Principles of serodiagnosis

Ms. Piyumali Arachchi

Mphil (USJP) BSc (UOC)
 Learning outcomes
• The types of antigen-antibody reactions

• Techniques for detection of antigen-antibody

reactions

• Interpretation of serological investigations in the

diagnosis of infectious diseases
 Methods of diagnosing infectious
disease in a patient
Clinical symptoms
Lab investigations
Detection of pathogen using culture
Detection of pathogen specific genes using
PCR
Detection of pathogen specific antigens or
antibodies (Serodiagnosis)
 Immune response
• When a viral or bacterial pathogen
Pathogen
infects the host, antigens are
displayed on MHC I and II and APC.
Antigen
• The response can be cell mediated or presenting
humoral cell

• The B and T cells produce antibodies

Naïve Naïve
specific T cell B cell

Cell Humoral
mediated immune
immune response
response
Humoral immune response
Immunological basis of serodiagnosis
Primary Secondary

1- Lag phase
2- Log phase
3- Plateu Ig G
4- Decline

Ig M
 How to detect an acute infection?

• Seroconversion – the time period during which a specific

antibody develops and becomes detectable by blood
• If there is four fold or more increase in the amount of
antibodies expressed (antibody titre) between acute and
convalescent samples is taken as an indication for recent
infection
• Presence of IgM antibodies – acute infection or recent
infection, vaccination
• Presence of IgG antibodies – past infection, vaccination
Performance and interpretation of
serological tests
Serological tests may be
•Qualitative
Is used to determine the presence or absence of
antibody or antigen

•Quantitative
Is used to determine the amount of antibodies or
antigens present in a sample.

8
 Procedure
1. Dilution of serum
•Equal amounts of physiological saline or buffer is
added to tubes.
•Add equal volume of serum to first tube.
•Mix well and transfer same volume of diluted serum
to next tube etc.
2. Allow to react with Ag/Ab
3. Determine the titre

Antibody titre - The reciprocal of the highest

dilution of the patients serum in which the antibody
is still detectable.

Eg : Dilution of 1/4 Antibody titre is 4

 Interpretation of serological test
• The increase in the number of antibodies can be
detected by comparing the antibody titres of paired
serum samples
• Most serological tests require collection of two samples
• Acute sample – taken at an early stage, no detectable
antibodies

• Convalescent sample – taken 10 to 14 days after acute

sample
• shows a 4 fold or more rise in antibody titre compared to an
acute sample
 Enzyme Linked Immuno Sorbant Assay
(ELISA)
• Commonly used to measure antibodies, antigens
proteins and glycoproteins in biological samples
• Carried out in 96 well plates
• Each ELISA measure a specific antigen
• Types of ELISA assays Ag coated on well
• Direct
Ab is added (in specimen)
• Indirect
Enzyme conjugated
anti - Ab is added
Ab coated with
enzyme Enzyme reacts
Ag in patient with substrate to
specimen develop a colured
product.
Enzyme Linked Immuno Sorbant Assay…
• The ELISA plate is coated with capture antibody
• Unbound antibody is washed from the plate
• Sample is added to the plate
• If the target antigen is in the specimen it will bind to the
antibody coating the plate
• Detection antibody is added
• Labeled with an enzyme
• Substrate is added to the plate
• The conversion of substrate in to its product is
measured
• Sandwich ELISA
• Two sets of antibodies are used to detect secreted
products

• Competitive ELISA
• Different in some respects to other ELISA
• Unlabeled primary antibody is incubated with the
antigen in the sample
• Ag/Ab complex is added to the ELISA plate precoated
with same antigen
• Unbound antibody is removed and secondary Ab is
added
 Major use of serological tests
• Diagnosis of infectious diseases
• Non culturable e.g: Treponima pallidum, Hepatitis
virus
• slow growing pathogens e.g: Mycoplasma
• highly infectious pathogens that are dangerous to
culture in a lab e.g: Rickettsia

• When patient has already been treated with

antibiotics
• To determine immunity/ past infection or
vaccination e.g: Hepatitis B
 Major use of serological tests…

• Epidemiological studies
• Diagnosis of autoimmune diseases
Rheumatoid artheritis (rheumatic factor, anti nuclear
antibodies)

• Determination of complement deficiencies

• Blood grouping (ABO and Rh) and HLA typing
Other serological assays
 Agglutination

• The antigen, when it is mixed with its corresponding

antigen, forms clumps or visible aggregates
• Uses of agglutination assay
• Lancefield grouping of Streptococci
• Identification of the causative agent of meningitis using latex
agglutination
• Serotyping of bacteria
• Detection of infections e.g: Influenza A, Hepatitis B, Dengue,
Japanese encephalitis, Chikungunya, Rubella
 Precipitation
• When antigen and antibody are present in optimal
concentrations, it gets precipitated
• Can be in solution, agar or in an electric field
• Interaction of soluble antigens with IgG or IgM antibodies
lead to precipitation reactions
• Precipitin ring test
 Complement fixation assay - CFA
Positive CFT Negative CFT
Ag
No Ab in serum
Ab
Unbound Ag
Ag-Ab complex
Ag Ab
Complement Free complement

Ag Ab
Sheep RBC coated with
anti sheep RBC
Ag Ab

RBC is lysed
RBC not lysed
Diagnosis of syphilis
diagnosis of viral infections – arboviruses, Respiratory
viruses

1/2 1/4 1/8 1/16

1/2 1/4 1/8 1/16

 Neutralization assay

Virus Antibody Ab neutralizes virus

Virus multiply in cell culture Cell culture not infected

 Solid phase assays
Radioimmuno assay
•Detects antigens/ antibodies using radioactive compounds
• To quantitate hormones, drugs, tumor markers, viral antigens

Plate coated with antibody

Addition of patients serum.

Antibody – Antigen reaction

Addition of Radiolabelled antibody

Determination of Radioactivity
Immunofluorescence assay
•A specific antibody tagged with a fluorescent dye is used
as an indicator system to detect antigens or antibodies in
serum
•An ultra violet light microscope is used to detect the
fluorescence
• Detection of Rabies antigen in animal brains

Direct IF
Specific Ab tagged with
fluorescent dye
Ag in patient tissue
Indirect IF
Immunochromatographic assay
•the immunological reaction is carried out on
chromatographic paper by capillary action.
•Useful as a rapid test (15 minutes).
•Antigens in the sample form a complex with the colloidal
gold labelled antibodies and move along the membrane by
capillary action.
•When this antibody-antigen complex reach the
immobilized antibody it will form an immunocomplex
generating a coloured line.