serology - prac. microbiology
DESCRIPTION
TRANSCRIPT
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