serology - prac. microbiology

Diagnosis of infectious diseases

DIRECT METHOD INDIRECT METHOD

(Serology)Specimen

Culture technique Non-culture technique

Antibiotic sensitivity

Detection of-IgM-rising titre of IgG

Microscopy

Identification e.g.-Microscopy-Bioch. reactions

- Serology- DNA probes.

- Molecular methods

- Serology.

ANTIGEN-ANTIBODY INTERACTIONS

Antigen-Antibody reactions in vitro = Serology

Diagnosis of diseases

Identification of Ag Ag //AbAbQuantitation of Ag Ag // AbAb

METHODS OF DETECTION OF ANTIGEN-ANTIBODY

REACTIONS

Reactions Accompanied by

Visible Phenomena

Reactions Detected by

Labeled Reagents

Reactions Accompanied by Visible Phenomena

Resulting Ag-Ab complexes seen directly• By naked eye• By microscope

A- AGGLUTINATION Ag: cellular or particulate

antigen = “agglutinogen” antibody = “agglutinin”

AGGLUTINATION

Agglutination

1 .Slide Agglutination 2 .Tube Agglutination

1. Slide agglutination (Qualitative Test)

Unknown Ag Add known

antiserum (Ab)

2. Tube agglutination (Quantitative Test)

Detection of Ab against a certain pathogen = indirect evidence for diagnosis of disease = serological diagnosis

Tube Agglutination

1/10 1/20 1/40 1/80 1/160 1/320 1/640

B- PASSIVE AGGLUTINATION

Soluble antigen + antibody → precipitation Not very sensitive!!

How can we convert:

precipitation agglutination ??

more sensitive !!!

Both Ag & Ab are soluble One known! The other not known!

Soluble known reactant is coated onto inert particles e.g. Latex particles

Known reactant becomes particulate (instead of soluble) Reaction becomes Agglutination (instead of precipitation)

+ ↔

Soluble known antigen coated on latex particle + Unknown antibody (IgM)

(Soluble) known antibody coated on latex particle + Unknown soluble antigen

Examples: Rheumatoid arthritis: (Patient produces Abs against IgG) Latex-IgG (known Ag) + serum from patient (Ab?)

C-Reactive protein: (inflammatory conditions) Latex-anti-CRP (known Ab) + serum (CRP?)

Pregnancy Test (Test for HCG) Latex-anti-HCG (known Ab) + urine (HCG?)

C- COOMBS (ANTIGLOBULIN) TEST

Non-agglutinating antibodies

Examples:

Anti-Rh antibodies Abs in autoimmune

haemolytic anaemia

Direct Coombs Test

Done for cases of:

- Newborn with erythroblastosis foetalis

- Patients with haemolytic anaemia

(RBCs have attached Abs already)

Indirect Coombs Test

Done to detect non-agglutinating antibodies in serum of Rh-negative mother sensitized with Rh antigen

D- HAEMAGGLUTINATION INHIBITION

Some viruses agglutinate RBCs in vitro.

Antibodies (if present) prevent haemagglutination = Haemagglutination inhibition

E- PRECIPITATION • Ag: soluble

Applications:Agar gel diffusion

a. Double diffusion b. Single radial immunodiffusion

Agar gel diffusiona. Double Immunodiffusion

Precipitation Line

Ag well

Abwell

Semi-solid medium

Elek’s Test(double diffusion)

Elek’s Test

b. Single radial immunodiffusion

• Method:

- Ab in media

- Ag in well

Precipitation Ring

Antibody

Ag

b-Single radial immunodiffusion

b. Single radial immunodiffusion

Note: Standards (1-4)of known concentrations are included in the test to create a curve.

Interpretation:Diameter of ring is proportional

to concentration of Ag in sample.

Ag Concentration

Dia

met

er2

4321

Ab in gel

• Used to quantitate various immunoglobulin classes

• Immunogloblin acts as an Ag.

• In well: patient’s serum (Ag?)

• In gel: anti-IgG, anti-IgM, ……etc

Standard 3

Standard 2

Standard 1Pt. 1

Pt. 2

Pt. 3

F- FLOCCULATION

Antigen: Small insoluble particulate

Examples:• Venereal Disease Research Laboratory (VDRL)

test

• Rapid plasma regain (RPR) test

VDRL: slide flocculation test for diagnosis of syphilis

Cardiolipin used as antigen instead of Treponema antigenAntibody detected is a heterophil Ab called Reagin

Negative Positive Microscopical aggregates

RPR rapid plasma reaginSmall carbon particles added → easier visibility (by naked eye) 

              -ve control +ve control -ve control +ve control

+ve Test -ve Test

H- Complement fixation:• Test depends on the fact that:

Ag - Ab → complement fixation• Sensitive• Used to: Detect and quantitate antibody Detect and quantitate antigen (less commonly)• Many applications in diagnosis of diseases caused by

bacteria, viruses, fungi, etc.• Famous Test: Wassermann Test for serologic diagnosis of

syphilis

I- Viral Neutralization: Certain viruses cause cytopathogenic (CPE) effect on certain cell cultures

Uninfected cells Viral-infected cells, showing cytopathic effect (CPE). Fusion of infected cells→

multinucleated giant cells

CPE are inhibited by virus-neutralizing Abs.

I. Viral Neutralization (cont.):

First step: Serum (virus-neutralizing Ab?) + known Virus

Second step: Cell culture inoculated with mixture:

No Ab (in serum) + Virus → no neutralization → CPE Ab ( in serum) + Virus → neutralization → No CPE

ANTIGEN-ANTIBODYREACTIONS

Reactions Accompanied by

Visible Phenomena

Reactions Detected by

Labeled Reagents

A. Immunofluorescence

B. ELISA

C. RIA

A. Immunofluorescent Techniques:

Principle: Fluorescent substances (e.g. Fluorescein isothiocyanate)

attached to known Ab → seen by fluorescence microscope using UV light → fluorescence = positive reaction

i) Direct Immunofluorescence

• Detects unknown Ag e.g.: rabies virus in

brain of dead animal• Tissue (Ag?) + labeled known Ab → binding → fluorescence

Positive test

ii) Indirect Immunofluorescence• Detects Ab in serum → indirect diagnosis of

disease e.g. Syphilis• Known antigen

(Treponema pallidum) bound to slide

+ serum (Ab against Treponema?)

• Labeled antihuman globulin added

→ fluorescence

B.Enzyme-Linked Immunosorbent Assay (ELISA)

• Very sensitive• Very specific• Used to detect Ag or Ab• Label used: Enzyme• Example: Horseraddish peroxidase or alkaline

phosphatase• Enzyme can be conjugated to Ag or Ab• Ag-enzyme or Ab-enzyme is called conjugate• Enzyme acts on colorless substrate → colored product

• Colour change = +ve test.

• Colour intensity (by spectrophotometer) is proportional to the amount of Ag or Ab (quantitative).

• Many variations in test procedure.

Direct Method(Double Antibody Technique) For detection and quantitation of Ag

Indirect MethodFor detection and quantitation of Ab in serum.

Known Ag

Unknown Ab in test serum

Enzyme labeled anti-human Ig

Substrate is added

ELISA

ELISA plates

C. Radioimmunoassay (RIA)• Same idea as ELlSA but different label: Radioactive

isotope (instead of enzyme)• Measurement of Degree of radioactivity (instead of degree of colour change) • As sensitive as ELISA• Disadvantage: Hazards of radioactivity • Applications:

– Measurement of biological substances (Ags) (e.g. drugs, hormones, tumour markers)– Measurement of antibodies

MCQs

1. If you mix bacteria with specific Abs, it would result in: a) Cell lysisb) Agglutination c) Haemagglutinationd) Precipitatione) Fluorescence 

2. In passive agglutination reaction, soluble antigens or antibodies coat:

a) Latex particlesb) White blood cellsc) Plateletsd) Carbon particlese) None of the above

3. Regarding direct Coomb's test:

a) Maternal RBCs are usedb) Group O Rh +ve RBCs are usedc) Anti-human globulins are added directly to foetal RBCsd) Mother's serum is usede) Foetal serum is used 

4. In Elek’s test:

a) The antigen is mixed with the agar gel.b)Lines of precipitate will form in positive cases.c) The antibody is incorporated in the agar. d) A strip of filter paper is soaked with the antigen.e) Agglutination can be observed. 

5. Single radial immunodiffusion test:

a) Is an example of an agglutination reactionb) Utilizes agar gel mixed with the antigenc) Depends on complement fixation during Ag-Ab reactiond) Is used to detect non-agglutinating antibodiese) Is a precipitation reaction

6. Direct ELISA requires:

a. Known Ab

b. Known Ag

c. Complement

d. Patient’s Ab

e. Sheep RBCs