Commonly Used Serological Techniques

MHC and Flow Cytometry

Methods for Ag-Ab detection & Applications

Applications:
Techniques:
 Microbial diseases
 Agglutination
 Early or presumptive
 Precipitation
diagnose
 Immunoflourescence
 Serotyping
 Radio-immunoassays
 Antibody levels
 Complement Fixation
 Antigen detection
 ELISA
 Agglutination
Definition – the visible clumping together of
antigen bearing cells, microorganisms or
particles in the presence of specific antibodies.
 Agglutination
 Particles may be RBCs (hemagglutination), bacterial cells
(Coagglutination) or inert particles such as latex or charcoal
coated with antigen or antibody.
 Precipitation tests
Definition: The antigen and antibody are in soluble
form which combine to form a visible precipitate. A
visible line of precipitation can be seen.

 Presence of electrolytes
Immunfluorescence
Principle: Use fluorescein isothiocyanate labeled-
immunoglobulin to detect antigens or antibodies.
 Fluorescent dyes illuminated by ultraviolet light are used to

show combination of antigen antibody.

 The end point antigen antibody complexes are seen
fluorescing against a dark background.
 Requires a fluorescent microscope.

V. Cholerae
 Radioimmunoassay (RIA)
 Radioactivity of the specific labeled antibody or antigen is
used to quantify the antigen or antibody in patient’s serum.
 Gamma-Spectrometer for reading.
 Complement Fixation
 To detect and quantify the antibodies which do not
agglutinate or precipitate.
 Detect the presence of either specific antibody or specific
antigen in a patient's serum, based on whether complement
fixation occurs.
 Can be made semi-quantitative by serial dilutions of serum.

Principal
When the complement takes part in the antigen antibody
reaction it is bound, or fixed, to the antigen antibody complexes.
When these complexes are on bacteria, red cells or other cells,
the complement brings about the lysis of the cells involved.
Two step process:
 Antibody (patient serum), antigen are mixed with fresh
complement an then sensitized sheep cells added.
 If the patient antibody is absent, the complement is free to
bind to the antibody coated sheep cells causing hemolysis
(means a Negative reaction).
 If the antibody is present, the antigen-antibody bind the
complement and no hemolysis (means a Positive reaction).
Enzyme Linked Immuno Sorbent Assay (ELISA)
Principal: The basic principle of ELISA technique is to use an
enzyme system to detect the binding of specific antigen (Ag)
with its antibody (Ab).
1. Sandwich/Double Antibody ELISA: (Detects Antigen)
2. Indirect ELISA: (Detects Antibodies)
 Major Histocompatibility Complex (MHC) or
Human Leukocyte Antigen (HLA) System
 Definition: A group of tissue antigens, controlled by
chromosomal region, carry a number of genetic Locus, each
with multi alleles, that have relevance to transplantation
rejection reaction & other immunological phenomena.
 In Human MHC Is Found on short arm of Chromosome 6.

Class I:
HLA-A, HLA-B,
HLA-C

Class II:
DP, DQ, DR
Human Leukocyte Antigen (HLA) Typing
 To match organ and tissue transplant recipients with

compatible donors.
 It identifies the major HLA genes a person has inherited and

the corresponding antigens (proteins) that are present on

the surface of their cells.
 You and potential donors will have blood drawn.

 The blood is tested in a lab to figure out your HLA type.

 Your HLA will be compared to potential donors to see if

there is a match.

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Matching of HLA

 You have many HLA markers. Half are inherited from your

mother and half from your father, so each brother and sister
who shares the same parents as you has a 25% chance (1
in 4) of being a close HLA match.

 Extended family members are not likely to be close HLA

matches. But about 70% (7 out of 10) of patients who need

a transplant won’t have a fully matched donor in their family.

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 Flow Cytometry
 Definition: The cells are stained with a light-sensitive dye,
placed in a fluid, and passed in a stream before a laser or
other type of light. The measurements are based on how the
light-sensitive dye reacts to the light.
 Measure the number of cells in a sample, percentage of live
cells, size, shape, and the presence of tumor markers on the
cell surface.
Applications in transplantation, hematology,
tumor immunology, chemotherapy, prenatal
diagnosis and genetics.

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