For More Medicine Free PPT - http://playnever.blogspot.com/ For Health benefits and medicine videos Subscribe youtube channel - https://www.youtube.com/playlist?list=PLKg-H-sMh9G01zEg4YpndngXODW2bq92w

1. BOND KING (SUNIL)
NITISH
RAHUL CHAUDHARY

2.  The simple combination of antigen and
antibody both in vivo and in vitro.

3. 1.Primary rxn (more sensetive)
 A specific recognization and combination of the
antigen with the binding site of its
corresponding antibody
 More sensitive than 2 deg or 3 deg
 They are not dependent on the variable that
control 2 deg. Or 3 deg.
 Quantitative test involve include
immunofluorescenes, RIA, immunoenzymatic
assay
 Required purified Ag/Ab preparation.

4. 2.Secondary Rxn
 Conformation of the amino acid chain resulting
from interchain hydrogen bonding.
 Include precipitation in solution or in gel,direct
agglutination on hemogglutination , complement
fixation.

5. 3.Tertiary Rxn
 Involve the folding of polypeptide chain through
hydrophobic and hydrogen bonds.
 Include phagocytosis,opsonization,
chemotaxis,immune adherence and cellular
degradation.
 Antibody molecules are considered to be capable
of recognization, binding and complexing with
specific antigen.

6. 3.Quaternary Rxn
 Involves the association of polypeptide
subunits to form one CHON (protein)

7.  Due to variation of behaviour of different
antibodies ,a broad spectrum of different
immunologic method has been developed some
and considerably more sensitive than others.
Examples
 Ag –Ab rxn are sensitive in the nanogarms to
picograms, milliliter range.

8. I.Double diffusion technique (ouchterlony -
technique)
 1st method used to establish the relationship of
HBsAg to type B hepatitis.
 Known as double diffusion because both Ag and
Ab diffuse.
 If the well size and shape,distance betwn wells,
temperature and incubation time are optimal it
will result to a visible precipitate.

9. 1.Identity Rxn
 Is revealed when the lines of the ppt come
together on the plate, forming a smooth curve
,indicating that the Ab is precipitating identical Ag
species.
2.Non –identity Rxn
 Is revealed when ppt lines cross one another
because they don’t have antigenic determinats in
common.
3.Partial – identity Rxn
 Is revealed when ppt. lines merges with spur
formation,which indicates that Ag are not identical
but do not possess common determinants.

10.  Involves the addition of anti-serum to a
liquefied gel, which is formed into a plate and
allowed to solidify by cooling at room
temperature.
 The Ag solution is added to wells to cut into
agar
 The Ag diffuses in all direction from the well
and ppt. occurs in a ring surrounding the wells.

11. 1.The Fahay (kinetic diffusion) method
 The diameter of the ppt rings measured at 18
hrs.the logarithm of conc. of the standards is
proportional to the diameter of precipitation
ring.
2.The mancini (end point diffusion) method
 The Ag is allowed to diffuse fully to achieve
ppt.
 A maximal ring diameter is obtained in about 24
hrs.by IgG in normal conc. and in 50-72 hrs. by
IgM in normal concentration

12.  Over or underfilling of the wells.
 Spilling the patient’s serum on the gel.
 Nicking the side of the well when filling
 Improper incubation time and temp.
 Specimen contamination

13.  Gel is poured onto a plate and cooled 2 columns
of wells are cut with the Ag in one well and Ab
in the other.
 The plate is placed in an electric field.
 At PH 8.6 the Ag migrates toward the anode
and the Ab migrates toward the cathode ppt.
occurs.
 Useful in the detection autoantibodies Abs to
infectious agents and certain microbial agents.

14.  Reversal of the wells, current is applied in the
wrong direction.
 Improper PH of the buffer.
 Prozone or post zone
 Wells are not parallel.

15. IV. Immunoelectrophoresis
 Useful for the identification of monoclonal
proteins
 Utilizes both electrophoresis and double
diffusion.
 Under and electrical current, Ag migrates
through a gel medium
 Example :- agar

16. V. Immunofixation Electrophoresis (IFE)
 A cellulose acetate strip impregnated with anti-
serum placed over proteins after serum,urine
or cerebrospinal are electrophoresed.
 Diffusion of the anti-serum in to the gel occurs
rapidly, resulting in ppt. of Ab-Ag complexes.

17. VI. Rocket Electrophoresis
 Used to quantiate Ag other than
immunoglobulins.
 The anti-serum is incorporated into the agar
and the unknown Ag is placed in the well and
electrophoresed.
 The Ag migrates through the gel, it combines
with Ab and ppt occurs in the shape of rocket.

18.  Agglutination rxn’s are similar to precipitin rxn’s
except the union of Ab occurs with suspended
particulate Ag rather with soluble Ag.
 Particles involved are cells (bacteria, yeast,
erythrocytes) or latex particles which in general
are large enough for direct observations.
 When these Ag combine with their specific Ag-
Ab aggregation, clumping of the particles is
seen. Agglutination results from the cross-
linking of cells or particles by Ab molecules.
Occurs in two phase
- specific Ag-Ab binding
- lattice formation

19. Factors influencing the rxn.
 Elevation or decrease of temperature
 Motion (shaking, stirring, centrifugation)
 PH
 Class of antibody( IgM/ IgG)

20. 1. Direct agglutination
 Uses Ag found naturally on the surface of the
cells (RBC,Bacteria)
 Direct agglutination of red cell may be used to
detect ABO and RhAg.
 May also be used to detect Ab to infectious
agent.
 Also used with the involvement of bacterial
natural Ag which demonstrate recent infection
by increasing the titer of Ab against a specific
bacteria.
 Can be used to detect febrile Abs (Widal test)

21. 2. Viral hemagglutination
 Occurs when a virus (rubella/ influenza)
agglutinates RBC by binding to receptors on the
RBC surface.
 The test most often used in the viral
hemagglutination inhibition test, which inhibits
the virus from agglutinating the RBC.

22. 3. Passive / Reverse passive agglutination
 In passive agglutination, the antigen is attached
(coated) to a particulate carrier. If the Ab is
attached to a particulate carrier, the technique is
referred to as reverse passive agglutination.
 The carrier used charcoal, latex, particles, gelatin
particles and RBC’s
Example
 Passive agglutination procedure can be used to
detect nontreponemal Ab in syphilis, RF , rubella Ab
and thymoglobulin Ab.
 Latex particles and red cells may be used to detect
Ag in the reverse passive agglutination technique can
be used to detect c-reactive CHON (protein).

23. 4. Antiglobulin Technique
 Generally used in the immunohematology laboratory.
 RBC have a net negative charge and therefore repel
one another since IgG molecules are small and
cannot link one Ag binding site on a 2nd RBC the
lattice is not formed and reaction is not visible.
Specific procedure
1. DAT
 When an in vivo attachment of Ab to an individual’s
red cells has occurred.
example:- HDN,AIHA ,HIR
2. IAT
 Used to determine compatibility btwn donar and
recipient.

24. 5. Agglutination Inhibition Rxn’s
 Can be used with direct or passive
agglutination.
 1st step, soluble Ag in the patient’s sample is
incubated with Known Ab reagent . If the
soluble Ag is present a rxn will take place but
will not be visible.
 2nd step, the Ag is added.
 If the Ag was present in the patient sample,
there is no free Ab to attach to the Ag,
agglutination is therefore inhibited which
represents as positive test.
example :- HCG detection in serum / urine

25.  Is based when on Ag combines with Ab in the +nce
of complement ,the complement is “fixed” by the
Ag-Ab complex and is unable to react with cell
sensitized will other Ag- Ab complexes.
 As an indicator of the presence of “unfixed”
comlement, RBC sensitized with specific Ab are
used.
 Lysis of RBC indicates the +nce of unfixed
complement, the lack of hemolysis indicates that
the complement has reacted with the test Ag-Ab
complex
Example
viral, rickettsial and fungal serology( rocky
mountain, spotted fever, herpes, simple infection
and infuenza)

26.  Rxn is based on the fact that the following the
primary specific Ag-Ab rxn; the Ag-Ab complex
develops an ability to adhere to particles such
as RBC, silica, starch granules and bacteria.
Examples
Trypanosomiasis and syphilis

27.  In case of viruses, a dose of viruses known to
be lethal to a test animal is mixed with the
serum to be tested for Ab against the virus.
 The mixture is injected in to the test animal
and observed for the rxn. If the lethal dose
fails to kill the test animal neutralizing Ab is
present in the test serum.
 In case of toxins, an individual who has been
exposed to toxin- producing bacteria is
injected with antitoxin. The toxin- antitoxin
rxn in vivo protects the recipient by
neutralizing the toxin.

28.  Faliure to protect the animal means that the
test, serum has no protective Ab against the
particular toxin.
 Anti- toxin potency can be measured by ppt and
by floculation techniques in vitro.
Examples
Schick test – detect Ab to diphteria toxin.
Dick test – detect Ab to scarlatinal toxin.

29.  Involves the study of Ag in tissue section by
the use of specific Ab that has been labeled
with color and is applied over a section of
tissue so that a micro precipitate is formed at
the site of Ag.
 The fluorescent dye usually used fluorescent
isocyanate or isothiocyanate is linked with
serum Ab and yields a blue green fluorescent
substance when illuminated with ultra violet
light.

30. 1. Indirect or Double- layered Technique
 Used to detect Ab in unknown sera
 Unlabelled Ab is exposed to Ag followed by the
addition of labeled antiglobulin sera, directed
against the globulin of species used in the
initial exposure.
2. Complement staining Technique
 The process is similar to indirect method
except that the conjugate is directed against
the species supplying the complement.
 Fixation of the complement to the complex.

31. 3. Inhibition Technique
 Is based on the procedure of blocking specific
Ag-Ab rxn by initial exposure to a different
aliquot of homologous Ab.
 Unlabelled Ab is added to Ag saturating the Ag
then labeled Ab is added.
 No fluorescence is seen, test is non- reactive .
no Ag site reacted with the labeled Ab.

32.  Refers to many techniques performed in clinical
lab.
 Any substance that will complex to another
substance is referred to as ligand.
 The ligand is the substance to be measured
(hormone combining with CHON, drug combining
with an Ab)

33. 1. Radio immunoassay (RIA)
 Widely used method
 Conc. Of Ag/Ab in serum is measured with the
use of radioisotopes.
 Extremely sensitive can detect trace amount of
Ag/Ab.
 Allows large nos. of test to be performed in
short period of time.
 Disadvantage is hazards and instability of
isotopes.

34. 2. ELISA (enzyme linked immuno sorbent assay)
 Principle is similar to that of
immunofluorescenes in that purified enzyme is
linked in a stable manner to a specific Ab.
 Horseradish peroxidases and alkaline
phosphatase are two common used enzymes.

35.  Refer to the no. of Ab molecules / unit volume
of the original serum.
 Gives as indication of the Ab conc. In a
patient’s serum
 The titer is read at the highest dilution of
serum that gives rxn with Ag.

36.  Based on (Ag-Ab) precipitation rxn.
 Photometric measurement of the quantity of
cloudiness or turbidity in a solution caused by
suspended particles.
 The interaction of light with the particles in
solution causes the light to be transmitted
through the solution and be reflected.

37. 1. Flow cell cytometry
 Large no. of cells pass through an aperture,
where thus exposed to light/ electric current
to generate a signal that is measured.
2. Latex bead Ingestion
 Used latex beads to assess monocyte number.
3. Lymphocyte transformation
 Used mitogen, specific Ag to determine the
ability of lymphocytes to respond to at
stimulus.

38. 4. Mixed – lymphocyte culture
 Used recipient donor lymphocytes to detect
compatibility or incompatibility with respect to
HLA-D Ag.
5. Migration Inhibition
 Uses monocyte and granulocytes to determine
the ability of the lymphocyte to produce
chemo-tactic factor.
6. Microcytotoxicity
 Used to detect HLA-B, HLA-C and HLA-DR Ag
and HLA-Ab

39. 7. Cell- mediated monoctolysis
 Used tumor cells to determine the ability of
monocytes to kill cells.
8. Ab-dependent Cell- mediated cytotoxicity
 Used tumor cells /cells containing bacteria to
determine the ability of natural killer cells to
lyse other cells.
9. Lympholysis
 Uses a labeled target cell to determine the
ability of cytotoxic T-cell to lyse cells.

40. 10. Boyden chamber
 Uses a chemo-tactic agent to determine the
ability of neutrophils to respond to chemo-
tactic Agent.
11. Phagocytosis
 Uses Escherichia coli polysaccharides with oil
red o stain to determine the ability of
neutrophil to phagocytize.
12. Nitroblue tetrazolium Test
 Uses nitro-blue tetrazolium to determine the
ability of neutrophils to effect intracellular
kill.