01.ENUMERATE THEANTlGEN-ANTIBODY REACTIONS USED INSEROLOGICAL DIAGNOSIS.
Types of antigen-antibody reactions.
Conventional Techniques
. Precipitation reaction
Agglutination reaction
. Complement fixation test
Neutralization test.
Newer Techniques (Labelled Assays)
Enzyme-linked immunosorbent assay (ELISA)
Immunofluorescence assay (IFA)
Radioimmunoassay (RIA)
Chemiluminescence-linked immunoassay (CLIA)
(immunochromatographic test), flow through assay.
napid test:Cassette ELISA. lateralflovw test
92Competency Based Practical Manual for Microbiofogy
REACTIONE
Q2. WHAT IS PRECIPITATION AND ENUMERATE VARIOUS PRECIPITATION
Definition
When a soluble antigen reacts with its specific antibody in the presence of an electrolyte at optimal
and pli, it leads to formation of antigen-antibody complex in the form ot: temperature
Insoluble precipitate band when gel containing medium is usedor insoluble floccules or precipitate ring when
liquidmedium is used.
Precipitation in liquid medium
king test: Ascoli's thermoprecipitin test, Streptococcalgrouping by Lancefield's technique
Slide test: Flocculation test - VDRL test for
syphilis
Tube test: Kahn's test for syphilis
Precipitation in gel (immunodiffusion)
Single diffusion inone dimension (Oudin procedure)
Double diffusion in one dimension (Oakley Fultrope procedure)
Single diffusion in two dimensions (Radial
immunodiffusion)
Double diffusion in two dimensions (Ouchterlony procedure)
Inmunoclectrophoresis
Electroimmunodiffusion
Countercurrent immunoelectrophoresis (CIEP)
IRocket electrophoresis.
Serology 93
03.DEFINE AGGLUTINATION AND ENUMERATE VARIoUS AGGLUTINATION REACTIONS.
Definition
particulateeor insoluble antigen is mixed with its specific antibody in the
When a presence of electrolytes at asuitable
temperature and pH, particles are clumped or agglutinated.
1.Direct Agglutination Test
Slide agglutination test:For blood grouping and cross matching
Tube agglutination test: Widal test for Enteric fever
Hetrophile agglutination test:Weil-Felix for typhus fever, Paul Bunnel test for infectious mononucleosis
Microscopic agglutination: For leptospirosis.
2. Passive Agglutination Test (For Antibody Detection)
Aprecipitation reaction can be converted into agglutination test by attaching soluble antigens to the
surface of
carrier such as latex particles, bentonite and red blood cells.
Latex agglutination test (LAT)-for detection of ASO (antistreptolysin antibody), CRP, RA
Indirect hemagglutination test.
3. Reverse Passive Agglutination Reaction
estimation of antigens.
When instead of antigen, antibody is absorbed on the carrier particles for
molecule. Specific antibody binds with
Coagglutination test: Staphylococcus aureus (protein A) acts as a carrier Salmonella typhi
cells are used for detection of antigen, e.g.,
Fcportion and Fab site remain free. Such sensitized
antigen in serum.
For detection of hepatitis B surface antigen (HBsAg).
PRINCIPLE AND APPLICATIONS OF COOMBS TEST.
Q4. DESCRIBE THE
It is performed to diagnose Rh incompatibilityby detecting Rh antibody from mother's and baby's serum. It is used
totest for autoimmune hemolytic anemia.
Direct Coombs test (also known as the direct antiglobulin testtor DAT) is used to detect if antibodies have bound to
RBC surface antigens in vivo. Ablood I sample is taken and the RBCs are washed and then incubated: with antihuman
globulin. If this produces agglutination of RBCs, the direct Coombs test is positive, e-g, erythroblastosisfetalis.
Indirect Coombs test (also known as the indirect antiglobulintest or IAT) is used to detect in vitro antibody-antigen
reactions. It is used to detect very low concentrations of antibodies present in a patient's plasma/serum prior to
a blood transfusion. In antenatal care, this test is used to screen pregnant women for antibodies that may cause
identification
hemolytic disease of the newborn. The IAT can also be used for compatibility testing,antibody
Direct Coombs test /direct antiglobulin test
Positive test result
Legend
Antigens on
the red
blood cell's
surface
Human
anti-RBC
antibody
Antihuman
Blood sample from a patient with
immune mediated hemolytic anemia
The patient's washed
RBCs are incubated with
RBCs agglutinate: Antihuman Y antibody
antibodies form links between
Antibodies are shown attached to antihuman antibodies RBCs by binding to the human (Coombs
antigens on the RBC surface (Coombs reagent) antibodies on the RBCs reagent)
Indirect Coombs test / Indirect antiglobulin test
Positive test result
Recipient's serurn
is obtained, Donor's blood
containing sample is added to Recipient'slg's that target
the donor's red
antibodies (lg's) the tube blood cells Anti-human lg's Agglutination of red blood
with serum form antibody-antigen (Coombs antibodies)
are added to the cells occurs, because
complexes solution human lg's are attached
to red blood cells
ABOUTCOMPLEMENT FIXATION TEST.
WRITE BRIEFLY ABOUT
Q.5
Principle: It is atwO way step. When antigen and antibodies are mixed, complement is fixed to the antigen-antibody
complex.Itcan be detected by adding amboreceptors (Sheep RBCs coated with anti-sheep RBC antibody).
First Step
Antigen(soluble or particulate) +test serum +guinea pig complement are all added together.
Fthe test serum is positivefor antibody-Ag-Ab complex is formed. Complement gets fixed to the complex,
so there will be nofree complements in the serum.
If the test serum is negative for antibody-there is no Ag-Ab complex. Complements are not fixed, hence
remain free in the serum.
Second Step
Ahemolysis indicator system is added. It consists of sheep RBCs coated with its antibodies called amboreceptors.
If thetest serum is positive forantibody-no free complement in serum for binding to amboreceptors-no
hemolysis.
If the test serum is negative for antibody-free complement attached to amboreceptors bound on sheep
RBCS--hemolysis.
9 8 Competency Based PrACIdl
08. MENTION THE PRINCIPLEOF ELISA. DESCRIBE THE DIFFERENT TYPES OF ELISA
DONE, LIST OUT THE VARIOUS APPLICATIONS.
Principle: ELISA can provide auseful measurement of antigen or antibody concentration.
There aretwocomponents:
1. Immunosorbent:An absorbing material (polyvinyl, polystyrene) is used that specifically absorbs the known
antigen or antibody present in serum.
2. Enzyme: Is used to label one of the components of immunoassay.
Following antigen- antibody reaction,chromogenic substrate specific to enzyme (o-phenylenediamine for peroxidase
and p-nitrophenylphosphate for alkaline phosphatase) is added. Reaction is detected by reading optical density.
(Ag + AB complex) - enzyme + substrate ’ activates the chromogen -’ color change ’
Detected by
spectrophotometry.
Types of ELISA
Direct ELISA: Used for detection of antigen
Indirect ELISA: Used for detection of
antibody/antigen
Sandwich ELISA: Used for detection of antigen - direct (single Ab) and
indirect (double Ab)
Competitive ELISA: Used for detection of antigen/antibody
Cassette ELISA (Cylinder ELISA): Used for detection of
antibody.
Applications
For antigen detection: Hepatitis B
surface antigen, hepatitis Binner core antigen, NSI
For antibody detection: Hepatitis B, C, dengue antigen
HIV, dengue, Epstein-Barr virus, Herpes Simplex virus, toxoplasmosis,
leishmaniasis.
(a) Direct ELISA
Wash
Wash
Antigen Add enzyme (E) Add substrate (S)
(Ag) coated conjugated and mneasure
well antibody (Ab) to color
be measured
(b) Indirect ELISA
-ES
Wash Wash Wash
Antigen Add antibody Add enzyme (E) Add substrate (S)
(Ag) coated (Ab) to be
well
-conjugated and mneasure
measured secondary antibody color
(c) Sandwich ELISA
IHE-S
YY Antigen
Wash
YYAdd antibody
Wash
Add enzyme (E)
Wash
Add substrate (S)
(Ab) coated (Ag) to be -Conjugated and measure
well measured antibody color
(d) Competitive ELISA
E rES
YY
S
Wash Wash
Incubate Add Ag - Ab Add enzyme (E) Add substrate (S)
antibody (Ab) mixture to conjugated and measure
with antigen (Ag) antigen-coated secondary Color
to be measured well antibody
Types of ELISA
UrIIunuWIIg cauneterisation.
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