مناعه عملي

1. ‫بالم‬ ‫العلمية‬ ‫المهن‬ ‫نقابة‬
‫ن‬
‫وفية‬ [1]
iagnostic erology
Basic concepts
Antigen
An antigen is any substance that causesthe immune system to produce antibodies against it.
An antigen may be a foreign substance from the environment such as bacteria and viruses or it may also be formed within the
body, as with bacterial toxins or tissue cells.
Epitope
An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by antibodies.
Antibodies
An antibody or immunoglobulin is a protein produced by the body's immune system when it detects antigens.

2. ‫بالم‬ ‫العلمية‬ ‫المهن‬ ‫نقابة‬
‫ن‬
‫وفية‬ [2]
Antigen-Antibody interactions
Antigen-antibody reaction is a specific chemical interaction between antibodies produced by B cells and antigens during immune
reaction.
Lock and key concept
The antibody recognizes a unique part of the antigen. Each tip of the "Y" of an antibody containsan antigen binding site paratope (a
structure analogous to a lock) that is specific for one particular antigenic determinant epitope (similarly analogous to a key) on an
antigen, allowing these two structures to bind together with precision.
Serology
It refers to using antigen-antibody reactions in the laboratory for diagnostic purposes. Its name comes from the fact that serum, the
liquid portion of the blood where antibodiesare found is used in testing. Serologic testing may be used in the clinical laboratory in two
distinct ways:
▪ To identify unknown antigens. This is called direct serologic testing. Direct serologic testing uses a preparation known
antibodies,called antiserum, to identify an unknown antigen such as a microorganism.
▪ To detect antibodies being made against a specific antigen in the patient's serum.This is called indirect serologic testing.
Indirect serologic testing is the procedure by which antibodies in a person's serum against an antigen associated with a
particular disease are detected using a known antigen.

3. ‫بالم‬ ‫العلمية‬ ‫المهن‬ ‫نقابة‬
‫ن‬
‫وفية‬ [3]
Agglutination assays
Agglutination is the clumping of particles. Agglutination occurs if a particulate antigen is mixed with its corresponding antibody
(known as isoagglutinin).
Direct agglutination test
Principle
The antibody is mixed with the particulate antigen and a positive test is indicated by the agglutination of the particulate antigens.
The reactions between Ag and Ab occur in two stages:
▪ Primary stage (Sensitization): This is rapid reaction involves formation of Ag-Ab complex (Attachment of a single antibody to
a single antigen)
▪ Secondary stage (Lattice formation): Random collisions of antibody-coated antigens lead to the formation of bridges between
these sensitized antigens to formthe lattice that constitutes visible agglutination.
Laboratory example
▪ ABO test
A patient's red blood cells can be mixed with antibody to a blood group antigen to
determine a person'sblood type.
▪ Widal test
The Widal test is a serological test for enteric fever whereby bacteria causing typhoid
fever (the regent is stained Salmonella antigens) are mixed with serum containing
specific antibodies obtained from an infected individual.
Direct flocculation test
Principle
It is a form of agglutination in which the Ag is found in colloidal form instead of being particulate. In the flocculation test the antigen –
antibody complex become aggregated in small flocculeswhich should be read microscopically using a low power objective.
Laboratory example
▪ RPR (Rapid Plasma Reagin) for syphilis:
It is a semi quantitative non-treponemal flocculation test for the detection of reagin antibodiesin human serum as a screening test
in syphilis serology.

4. ‫بالم‬ ‫العلمية‬ ‫المهن‬ ‫نقابة‬
‫ن‬
‫وفية‬ [4]
The term "reagin" means that this test does not look for antibodies against the actual bacterium, but rather for antibodies
against substances released by cells when they are damaged by T. pallidum
The reagin antibody binds with the antigen that is composed of a complex of cardiolipin, and cholesterol particles with
activated charcoal; the result of this antigen-antibody reaction is macroscopic flocculation.
Indirect latex agglutination
Latex particles
Usually prepared by mixing styrene with a surfactant (sodium dodecyl sulfate) solution, resulting in billions of emulsified micelles
extremely uniform in diameter
Principle
Latex test is an agglutination technique used to detect antibodies or antigens.For example, in a latex test, a sample is mixed with latex
beads coated with antibodies. If the antigen is present, it will react with the antibodiescausing the latex beadsto clump.

5. ‫بالم‬ ‫العلمية‬ ‫المهن‬ ‫نقابة‬
‫ن‬
‫وفية‬ [5]
Laboratory example
▪ ASOT test: is a stabilized buffered suspension of polystyrene latex particlesthat have been coated with streptolysin O. when the
latex reagent is mixed with a serum containing antibodiesto streptolysin O, agglutination occurs.
▪ CRP test: Latex particles coated with goat anti-human CRP antibodies are agglutinated when mixed with samplescontaining CRP.

6. ‫بالم‬ ‫العلمية‬ ‫المهن‬ ‫نقابة‬
‫ن‬
‫وفية‬ [6]
Indirect hemagglutination
Synthesized RBCs
A cell that has combined with antigen to form a complex capable of reacting with specific antibody
Principle
This is called passive hemagglutination. The agglutination test only works with particulate antigens. However, it is possible to coat
erythrocytes with a soluble antigen and use the coated red blood cells in an agglutination test for antibody to this soluble antigen
Laboratory example
Anti-Schistosomal antibody: the detection of Schistosoma antibody by IHA
Procedure
1. Allow reagents and samples to return to room temperature
2. Dilute serum using buffer reagent (1:40)
3. By meaning of a micropipette deliver 50 ml buffer in a well of the
microplate
4. Add 50 ml diluted sample to the well and mix
5. Carefully shake the RBCs suspension and distribute one drop (about 50
ml) in the well
6. Very carefully homogenize well content (by lateral thrumming on the
edges of the microplate)
7. Allow the plate to remain motionless, protected from vibrations for
about 3 hours then read result
Results
If the specific antibodies are present in the serum, they will agglutinate with
the red blood cells and a red/brown deposit will appearon the bottom of the
well. With a non-reactive serum, no agglutination occurs with the sensitized
cells and they drop to the bottom of the well, forming a compact button.

7. ‫بالم‬ ‫العلمية‬ ‫المهن‬ ‫نقابة‬
‫ن‬
‫وفية‬ [7]
Agglutination test results
Qualitative results
Agglutination tests can be used in a qualitative manner to assay for the presence of an antigen or an antibody.The antibody is mixed
with the antigen and a positive test is indicated by the agglutination.
Semi-quantitative results
Agglutination tests can be used to measure the level of antibodies or antigens.In this test, serial dilutions are made of a sample to be
tested and then a fixed amount of the reagent is added.
▪ Serial dilution
A serial dilution may be defined as multiple progressive dilutions ranging from more-concentrated solutions to less-concentrated
solutions
▪ Procedure
1. Prepare test tubes, each contains 50 microliter saline
2. To the first tube add 50 microliter serum
50 serum + 50 saline (1:1) >>> mix well then add 50 micro to the next tube (1:4) >>> mix well then add 50 micro to the next
tube (1:8) >>> mix well then add 50 micro to the next tube (1:16) >>> mix well then add 50 micro to the next tube (1:32)
>>> mix well then add 50 micro to the next tube (1:64) >>> discard the last 50 microliter
3. From each tube agglutination test is done (using volume to volume)
▪ Titer
A titer is a way of expressing concentration. The titer corresponds to the highest dilution factorthat still yields a positive reading

8. ‫بالم‬ ‫العلمية‬ ‫المهن‬ ‫نقابة‬
‫ن‬
‫وفية‬ [8]
▪ Prozone effect
The prozone effect is a lack of agglutination or clumping during tests for antibodies.It may result when the concentrations of
antibodies orantigensare too high.
Mechanism: In a typical immunoassay, antigens and antibodies bind to create a conjugate, which is detected and measured.When
prozone effect occurs, excess antigensor antibodiesbind all of the receptor sites, leaving nothing available to become a conjugate.
Clinical significance: Since the results of the agglutination test are not accurately displayed, the prozone effect may lead to a false
negative result.
Avoiding the prozone effect: While the prozone effect may have serious consequencesconcerning the results of serum testing,
proper dilution of the serum can help prevent its likelihood.
‫ال‬ ‫شغل‬ ‫نتائج‬ ‫لدقه‬ ‫احتياطات‬
serology
1
.
( ‫الثالجه‬ ‫فى‬ ‫السيرولوجي‬ ‫كواشف‬ ‫حفظ‬ ‫دائما‬ ‫يتم‬
2
-
8
‫ضوء‬ ‫او‬ ‫عاليه‬ ‫حراره‬ ‫لدرجه‬ ‫تعريضها‬ ‫او‬ ‫تجميدها‬ ‫ممنوع‬ ‫و‬ ‫درجه‬ )
‫المباشر‬ ‫الشمس‬
2
.
‫للرئيجنت‬ ‫خلط‬ ‫عمل‬ ‫من‬ ‫البد‬ ‫و‬ ‫الشغل‬ ‫قبل‬ ‫الغرفه‬ ‫حراره‬ ‫لدرجه‬ ‫الكواشف‬ ‫تصل‬ ‫من‬ ‫البد‬
mixed gently)
‫توزيع‬ ‫العاده‬ )
( ‫الالتكس‬ ‫جزيئات‬
re-suspend latex
)
3
.
( ‫الــ‬ ‫على‬ ‫دالله‬ ‫يعتبر‬ ‫الرئيجنت‬ ‫فى‬ ‫ترسيبات‬ ‫او‬ ‫تجمعات‬ ‫اى‬ ‫وجود‬
(deterioration
‫استخدامه‬ ‫يمكن‬ ‫ال‬ ‫بالتالي‬ ‫و‬
4
.
‫الـ‬ ‫تنظيف‬ ‫المستحسن‬ ‫من‬
test slide)
( ‫قطن‬ ‫او‬ ‫قماش‬ ‫بقطعه‬ )
non-abrasive material
‫و‬ ‫مقطر‬ ‫بماء‬ ‫غمرها‬ ‫ثم‬ )
‫رشها‬ ‫ثم‬ ‫تجف‬ ‫تركها‬
spray)
( ‫بـ‬ )
70% alcohol solution
‫الشغل‬ ‫قبل‬ ‫تجف‬ ‫تركها‬ ‫و‬ )
5
.
‫البالزما‬ ‫عينات‬ ‫استخدام‬ ‫يصح‬ ‫ال‬ ‫و‬ ‫السيرولوجي‬ ‫لشغل‬ ‫المفضله‬ ‫العينات‬ ‫هي‬ ‫السيرم‬ ‫عينات‬
6
.
‫مده‬ ‫الحضانه‬ ‫فى‬ ‫العينه‬ ‫بترك‬ ‫الجلطه‬ ‫لتكون‬ ‫فرصه‬ ‫اعطاء‬ ‫من‬ ‫البد‬
20
( ‫االقل‬ ‫على‬ ‫دقيقه‬
allow a clot to form and retract
)
( ‫علي‬ ‫للحصول‬
clear serum
)
( ‫ال‬ ‫لتفادي‬
False positive
)
7
.
( ‫الدهنيه‬ ‫العينات‬ ‫استخدام‬ ‫ممنوع‬
lipemic serum
( ‫ال‬ ‫العينات‬ ‫و‬ )
haemolysed
)
8
.
( ‫ال‬ ‫لتفادي‬ )‫دقيقتين‬ ‫(خالل‬ ‫النتيجه‬ ‫لقراءه‬ ‫المحدد‬ ‫بالوقت‬ ‫تماما‬ ‫التزم‬
False positive
( ‫ال‬ ‫عن‬ ‫الناتج‬ )
Drying
)

9. ‫بالم‬ ‫العلمية‬ ‫المهن‬ ‫نقابة‬
‫ن‬
‫وفية‬ [9]
mmunochromatographic ssays
Immunochromatographic tests are rapid strip-based immunoassays whose physical principle relieson the migration of nanoparticles
along a membrane.
Basic concepts
▪ Polyclonal antibodies: They are a collection of immunoglobulin moleculesthat react against a specific antigen,each identifying a
different epitope.
▪ Monoclonal antibodies: Mono-specific antibodies that are the same because they are made by identical immune cells and have
monovalent affinity, in that they bind to the same epitope.
Test format
forantigen detection
Strip formats contain five main components: sample pad, conjugate pad, test line, control line and absorbing pad

10. ‫بالم‬ ‫العلمية‬ ‫المهن‬ ‫نقابة‬
‫ن‬
‫وفية‬ [10]
Assay Sample pad Conjugate pad Test line Control line
HCG
(Pregnancy test)
Urine or
serum
Anti-beta hCG monoclonal
antibody -gold colloid
Anti-alpha hCGmonoclonal
antibody
Anti-antibody detection
reagent (Anti- IgG antibody)
Principle
▪ Sample containing the Analyte (HCG) is applied to the sample application pad and it subsequently migrates to the other parts
of strip.
▪ At conjugate pad, target HCG is captured by the gold labeled analyte antibody conjugate and results in the formation of
labeled antibody conjugate/analyte complex.
▪ This complex moves under capillary action and at test line, this label complex is captured by another analyte antibody
forming labeled antibody conjugate/analyte/ immobilized antibody complex.
▪ Excess labeled antibody conjugate will be captured at control zone by anti- IgG antibody.
In case of a positive reaction, a red colored band is formed in the test region (due to the formation of antibody
conjugate/analyte/ immobilized antibody complex). The absence of a colored band in the test region (T) indicates a negative
result. A color band will always appear in the control region (C)
HBsAg Serum
Anti-HBsAg antibody -gold
colloid
Anti-HBsAg capture antibody Anti- IgG antibody
H. pylori Ag
Stool
Anti-H. pylori specific antibody
conjugated with colloidal gold
Anti-H. pylori capture
antibody
Anti- IgG antibody
Fecal occult
blood
Stool
Hemoglobin antibody
conjugated with colloidal gold
Immobilized anti-hemoglobin
antibody
Anti- IgG antibody

11. ‫بالم‬ ‫العلمية‬ ‫المهن‬ ‫نقابة‬
‫ن‬
‫وفية‬ [11]
forantibodydetection
Assay Sample pad Conjugate pad Test line Control line
HAV Serum
HAV antigen conjugated with
colloidal gold
with IgG conjugated to
colloidal gold
Anti-human IgG monoclonal
antibody
Anti-antibody detection
reagent (Anti-IgG antibodies)
Principle:
▪ The sample is applied to the sample well followed by the addition of an assay diluent.
The assay diluent facilitates the lateral flow of the released products as well as promoting the binding of antibodies and antigen.
▪ If there is a certain concentration of HAV antibody in sample, the antibody will combine with gold-labeled HAV antigen on the
detection card and form a gold-labeled antigen-antibody complex. The complex will then travels on the membrane due to
capillary action along with the rabbit IgG colloidal gold conjugate and react with the anti-human IgG monoclonal antibody on the
detection line (T-line). Gold-labeled antigen-antibody-second antibody complex will accumulate in the detection area and
indicates a red line.
▪ The rabbit IgG colloidal gold conjugate move further on the membrane and are subsequently immobilized by the anti-antibody
detection reagent coated on the membrane at the control region forming a pink colored band.
Toxoplasma
IgM
Serum
T. gondii antigens conjugated
with colloidal gold
Pre-coated with monoclonal
anti-human IgM
Anti-antibody detection
reagent
H. pylori Serum
H. pylori antigen conjugated
with colloidal gold
Immobilized anti-human IgG
Anti-antibody detection
reagent
HCV (right sign) Serum
Recombinant HCV antigen
conjugated colloid gold
co-dispensed with IgG
colloidal gold conjugate
The membrane is pre-coated
with recombinant HCV antigen
on the test line region
Anti-antibody detection
reagent (Anti-IgG antibodies)
Principle
▪ It utilizes the principle of lateral flow immunochromatography, a unique two site double antigen sandwich immunoassay on a
membrane. As the test specimen flow through the test device, the colored HCV specific recombinant antigen-colloidal gold
conjugate complexes with HCVantibodies in the sample.
▪ This complex moves furtheron the membrane to the test region ‘T’ where it is immobilized by the HCV specific recombinant
antigens coated on the membrane leading to formation of a colored band, which confirms a positive test result.
▪ The unreacted conjugate and unbound complex, if any,along with rabbit IgG gold conjugate move further on the membrane
and are subsequently immobilized by the goat anti-rabbit antibodies coated on the membrane at the control region ‘C’,
forming a colored band.

12. ‫بالم‬ ‫العلمية‬ ‫المهن‬ ‫نقابة‬
‫ن‬
‫وفية‬ [12]
forantibody detection
Laboratory example: HCV Tri dot
Test reagents
▪ Test dots: Contain highly purified HCV antigensfor Core (protease/helicase),NS3, NS4,NS5 in the form of two test dots “T1” &
“T2” to provide a highly sensitive and specific diagnostic test.
▪ Protein-A conjugate: it is a single polypeptide chain conjugated to gold nanoparticles.It has a high affinity binding sites for the
Fc region of IgG which enables binding to a wide spectrum of antibodiesand their subclasses from different species.
The protein A Conjugate should not be subjected to frequent temperature fluctuations
▪ Buffer solution: an aqueous solution consisting of a mixture of a weak acid and its conjugate base
Principle
▪ Sample and the reagents pass through the membrane and are absorbed into the underlying absorbent pad.
▪ As the patient's sample passes through the membrane, HCV antibodies if present in serum/plasma, bind to the immobilized
antigens. In the subsequent washing step, unbound serum/plasma proteins are removed.
▪ In the next step, the protein-A conjugate is added which binds to the Fc portion of the HCV antibodies to give distinct pinkish
purple dot against a white background at the test region (“T1”&/ or “T2”).
At the control region a “Built-in Quality Control Dot” hasbeen devised to confirm the proper functioning of the device,reagent
and correct procedural application.
Procedure
▪ Step No. 1: Add 3 drops of buffer Solution to the center of the device.
▪ Step No. 2 Hold the dropper vertically downwards and add 1 drop of patient'ssample (50 µl serum or
plasma) using the sample dropper provided. (Use a separate sample dropper foreach specimen to be
tested).
▪ Step No. 3: Add 5 drops of buffer Solution.
▪ Step No. 4 Add 2 drops of protein- A conjugate.
▪ Step No. 5: Add 5 drops of buffer Solution.
▪ Step No. 6: Read result immediately and discard the device considering it to be potentially infectious.
It is important to allow each solution to soak in the test device before adding the next solution.

13. ‫بالم‬ ‫العلمية‬ ‫المهن‬ ‫نقابة‬
‫ن‬
‫وفية‬ [13]
Advantage
Based on “Flow through technology”, which is similar to ELISA technology because of involvement of washing steps at various levels
to enhance the specificity.
for antigen detection
▪ Test format: Strip formats contain four main components
▪ Sample pad: Serum or urine
▪ Conjugate pad: Analyte-antibody-gold conjugate
▪ Test line: Analyte-antigen (same analyte to be detected)
▪ Control line: Anti-antibody detection reagent
▪ Principle
▪ When liquid sample reachesat the test line, pre-immobilized antigen will bind to the labeled conjugate if target analyte in
sample solution is absent.
Absence of color at test line is an indication for the presence of analyte while appearance of color both at test and controllines
indicates a negative result.
▪ Laboratory example: Such format suits best for low molecular weight compounds which cannot bind two antibodies
simultaneously such as drugs (toxicology screen).

14. ‫بالم‬ ‫العلمية‬ ‫المهن‬ ‫نقابة‬
‫ن‬
‫وفية‬ [14]
General precaution
▪ The test device should be stored at temperature (2 -30oC).
The test device is sensitive to humidity as well as heat so do not expose the kit to temperature over 30oC and do not freeze
▪ The test device must be used immediately after removing from the pouch
▪ Separate the serum or plasma from blood as soon as possible to avoid hemolysis
▪ Serum samples demonstrating gross lipemia, gross hemolysis or turbidity should not be used
▪ Refrigerated samples must be equilibrated to room temperature before testing to avoid invalid results
▪ Do not perform the test in a room with strong air flow,i.e. an electric fan or strong air-conditioning.
▪ In non-competitive assay, the intensity of the color in the test line region may vary depending on the concentration of analyte
present in the sample so any shade of color in the test line region should be considered positive
▪ Some tests will produce a faint positive test result if read after the instructed time. Most manufacturers suggest to read th eir
test after 3 minutesand to NOT READ it after 10-15 minutes (this is different from kit to kit). Any positive test appearing
after that time is inaccurate and cannot be considered positive (known as evaporation lines).
To avoid confusion, discard the test device after interpreting the result.
• On the other hand, the intinisty of color line is not a factor in competitive assay so a drug test is negative no matter how strong
or faint the line is

15. ‫بالم‬ ‫العلمية‬ ‫المهن‬ ‫نقابة‬
‫ن‬
‫وفية‬ [15]
Quantitative immunochromatographic analyzers
It is an immunochromatographic analyzer providing immediate point-of-care testing (POCT) at patient care settings or clinical
laboratories.
It is used for quantitative and qualitative determination of analytesby capturing and analyzing the image of test result for lateral flow
immunoassay.
The antigen concentration in the blood appears as the intensity on the test line on the test cassette

16. ‫بالم‬ ‫العلمية‬ ‫المهن‬ ‫نقابة‬
‫ن‬
‫وفية‬ [16]
Colloidal gold-based immunochromatographic assay
Specific contents are as follows: Two antibodiesbinding distinct epitopes present on the antigen molecule are used. One ( coating
antibody) labeled with a colloidal gold nanoparticles and the other(capture antibody) fixed onto surfaces of NC membrane. The
coating antibody is in a dehydrated state within the conjugate pad. When standard solution or sample were added onto the sample pad of
the test strip, the antibody formed a complex with the antigen in the liquid phase and moved forward continuously until it was captured
by the antibody fixed on the surfaces of NC membrane, which generated a signal in proportion about the antigen concentration.
A visible color appeared in less than 10 min, and the intensity determines the amount of the antigen. In other word, the
more antigen that was present in the sample, the more noticeable the red band appeared.
Colloidal gold is a colloidal suspension of nanoparticles of gold in a fluid, usually water. The colloid is usually either an
intense red color (for spherical particles lessthan 100 nm) or blue/purple (for larger spherical particles or nanorods)

17. ‫بالم‬ ‫العلمية‬ ‫المهن‬ ‫نقابة‬
‫ن‬
‫وفية‬ [17]
Quantitative test can be realized by using a reflectance photometry.For reflectance measurements,the photometer
quantitatively compares the fraction of light that reflectsfrom the reference and test samples.
Fluorescence labeled immunochromatography
It is an improved immunochromatography assay using detection antibodies conjugated with fluorescent latex beads for specific
antigens. The fluorescent signal was detected by the specific fluorescent reader.
The limit of detection is significantly improved over that of conventional Immunochromatography methods