Antigen Antibody

reactions (2)
complement fixation test
• The complement fixation test (CFT) was extensively used in syphilis
serology after being introduced by Wasserman in 1909

• The demand on equipment and reagents is small

• A large variety of test antigens are readily available

 Principle of CFT

• The complete fixation test (CFT) is used to detect the presence of specific
antibodies in the patient’s serum.

• This test is based on the use of complement, a Biologically labile serum

factor that causes the immune cytolysis i.e. lysis of antibody coated cells.
principle

First step (Complement fixation stage): 

• A known antigen and inactivated patient’s serum are incubated with a
standardized, limited amount of complement.

• Note: patient’s serum is heated at 56°C for 30 minutes to inactivate

endogenous complement which may disturb the test calibration.
• If the serum contains specific complement activating antibody, the
complement will be activated or fixed by the antigen-antibody complex.
• However, if there is no antibody in the patient’s serum, there will be no
formation of antigen-antibody complex, thus complement will not be
fixed but will remain free (In the indicator stage this complement will react
with RBC coated with antibody to sheep RBC ).
 principle
Second step (Indicator Stage): 
• The second step detects whether complement has been utilized in the first
step or not. This is done by adding the indicator system.

• If the complement is fixed in the first step owing to the presence of

antibody there will be no complement left to fix to the indicator system. There
won’t be any lysis of RBCs.

• However, if there is no specific antibody in the patient’s serum, there will be no

antigen-antibody complex,  therefore, complement will be present free or
unfixed in the mixture. This unfixed complement will now react with the
antibody- coated sheep RBCs to bring about their lysis.
 Interpretation
• In the positive test : The available complement is fixed by Ag-Ab
complex and no hemolysis of sheep RBCs occurs. So the test is positive
for presence of antibodies.

• In the negative test : No Ag-Ab reaction occurs and the complement is

free. This free complement binds to the complex of sheep RBC and it’s
antibody to cause hemolysis, causing the development of pink color.

• Controls should be used along with the test to ensure that

• Antigen and serum are not anti complimentary
• The appropriate amount of complement is used and
• The sheep red blood cells do not undergo autolysis
 Positive Test
• Step 1:
At 37°C
Antigen + Antibody + Complement complement fixed
(from serum) 1 Hour

• Step 2:
At 37°C
Fixed Complement complex + Haemolytic system No haemolysis
1 Hour
(Test Positive)
 Negative Test

• Step 1:
At 37°C
Antigen + Antibody absent + Complement complement not fixed
1 Hour

• Step 2:
At 37°C
Free Complement + Haemolytic system Haemolysis
1 Hour (Test Negative)
Materials and Reagents
• The test requires five reagents and is carried out in two steps.

Test System
• Antigen: It may be soluble or particulate.

• Antibody: Human serum (May or may not contain Antibody towards specific
Antigen)

• Complement: It is pooled serum obtained from 4 to 5 guinea pigs. It should be

fresh or specially preserved as the complement activity is heat labile (stored at
-30 °C in small fractions). The complement activity should be initially
standardized before using in the test.
Indicator System (Haemolytic system)
• Erythrocytes: Sheep RBC
Amboceptor (Hemolysin): Rabbit antibody to sheep red cells prepared by
inoculating sheep erythrocytes into rabbit under standard immunization
protocol.
Advantages and disadvantages of CFT
Advantages
1. Ability to screen against a large number of viral and
bacterial infections at the same time.
2. Economical.
Disadvantages
3. Not sensitive - cannot be used for immunity screening
4. Time consuming and labor intensive
5. Often non-specific e.g. cross-reactivity between HSV and
VZV
Immune adherence
• When some bacteria (such as vibrio cholerae or treponema pallidum

combine with their specific antibody in the presence of complement

and some particles such as erythrocytes or platelets, they adhere to the

erythrocytes or platelets. This is called immune adherence.
Immobilisation test
• Here antigen is incubated with patient’s serum in presence of complement.

• If specific antibody is present it would immobilize the antigen.

e.g.Treponema palladium immobilization test, considered gold standard for
the serodiagnosis of syphilis
 Neutralization
• A procedure in which the chemical or biological activity of a reagent
or a living organism is inhibited, usually by a specific neutralizing
antibody.
• As an example, the lethal or the dermonecrotic actions of
diphtheria toxin on animals may be completely neutralized by an
equivalent amount of diphtheria antitoxin—an antibody produced
in animals or in humans after contact with diphtheria toxin or
toxoid.
Neutralization
• Lesser amounts of antitoxin provide intermediate degrees of
inhibition.
• These facts provide the basis for the Schick test for
susceptibility to diphtheria.
• Tetanus and botulinum toxins may be similarly inhibited by their
specific antitoxins.
• In contrast, the typical toxins of dysentery and other gram-
negative bacteria are only slightly neutralized, even by large
excesses of antibody.
 Immunochromatography

Simple device intended to detect the presence (or absence) of a •

target analyte in sample (matrix) without the need for specialized
.and costly equipment
A widely spread and well known application is the home •
.pregnancy test
 Principle of Immunochromatography Kit:

• The liquid sample is dropped on the sample pad, the antigen in the
sample forms an immuncomplex with the antibody labeled with
colloidal gold.
• Its complex moves along with the liquid sample, and makes a contact
with the antibody immobilized on the membrane, followed by forming
an immuncomplxes with the immobilized antibody, resulting in
generating a colored red purple line.
Principle of Immunochromatography Kit:

• Appearance of red purple line on the membrane indicates the

presence of antigen in interest in the sample. Since the liquid of
the sample migrates through the membrane very fast, it makes it
possible to detect the presence or absence of antigen within 15
minutes.

• An Immunochromatographic unit is completed by attaching the

sample pad at the end of the membrane.
How they work:

• Sample placement
• To perform the test, a sample is placed on the
sample pad at the end of the strip.
• The sample may be used alone as commonly done
with urine or serum or whole blood or plasma
compatible tests, or it may be mixed with a buffer
specific to the test
• : Capillary action
• Then capillary action draws the fluid mixture up the sample pad and into the membrane.
• The sample/detector molecule mix continues to move up the membrane until it reaches
the analyte capture molecule.
• In these lines a second (and third) antibody or antigen, immobilized as a thin stripe in the
nitrocellulose will then capture the complexes if it is positive for the target analytes.
• The control line should always show as a visible line, otherwise the test is invalid and must
be repeated. If the test is positive, a colored (typically pink or purple ) line develops along
with the control line
Advantages:

1. Can detect antigen or antibody

2. Commercially available.
3. Easy to perform
4. Limited / no instrumentation.
5. Single use, rapid test.
6. User friendly format
7. Very short time to get test result.
8. Long-term stability over a wide range of climates
9. Relatively inexpensive to make.
Applications of Immunochromatographic
assays:
• It can be applied for multiple test platforms for liver, sexually
• transmitted diseases, cardiac markers, as well as women’s and men’s
ss
• health (hCG, VDRL, CMV, PSA, H. Pylori, Troponin I, TORCH, HIV, HBs Ag
• , HBV, HCV, RA, CRP, ASO, SLE, IM, salmonella IgM & IgG, …).
 ELISA TEST

• Elisa is so named because it involves the use of an

immunoabsorbent, an absorbing material specific for one of the
components of the reaction—the antigen or antibody. This may be a
particulate—cellulose or agarose or a solid phase such as
polystyrene,polyvinyl or polycarbonate tubes or microwells
or membranes or discs of polyacrylamide, paper or plastic.
• Elisa is usually done using 96-well microtitre plates.
• Types of elisa---Sandwich elisa
• Competitive elisa
• Capture elisa
• Cassette elisa
• Uses of elisa---diseases like Japanese encephalitis,Dengue,hepatitis
virus,Influenza virus,CMV,EBV,TORCH.
• SyphilisIgG,IgM.food adultarent ,food toxin also
• HIV ----antigen and antibody detection.
IMMUNOFLUORESCENCE

FLUORESCENCE is the property of absorbing light rays of one

particular wave length and emitting rays with a different wave length.
It is a powerful technique that utilizes fluorescent labeled antibodies
to detect specific target antigens
The fluorescent antibody or immunofluorescence technique has
several diagnostic and research applications.
• Direct immunofluorescence—this can be used for identification of
bacteria,viruses or other antigens using specific antiserum labelled
with a fluorescent dye. Example – for diagnosis of rabies.

• A disadvantage of this method is that separate fluorescent

conjugates have to be prepared against each antigen to be tested.
Indirect immunofluorescence----this test overcomes the difficulty
mentioned in direct method by using an antiglobulin fluorescent
conjugate. Example– fluorescent treponemal antibody test for the
diagnosis of syphilis.
Flow cytometry---this is the fluorescence technique used to identify
and enumerate cells bearing a particular antigen or the surface
markers by suspending them in astream of fluid and passing them
through an electronic detection apparatus.
• Application of flow cytometry----It is widely used in research and
diagnostics—to count blood cells including DLC, to isolate Tcell
subsets—CD4 and CD8 counts in HIV patients,for diagnosis, treatment
and prognosis in cancer(leukemias), to study the cell cycle and
apoptosis etc.