Serological diagnosis of virus
Bibek Raj Bhattarai
Bsc. MLT 2nd year
2075
Conventional Serological Methods
• 1. Precipitation
• 2. Agglutination
• 3. Haemagglutination
• 4. Haemagglutination inhibition
• 5. Complement Fixation Test
• 6. Fluorescent Antibody Test
• 7.Neutralization test
Precipitation:
• Reaction between soluble antigen and
antibody = Insoluble PPT
• Carried out either in liquid media / in gels e.g.
agar, agarose, polyacrylamide
• Can be Qualitative or quantitative
• Sensitive, can detect as little as 1μg of protein
• For detection of HBsAg of HBV
Latex Agglutination
• Reaction of antibodies with particulate or insoluble
antigens in presence of an electrolyte at suitable pH
& temp.= formation of visible clumps
• Applications-
– HIV antibody test by latex agglutination
Haemagglutination
• Agglutination of RBCs
– Useful for diagnosis of viral infections e.g.
influenza, mumps & measles.
Complement Fixation Test
• Complement has ability to fix on Ag-Ab complex
• In presence of appropriate Ab, ‘Complement’ causes
lysis of RBCs
• Two steps –
– 1.Complement Fixation Step
– Inactivated serum of patient + Ag + C
– incubation at 37o C for 1 hr.
• 2. Indicator Step
– Addition of sheep RBCs & antisheep RBC
antibodies )
– No hemolysis = Positive test
– Hemolysis = Negative Test
• Common viruses detected by CFT are
Influenza A, Influenza B, Influenza C,
Adenovirus, RSV, Measles, etc
Fluorescent Antibody Test
• Use antibodies labeled with fluorescent dyes e.g.
fluorescein isothiocynate (Green fluorescence ),
Rhodamine B ( Orange red )
• Used in rabies virus identification in brain tissue
• Two types –
• 1. Direct FAT : Specimen ( Ag) is fixed on slide +
labelled Abs = examined under fluorescent
microscope =
• If fluorescene = + ve test.
• 2. Indirect FAT :
• Known Ag fixed on slide + test serum +
labelled antiimmunoglobulin = observation
under fluro microscope
• * If fluorescence = +ve test ( Abs are present )
Enzyme linked immunosorbant assay
( ELISA )
• Uses antibodies linked to an enzyme E.g. horseradish
peroxidase or alkaline phosphatase
• Antigen – antibody reactions are detected by enzyme
activity.
• The specific Antigen is added to the test well.
• An antibody linked to the enzyme is added to it.
• It will bind if the antigen specific for it is present.
Enzyme Immunoasssay(EIA)
• Routine ELISA tests done in our laboratory for viral
diagnosis are HIV 1&2 Ab, HBsAg, HCV Ab, Rubella
IgM/IgG Ab, CMV Ab, HSV Ab ,etc..
• Also CLIA and ECLIA can be done instead of EIA.
Types of ELISA
EIA (Enzyme Immuno Assay)…
• Generations of EIA
1. First : Whole virus particles were used
2. Second: Lysed virus particles were used
3. Third: Recombinant Ag or Ab are used
4. Fourth: Synthetic peptides are used
5. Fifth: Streptavidine and biotin are used
Negative Control
Positive Control
Positive tests
ELISA Reader
Radioimmunoassay ( RIA )
• Steps are very similar with ELISA
• In RIA, instead of enzyme linked Abs ,
radiolabelled Abs i.e. antiglobulin labelled
with a radioactive compound is added.
• The amount of radioactivity in wells provides
an estimate of the titer of target antibody.
Western blotting
• Confirmatory test for HIV infection
• Virus are lyzed and antigens are separated by
polyacrylamide gel electrophoresis
• Separated molecules are transferred to
another matrix e.g. nitrocellulose membrane.
• Serum added and incubated
• Enzyme conjugate is added and then sbstrate
is added for visualization.
Immunochromatography…
• Tests commonly done
with IC are
– HIV 1&2 antibody,
– Hepatitis B surface
antigen
Negative Positive Invalid
– Hepatitis C antigen
– Dengue Antibody, etc. Fig. Results and interpretation of
Immunochromatography test
Immunochromatography
Fig. Principle of Immunochromatography
Neutralization Test
• For confirmation of Hepatitis B virus infecttion
• HBsAg in serum is mixed with specific anti
HBsAb
• HBsAg antigen is neutralized
• ELISA is performed from neutralized serum
• IF ELISA negative: Confirm HBsAg positive
