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Exp.2  Measurement of Antibody
Antigen (Ag) and antibody (Ab)
meet and bind specifically. Under certain circumstances some reaction
phenomenon would occur, such as agglutination and precipitation. The principle
could be used to monitor the unknown antibody (or antigen) by known antigen (or
antibody). This is called serological reaction, for the antibody in experiment
is always in serum.












































Results


If RBC agglutination occurred,
the mixed fluid turned from turbid to transparent gradually and red
agglutination clot appeared. If the mixed fluid keeps turbid, then no agglutination
occurs. Blood type could be determined.


Table 2-1  Blood
Type Result



  
  
            Blood type  
  
Serum
  
  
  
A
  
  
  
B
  
  
  
AB
  
  
  
O
  


  
  
Anti-A
  serum
  
  
  
+
  
  
  
-
  
  
  
+
  
  
  
-
  


  
  
Anti-B
  serum
  
  
  
-
  
  
  
+
  
  
  
+
  
  
  
-
  




Attentions


1.Mark the two side of the
slide with A and B. Keep the two serum fluid separated.


2.Disinfect finger before
taking blood. Do not stab finger before the finger is dry in case ethanol would
destroy RBC.


3.Do not confuse the two side
of wooden stick when mixing.


4.Observe result in time.


5.Before discard the blood
stained material, put it in certain places and disinfect strictly to avoid
disseminating disease. For example, Slide should be put in 84 disinfect fluid.
Stick, needle and tampon should be disinfected under high pressure.





Tube Agglutination Test




Principle


Dilute the examinee’s serum
consecutively. Mix it with antigen suspension of known concentration in test
tube. Observe the agglutination phenomenon in certain reaction time.


In this experiment, typhoid
bacterium of certain quantity acts as antigen. Whether serum contains
corresponding antibody or not is up to the agglutination reaction to decide.
Furthermore, we could decide the titer of antibody by the agglutination extent
in different tubes. We could diagnose and tell patient condition and prognosis
by this principle.


Materials


1.Inactivated typhoid
bacteria (7´10⁸/ml)


2.Animal serum to be examined (Immunized by typhoid
bacteria ‘H’ antigen)


3.Normal saline(NS)


Methods


1.Take 6 test tubes. Mark them and put into rack in
turn.


2.Add NS 0.9ml into No.1 test tube. Add 0.5ml NS into
other tubes. Then add typhoid immunized serum 0.1 ml to No.1 tube and mix it
with NS evenly. Draw 0.5 ml mixed fluid from No.1 tube to No. 2 tube. Mix the
fluid evenly in No.2 tube and draw 0.5 ml to No.3 tube. Also in such way the
serum is diluted in other tubes. When it turns to No. 5 tube, after the
mixation of fluid, draw 0.5 ml and discard it. So the No. 6 tube contains no
typhoid immunized serum and acts as the negative control tube in this
experiment. At last, add 0.5 ml typhoid bacteria fluid in each tube. The table
below indicates the whole process.


Table
2-2  Adding sample table of tube
agglutinatin test



  
  
Tube
  number
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  


  
  
Normal
  saline
  
  
  

  
    
    
    
    
    
    
    
    
    
    
    
    
  
  
  
    
    
  
  
  
  0.9
  
  
  

  
  0.5
  
  
  

  
  0.5
  
  
  

  
  0.5
  
  
  

  
  0.5
  
  
  
0.5
  


  
  
Typhoid
  immunized serum
  
  
  
0.1
  
  
  
0.5
  
  
  
0.5
  
  
  
0.5
  
  
  
0.5
  
  
  
Discard
  0.5
  


  
  
Typhoid
  bacteria suspension
  
  
  
0.5
  
  
  
0.5
  
  
  
0.5
  
  
  
0.5
  
  
  
0.5
  
  
  
0.5
  


  
  
Final
  serum  dilution
  
  
  
1∶20
  
  
  
1∶40
  
  
  
1∶80
  
  
  
1∶160
  
  
  
1∶320
  
  
  
control
  




3.shake
rack gently to mix the fluid in test tubes. Put rack in 56℃
water bathe for 1~2
hour.


Results


1.Control tube (No.6 tube):
Supernatant is turbid. Precipitated bacteria lie in the tube bottom. Shake
gently and bacteria disperse into evenly turbid fluid.


2.Experiment tube: Observe
result in turn from tube 1 to tube 5. Positive result tube bottom has irregular
and loose agglutination clot. Negative result tube has same result with control
tube.


3.Classification of agglutination
extent.


++++：Fluid is clear and transparent. Bacteria agglutinate
completely to the tube bottom. Shake gently and large agglutination clot could
be seen.


+++：Fluid is slightly turbid. Bacteria agglutinate
mostly to the tube bottom. Shake gently and comparatively smaller agglutination
clot could be seen.


++：Fluid is obviously turbid. Shake gently and
agglutination clot could be seen obviously. Agglutination clot is smaller.


+：Fluid is turbid. Observed carefully, small
agglutination particle could be seen.


-:  Same as control tube.


4. Judgement of
agglutination titer.


The titer is customarily
reported as the reciprocal of the highest dilution that causes an obvious
agglutination (++).


Attentions


1.Mark the tubes in turn.
Pipette of different reagent should not be mixed up.


2.Be sure to mix evenly when
dilute the antiserum and then add to next tube.


3.Add reagent accurately.
Avoid air bubble.


4.Shake gently after all
reagents are added and then have water bathe.


5.During water bathe, keep
the tubes from hot water dropping into them.


6.After experiment, take out
the tubes gently, in case agglutination would disperse and result is hard to
observe.


7.Observe the control tube
to decide the experiment credibility. Then observe the experiment tubes.


II Indirect Agglutination Reaction


The reaction of particle
antigen and corresponding antibody could be determined qualitatively and
quantitatively. While the reaction of soluble antigen and corresponding
antibody could not yield visible direct agglutination phenomenon. According to
the principle of direct agglutination test, we could adhere soluble antigen to
certain particle object, carrier, to make it to be an artificial particle
antigen. And then combine the artificial particle antigen with corresponding
antigen to observe indirect agglutination phenomenon.


Indirect agglutination
reaction refers to adhere of soluble antigen(or antibody) to particle object
(carrier) that has nothing to do with immune and has certain size, and then
react with corresponding antibody (or antigen) to yield agglutination reaction.
The common carriers include RBC, polystyrene latex particle, SPA etc. According
to the carrier, indirect agglutination reaction could be classified into
different types.



Precipitation Reaction




Precipitation reaction
refers to the reaction of soluble antigen and corresponding antibody and yield
precipitation line or circle. Common precipitation reactions include circle
precipitation reaction, floccule precipitation reaction, agar diffusion and
immune electrophoresis, etc.


Agar diffusion test refers to soluble antigen and
antibody diffuse in agar and yield white precipitation line or circle, given that
they correspond to each other and with appropriate proportion. Agar is an amylose of large molecule. Agar could be
melt at the temperature above 100℃ and concrete under 45℃
to form reticulation formation, which allows antigen and antibody diffuse
freely inside it. Agar diffusion test could be divided into single diffusion
and double diffusion. The single diffusion refers to only diffusion of antigen
or antibody, so it could be used as quantitative monitor. Double diffusion
refers to diffusion of both antigen and antibody, so it could be used as
qualitative analysis for antigen or antibody.






ⅠSingle agar diffusion test




Principle


Single agar diffusion test is quantitative test.
Usually it is used to measure unknown antigen by known antibody. Mix antibody
of certain quantity with agar and layer on slide to make an agar plate contains
antibody. Cut wells after agar concretion. Then add unknown antigen into wells.
Antibody concentration is even for antibody and agar mixed before concretion.
So antigen diffuses outward from wells. The farther from the well, the lower is
the antigen concentration. So white precipitation circle is formed at the place
where antigen and antibody are of appropriate proportion. This is called single
diffusion for only antigen diffuses.


Observing the result, we can find the diameter of
precipitation circle is proportional to the antigen concentration. If we define
the diameter of precipitation circle, which is the reaction result of different
concentration standard antigen and certain concentration anti-serum, as y-axis,
and define the antigen concentration as x-axis, we can draw a standard curve.
If we measure the diameter of precipitation circle of the unknown antigen, we
can tell its concentration from the standard curve. This experiment could be
used to monitor the concentration of immunoglobulin (Ig) or serum complement of
the sample.


Materials


1.Diagnostic serum of human immunoglobulin IgG( freeze-dry
sheep anti human IgG)


2.Human Ig standard serum, examinee’s serum


3.Agar, slide, cutter, micropipette, moist chamber, 37℃-temperature
machine


Methods and Results


1.Make plate: According to half of the serum titer, dilute
anti-serum with 56℃ pre-warm
normal saline. Then add agar that cooled off to 56℃
and mixed evenly. Draw 3 ml agar that contains anti-serum with pipette and
layer the agar onto slide evenly. After agar concretion, put the slide in 4 ℃
refrigerator for 5 min.


2.Cut well: Cut well with cutter. The diameter of well is 3mm. Keep the distance among wells above 1cm. Cut 5 wells every row.


3.Dilute standard serum and sample serum following
protocol. Dilute the sample to 1:50, Standard serum dilute serially to 1:12.5,
1:25, 1:50, 1:100, 1:200.


4.Add sample: Add standard serum of different dilution 10μl
into wells of first row in turn, respectively. Add sample serum 10μl into wells
of second row, respectively.


5.Keep agar slide in 37℃ moist
chamber for 24h. Then measure the diameter of precipitation ring.


6.Draw standard curve on semilogarithmic paper. Define the
diameter of precipitation ring as y-axis and IgG concentration of corresponding
well as x-axis. Refer to the standard curve according to the precipitation
circle of unknown serum. We could get the IgG concentration of unknown serum by
counting the IgG concentration shown in curve with the dilution ratio.


Attentions


1.Layer the slide quickly and avoid air bubble. Anti-serum
should be pre-hotted and agar should be cooled off to 56℃.


2.Do not destroy the wall of wells when add sample.


3.Distance among wells should be no less than 1cm.






ⅡDouble Agar Diffusion Test




Principle


Double agar diffusion test is a qualitative test. Add
soluble antigen and corresponding antibody into wells on agar plate. Both
antigen and antibody could diffuse. So visible precipitation line appears where
antigen and antibody are of appropriate proportion. If antigen and antibody is
not corresponding, precipitation line would not appear. This test is often used
to analyze the purity and reciprocal relationship of antigen and antibody.


Materials


1.Normal human serum


2.Sheep anti human IgG , unknown Ab1 and Ab2


3.1% agar in normal saline


3.Slide, cutter, moist chamber, 37℃-temperature
machine


Methods


1.Make plate: Layer the 1% melt agar 3ml onto a slide.


2.Cut wells: Cut on agar plate after the agar is concrete.


3.Add sample: Add sheep anti human IgG (Marked as Ab) into
central well. Add Ag1 into wells up and down. Add Ag2 into wells left and
right.


4.Put agar plate into 37℃ moist
chamber. Observe result after 24h.







  
  
  
    
    

    

    
    
      
      
      
      
      
      
      
      
      
      
      
      
    
    
    
    
    
    A
    

    
    
    
    
  
  
  
                        









  
  
    
    

    

    

    
       Ab          Ag2
    
          
    

    
          Ag1
    
      
    
  
    
1cm  Ab           Ag2
    

    

    
Ag1
    
    
  
  
                             Ag1











  
  
  
    
    

    

    
    
    
    
  
  
  
      
Ag2





















            Fig 2-3    Photo
Indicating of adding sample


Add
Ab into central well.


Add
Ag1 into wells up and down.


Add
Ag2 into wells left and right.





Results


Precipitation line appears among central well and
surrounding wells, which means positive reaction. Positive result indicates
that antibody reacted with corresponding antigen. If no precipitation line
appears, the result is negative. Negative result indicates antigen have not met
corresponding antibody.


Attentions


1.Agar plate should be layered once to be even and
smooth.


2.Convert the bottle and hang in the air when add
sample. Squeeze gently and avoid too much fluid overflow the wells, which would
affect the precipitation line formation.


Complement Fixation Test


Principle


Complement (C) has no specificity and could be
activated by any antigen-antibody complex. But complement could not be
activated by dissociative antigen or antibody.


Complement fixation test (CF) is a kind of reaction
system that complement participated in. In CF, sheep red blood cell (SRBC) and
hemolysin (anti-SRBC antibody) act as indicating system. The combination of
SRBC and hemolysin could activate complement and lead to RBC destruction,
hemolysis occurs.


So the five components in CF test could be divided
into 2 system:① test
system：known
antigen (or antibody) and unknown antibody (or antigen) ②
indicating system: SRBC and hemolysin. Test system reacts with complement at
first. Then indicating system is added. No hemolysis indicates that antigen is
corresponding to antibody and the immune complex of the testing system
activated and used up complement. So no complement combine with indicating
system and no hemolysis occurs. This is defined as CF test positive result. On
the contrary, if hemolysis occurs, the CF test has negative result.


Results


Tube 2、3、4、5 are control tubes, which should
appear hemolysis、hemolysis、hemolysis and no hemolysis, respectively. These control results
indicating that control tubes are normal. Tube 1 is experiment tube. No
hemolysis indicates Positive result of complement fixation test (+). Hemolysis
indicates negative result (-).


Attentions


1.Shake gently before sheep
blood usage. Avoid severe shake in case hemolysis.


2.Do not mix up pipette of
different reagents.


3.Complement is not stable and
should be kept at low temperature. Take it out from refrigerator just before
adding sample.


4.Avoid water dropping into
tube during water bathe.


5.Reaction situation of control
tubes is the reference for judging the experiment credibility, as a series of
factors may affect the experiment.





Exp.3  Enzyme-Linked
Immunosorbent Assay (ELISA)







Principle


ELISA (Enzyme-Linked Immunosorbent Assay) was
established by Van Weeman,Schuur,Enyvall and Perlmann in 1971. An enzyme is
linked to an antibody in such away that it does not affect the enzymatic
activity or the antibody specificity. First, the known antibody or antigen is
fixed on a solid carrier. Then a sample and the corresponding enzyme-linked
Ab/Ag are added to bind to the Ag/Ab attached to the solid carrier. The
resulting Ag-Ab complex and the excess Ab/Ag are separated by washing. Finally
the substrate is added and catalysis cause a color change which can be
measured.


ELISA has
the advantages of high sensitivity and specificity. It combines the  specificity of the Ab-Ag reaction with the
catalysis of enzymes.


Methods


1．  Qualitative method：


（1）Coating：sheep blood cells from sheep blood that was aseptically defribinated are
shattered after washing in normal saline and diluted in coating buffer. Add 100
μl to each well of a ELISA plate and
incubate overnight at 4℃. The next day, wash the
plate with PBS-Tween using a wash bottle. Soak the wells for 3 minutes and
repeat the wash two times.




(3)Wash the plate 3-4 times (3 min each time), then
add sheep anti-rabbit IgG linked to enzyme to the wells (100μl/well). Incubate at 37℃
for 30min.


（4）Wash the plate 3-4 times (3 min each time), then add freshly prepared
substrate to wells (100μl/well), place the plate in a dark
place. After 20 min, add 2M H₂SO₄ to quench the reaction.


2．  Quantitative method：


（1）Coating as qualitative method.


（2）Add a group of serially diluted
primary antibodies (hemolysin), test samples and a negative control (PBS) (100μl/well). Incubate at 37℃
for 30min.


（3）Wash the plate 3-4
times (3 min each time), then add sheep anti-rabbit IgG linked to enzyme to the
wells (100μl/well). Incubate at 37℃
for 30min.


（4）Wash the plate 3-4 times (3 min each time), then add freshly prepared
substrate to wells (100μl/well). Place the plate in a dark
place. After 20 min, add 2M H₂SO₄ to quench the reaction.


（5）Assess results by spectrophotometric
measurement of the absorbance at 492 nm. Use
the concentration of serially diluted primary antibodies as
the X values and their OD as the Y values to construct a standard curve, which
is used to determine the concentration of the test samples.


Attentions


1．It takes at least 18 h
for coating.


2．A wet box should be
applied for coating and incubating


3．Efficent washing is
necessary for good results.  








Exp.4  Analysis of antibody-forming
cells in vitro









Principle


The B lymphocytes from the
spleens of mice immunized with sheep blood cells (SRBC) react with SRBC and
complement in a liquid medium. The Ag-Ab complexes result from the binding of
the SRBC and the IgM secreted by the spleen cells. The complexes activate the
complement system, inducing hemolysis. The quantity of the released haemachrome
can be determined by spectrophotometry.


Part Three Specific Cellular Immune Function Assay



Exp.5  Separation of Mononuclear
Cells from        Peripheral Human Blood




      


When detecting specific
cellular immune function, we need to separate and purify human mononuclear
cells from peripheral blood (PBMC).  The
commonly used  techniques include
physical methods (such as density gradient centrifugation) and chemical methods
(such as low osmotic saline lysis of red blood cells, NH₄Cl lysis of
red blood cells).  In this part we
will only introduce the most common method (Ficoll-Hypaque density gradient
centrifugation), which is simple and yields a large quantity of highly purified
cells.  Additionally, it doesn’t affect
the activity of the cells.


Principle


       The separation medium commonly used to
separate PBMC is composed of Ficoll and Hypaque mixed in certain
proportions.  Its specific gravity is
1.077±0.001 g/L at 20^oC,
the gravity of lymophocytes and moncytes is lower than that of the separation
medium.  It is about 1.070g/L, but the gravity of the granulocytes
and erythrocytes is the highest.  It is
about 1.092g/L.  Following centrifugation, the cells
distribute according to their specific gravity in the density gradient.
Lymphocytes and monocytes are located upper levels of separation medium,but
granulocytes and erythrocytes sink to the bottom of the tube. Thus, lymphocytes
and monocytes are separated.


Ficoll-Hypaque density gradient centrifugation


Attentions


1.Pay attention to aseptic manipulation when drawing peripheral
veinous blood.


2.Take care to protect yourself from blood borne
infectious diseases.


3.Complete the whole process in the shortest time
possible to decrease the number of dead cells.


4.It is necessary to use a horizontal centrifuge when
separating PBMC with lymphocyte separation medium.









Exp.6  Erythrocyte Rosette Forming Cell Assay







Principle


       The surface of peripheral T lymphocytes contains the receptor
CD2, which binds to sheep erythrocytes (E receptor).  CD2 is not present on the surface of B lymphocytes.  Therefore, when mixed with sheep erythrocytes
in vitro, only T lymphocytes can form E rosettes with sheep erythrocytes using
E receptors.  This test can be used to
detect the number and ratio of T lymphocytes in peripheral blood.


Results


       If
three or more erythrocytes are adherent to one lymphocyte, E –rossete formation
is positive.  The percentage of E
–rossete formation is calculated by counting the number of rossete forming
cells out of 200 lymphocytes.


                                 Lymphocytes that formed rossetes


The percentage of E–rossete formation=                                          ×100%                       Lymphocytes
(formed rossetes+didn’t form rossetes)


Normal value:  The percentage of E –rossete formation in human peripheral
blood is 60-80%.


Attentions


1.The sheep blood
used in the E-rossete forming test must be fresh.  It can be stored for less than two weeks.


2.Before the fetal
calf serum is used, the complement must be inactivated by incubation at 56^oC.


3.After the E-rossete
formation, manipulation of the cells must be gentle to avoid separating the
formed rossetes.









Exp.7 Lymphocyte
Proliferation Assay




      


A commonly used
method to measure cellular immune function analyzes in vitro lymphocyte proliferation.
The substances which stimulate lymphocyte proliferation are divided into
two categories:  (1) nonspecific
stimulators or mitogens, such as PHA, or ConA; (2) specific stimulators or
antigens, such as the purified protein derivative (PPD) from
M.tuberculosis.  Among these stimulators,
PHA is widely used. The receptor for PHA is expressed only on the surface of T
lymphocytes.  The extent to which PHA stimulates T lymphocytes to
proliferate is compared to the proliferation stimulated by a specific
antigen.  The common methods for
detecting lymphocyte proliferation in vitro include morphologic counting, ³H-thymidine
incorporation.Morphologic counting is simple，but it is affected by subjective factor.   ³H-thymidine incorporation has higher
sensitivity and accuracy，though hving radioactive contamination，it is still a generally acknowlleged method for us.


       When T lymphocytes are incubated with PHA in vitro, they are stimulated to increase the synthesis of nucleic
acid and protein.  At the same time, the morphology
of cells is transformed to a lymphoblast.
Thus, this test is also called lymphocyte transformation test.


I. Morphologic Counting


Principle


       Peripheral blood or separated
lymphocytes are incubated with PHA in
vitro for 72h.   The cells are
smeared onto a slide and stained.  Under
the microscope large naïve lymphocytes can be observed.  Only T lymphocytes express the receptor for
PHA, so PHA can only stimulate T lymphocytes.
The percent of T cells that transform is calculated by counting 100～200 lymphocytes.
This is correlated with the cellular immune function, so it is used as
one of the guidelines for cellular immunity function assays.


Results


       Observe the results noticing the sizes and morphology of the
cells.  During the
transformation the cells that are commonly seen are:  lymphoblasts, transitional lymphocytes, cells
in which the nucleus is dividing, mature lymphocytes, etc.  


Principle


       T lymphocytes
that are stimulated by PHA undergo active cell division.  As the cells continuously enter the S phase
of the cell cycle their DNA synthesis increases compared to nondividing cells.  ³H-thymidine a precursor of DNA is
added to the culture medium.  It is taken
into the cells and incorporated into the newly synthesized DNA. ³H
isotope can release βray,the ray is
detected with a liquid scintillation counter.
By measuring the quantity of the isotope incorporated in the cell, we
can estimate the percentage of lymphocytes that are stimulated by PHA.  This method is less subjective and easy to
repeat.

  

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