tests based on Ag/Ab reactions will 
have to depend on lattice formation itself or 
• we will have to utilize ways to detect small 
immune complexes 
• All tests based on Ag/Ab reactions can be 
used to detect either Ag or Ab
Antigen antibody tests 
• Used in both directions 
– Qualitative 
– Quantitative
Uses of antigens/antibody reactions 
• Diagnosis Infectious diseases 
• Diagnosis of autoimmune diseases 
• Determination of blood type and HLA types 
• Determination of chemical levels
Diagnosis of infectious 
diseases 
• When the organism cannot be cultured-or 
difficult to culture hep A B C Syphilis 
• When the organism is too dangerous to 
culture- rickettsial disease 
• When the culture technique is not readily 
available-HIV,EBV 
• When the organism takes too long to grow 
e.g Mycoplasma
Qualitative /quantitative 
• Qualitative 
– determines antigen or antibody is present or 
absent 
• Quantitative 
– determines the quantity of the antibody 
– Titer 
– The highest dilution of the specimen usually 
serum which gives a positive reaction in the 
test
Antigen and antibody reactions in 
the lab 
• Precipitation tests 
• Agglutination 
• Elisa 
• Radioimmunoassay 
• Immunofluorescence 
• Complement Fixation
Precipitation tests 
• The antigen and antibody are in, soluble 
form 
• Combine to form a visible precipitate 
• Presence of electrolytes 
• Positive controls and negative controls
Precipitation tests 
• Precipitation techniques 
– Tube precipitation test 
– Gel diffusion 
• Double 
• Single radial 
• precipitation in agar with an electric field 
– Immuno electrophoresis 
– Countercurrent electrophoresis (CEP),
Precipitation in a capillary tube 
• Streptococcus grouping
Double Diffusion 
• antigen and antibody are placed in different 
wells in agar and 
• allowed to diffuse and form concentration 
gradients. 
• Where optimal proportions occur, lines of 
precipitate form
Double diffusion method (Ouchterlony) 
• indicates whether 
– antigens are identical, 
– Antigens not identical 
– Partially identical
Single immunodiffusion/Radial 
immunodiffusion 
• Method 
– Ab in gel 
– Ag in a well
Radial Immunodiffusion (Mancini) 
• Interpretation 
– Diameter of ring is 
proportional to the 
concentration 
• Quantitative 
– Ig levels 
Ag Concentration 
Ab in gel 
Diameter2 
Ag Ag Ag Ag
Precipitation in agar with an 
electric field
Immunoelectrophoresis 
• Application of electric current 
• Separation of proteins
Interpertation 
• serum proteins are characterized in terms 
of their 
• presence, 
• absence 
• unusual pattern (e.g., human myeloma protein).
Counter- 
Immunoelectrophoresis 
• Method 
– Ag and Ab migrate toward each other by 
electrophoresis 
– Used only when Ag and Ab have opposite charges 
- + 
• Qualitative 
–Rapid 
Ag Ab
uses 
• The meeting of the antigen and antibody is 
greatly accelerated 
• made visible in 30–60 minutes. 
• detection of bacterial and fungal 
polysaccharide antigens in cerebrospinal 
fluid.
Agglutination 
• Visible clumping together of particulate matter by 
antigen combining with its specific antibody. 
• The clumps will be called agglutinates 
• Performed 
– Slide 
– Tube 
– Tile 
– Micrtitration plates
Agglutination 
• Used in both directions 
• Antigen part of a particulate matter 
Salmonella 
• Particulate matter 
– Latex 
– Carbon particles 
– cells 
– Bacteria 
• Stablised staphylococcal cells
Agglutination 
• Active agglutination 
test 
• The antigen part of a 
particulate matter per 
se 
• examples 
• Salmonella, vibrio,
Agglutination 
• Passive agglutination test 
• Antigen or antibody are not part of 
particulate matter but are attached (rided on 
inert particles like latex, carbon,) 
+  Particulat 
matter
coagglutination 
• stabilized staphylococcal cells-protein 
affinity for FC fragment of antibody – 
protein A
Prozone phenomenon 
• Tube agglutination 
• The lower dilutions do not show agglutination 
• the tubes (prior/before the optimum zone ) 
• The tubes in higher dilutions show agglutination 
• Reasons/factors 
– Antibody excess:High level of antibody 
– Non specific inhibitory factors
Haemagglutination 
• Active Haemagglutination test 
– (blood group) 
• Passive haemagglutination (TPHA) 
– Known antigen coated on to treated RBC,s 
– Treated To remove their own antigens 
– Turkey,s RBC,s are used
Immunofluorescence 
• Fluorescent dyes illuminated by ultraviolet 
light are used to show combination of 
antigen antibody . 
• The end point antigen antibody complexes 
are seen fluorescing against a dark 
background 
• Direct 
• Indirect
Immunofluorescence 
• Direct 
– Ab to tissue Ag is labeled with fluorochrome
Immunofluorescence 
• Indirect 
– Ab to tissue Ag is 
unlabeled 
– Fluorochrome-labeled 
anti-Ig is used to detect 
binding of the first Ab. 
• Qualitative to Semi- 
Quantitative
Fluorescence-Activated Cell Sorting (Flow 
Cytometry) 
• the patient's cells are labeled with monoclonal 
antibody to the protein specific to the cell of 
interest, e.g., CD4 protein 
• The monoclonal antibody is tagged with a 
fluorescent dye, such as fluorescein or rhodamine. 
• Single cells are passed through a laser light beam, 
• the number of cells that fluoresce is counted by 
use of a machine called a fluorescence-activated 
cell sorter (FACS).
Immunofluorescence 
• Flow Cytometry 
– Cells in suspension are labeld with fluorescent tag 
•Cells analyzed on a flow cytometer
Assays Based on Complement 
Lattice formation not required
CFT 
• The CFT is used to detect antibodies or 
antigens 
• inference is made by the ability of the 
antibodies to fix the complement 
• The fixation of the complement is measured 
by adding the indicator system 
• Used in diagnosis of viral parasitic and 
rickettsial diseases
Complement Fixation 
• Methodology 
– Antibody known mixed with test material to be assayed for Ag 
– Standard amount of complement is added 
– Erythrocytes coated with Abs is added 
– Amount of erythrocyte lysis is determined 
Ag NO Ag 
Ag 
Ag
RadioImmunoassay 
• The radioactivity of the specific labeled 
antibody or antigen is used to quantify the 
antigen or antibody in patient ,s serum 
• HbsAg 
• Hav IGM
ELISA 
• Uses an enzyme system to show the specific 
combination of antigen antibody 
• An enzyme labeled or linked to a specific antigen 
• A substrate 
• A color reader 
• Double antibody technique to detect and assay 
antigen 
• Indirect technique to Assay antibody
Direct Elisa 
• Ag detection 
– Add labeled antibody 
– Amount of labeled Ab 
bound is proportional 
to the amount of Ag in 
the sample
Indirect ELISA 
• Ab detection 
– Immobilize Ag 
– Incubate with sample 
– Add labeled anti-Ig 
– Amount of labeled Ab 
bound is proportional 
to amount of Ab in the 
sample 
• Quantitative 
Immobilized Ag 
Solid 
Phase 
Ab in 
Patient’s 
sample 
Labeled 
Anti-Ig
Western blot 
• HIV proteins are separated electrophoretically in a gel, 
• discrete bands of viral protein. 
• These proteins are then transferred from the gel, i.e., blotted, 
onto filter paper, and 
• the person's serum is added. 
• If antibodies are present, they bind to the viral proteins 
(primarily gp41 and p24) and can be detected by adding 
antibody to human IgG labeled an enzyme, 
• produces a visible color change when the enzyme substrate is 
added.
Neutralization Tests 
• These use the ability of 
• antibodies to block the effect of toxins or 
the infectivity of viruses. 
• They can be used in cell culture ( inhibition 
of cytopathic effect 
• in host animals ( mouse protection tests).
Extras
Ag-Ab reactions 
Tests for Ag-Ab reactions
Nature of Ag/Ab Reactions 
• Lock and Key Concept 
• Non-covalent Bonds 
– Hydrogen bonds 
– Electrostatic bonds 
– Van der Waal forces 
– Hydrophobic bonds 
• Multiple Bonds 
• Reversible 
http://www.med.sc.edu:85/chime2/lyso-abfr.htm 
Source: Li, Y., Li, H., Smith-Gill, S. J., 
Mariuzza, R. A., Biochemistry 39, 6296, 2000
Coombs (Antiglobulin)Tests 
• Incomplete Ab 
• Direct Coombs Test 
– Detects antibodies on erythrocytes 
+  
Patient’s RBCs Coombs Reagent 
(Antiglobulin)
Coombs (Antiglobulin)Tests 
• Indirect Coombs Test 
– Detects anti-erythrocyte antibodies in serum 
Patient’s 
Serum 
+  
Target 
RBCs 
Step 1 
+  
Coombs Reagent 
(Antiglobulin) 
Step 2
Immunofluorescence 
• Flow Cytometry cont. 
– Data displayed 
One Parameter Histogram 
Unstained cells 
Green Fluorescence Intensity 
Number of Cells 
FITC-labeled cells 
Red Fluorescence Intensity 
Green Fluorescence Intensity 
Two Parameter Histogram
Agglutination Tests 
Lattice Formation
Active Agglutination 
• Definition - tests that have as their endpoint 
the agglutination of a particulate antigen 
– Agglutinin
Agglutination/Hemagglutination 
• Quantitative agglutination test 
– Titer 
– Prozone 
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
Agglutination/Hemagglutination 
• Definition 
• Qualitative test 
• Quantitative test 
• Applications 
– Blood typing 
– Bacterial infections 
–Fourfold rise in titer 
• Practical considerations 
– Easy 
– Semi-quantitative 
1/2 
1/4 
1/8 
1/16 
1/32 
1/64 
1/128 
1/256 
1/512
Passive Agglutination/Hemagglutination 
• Definition - agglutination test done with a 
soluble antigen coated onto a particle 
• Applications 
– Measurement of antibodies to soluble antigens
Coombs (Antiglobulin)Tests 
• Applications 
– Detection of anti-Rh Ab 
– Autoimmune hemolytic anemia
Precipitation Tests 
Lattice Formation
Radioimmuoassays (RIA) 
Enzyme-Linked Immunosorbent 
Assays (ELISA) 
Lattice formation not required
Competitive ELISA for antigen 
• Method 
– Determine amount 
of Ab needed to bind 
to a known amount 
of labeled Ag 
+  
Prior to Test 
Labeled 
Ag 
+  
Test 
+ 
Patient’s 
sample 
Labeled 
Ag 
+ 
– Use predetermined 
amounts of labeled 
Ag and Ab and add a 
sample containing 
unlabeled Ag as a 
competitor
Competitive ELISA for Ag 
• Method cont. 
– Determine amount 
of labeled Ag bound 
to Ab 
  NH4SO4 
  anti-Ig 
• Immobilize the Ab 
• Quantitative 
– Most sensitive test 
+  
Test 
+ 
Patient’s 
sample 
Labeled 
Ag 
+ 
Solid 
Phase 
Solid 
Phase 
– Concentration determined from a standard curve 
using known amounts of unlabeled Ag
• Extras
Solid Phase RIA for antigen/direct 
• Ag detection 
– Immobilize Ab 
– Incubate with sample 
– Add labeled antibody 
– Amount of labeled Ab 
bound is proportional to 
the amount of Ag in the 
sample 
• Quantitative 
Ag 
Solid 
Phase 
Ag in 
Patient’s 
sample 
Immobilized 
Labeled 
Ab
Solid Phase RIA for antibody/indirect 
• Ab detection 
– Immobilize Ag 
– Incubate with sample 
– Add labeled anti-Ig 
– Amount of labeled Ab 
bound is proportional 
to amount of Ab in the 
sample 
• Quantitative 
Immobilized Ag 
Solid 
Phase 
Ab in 
Patient’s 
sample 
Labeled 
Anti-Ig
Factors Affecting Measurement of 
Ag/Ab Reactions 
• Affinity 
• Avidity 
• Ag:Ab ratio 
• Physical form of Ag 
Ab excess Ag excess 
Equivalence – Lattice formation
Cross Reactivity 
• The ability of an individual Ab combining site to 
react with more than one type of antigenic 
determinant. 
• The ability of a Ab molecules to react with more 
than one Ag 
Anti-A 
Ab 
Ag A 
Cross reactions 
Anti-A 
Ab 
Ag B 
Shared A.D 
Anti-A 
Ab 
Ag C 
Similar A.D
Immunoelectrophoresis 
• Method 
– Ags are separated by electrophoresis 
– Ab is placed in trough cut in the agar 
+ - 
Ag 
• Interpretation 
Ag 
Ab 
Ag 
Ab 
– Precipitin arc represent individual antigens
Haemagglutination inhibiotion test 
• Certain viruses measles and influenza 
arboviruses agglutinate RBC,s 
• If specific antibodies are included in the 
system a virus is identified
Tests for Cell Associated 
Antigens 
Lattice formation not required
Immunofluorescence 
• Flow Cytometry 
– Cells in suspension are labeld with fluorescent tag 
•Cells analyzed on a flow cytometer
Double Antibody ELISA 
• Ag detection 
– Immobilize Ab 
– Incubate with sample 
– Add labeled antibody 
– Amount of labeled Ab 
bound is proportional to 
the amount of Ag in the 
sample 
• Quantitative 
Ag 
Solid 
Phase 
Ag in 
Patient’s 
sample 
Immobilized 
Labeled 
Ab

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Antigen-Antibody Reaction Tests

  • 2. plasma • Albumin • Fibrinogen • Gobulins – Alpha – Beta – Gammaglobulis
  • 3. Gammaglobulis • Five types – IgG – IgA – IgM – IgE – IgD
  • 4.
  • 5.
  • 6.
  • 7.
  • 8.
  • 9. functions of antibodies • to neutralize – toxins – viruses, • to opsonize microbes – so they are more easily phagocytosed, • to activate complement • to prevent the attachment of microbes to mucosal surfaces. • antibodies have a catalytic (enzymatic) capability
  • 10. • Strength of the reaction between an antigenic determinant (antigen) and an antigen combining site (antibody) High Affinity Ab Ag Affinity Low Affinity Ab Ag Affinity =  attractive and repulsive forces
  • 11. Avidity • The overall strength of binding between an Ag with many determinants and multivalent Abs Keq = 104 Affinity 106 Avidity 1010 Avidity
  • 12. Specificity • The ability of an antibody combining site to react with only an antigenic determinant. • The ability of antibody to combine with an antigen
  • 13. Cross Reactivity • The ability of an individual Ab combining site to react with more than one type of antigenic determinant. • The ability of a Ab molecules to react with more than one Ag Anti-A Ab Ag A Cross reaction Anti-A Ab Ag C Similar A.D
  • 14. Factors Affecting Happening of Ag/Ab Reactions • Affinity • Avidity • Ag:Ab ratio • Physical form of Ag Ab excess Ag excess Equivalence – Lattice formation
  • 15. Tests Based on Ag/Ab Reactions • All tests based on Ag/Ab reactions will have to depend on lattice formation itself or • we will have to utilize ways to detect small immune complexes • All tests based on Ag/Ab reactions can be used to detect either Ag or Ab
  • 16. Antigen antibody tests • Used in both directions – Qualitative – Quantitative
  • 17. Uses of antigens/antibody reactions • Diagnosis Infectious diseases • Diagnosis of autoimmune diseases • Determination of blood type and HLA types • Determination of chemical levels
  • 18. Diagnosis of infectious diseases • When the organism cannot be cultured-or difficult to culture hep A B C Syphilis • When the organism is too dangerous to culture- rickettsial disease • When the culture technique is not readily available-HIV,EBV • When the organism takes too long to grow e.g Mycoplasma
  • 19. Qualitative /quantitative • Qualitative – determines antigen or antibody is present or absent • Quantitative – determines the quantity of the antibody – Titer – The highest dilution of the specimen usually serum which gives a positive reaction in the test
  • 20.
  • 21. Antigen and antibody reactions in the lab • Precipitation tests • Agglutination • Elisa • Radioimmunoassay • Immunofluorescence • Complement Fixation
  • 22. Precipitation tests • The antigen and antibody are in, soluble form • Combine to form a visible precipitate • Presence of electrolytes • Positive controls and negative controls
  • 23. Precipitation tests • Precipitation techniques – Tube precipitation test – Gel diffusion • Double • Single radial • precipitation in agar with an electric field – Immuno electrophoresis – Countercurrent electrophoresis (CEP),
  • 24. Precipitation in a capillary tube • Streptococcus grouping
  • 25. Double Diffusion • antigen and antibody are placed in different wells in agar and • allowed to diffuse and form concentration gradients. • Where optimal proportions occur, lines of precipitate form
  • 26.
  • 27.
  • 28. Double diffusion method (Ouchterlony) • indicates whether – antigens are identical, – Antigens not identical – Partially identical
  • 29. Single immunodiffusion/Radial immunodiffusion • Method – Ab in gel – Ag in a well
  • 30. Radial Immunodiffusion (Mancini) • Interpretation – Diameter of ring is proportional to the concentration • Quantitative – Ig levels Ag Concentration Ab in gel Diameter2 Ag Ag Ag Ag
  • 31. Precipitation in agar with an electric field
  • 32. Immunoelectrophoresis • Application of electric current • Separation of proteins
  • 33.
  • 34. Interpertation • serum proteins are characterized in terms of their • presence, • absence • unusual pattern (e.g., human myeloma protein).
  • 35. Counter- Immunoelectrophoresis • Method – Ag and Ab migrate toward each other by electrophoresis – Used only when Ag and Ab have opposite charges - + • Qualitative –Rapid Ag Ab
  • 36. uses • The meeting of the antigen and antibody is greatly accelerated • made visible in 30–60 minutes. • detection of bacterial and fungal polysaccharide antigens in cerebrospinal fluid.
  • 37. Agglutination • Visible clumping together of particulate matter by antigen combining with its specific antibody. • The clumps will be called agglutinates • Performed – Slide – Tube – Tile – Micrtitration plates
  • 38.
  • 39. Agglutination • Used in both directions • Antigen part of a particulate matter Salmonella • Particulate matter – Latex – Carbon particles – cells – Bacteria • Stablised staphylococcal cells
  • 40. Agglutination • Active agglutination test • The antigen part of a particulate matter per se • examples • Salmonella, vibrio,
  • 41. Agglutination • Passive agglutination test • Antigen or antibody are not part of particulate matter but are attached (rided on inert particles like latex, carbon,) +  Particulat matter
  • 42. coagglutination • stabilized staphylococcal cells-protein affinity for FC fragment of antibody – protein A
  • 43. Prozone phenomenon • Tube agglutination • The lower dilutions do not show agglutination • the tubes (prior/before the optimum zone ) • The tubes in higher dilutions show agglutination • Reasons/factors – Antibody excess:High level of antibody – Non specific inhibitory factors
  • 44. Haemagglutination • Active Haemagglutination test – (blood group) • Passive haemagglutination (TPHA) – Known antigen coated on to treated RBC,s – Treated To remove their own antigens – Turkey,s RBC,s are used
  • 45. Immunofluorescence • Fluorescent dyes illuminated by ultraviolet light are used to show combination of antigen antibody . • The end point antigen antibody complexes are seen fluorescing against a dark background • Direct • Indirect
  • 46.
  • 47. Immunofluorescence • Direct – Ab to tissue Ag is labeled with fluorochrome
  • 48.
  • 49.
  • 50. Immunofluorescence • Indirect – Ab to tissue Ag is unlabeled – Fluorochrome-labeled anti-Ig is used to detect binding of the first Ab. • Qualitative to Semi- Quantitative
  • 51. Fluorescence-Activated Cell Sorting (Flow Cytometry) • the patient's cells are labeled with monoclonal antibody to the protein specific to the cell of interest, e.g., CD4 protein • The monoclonal antibody is tagged with a fluorescent dye, such as fluorescein or rhodamine. • Single cells are passed through a laser light beam, • the number of cells that fluoresce is counted by use of a machine called a fluorescence-activated cell sorter (FACS).
  • 52. Immunofluorescence • Flow Cytometry – Cells in suspension are labeld with fluorescent tag •Cells analyzed on a flow cytometer
  • 53. Assays Based on Complement Lattice formation not required
  • 54. CFT • The CFT is used to detect antibodies or antigens • inference is made by the ability of the antibodies to fix the complement • The fixation of the complement is measured by adding the indicator system • Used in diagnosis of viral parasitic and rickettsial diseases
  • 55.
  • 56.
  • 57.
  • 58.
  • 59.
  • 60.
  • 61. Complement Fixation • Methodology – Antibody known mixed with test material to be assayed for Ag – Standard amount of complement is added – Erythrocytes coated with Abs is added – Amount of erythrocyte lysis is determined Ag NO Ag Ag Ag
  • 62. RadioImmunoassay • The radioactivity of the specific labeled antibody or antigen is used to quantify the antigen or antibody in patient ,s serum • HbsAg • Hav IGM
  • 63.
  • 64. ELISA • Uses an enzyme system to show the specific combination of antigen antibody • An enzyme labeled or linked to a specific antigen • A substrate • A color reader • Double antibody technique to detect and assay antigen • Indirect technique to Assay antibody
  • 65. Direct Elisa • Ag detection – Add labeled antibody – Amount of labeled Ab bound is proportional to the amount of Ag in the sample
  • 66. Indirect ELISA • Ab detection – Immobilize Ag – Incubate with sample – Add labeled anti-Ig – Amount of labeled Ab bound is proportional to amount of Ab in the sample • Quantitative Immobilized Ag Solid Phase Ab in Patient’s sample Labeled Anti-Ig
  • 67. Western blot • HIV proteins are separated electrophoretically in a gel, • discrete bands of viral protein. • These proteins are then transferred from the gel, i.e., blotted, onto filter paper, and • the person's serum is added. • If antibodies are present, they bind to the viral proteins (primarily gp41 and p24) and can be detected by adding antibody to human IgG labeled an enzyme, • produces a visible color change when the enzyme substrate is added.
  • 68.
  • 69. Neutralization Tests • These use the ability of • antibodies to block the effect of toxins or the infectivity of viruses. • They can be used in cell culture ( inhibition of cytopathic effect • in host animals ( mouse protection tests).
  • 71. Ag-Ab reactions Tests for Ag-Ab reactions
  • 72. Nature of Ag/Ab Reactions • Lock and Key Concept • Non-covalent Bonds – Hydrogen bonds – Electrostatic bonds – Van der Waal forces – Hydrophobic bonds • Multiple Bonds • Reversible http://www.med.sc.edu:85/chime2/lyso-abfr.htm Source: Li, Y., Li, H., Smith-Gill, S. J., Mariuzza, R. A., Biochemistry 39, 6296, 2000
  • 73.
  • 74. Coombs (Antiglobulin)Tests • Incomplete Ab • Direct Coombs Test – Detects antibodies on erythrocytes +  Patient’s RBCs Coombs Reagent (Antiglobulin)
  • 75. Coombs (Antiglobulin)Tests • Indirect Coombs Test – Detects anti-erythrocyte antibodies in serum Patient’s Serum +  Target RBCs Step 1 +  Coombs Reagent (Antiglobulin) Step 2
  • 76. Immunofluorescence • Flow Cytometry cont. – Data displayed One Parameter Histogram Unstained cells Green Fluorescence Intensity Number of Cells FITC-labeled cells Red Fluorescence Intensity Green Fluorescence Intensity Two Parameter Histogram
  • 78. Active Agglutination • Definition - tests that have as their endpoint the agglutination of a particulate antigen – Agglutinin
  • 79. Agglutination/Hemagglutination • Quantitative agglutination test – Titer – Prozone 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
  • 80. Agglutination/Hemagglutination • Definition • Qualitative test • Quantitative test • Applications – Blood typing – Bacterial infections –Fourfold rise in titer • Practical considerations – Easy – Semi-quantitative 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512
  • 81. Passive Agglutination/Hemagglutination • Definition - agglutination test done with a soluble antigen coated onto a particle • Applications – Measurement of antibodies to soluble antigens
  • 82. Coombs (Antiglobulin)Tests • Applications – Detection of anti-Rh Ab – Autoimmune hemolytic anemia
  • 84. Radioimmuoassays (RIA) Enzyme-Linked Immunosorbent Assays (ELISA) Lattice formation not required
  • 85. Competitive ELISA for antigen • Method – Determine amount of Ab needed to bind to a known amount of labeled Ag +  Prior to Test Labeled Ag +  Test + Patient’s sample Labeled Ag + – Use predetermined amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor
  • 86. Competitive ELISA for Ag • Method cont. – Determine amount of labeled Ag bound to Ab   NH4SO4   anti-Ig • Immobilize the Ab • Quantitative – Most sensitive test +  Test + Patient’s sample Labeled Ag + Solid Phase Solid Phase – Concentration determined from a standard curve using known amounts of unlabeled Ag
  • 88. Solid Phase RIA for antigen/direct • Ag detection – Immobilize Ab – Incubate with sample – Add labeled antibody – Amount of labeled Ab bound is proportional to the amount of Ag in the sample • Quantitative Ag Solid Phase Ag in Patient’s sample Immobilized Labeled Ab
  • 89. Solid Phase RIA for antibody/indirect • Ab detection – Immobilize Ag – Incubate with sample – Add labeled anti-Ig – Amount of labeled Ab bound is proportional to amount of Ab in the sample • Quantitative Immobilized Ag Solid Phase Ab in Patient’s sample Labeled Anti-Ig
  • 90.
  • 91. Factors Affecting Measurement of Ag/Ab Reactions • Affinity • Avidity • Ag:Ab ratio • Physical form of Ag Ab excess Ag excess Equivalence – Lattice formation
  • 92. Cross Reactivity • The ability of an individual Ab combining site to react with more than one type of antigenic determinant. • The ability of a Ab molecules to react with more than one Ag Anti-A Ab Ag A Cross reactions Anti-A Ab Ag B Shared A.D Anti-A Ab Ag C Similar A.D
  • 93.
  • 94. Immunoelectrophoresis • Method – Ags are separated by electrophoresis – Ab is placed in trough cut in the agar + - Ag • Interpretation Ag Ab Ag Ab – Precipitin arc represent individual antigens
  • 95.
  • 96.
  • 97. Haemagglutination inhibiotion test • Certain viruses measles and influenza arboviruses agglutinate RBC,s • If specific antibodies are included in the system a virus is identified
  • 98. Tests for Cell Associated Antigens Lattice formation not required
  • 99. Immunofluorescence • Flow Cytometry – Cells in suspension are labeld with fluorescent tag •Cells analyzed on a flow cytometer
  • 100.
  • 101. Double Antibody ELISA • Ag detection – Immobilize Ab – Incubate with sample – Add labeled antibody – Amount of labeled Ab bound is proportional to the amount of Ag in the sample • Quantitative Ag Solid Phase Ag in Patient’s sample Immobilized Labeled Ab

Editor's Notes

  1. Counter immuno electrophoresis immuno electro osmo phoresis (IEOP)
  2. Reverse passive Haemagglutination test(RPHA) Antibody is attached to RBC,s identification of viruses which will not cause haemagglutination per se.