Quantifying Antigen-Antibody reactions• Ag-Ab interaction is a bimolecular association• It involves various non-covalent interactions between

the epitope of the antigen & the variable-region (VH/VL) domain of the antibody molecule.

Steps• Follow the patient from early stage of infection to

later stage• Ab. level (titer) will increase in response to disease• A rise in titer is characteristic of an active infection• To quantify antibodies- perform serial dilutions• Use microtiter plates- small sample volume

Antibody Titer• Antibody titre is the

concentration of antibodies against a particular antigen

• Serology test is usually done using micro well plate.

• So that the test sample can be done in a very small sample.

• Uses serology-study and diagnostic use of antigen-antibody interactions in blood serum

• Use immunological processes in two general diagnostic ways– Use known antibodies to detect antigens associated

with an infectious agent– Use antigens to detect specific antibodies in a

patient’s blood to determine exposure to a specific pathogen (to manage the treatment of a disease).

• Test chosen based on the suspected diagnosis, cost to perform the test, and the speed with which a result can be obtained

Immune Testing

• Numerous types of serologic test -differ in their speed and sensitivity.

• Some are strictly qualitative, others are quantitative.

1. Precipitation tests (a) Immuno diffusion

(b) Immunoelectrophoresis

2. Agglutination tests

3. Neutralization

4. Complement fixation

5.5. Immuno fluorescenceImmuno fluorescence

6.6. Radioimmunoassay (RIA)Radioimmunoassay (RIA)

7.7. Enzyme-Linked Immuno sorbent Assay (ELISA)Enzyme-Linked Immuno sorbent Assay (ELISA)

8.8. Western BlottingWestern Blotting

Immune Testing

• One of the easiest of serological tests• Soluble antigens interact with

antibodies and form a lattice that eventually develops into a visible precipitate.

• Occur best when antigen and antibody are present in optimal proportions.

• Precipitin ring test is performed in a small tube.

• Antibodies that aggregate soluble antigens are called precipitins.

1. Precipitation Tests

FIGURE- Precipitation reactions in fluids yield a precipitin curve.

( Lattices or large aggregates )

( no precipitate is formed if an Ag contains only a single copy of each epitope )

Precipitation ReactionsPrecipitation Reactions

Precipitation reactions:(a) Polyclonal antibodies can form lattices, or large aggregates.

However, monoclonal antibody can link only two molecules of antigen and no precipitate is formed.

(b) Precipitation curve - for a system of one Ag and its antibodies. • This plot of the amount of antibody precipitated versus increasing

antigen concentrations (at constant total Ab) reveals 3 zones: • 1. Zone of antibody excess - precipitation is inhibited

and antibody not bound to antigen can be detected in the supernatant;

• 2. Zone equivalence - maximal precipitation in which antibody and antigen form large insoluble complexes and neither antibody nor antigen can be detected in the supernatant; and

• 3. Zone of antigen excess - precipitation is inhibited & Ag. not bound to Ab. can be detected in the supernatant.

Precipitation Reactions(a). Immunodiffusion Tests• Precipitation tests - done in gels• Immunodiffusion procedures are carried out in an agar gel medium.• The precipitate is easily seen in gels yield visible precipitin lines• But no visible precipitate forms in regions of Ab or Ag excess.

(b). Immunoelectrophoresis (electrophoresis & immunodiffusion) analyzes serum proteins.

Precipitation ReactionsPrecipitation Reactions

Diagrammatic representation of radial & double immunodiffusion.: Precipitation reactions in gels yield visible precipitin lines; no visible precipitate forms in regions of Ab or Ag excess.

in the Ab-containing semisolid medium

The region of equivalence

-> The area is proportional to the conc. of Ag.

Precipitation reactions: (a) Radial Immunodiffusion (Mancini)

• Interpretation– Diameter of ring is

proportional to the Ag concentration

• Quantitative– Ig levels

• Method– Ab in gel– Ag in a well

Ag Concentration

Dia

met

er2

AgAgAgAg

Ab in gel Precipitin Rings

(b) Immunoelectrophoresis• Method

– Ags are separated by electrophoresis

• Interpretation- Precipitin arc represent individual antigens• Qualitative - Relative concentration

Ag-+

Ag

Ab

Ag

Ab

– Ab is placed in trough cut in the agar

(c) Rocket Immunoelectrophoresis• Antigen is electrophoresed into gel containing

antibody. The distance from the starting well to the front of the rocket shaped arc is related to antigen concentration.

• Agglutination occurs due to the cross-linking of particulate antigens by antibody molecules.

• Agglutination is the visible clumping of insoluble particles, whereas precipitation involves the aggregation of soluble molecules

Types of Agglutination Reactions

(a) Direct agglutination reactions

(b) Indirect or passive agglutination tests

(c) Hemagglutination reactions

2. Agglutination Tests

(2a) Direct Agglutination Tests• Used to determine antibody titer against large

cellular antigens (Bacteria, Fungi or RBCs) • Particulate antigen reacts directly with

antibodies.• Antigen found naturally on particle (cell). • Blood Grouping is an example

ABO blood types

-

(2b) Indirect Agglutination Tests• Soluble antigen is coated onto particles to give

indirect agglutination

or antibody can be placed on particles to test for antigen presence

• Antibodies cause visible agglutination (clumps) of soluble antigens affixed to latex spheres.

Figure- Antibody coated latex particles

Indirect Coombs (Antiglobulin)Tests • Detects anti-erythrocyte antibodies in serum• Applications

– Detection of anti-Rh Ab

– Autoimmune hemolytic anemia

Patient’s Serum

TargetRBCs

+ Step 1

+

Coombs Reagent(Antiglobulin)

Step 2

(2c) Hemagglutination test– Agglutination reactions using red blood cells.

Qualitative agglutination test - Antigen or Antibody.

+

• Applications – Blood group typing– The identification of viruses.– The diagnosis of certain diseases (Bacterial infection)

• Practical considerations– Easy & Semi-quantitative test

Agglutination Inhibition• Test based on the inhibition of agglutination due

to competition with a soluble antigens for limited antibody combining sites.

• Patient sample added to reagent antibody specific for antigen being tested, if antigen is present it binds to reagent antibody.

• Reagent particles (latex or RBCs) coated with the same antigen are added, if antigen was present in the sample all reagent antibody binds to it so no antibody is present to react with antigens coating the particles

• Applications - Measurement of soluble Ag

Agglutination/Hemagglutination Inhibition

• Applications - Measurement of soluble Ag

• Practical considerations- Same as agglutination test

+ Prior to Test

+ +

Test

Patient’s sample

FIGURE 6-8-The original home pregnancy test kit employed hapten inhibition (agglutination inhibition) to determine the presence or absence of human chorionic gonadotropin (HCG) >>> The kits currently on the market use ELISA-based assays.-Also used to determine the use of illegal drugs, & immunity (Ab) to virus (rubella).

Agglutination inhibitionAgglutination inhibition

ReactionsReactions

• Viruses introduced into appropriate cell cultures will invade and kill the cells, a phenomenon called cytopathic effect

• The ability of a virus to kill culture cells is neutralized when the virus is first mixed with antibodies against it

• Absence of cytopathic effect indicates the presence of antibodies against the virus

• This test can also be used to test toxins• Advantages - Sensitive and specific enough to identify

whether an individual has been exposed to a particular virus or viral strain

3. Viral Neutralization

3. NEUTRALIZATION

4. Complement-Fixation Reactions• Complement-fixation:

– Complement (group of serum proteins) binds to antigen-antibody complex and is used up.

• Complement-fixation can be used to detect very small amounts of antibody.

– Wasserman test for syphilis (in the past)

– Certain viral & fungal diseases.

Complement Fixation

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

• Uses fluorescent dyes as labels

• Fluorescein is the most important dye used in these test– Dyes, chemically linked to an antibody without

affecting antibody’s ability to bind antigen– Glows bright green when exposed to fluorescent

light

• Fluorescein-labeled antibodies used in 2 types of tests– Direct fluorescent antibody test– Indirect fluorescent antibody tests

5. Fluorescent Antibody Test

Fluorescent Antibody Techniques

(5a).Direct Immunofluorescence tests

Ag

FluorochromeLabeled Ab

Tissue Section

1. Microbe (antigen) fixed to a slide2. Known Antibody to tissue Ag is labeled with fluorochrome3. Incubated and rinsed (remove free Ab)4. Labeled Antibodies that attach can be viewed using a

fluorescence microscope (yellow green fluorescence).5. Used to detect specific antigen under a fluorescent microscope.

SlideAntigen

YAntibody

Fluorescent dye Not a quantitative test

(5b).Indirect Immunofluorescence test

• Used to demonstrate the presence of antibody in serum.

• Ab to tissue Ag is unlabeled• Fluorochrome-labeled anti-Ig

is used to detect binding of the first (serum) Antibody

Ag

FluorochromeLabeled Anti-Ig

Tissue Section

UnlabeledAb

• Qualitative to Semi-Quantitative

• A fluorescence-activated cell sorter can be used to and

count cells labeled with fluorescent antibodies.

Immunofluorescence Tests - Tests for Cell Associated antigens

• Flow Cytometry– Cells in suspension are labeld with fluorescent dyes (fluorescein isothiocyanate).– Direct or Indirect Fluorescence– Cells analyzed on a flow cytometer

FlowTip

Laser

FLDetector

LightScatter

Detector

Green Fluorescence Intensity

Nu

mb

er o

f C

ells

Unstained cells

FITC-labeled cells

One Parameter Histogram

6. Radioimmunoassay (RIA)

• Check sample for presence of antigen.

• Known amounts of radioactively labeled antigen, known antibody and unknown sample mixed together.

• If sample has high level of antigen, it will compete with labeled antigen and little radioactive antigen will bind.

• If sample has low level of antigen, it will not be able to compete with labeled antigen and much radioactive antigen will bind.

A solid-phase radioimmunoassay (RIA) to detect hepatitis B virus in blood samples & A standard curve to determine the conc. of HBsAg in unknown serum.

Radioimmuno Radioimmuno

AssayAssay - One of the most sensitive technique for measuring hormones, drugs, & vitamins at conc. Of<0.001 ㎍ / ㎖ first discovered by Dr. Berson & Yalow in 1960 (1977 Novel prize to Yalow)- The principle involves competitive binding of radiolabeled Ag and unlabeled Ag to the limited supply of a high affinity Ab.

Assay Procedure• Add known amounts of the test sample + labelled

antigen into the microtitre wells• Incubate allow the reaction to reach completion• Decant & wash contents of the well removes all

unbound antigens• Radioactivity remaining in the Microtitre wells

measured by a Counter [GM counter , Scintillation counter etc]

• Intensity of radioactivity is inversely correlated with the conc of antigens in the test sample

• Sensitive to very low conc of antigens

Advantages & Disadvantages of RIA• Advantages

– Immune reactions highly are specific– Immune reactions are highly sensitive

• Disadvantages – Radiation hazards: Uses radiolabelled reagents– Requires specially trained persons– Labs require special license to handle radioactive

material– Requires special arrangements for

• Requisition, storage of radioactive material • Radioactive waste disposal.

7. Enzyme-Linked Immunosorbent Assay• Principle of ELISA :

– Uses an immune reaction like RIA, differs in detection method– Detection based on

• Enzyme catalysed reaction or Fluorescent probe• NOT radioactivity [great advantage!]

• ELISA techniques use antibodies linked to an enzyme as horse radish peroxidase or alkaline phosphatase.

• Ag-Ab reactions are detected by the enzyme-substrate reaction. A color change indicates an antigen-antibody reaction has occurred.Types• The direct ELISA- Sandwich or Capture Assays &

Competitive ELISA - Used to detect antigens • The indirect ELISA -Used to detect specific antibodies

(i.e. HIV in serum) against antigen bound in a test well.

Variations in the enzyme-linked immunosorbent assay (ELISA) technique, similar to RIA except using an Enzyme (alkaline , ⓟ horseradish peroxidase, & β-galactosidase) : safer & less costly.

to detect Ab (HIV, HCV)

to detect Ag , For e.g- HCG

to detect Ag

ELISAELISA

Noncompetitive ELISA or “Indirect” ELISA

• Inactivated HIV antigens pre-coated onto an ELISA plate• Add patient serum (Ab). Incubate: till antigen antibody

reaction is complete, Wash remove unbound antibody• Add known labeled Anti-human immunoglobulin

coupled to an enzyme. It binds to human antibodies. • Add substrate which changes color when cleaved by the

enzyme.Colour proportional to antibody in patient sample

A 96 well ELISA plate

Serological TestsNoncompetitive ELISA or “Indirect” ELISA

Direct ELISASandwich or Capture Assays• Used for antigens with multiple epitopes• Antibody to one epitope fluid, antibody to second

epitope fixed.• Enzyme label used to detect reaction

Competitive ELISA• Titrewells coated with antibodies• Add known quantities of patient sample containing

antigen + antigen labelled with enzyme• Unknown antigen competes with labeled known

antigen• Enzyme + Substrate Product measure colour• Colour inversely related to antigen in patient sample

Applications of Immunoassays[RIA & ELISA]

• Analysis of hormones, vitamins, metabolites, diagnostic markers– Eg. ACTH, FSH, T3, T4, Glucagon, Insulin,

Testosterone, vitamin B12, prostaglandins, glucocorticoids,

• Therapeutic drug monitoring: – Barbiturates, morphine, digoxin,

• Diagnostic procedures for detecting infection – HIV, Hepatitis A, Hepatitis B etc.,

Advantages of ELISA• Sensitive: Nanogram levels or lower

• Minimal reagents

• Qualitative & Quantitative – Qualitative E.g. HIV testing – Quantitative assays E.g. Theraputic Drug

Monitoring

• Greater scope : Wells can be coated with Antigens or Antibodies

• Suitable for automation high speed

• NO radiation hazards

8. Western Blot

• Combines Electrophoresis with ELISA to separate and identify antigens

• Confirms positive ELISA results (ELISA may give some false +ve)

Steps:

• Antigen mixture separated by gel electrophoresis

• Blotted onto a filter paper• Serum sample applied to filter• Labels used to interpret results• Used to confirm positive HIV test

Western blotting

: separates the components according to their molecular weight.

: the proteins in the gel are transferred to the sheet of nitrocellulose or nylon by the passage of an electric current.

: probed with Ab & then radiolabeled or enzyme-linked 2nd Ab.

: a position is visualized by means of an ELISA reaction.

Expose x-ray film and develop

Western Blotting

Serological Tests

• Agglutination: Particulate antigens

• Hemagglutination: Agglutination of RBCs

• Precipitation: Soluble antigens

• Fluorescent-antibody technique: Antibodies linked to fluorescent dye

• Complement fixation: RBCs are indicator

• Neutralization: Inactivates toxin or virus

• ELISA: Peroxidase enzyme is the indicator

Immunological Tests & Some of Their Uses