Ag-Ab reactionsTests 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

• Reversible

• Multiple Bonds

Source: Li, Y., Li, H., Smith-Gill, S. J., Mariuzza, R. A., Biochemistry 39, 6296, 2000

http://www.med.sc.edu:85/chime2/lyso-abfr.htm

Affinity = ∑ attractive and repulsive forces

Ab

Ag

High Affinity

Ab

Ag

Low Affinity

Affinity• Strength of the reaction between a single antigenic

determinant and a single Ab combining site

Calculation of Affinity

Ag + Ab Ag-Ab

Keq = [Ag-Ab]

[Ag] x [Ab]

Applying the Law of Mass Action:

Avidity• The overall strength of binding between an Ag

with many determinants and multivalent Abs

Keq = 104

Affinity106

Avidity1010

Avidity

Specificity

• The ability of an individual antibody combining site to react with only one antigenic determinant.

• The ability of a population of antibody molecules to react with only one antigen.

Cross Reactivity• The ability of an individual Ab combining site to

react with more than one antigenic determinant.• The ability of a population of Ab molecules to

react with more than one Ag

Anti-A Ab

Ag A

Anti-A Ab

Ag B

Shared epitope

Anti-A Ab

Ag C

Similar epitope

Cross reactions

Factors Affecting Measurement 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 or they 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

Agglutination Tests

Lattice Formation

Agglutination/Hemagglutination

• Definition - tests that have as their endpoint the agglutination of a particulate antigen– Agglutinin/hemagglutinin

+ ↔

• Qualitative agglutination test– Ag or Ab

Agglutination/Hemagglutination• Quantitative agglutination test

– Titer– Prozone

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

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

1/12

8

1/25

6

1/51

2

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

• 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

TargetRBCs

+ ↔Step 1

+ ↔Coombs Reagent

(Antiglobulin)

Step 2

Coombs (Antiglobulin)Tests • Applications

– Detection of anti-Rh Ab– Autoimmune hemolytic anemia

Agglutination/Hemagglutination Inhibition• Definition - test based on the inhibition of

agglutination due to competition with a soluble Ag

+ ↔

Prior to Test

+ ↔+

Test

Patient’s sample

Agglutination/Hemagglutination Inhibition

• Applications– Measurement of soluble Ag

• Practical considerations– Same as agglutination test

• Definition

Precipitation Tests

Lattice Formation

Radial Immunodiffusion (Mancini)

• Interpretation– Diameter of ring is

proportional to the concentration

• Quantitative– Ig levels

• Method– Ab in gel– Ag in a well

Ag Concentration

Dia

met

er2

AgAgAgAg

Ab in gel

Immunoelectrophoresis• Method

– Ags are separated by electrophoresis

• Interpretation– Precipitin arc represent individual antigens

Ag-+

Ag

Ab

Ag

Ab

– Ab is placed in trough cut in the agar

Immunoelectrophoresis

• Method• Interpretation• Qualitative

– Relative concentration

Countercurrent electrophoresis• Method

– Ag and Ab migrate toward each other by electrophoresis

– Used only when Ag and Ab have opposite charges

• Qualitative–Rapid

Ag Ab- +

Radioimmuoassays (RIA)Enzyme-Linked Immunosorbent

Assays (ELISA)

Lattice formation not required

Competitive RIA/ELISA for Ag • Method

– Determine amount of Ab needed to bind to a known amount of labeled Ag

+ ↔

Prior to Test

Labeled Ag

+ ↔

Test

+Patient’ssample

LabeledAg

+

– Use predetermined amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor

Competitive RIA/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’ssample

LabeledAg

+

– Concentration determined from a standard curve using known amounts of unlabeled Ag

SolidPhase

SolidPhase

Solid Phase Non-Competitive RIA/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

SolidPhase

AgImmobilized

Ab in Patient’s

sample

LabeledAnti-Ig

Solid Phase Non-Competitive RIA/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

SolidPhase

AgImmobilized

Ag in Patient’s

sample

LabeledAb

Tests for Cell Associated Antigens

Lattice formation not required

Immunofluorescence

• Direct– Ab to tissue Ag is labeled with fluorochrome

Ag

FluorochromeLabeled Ab

Tissue Section

Immunofluorescence

• Indirect– Ab to tissue Ag is

unlabeled– Fluorochrome-labeled anti-

Ig is used to detect binding of the first Ab.

Ag

FluorochromeLabeled Anti-Ig

Tissue Section

UnlabeledAb

• Qualitative to Semi-Quantitative

Immunofluorescence

• Flow Cytometry– Cells in suspension are labeld with fluorescent tag

• Direct or Indirect Fluorescence– Cells analyzed on a flow cytometer

FlowTip

Laser

FLDetector

LightScatter

Detector

Immunofluorescence

• Flow Cytometry cont.– Data displayed

Green Fluorescence Intensity

Num

ber

of C

ells

Unstained cells

FITC-labeled cells

One Parameter Histogram

Red Fluorescence Intensity

Gre

en F

luor

esce

nce

Inte

nsity

Two Parameter Histogram

Assays Based on Complement

Lattice formation not required

Complement Fixation

– Ag mixed with test serum to be assayed for Ab– Standard amount of complement is added– Erythrocytes coated with Abs is added– Amount of erythrocyte lysis is determined

Ag

Patient’sserum

Ag No Ag

Ag

• Methodology