Antigen -Antibody Interactions Principles and Applications

Pathology

2. The Ag-Ab interaction is a biomolecular
association similar to enzyme-substrate
interaction but with few differences as:
1.It does not lead to irreversible chemical alteration
in either the Ag or the Ab.
2.It is a noncovalent interaction which include
hydrogen bond, ionic bond, hydrophobic bond
and van der Waals interaction.
Strong Ag-Ab interaction depend on a very close fit
between the Ag & Ab.

3. Ab affinity:
It is the strength of the total noncovalent interaction
between a single Ag-binding site on an Ab & a single
epitope, so
low affinity week binding dissociate easily.
→ →
high affinity strong binding difficult to dissociate.
→ →
Ab avidity:
Is the strength of multiple interaction between a
multivalent Ab & Ag. High avidity can compensate for
low affinity for e.g. IgM has low affinity than IgG but
has high avidity bacause it is pentamer.

4. Cross reactivity:
In which Ab elicited by one Ag can cross react with an
unrelated Ag. This is because the 2 different Ags share
an identical epitope as for e.g.
1.ABO blood- group Ag cross react with microbial Ags
present on the common intestinal bacteria which
induce the formation of Ab in individuals lacking the
similar blood-group Ag on their red blood cells.
2.Streptococcal pyogenes cell protein(M protein),of
which Ab against it will cross-react with several
myocardial & skeletal muscle proteins resulting in
development of Rheumatic fever and
glomerulonephritis.

8. Many tests based have been developed to
determine the presence of antibodies or
antigens in a patient in the diagnose
Is done in many areas of the clinical
laboratory-microbiology, chemistry,
toxicology, immunology, hematology,
cytopathology, immunohematology(blood
banking)-and great variety of specimens are
tested.

9. Types of diagnostic tests
Many types of diagnostic tests are performed in the
immunology laboratory. Most of these tests can be designed to
determine the presence of either the Ag or the Ab.
Major uses of serologic (Ab – based ) tests:
1. Diagnosis of infectious diseases:
•When the organism cannot be cultured as in syphilis, hepatitis
A,B, and C.
•When the organism is too dangerous to culture e.g. rickettsial
disease.
•When culture technique are not readily available, e.g. HIV.
•When the organism takes long time to grow, e.g. Mycoplasma.
2.Diagnosis of autoimmune disease, e.g. Ab against DNA in SLE.
3.Determination of blood type and HLA.

10. ANTIGEN-ANTIBODY INTERACTIONS
Many diagnoses would not be possible without laboratory
procedures that identify antibodies or antigens in the
patient
Interaction of antigen and antibody occurs in vivo, and
in clinical settings it provides the basis for all
serologically based tests.
The formation of immune complexes produces a
visible reaction that is the basis of precipitation and
agglutination tests.

11. Antigen-Antibody Interaction
Precipitation
reaction
Agglutination
reaction
Chromatography
Immunoassay
Labeled Ags
OR Abs
Indirect
(passive)
Direct:
-Blood group
-Bacterial aggl.
PPT in gel
PPT in
Soluble
Single Radial
diffusion
Double
diffusion
PPT in Agar with
electric field
Immunoelectrophoresis
Counter immuno electroph.
Rocket electrophoresis

12. Specimen collection and preparation
Obtain a sample of venous blood from the patient
and allow a clot to form. Centrifuge clotted blood
sample and collect clear serum .

13. It is an interaction between Ab & particulate Ag resulting in
visible clumping called (agglutination).
The Ab that produce such a reaction is called (agglutinin).This
reaction need a polyvalent Ag. Just as an excess of Ab inhibit ppt
reaction, it will also inhibit agglutination and this is called (Prozone
effect).
Causes of Prozone effect:
1.High Ab concentration. This will result in univalent binding of the
Ab to Ag, so no cross-linking of one Ag to another. To overcome this
you most dilute the serum .
2.Incomplete Ab (often of IgG). This Ab may occupy most of the Agic
site, this block the access by IgM, which is a good agglutinin.

14. Types of agglutination reactions
[1] Rose-Bengal test:
-Principle of the test:
The test depends on the ability of the Ab in
the patient’s serum to agglutinate the stained
bacterial Ag. When this occurs the aggregates
become clearly visible to the naked eye .
Ag : stained bacterial Ag (killed suspension of
Brucella abortus )
Ab : Abs to Brucella in the patients serum .
Direct agglutination (Bacterial agglutination)

15. Assay procedure :
1- Qualitative method :
1- Allow kit and patient serums to come to room
temperature .
2- Transfer one drop (50 µl) of patient’s serum on
a test slide.
3- Shake the reagent then using the dropper and
add one drop to the test slide and mix and gently
rotate the mixture for 4 minutes.

16. 2- Semi-quantitative method :
1- Using isotonic saline prepare serial dilution of
the patient serum (1/2, 1/4, 1/8, 1/16, 1/32, 1/64,
and so on ).
2- Transfer one drop (50 µl) of each serum dilution
to the test circle slide .
3- Shake the reagent, add one drop of suspension
to the test slide then mix and gently rotate the
test slide for 4 minutes .

17. - Results :
A positive result is indicated by the obvious
agglutination pattern of the suspension on the
test slide.
A negative result is indicated by no change in
the suspension on the test slide .
Agglutination of the Ag with undiluted sample
indicates the presence of Ab at a concentration
of greater than or equal to 25 IU/ml .
A comparison between samples taken 10-14 days
apart may be of value in acute illness .

18. 2- Widal Test :
-Principle of the test :
The test depends on the ability of Ab in the
patient’s serum to agglutinate the stained
bacterial Ags. When this occurs the
aggregates become clearly visible to the
necked eye.
*Ags :Suspension of bacteria stained and
killed. *Salmonella O-Group A(Paratyphi A-O).
*Salmonella O-Group B(Paratyphi B-O).
*Salmonella H-Group a(Paratyphi A-H).
*Salmonella H-Group b(Paratyphi B-H).
*Salmonella O-Group d(Typhi O).
*Salmonella H-Group d(Typhi H).
*Ab : Ab to bacterial Ags in patient’s serum .

19. Assay procedure :
1- Rapid slide test :
1- Using a graduated pipette add the following
amounts of serum to consecutive circles on a slide
for each dilution under test.
0.08 ml, 0.04 ml, 0.02 ml, 0.01 ml, 0.005 ml
2- Thoroughly resuspend the Ag and add a drop to
the appropriate circle on the slide.
3-Mix, and gently rotate the test slide for 1 minute
whilst examining the test slide for agglutination.
4- Results obtained correspond to tube agglutination
titers of 1:20, 1:40, 1:80, 1:160, 1:320 respectively.
Any sample showing agglutination should be tested
in the tube agglutination test.

20. Is useful with soluble Ag. This test done with the use
of Ag coated RBC, or the use of synthetic particales,
such as latex beads.
[1] Qualitative determination of anti-
streptolysin O (ASO).
-Clinical finding :
Streptolysin O is toxic immunogenic exoenzyme produced by β –
hemolytic streptococci of group A, C, and G.
Measuring the ASO Abs are useful for the diagnostic of rheumatoid
fever, acute glomerulonephritis and streptococcal infections.
Rheumatic fever is an inflammatory disease affecting connective
tissue from several parts of human body as skin, heart, joints,
etc….and acute glomerulonephritis is a renal infection that affects
mainly to renal glomerulus.
Indirect ( Passive ) agglutination:

21. -Principle of the test :
The ASO-Latex is a slide agglutination test for the
qualitative and semi-quanitative detection of
anti-streptolysin O(ASO) Abs. Latex particles with
streptolysin O are agglutinated when mixed with
samples containing ASO Abs.
*Ag :Latex particles coated with streptolysin O.
*Ab :Antistreptolysin O (ASO ) in patient’s serum .

22. -Procedure :
1- Qualitative method:
1- Allow the reagents and samples to reach to room
temperature .
2- Place 50µl of the sample and one drop of each
positive and negative controls into separate
circles on the slide test .
3- Shake the reagent gently befor using and add
one drop (50µl) next to the samples to be tested.
4-Mix the drops with a stirrer, spreading them
over the entire surface of the circle, and read
the result within 2 minutes .
2- Semi-Quantitative method:
1- Make serial two fold dilutions of the sample .
2- Proceed for each dilution as in the qualitative method .
The presence of agglutination indicates an ASO
concentration equal or greater than 200 IU/ml.

23. [2] Qualitative determination of Rheumatoid
Factors (RF):
-Clinical significance :
Rheumatoid factors are a group of Abs directed
to determinants in the FC portion of the
immunoglobulin G molecule .
Although rheumatoid factors are found in a
number of rheumatoid disorders, such as,
Systemic Lupus Erythematosus (SLE) ,and
Sjögren’s syndrome, as well as in nonrheumatoid
arthritis (RA), utility as an aid in the diagnosis of
rheumatoid arthritis (RA).

24. Principle :
The RF-latex is a slide agglutination test for the
qualitative and semi-quantitative detection of
RF in human serum. Latex particles coated
with human gammaglobulin are agglutinated
when mixed with samples containing RF.
*Ag : Latex particles coated with human
gammaglobulin .
*Ab :Serum containing RF .

25. -Procedure :
1- Qualitative method:
1- Allow the reagents and samples to reach to room
temperature .
2- Place 50µl of the sample and one drop of each positive
and negative controls into separate circles on the
slide test .
3- Shake the RF-latex reagent gently before using and
add one drop (50µl) next to the samples to be tested.
4-Mix the drops with a stirrer, spreading them over the
entire surface of the circle, and read the result within
2 minutes .
2- Semi-Quantitative method:
1- Make serial two fold dilutions of the sample .
2- Proceed for each dilution as in the qualitative method .
The presence of agglutination indicates a RF concentration equal
or greater than 8 IU/ml.

26. [3]A rapid latex slide test for the detection of CRP in
serum :
Tissue-damaging associated with inflammatory
diseases, infection and neoplasms are associated
with a major accute phase response of the C-reactive
protein (CRP) and other acute phase reactants. The
CRP response frequently precedes clinical symptoms,
including fever, Measuring changes in the
concentration of CRP provides useful diagnostic
information about how acute and how serious a
disease is. It also allows the assessment of
complications during the disease and judgment
about the disease genesis.

27. -Principle of the test :
CRP latex reagent is a suspension of polystyrene
particles sensitized with anti-human CRP. When
the latex reagent is mixed with a serum
containing C-reactive protein, visible
agglutination occurs. The latex reagent has been
produced so that agglutination will take place
only when the level of CRP is greater than 6
mg/L.

28. [1]Toxoplasmosois Latex test kit :
-Toxoplasma is an infectious disease affecting various
mammalian species including man. Infection is caused
by the protozoan parasite Toxoplasma Gondii . Infection
is usually acquired from faeces of infected cats.
The acquired disease is usually mild and not diagnosed.
In pregnant women patients can transmit the infection
across the placenta to the fetus causing congenital
Toxoplasmosis.
Disease manifestation depend on the stage of fetal
development; although the fetus is less likely to be
infected early in pregnancy, the effects on those
infected are more severe during the first trimester of
pregnancy. The consequences of congenital
toxoplasmosis range from spontaneous abortion and
prematurely to neurological congenital abnormalities.

29. -Principle :
The Toxoplasmosis latex reagent is a
suspension of polystyrene particles sensitized
with Toxoplasma Gondii When serum from
an infected individual is mixed with the latex
particles a distinct agglutination pattern is
observed as a result of the formation of Ag-Ab
complexes. In the absence of infection no
agglutination will be observed. A positive
result indicates a level of infection greater
than 4 IU/ml .

30. -Procedure :
1-Bring reagents and serum samples to room
temperature .
2-Dilute samples 1/2-1/8in physiological saline.
3-Place one drop of diluted serum onto a test slide.
4-Mix well the latex reagent and add one drop over
each serum drop.
5-Mix and rotate the slide for 3 minutes .
6-Observe presence or absence of agglutination .
7-When the results are positive ,prepare serial two-fold
dilutions from(1/16 to 1/640) and repeat steps 3-6.
The titer is the highest dilution with a clear
agglutination .

31. Chromatographic immunoassay :
Pregnancy test strip (Urine/Serum):
Human chorionic gonadotropin (hCG) is a
glycoprotein hormone produced by the
developing placenta shortly after fertilization.
In normal pregnancy, hCG can be detected in
both urine and serum as early as 7 to 10 days
after conception.
-Principle:
The hCG one step pregnancy test strip
(Urine/serum) is a rapid chromatographic
immunoassay for the qualitative detection of
hCG to aid in the early detection of pregnancy .

32. Principle :
The test uses two lines to indicate results. The
test utilized a combination of Abs including a
monoclonal hCG Ab to selectivesly detect
elevated levels of hCG.
The control line is composed of goat polyclonal
Abs and colloidal gold particles. The assay is
conducted by immersing the test strip in a
urine or serum specimen and observing the
formation of colored lines. The specimen
migrates via capillary action along the
membrane to react with the colored conjugate .
Ag : hCG in urine or serum samples .
Ab : anti-hCG coated on the membrane .

33. Procedure :
Immerse the test strip vertically in the urine or serum
specimen for at least 10-15 seconds.
Place the test strip on a non-absorbent flat surface,
start the timer and wait for the colored line(s) to
appear .
Read the result at 3 minutes when testing a urine
specimen, or at 5 minutes when testing a serum
specimen .
Positive : Two distinct colored lines appear. One line
should be in the control line region(C) and another line
should be in the test line region .
Negative : One colored line appears in the control
line region (C). No apparent colored in appears in the
test line region (T) .

34. [2] Rubella IgM antibodies :
Principle: It is qualitative immunoassay for the
detection of IgM Abs to Rubella virus in serum or
plasma specimens.Ags of Rubella are coated in the
test line region of the test. During testing, the serum
or plasma specimen reacts with Goat anti-human
IgM coated particals in the test strip. The mixture
then migrates upward on the membrane by capillary
action and reacts with Rubella specific Ags on the
membrane test line region. The presence of a colored
line in the test line region indicates a positive result
for rubella virus infection, while it’s absence
indicates a negative result for infection.

35. [3]One step Anti-TP( Treponema Pallidum/ Syphilis )
Test :
Principle of the test :
It uses for the detection of TP antibody in
human whole blood, serum, or plasma. TP Ag ,
coupled with colloidal gold particles, is dried
on a conjugate pad. During the assay, the
specimen is allowed to react with the colored
conjugate (Ag-colloid gold conjugate); the
mixture then migrates chromatographically
along the membrane by capillary action .

36. C
T
C
T
C
T
C
T
C
T
Positive Negative
Invalid