Transcript Agglutination Tests

LAB 1
Immunology Laboratory Equipment
1- Centrifuge
• Is equipment used to separate substances of greater and lesser density.
• Simple centrifuges are used in chemistry, biology, and biochemistry for
isolating and separating suspensions. They vary widely in speed and
capacity. They usually comprise a rotor containing two, four, six, or many
more numbered wells within which the samples, contained in centrifuge
tubes, may be placed.
Microcentrifuge tube
Microcentrifuge tubes are small, cylindrical plastic containers with conical
bottoms, typically with an integral snap cap. They are used in molecular biology
and biochemistry to store and centrifuge small amounts of liquid.
2- Incubator
•
•
In biology, an incubator is a device used to
grow and maintain of course
microbiological cultures or cell cultures.
The incubator maintains optimal
temperature, humidity and other
conditions such as the carbon dioxide
(CO2) and oxygen content of the
atmosphere inside. Incubators are
essential for a lot of experimental work in
cell biology, microbiology and molecular
biology and are used to culture both
bacterial as well as eukaryotic cells.
Most incubators include a timer; some can
also be programmed to cycle through
different temperatures, humidity levels,
etc. Incubators can vary in size from
tabletop to units the size of small rooms.
3- Spectrophotometer
• Is a photometer (a devise for measuring light intensity)
that can measure intensity as a function of the color, or
more specifically, the wavelength of light.
4- Micro plate reader
(ELISA plate reader)
• Microplate Readers (also known
as plate readers) are laboratory
instruments designed to detect
biological, chemical or physical
events of samples in microtiter
plate. Sample reactions can be
assayed in well format microtiter
plates. The most common
microplate format used in
academic research laboratories or
clinical diagnostic laboratories is
96-well. In most cases, a highintensity lamp passes light to the
microtiter well and detector
quantifies the light emitted by the
reaction happening in the
microplate well.
Micro plate detection uses:
• ELISAs (Enzyme-linked immunosorbent assay, a biochemical
technique used mainly in immunology to detect antibodies or
antigens).
• Protein and cell growth assays
• Nucleic acid quantitation
• Molecular interactions
• Enzyme activity
• Cell toxicity, proliferation, and viability
• ATP quantification
• Immunoassays
• High throughput screening of compounds and targets in drug
discovery such as FLIPR assays.
5- Microtiter plate (ELISA plate -8x12) and ELISA pipette
• Is a flat plate with
multiple ‘’wells’’ used as
small test tubes. The
microplate has become a
standard tool in analytical
research and clinical
diagnostic testing
laboratories. A very
common usage is in the
enzyme-linked
immunosorbent assay
(ELISA).
6- Autoclave
• An autoclave is a pressurized device designed
to heat aqueous solutions above their boiling
point to achieve sterilization.
7- pipette
• Also called a pipet, pipettor or medicine dropper.
• Is a laboratory instrument used to transport a
measured volume of liquid.
8- Homogenizer
•
A homogenizer is a piece of laboratory equipment used for the homogenization of
various types of material, such as tissue, plant, food, soil, and many others.
Homogenization is a very common sample preparation step prior to the analysis of
nucleic acids, proteins, cells, metabolism, pathogens, and many other targets.
 For small cell culture laboratories, a domestic
refrigerator (preferably one without an autodefrost
freezer) is an adequate and inexpensive piece of
equipment for storing reagents and media at 2–8°C.
 For larger laboratories, a cold room restricted to cell
culture is more appropriate. Make sure that the
refrigerator or the cold room is cleaned regularly to
avoid contamination.
 Most cell culture reagents can be stored at –5°C
to –20°C; therefore an ultradeep freezer (i.e., a –
80°C freezer) is optional for storing most
reagents.
 While most reagents can withstand temperature
oscillations in an autodefrost (i.e., self-thawing)
freezer, some reagents such as antibiotics and
enzymes should be stored in a freezer that does
not autodefrost.
• is an electronic instrument used for measuring the pH
(acidity or alkalinity) of a liquid
• . A typical pH meter consists of a special measuring probe (a
glass electrode) connected to an electronic meter that
measures and displays the pH reading.
Leukocyte Total and Differential
Count
Lab 2
WBC TOTAL COUNT
Can be determined:
•manually by haemocytometer and
microscope
•automated by haematological analysers
•by estimation from a blood smear
17
-Leukocyte estimation in a blood smear
(400x):
Normal number
Increased
18
Decreased
DIFFERENTIAL LEUKOCYTE COUNT
Can be determined:
•from a stained blood smear
•automated by some haematological analysers,
but these need prior validation
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DIFFERENTIAL COUNT IN A STAINED BLOOD
SMEAR: BATTLESHIP METHOD
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How to perfom a differential count:
• Count 100-200 leukocytes, note each
cellular type identified
• Avoid counting the same field twice
• Do not focus on one section of the smear
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TYPES OF LEUKOCYTES
- GRANULOCYTES:
Neutrophils, Eosinophils, Basophils
- MONOCYTES
- LYMPHOCYTES
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Normal neutrophils and a lymphocyte
Neutrophils are similar in most domestic species, characterised by pale cytoplasm and dark nucleus
segmented into 2-5 lobes, although granules are more
23 readily seen in ruminants.
Small lymphocytes, dog
Small and medium lymphocyte, cow
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Normal neutrophil and monocyte
Monocytes are similar in most species; large cells with basophilic cytoplasm
(often vacuolated or with tiny pink granules), and a pale irregularly clefted
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or folded nucleus with clumped chromatin.
Normal eosinophil and basophil
Morphology of eosinophils varies widely with species; granules are large
in horses, small & numerous in ruminants, accompanied by vacuoles in
dogs, and rod-shaped in cats.
Basophils have purple-black granules, are numerous in large animals and
may be almost absent in healthy dogs and
cats.
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Normal WBCs in different species, some examples
(1)
(3)
(2)Eosinophil
Eosinophil and neutrophil
Basophil
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(4)
Eosinophil and neutrophils
REPORTING A DIFFERENTIAL COUNT
By relative % of each WBC type
(INCORRECT)
Ways to
report the
values
By absolute number of each WBC type
(CORRECT)
(%of each cell type x total WBC number)
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LAB 3
Serological test
Serological test
(Antigen antibody interactions)
• Serology is a blood test to detect the presence of
antibodies against a microorganism (antigen).
Certain microorganisms stimulate the body to
produce antibodies during an active infection.
• Blood is drawn from a vein (venipuncture), usually
from the inside of the elbow or the back of the hand.
A needle is inserted into the vein, and the blood is
collected in an air-tight vial or a syringe. Preparation
may vary depending on the specific test.
There are several serology techniques that can be used
depending on the suspected antibodies. Serology techniques
include
• Primary serological tests: (Marker techniques) e.g.
– Enzyme linked immuono sorben assay (ELISA)
– Immuno flurescent antibody technique (IFAT)
– Radio immuno assay (RIA)
• Secondary serological tests: e.g.
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–
–
–
–
Agglutination tests
Complement fixation tests (CFT)
Precipitation tests
Serum neutralization tests (SNT)
Toxin-antitoxin test
• Tertiary serological test: e.g.
– Determination of the protective value of an anti serum in an animal.
Agglutination tests

Agglutination is visible clumping of particles, cells and
bacteria by an antigen combining with its specific
antibody. Such antibodies called agglutinating
antibodies and the reaction is called agglutination. The
resulting clumps are referred to as agglutinates.
There are three types of Agglutination
tests:
First: Direct whole pathogen Agglutination:
When the antigen is particulate, the reaction of an antibody with the
antigen can be detected by agglutination (clumping) of the
antigen. The general term agglutinin is used to describe
antibodies that agglutinate particulate antigens. When the
antigen is an erythrocyte the term heamagglutination is used.
1.
To assess bacterial infections:
For antibody detect, bacterial agglutination tests can be performed on the surface
of glass slides or in tubes. Tube agglutination tests are often more sensitive,
since a long incubation period (allowing more antigen and antibodies to
interact) can be used. The small volume of liquid used for slide tests requires
rather rapid reading of the result before the liquid has evaporated.
e.g. A rise in titer of an antibody to a particular bacterium indicates an infection
with that bacterial type.
2.
Determination of blood types or antibodies to blood group antigens + Rh:
A blood group is a classification of blood based on the presence or absence
of inherited antigenic substances on the surface of red blood cells (RBCs).
These antigens may be proteins, carbohydrates, glycoproteins, or glycolipids,
depending on the blood group system.
- ABO test
ABO test
The ABO test shows that people have one of four blood types:
A, B, AB, or O. If your red blood cells have:
• The A antigen, you have type A blood. The liquid portion of
your blood (plasma) has antibodies that fight against type B
blood.
• The B antigen, you have type B blood. Your plasma has
antibodies that fight against type A blood.
• Neither the A nor B antigen, you have type O blood. Your
plasma has antibodies that fight against both type A and
type B blood.
• Both the A and B antigens, you have type AB blood. Your
plasma does not have antibodies against type A or type B
blood.
How to read your results
No Agglutination
-ve result
Agglutination
+ve result
Second: Particle ( indirect ) Agglutination Tests:
• These tests based on the detection of antibody via the agglutination of
an artificial carrier particle with antigen bound to its surface.
• The carrier particle may be latex, treated erythrocytes and
staphylococcal particle agglutination system (coagulation).
• When the carrier is erythrocyte the name of the test will be indirect or
Passive Hemagglutination test:
it is possible to coat erythrocytes with a soluble antigen (e.g. viral antigen,
a polysaccharide or a hapten; A hapten is a small molecule that can elicit
an immune response only when attached to a large carrier such as
a protein) and use the coated red blood cells in an agglutination test for
antibody to the soluble antigen. This is called passive hemagglutination.
•
•
Antibody detection to Cytomegalovirus, Rubella virus, Varicella-zoster virus and
others.
The treponema pallidum particle agglutination assay (also called TPPA test) is an
indirect agglutination assay used for detection and titration of antibodies against
the causative agent of syphilis.
LAB 4
Serological test (cont.)
Third : Coombs Test (Antiglobulin Test)
• This test used to detect antibodies which produced by the
body itself and analyze the red blood cells. This condition
happens in autoimmune hemolytic anemia or in hemolytic
disease of new born (RH disease).
• There are two types of coombs test:
1. The direct coombs test.
2. The indirect coombs test.
The direct coombs test
•
•
•
Also known as the direct antiglobulin test or DAT.
The Direct Coombs test is used
in vivo to test for
autoimmune hemolytic anemia; ie, a condition of a low count
of red blood cells (aka RBCs) caused by immune system lysis or
breaking of RBC membranes causing RBC destruction.
In certain diseases or conditions an individual’s blood may contain
IgG antibodies that can specifically bind to antigens on the red
blood cell surface membrane, and their circulating red blood cell
can become coated with IgG autoantibodies. The direct Coombs
test is used to detect these antibodies that are bound to the
surface of red blood cells; a blood sample is taken and the RBCs
are washed (removing the patient’s own plasma) and then
incubated with antihuman globulin (also known as “Coombs
reagent”. If this produces agglitination of RBCs, the direct Coombs
test is positive, a visual indication that antibodies are bound to the
surface of red blood cells.
http://en.wikipedia.org/wiki/File:Coombs_test_schematic.png
The indirect coombs test.
•
•
•
Also known as the indirect antiglobulin test or IAT. Is used in
prenatal testing of pregnant women and in testing blood prior
to a blood transfusion (to detect in-vitro antibody-antigen
reactions).
It is used to detect very low concentrations of antibodies
against RBCs that are present unbound in the patient's
plasma/serum prior to a blood transfusion. In antenatal care,
the IAT is used to screen pregnant women for antibodies that
may cause hemolytic disease of the newborn. In this case,
serum is extracted from the blood, and the serum is incubated
with RBCs of known antigenicity. Then, the coombs reagent is
added. If agglutination occurs, the indirect Coombs test is
positive.
The IAT can also be used for compatibility testing, antibody
identification, RBC phenotyping, and titration studies.
Hemagglutination Inhibition test
• The agglutination test can be modified to be used for the measurement of
soluble antigens. This test is called hemagglutination inhibition. It is called
hemagglutination inhibition because one measures the ability of soluble
antigen to inhibit the agglutination of antigen-coated red blood cells by
antibodies. In this test, a fixed amount of antibodies to the antigen in
question is mixed with a fixed amount of red blood cells coated with the
antigen. Also included in the mixture are different amounts of the sample
to be analyzed for the presence of the antigen.
 If the sample contains the antigen, the soluble antigen will compete
with the antigen coated on the red blood cells for binding to the
antibodies, thereby inhibiting the agglutination of the red blood
cells.
Hemagglutination inhibition test
Many human viruses have the ability to
bind to surface structure on red blood cells
from different species and cause
agglutination. Patients sera are added to a
system that contains the virus suspected of
causing disease. If antibodies to the virus
are present they will form complexes and
block the binding sites on the viral
surfaces. When the proper red blood cells
are added to the solution, all of the virus
particles which are bounded with
antibodies will prevent the virus from
agglutinate the red blood cells. Thus the
patient serum is positive for
hemagglutination inhibition test
• How to Test for the
Inhibition of Latex
Agglutination From human
chorionic
gonadotrophin(HCG)=>http:
//www.ehow.co.uk/how_85
76499_test-inhibition-latexagglutination-hcg.html
• HCG test (pregnancy test) :
to detect human chorionic
gonadotrophin hormone.
LAB 5
Flow cytometry
What is flow Cytometry?
Process for quantifying cells
•Measures different property of cells
•Able to categorize and quantify
•Even able to separate out
subpopulations of cells
What is Flow Cytometry?
• Flow ~ cells in motion, Cyto ~ cell , Metry ~
measure, Measuring properties of cells while in a
fluid stream.
• Flow cytometry (abbreviated: FCM) is a technique
for counting and examining microscopic particles,
such as cells and chromosomes, by suspending
them in a stream of fluid and passing them by an
electronic detection apparatus.
• It allows simultaneous multiparametric analysis
of the physical and/or chemical characteristics of
up to thousands of particles per second.
How it works
1. Draw cells, with excess fluid, from test tube into machine.
2. Cells pass in single file past laser.
3. Laser hits cell and light is scattered.
4. Photomultiplier multiplies light intensity and a light sensor measures the
amount of light and scatter pattern.
5. Based on cell characteristics (size and shape), the computer categorizes
and quantifies the cells.
Flow-Cytometer
Image Source: http://www.mybloodyourblood.org/biology_white.htm
Neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils
White Blood Cells, Platelets
(stained purple), a T-Lymphocyte
white cell (stained green), and a
Monocyte white cell (stained
gold) as seen through a scanning
electron microscope. ©2000
Dennis Kunkel, Ph.D.
Uses of Flow cytometry
• Flow cytometry is routinely used in the diagnosis of health
disorders, especially blood cancers.
• The technology has applications in a number of fields,
including molecular biology, pathology, immunology, plant
biology and marine biology.
• It has broad application in medicine (especially in
transplantation, hematology, tumor immunology and
chemotherapy, genetics and sperm sorting for sex
preselection).
• In protein engineering, flow cytometry is used in
conjunction with yeast display and bacterial display to
identify cell surface-displayed protein variants with desired
properties.
How Is It Done:
http://biology.berkeley.edu/crl/flow_cytometry_basic.html
Flow Cytometry Data Analysis
• The data generated by flow-cytometers can be
plotted in a single dimension, to produce a
histogram, or in two-dimensional dot plots or even
in three dimensions.
• The regions on these plots can be sequentially
separated, based on fluorescence intensity, by
creating a series of subset extractions, termed
"gates." Specific gating protocols exist for diagnostic
and clinical purposes especially in relation to
hematology.
• Data accumulated using the flow cytometer can be
analyzed using software, e.g., WinMDI(depricated),
Flowjo, or CellQuest Pro.
Fluorescence And Antibodies
To The Rescue
histogram
gating
plots
Contour Plot
Greyscale Density
Density Plot
Dot Plot
LAB 6
Allergy tests
Allergy
• Allergy is an appropriate and harmful immune
response to a normally harmless substance. It is
usually caused by proteins called Allergen, so we
can define allergen: the substance which causes
allergy and it is usually proteins.
• Types of allergens:
– House dust mites, fungi, pollen, animal dander and
saliva, food, insects, drugs, chemicals and latex.
– They may be indoors or outdoors.
– They enter the body through skin, digestive and
respiratory systems.
Mechanism of allergic reaction
• When an allergen gains entry the body, it is
recognized as foreign by the immune system.
The allergen stimulates B cells to proliferate
and produce specific IgE antibodies. The
antibodies bind to surface receptors of mast
cells, which are found in most tissues. On
subsequent exposure, the allergen react with
antibodies leading to lysis of the mast cell. This
causes the release of histamine and other
substances which cause immediate symptoms
of allergy.
Allergic diseases and symptoms
1.
2.
3.
4.
5.
Respiratory diseases:
A.
Asthma: wheezing ‫صعوبة التنفس‬, coughing and hard breathing.
B.
Allergic rhinitis ‫التهاب مخاطية األنف‬: sneezing ‫عطس‬, rhinorrhoea
‫إفرازات أنفية‬, nasal blockage and itching in nose, mouth, ears and
eyes.
C.
Sinusitis ‫التهاب الجيوب االنفية‬: face and teeth pain, headache, nasal
discharge and blockage.
D.
Nasal polyps: nasal discharge and blockage, headache, hard
breathing, snorting ‫ شخر‬and decrease the sense of smell.
Dermatological diseases:
A.
Urticaria: redness and itching.
B.
Eczema: skin rash, redness and itching.
C.
Angioedema: swelling nose, eyes, tongue, neck, throat and lips.
Allergic conjunctivitis: redness of eyes, itching, prickling and eye teary
eyes.
Gastroenteropathy: nausea, vomiting, diarrhea and abdomen or
stomach pain.
Anaphylactic shock: severe whole body allergic reaction can result in
death (hypotension, trachea spasm, wheezing, hard breathing, fainting
spell fast heart beating, sneezing, abdomen pain, urticaria,
angioedema, diarrhea, nausea, vomiting and other symptoms). It can
be treated with immediate adrenaline injection.
Allergy diagnosis
1. Patient history.
2. Physical examination.
3. Laboratory tests (in vivo – in vitro): involves
having a skin (in vivo) or blood (in vitro) test to
find out what substance, or allergen, may trigger
an allergic response in a person. Skin tests are
usually done because they are rapid, reliable,
and generally less expensive than blood tests,
but either type of test may be used.
Skin tests
A small amount of a suspected allergen is placed on or below the skin
to see if a reaction develops.
There are three types of skin tests:
1. Skin prick test. This test is done by placing a drop of a solution
containing a possible allergen on the skin, and a series of
scratches or needle pricks allows the solution to enter the skin. If
the skin develops a red, raised itchy area (called a wheal), it
usually means that the person is allergic to that allergen. This is
called a positive reaction.
2. Intradermal test. During this test, a small amount of the allergen
solution is injected into the skin. An intradermal allergy test may
be done when a substance does not cause a reaction in the skin
prick test but is still suspected as an allergen for that person. The
intradermal test is more sensitive than the skin prick test but is
more often positive in people who do not have symptoms to that
allergen (false-positive test results).
3. Skin patch test. For a skin patch test, the allergen solution is
placed on a pad that is taped to the skin for 24 to 72 hours. This
test is used to detect a skin allergy called contact dermatitis.
Skin prick test
Skin patch test
Blood test
• Allergy blood tests look for substances in the blood called
antibodies. Blood tests are not as sensitive as skin tests but
are often used for people who are not able to have skin
tests.
• The most common type of blood test used is the enzymelinked immunosorbent assay (ELISA, EIA). It measures the
blood level of a type of antibody (called immunoglobulin E,
or IgE) that the body may make in response to certain
allergens. IgE levels are often higher in people who have
allergies or asthma.
• Other lab testing methods, such as radioallergosorbent
testing (RAST). In this test the serum to be tested for
specific IgE is added to a solid phase allergen
immunosorbent. After wash, this is incubated with
radiolabelled purified or monoclonal anti IgE. The amount
of radioactivity reflects the quantity of allergen specific
antibodies in the patient serum.
radioallergosorbent testing (RAST)
Advantages of allergy blood tests include:
• Can be done at any time, regardless of any medications
you are taking.
• Requires only one needle stick (unlike skin testing). This
may be more attractive for people who are afraid of
needles. Allergy blood testing is the preferred test for
infants and very young children.
Disadvantages of allergy blood tests include:
• More expensive than skin testing. Many health insurers
do not cover allergy blood tests.
• May be less sensitive than skin tests.
• Takes days or weeks to get results because the blood
sample must be sent to a laboratory for evaluation.
Skin testing provides immediate results.
LAB 7
ELISA
Enzyme Linked Immunosorbent Assay
(ELISA)
ELISA
• Enzyme Linked Immunosorbent Assay (ELISA)
• Term Was Coined By Engvall and Pearlmann in 1971
• Different Types
– Sandwich
– Indirect
– Competitive
•
•
•
•
Similar To RIA, Except No Radiolabel
Can Be Used To Detect Both Antibody and Antigen
Very Sensitive, pg/mL
Relies on Monoclonal Abs
Sandwich ELISA
•
•
•
•
•
•
2 Antibodies Required
Must Recognize Different Epitopes
1st Antibody Is Referred To As Capture Ab
2nd Antibody Detection Ab
2nd Antibody Is Biotinylated
Enzymes Commonly Used: HRP (Horse Radish
Peroxidase) And AKP (Alkaline Phosphatase)
• Substrate is TMB (Chromogen)
ELISA Plate
• 96 well plate
• Made of plastic on which protein
can be adsorbed (bind) easily
• Usually done overnight @ 4C
• Special buffer used that will not
denature Ab and maximize binding
• Blocking step ensures no empty
spaces are left
• Blocking reagent is often 10% FBS
Sensitivity Of Elisa
• Typically the lowest cytokine concentration
that can be detected above negative control
• 2-3 S.D Above Mean Background Signal
• Depending On Antibody Pair Used Sensitivity
Varies
• Ex. 10 pg/mL
General Protocol
• Dilute capture Ab @ 1-4 g/mL In Binding Solution
• Ex. Stock Solution Of Capture Ab: 0.5 mg/mL And Capture Ab Recommended
Conc. 2 g/mL
• First Question To Ask Yourself ?
– How much volume would I use?
– Count 16 wells for S.C+
– 3 wells for Negative Controls
– Your Samples (usually in triplicates)
– Add them up and multiply by 100 L (typical volume used per well)
• Let’s Say 4 mL Needed
– You will need 16 L of capture Ab
• Add capture Antibody, Seal plate (minimize evaporation)
• Incubate overnight at 4C
Binding Solution
• Pharmingen Recommended Reagent
• 0.1 M Na HPO4, adjust to pH 9.0 or to pH
6.0 with 0.1 M NaH2PO4
• pH Is Very Important, If Wrong No Binding
• Some Antibodies Require pH 6.0
– Ex. Antibodies for mIL-10, mMCP-1, mTNF,
rGM-CSF).
Blocking
•
•
•
•
•
•
•
Blocking Reagent 10% FBS in PBS
Alternatively 1% BSA (Immunoassay Grade)
Filter To Remove Particulates
Plate Is Brought To R.T
Add 200 L per well Blocking Buffer
Wait For 2 Hours At R.T
Why Do We Block?
After Blocking
• Wash x3 With PBS/Tween (detergent)
• Add Standards + Samples
• Samples Are Typically Supernatants From
Cultures Or Patient Serum/Plasma
• Use 100 L
• Often Dilution Is Required If Signal Is Too
Strong
• Standards?
Standard Preparation
• Standards Are Diluted in Blocking
Buffer/Tween
• Start By Labeling eight, 1 mL Eppendorf Tubes
• Prepare Highest Conc. Tube (1 mL)
• Fill The Remaining Tubes with 0.5 mL Blocking
Buffer
• Serially Dilute From Top To Lowest
Assume You Have A Stock Tube @ 2ng/L, Volume 5 L
Usually Remaining Standard Cytokine Is Thrown Away
Thawing-Unthawing Affects Cytokine
After Standard Preparation
• Add Samples, Standards, Negative Control
– Negative Control Should Be The Buffer You Use
Dilute Standard or Culture Medium
• Incubate For 2 Hrs at R.T
• Aspirate And Wash 5x
Addition Of Detection Ab
•
•
•
•
Avidin is a Hen Oviduct Protein
Avidin has very high affinity for biotin (B vitamin)
B vitamin is conjugated on the detection Ab
Add Working Detector @ 100 L/well
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Ex. Stock Detection Antibody=0.5mg/mL
You need to prepare 5 mL @ 1  g/mL
Use 10  L of Stock Antibody
Add 5 L of Enzyme (Avidin-HRP)
Dilution is 1:1000
• Incubate for 60 mins @ R.T
• Wash 6x
Addition Substrate
• Prepare Substrate by Mixing 1:1 volume
• Add 100 L/well
• Incubate for 10 mins, Avoid Formation of
Excessively Bright Color (Spec will not be able
to read)
• Terminate Reaction by Adding 0.5 M H2SO4
(color changes from blue to yellow)
• Read Plate At Appropriate Wavelength (=450
nm)
LAB 8
Tissue Typing
Everyone has several antigens
located on the surface of his/her
leukocytes:
• One particular group of these antigens
is called the HLA (Human Leukocyte
Antigens).
The HLA
Is responsible for stimulating the immune
response to recognize tissue as self versus
non-self.
Is controlled by a set of genes located next
to each other on chromosome 6 called the
Major Histocompatibility Complex (MHC).
• The test that determines which HLA
antigens are present is called tissue
typing or HLA typing.
–Tissue typing identifies the similarity of
the antigens present in both the donor
and the recipient.
• The closer the HLA antigens on the transplanted
organ match the recipient, the more likely that
the recipient’s body will not reject the transplant.
• For this reason, tissue typing of the kidney donor
and recipient is necessary before a kidney
transplantation.
There are two main classes of HLA antigens:
• Class I (HLA-A, HLA-B, and HLA-Cw)
• Class II (HLA-DR, HLA-DQ, and HLA-DP)
• Every person inherits each of the
following antigens from each parent:
– HLA-A antigen
– HLA-B antigen
– HLA-Cw antigen
– HLA-DR antigen
– HLA-DQ antigen and
– HLA-DP antigen
• The set of HLA antigens received
from a parent is called a haplotype.
–There are a variety of alleles for each
of these HLA antigens.
–The large number of possible variations
and combinations of HLA antigens
make finding a match in a family more
likely than finding a match in the
general public.
• When performing an HLA typing test for a
kidney transplant, the following HLA
antigens are looked at:
– HLA-A
– HLA-B
– HLA-DR
• The MHC genes are the most polymorphic
known.
– There are hundreds of known alleles for each HLA
Antigen.
• Each allele is identified by a number (i.e. HLA-A1 or HLA-A2).
• Six HLA antigens are looked at for each person.
– Remember each person has two of each of the
antigens (one inherited from the mother and one
inherited from the father).
• By analyzing which six of these HLA-antigens
both the donor and recipient have, scientists
are able to determine the closeness of tissue
matching.
– A six-antigen match is the best compatibility
between a donor and recipient.
• This match occurs 25% of the time between siblings ‫االشقاء‬who have the
same mother and father.
HLA Typing Techniques
• Traditionally, HLA typing was done using serological
techniques:
– Blood from the patient was mixed with serum containing
known antibodies to determine which antigens were
present.
• HLA typing now is predominantly done using
molecular techniques:
– Patient’s DNA is isolated.
– PCR is used to amplify specific HLA genes.
– Genes are sequenced to determine which alleles are
present.
• Once the donor and recipient have been
tested for tissue compatibility, the next step is
an Antibody Screening (also called a Panel
Reactive Antibody or PRA).
– A small amount of the organ recipient’s serum is
mixed with cells from 60 different individuals
(each test is done separately).
Purpose of Antibody Screening
• Scientists can determine how many different
HLA antibodies a patient has in his/her
blood.
– If a patient reacts with 30/60 cells, he/she is said
to have 50 Percent Reactive Antibody (also
known as PRA).
– The lower a person’s PRA, the less likely he/she is
to reject a transplant.
Crossmatch Test
• After tissue typing and antibody screening are
complete and a potential donor has been
identified, the final test is called a crossmatch
test.
– Crossmatch Test: A small amount of the potential
donor’s white cells is mixed with a small amount of
the recipient’s serum.
• By exposing the donor’s HLA to the recipient’s serum, scientists can
determine if the recipient has antibodies to any of the donor’s HLA.
• Positive Crossmatch: A reaction between the
donor’s and recipient’s samples occurs.
– Indicates that the recipient’s body will likely reject
the implanted kidney.
– Indicates the transplant cannot be performed.
• Negative Crossmatch: No reaction between the
donor’s and recipient’s samples occurs.
– Indicates that the recipient’s body will most likely
not reject the implanted kidney.
– Indicates the transplant can be performed.