Serological tests involved in identification of infectious agents

Prof M.I.N. Matee

Precipitation Tests

Lattice Formation

PRECIPITATION

Precipitation reactions in fluids (p. 142)

Precipitation reaction can be seen

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

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- +

Immunoelectrophoresis

• Method

• Interpretation

• Qualitative– Relative concentration

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

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

1/12

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

Hemagglutination

Used to identify blood group antigens or antibodies to them

Variations: Chemically couple haptens to RBC

Use other particles - bacteria, antigen-coated latex beads

Agglutination Inhibition Assay for HCG

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

Immunofluorescence

Antibodies can be labeled with fluorescent dye

Can localize binding sites on cells

Dyes: Fluorescein, rhodamine, phycoerythrincan be conjugated to Fc region of Ab

(so antigen binding is unaffected)

Absorb at one wavelength and emit at another

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

ELISA- enzyme-linked immunosorbent assay

Based on RIA

Nearly as sensitiveCheaper and safer

Many detection systems have been developed

Many variations of the assay have been developed

All ELISAs use an antibody conjugated with anenzyme that turns a colorless substrateinto a colored product

Direct- detects antigens using a single labeledantibody against that antigen

Relatively few applications and permutations

ELI SA Enzyme-Linked Immunosorbent Assay (ELISA)

© 1998 Gold Standard Multimedia Inc.

1. Antigen HIV Protein2. Test antibody Human serum3. Developing Ab Enzyme-Goat anti-Human IgG4. Substrate Colorless > Blue

Enzyme-Linked Immunosorbent Assay (ELISA)

HIV Ag HumanSerum

AP-Goat Anti-Human IgG

Colorless >Yellow

AP = alkaline phosphatase

Western Blot for HIV Antibodies

+ - Patient sera

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

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

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

• 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

Ag

Immobilized

Ag in Patient’s

sample

LabeledAb

Tissue Immunofluorescence/Immunohistochemistry

NEUTRALIZATION

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

CD4

CD8

Immunofluorescence

• Flow Cytometry cont.– Data displayed

Green Fluorescence Intensity

Nu

mb

er o

f C

ells

Unstained cells

FITC-labeled cells

One Parameter Histogram

Red Fluorescence Intensity

Gre

en F

luor

esce

nce

In

ten

sity

Two Parameter Histogram