Experiment of Medical Immunology

The Department of Immunology

September, 2014
Introduction to the experiment
1. Every one should enter the lab with the white cloak.
2.The used recoverable material, such as used slide,
should be put in the jar, but the used disposable
material should be put into the wastebin or shovel.
3.You should tell the teacher immediately when some
unexpected thing is taken place.
4.Every one should not waste any material and abuse
the apparatus.
5.You should clean the desk and the lab, and wash
you hand when you finish the experiment.
6.The purpose of the experiment is to review the
theory knowledge and train your capability to
work independently.
Experiment report
1. The name of the experiment.
2. Materials: some important reagent.
3. Techniques and Procedures.
4. Results.
5. Conclusion.
6. Discussion.
Note: Scores=30% report + 70% examination
Experiment 1 Agglutination Reaction
[content]
1. Slide agglutination test
----Blood group examination (every one do it);
2. Indirect agglutination reaction
----Latex agglutination test for detection of RF
(Group do it).
[Purpose]
1. Understand the nature of Ag-Ab reactions;
2. Master the definition and categories of agglutination;
3. Master the way of blood group examination;
4. Know Latex agglutination test for detection of RF.
[Principle]
. Theory about antigen-antibody reaction
Antigen-antibody reaction in vitro
----Serologic reaction
1. Nature of Ag-Ab reactions
(1)Specificity
(2)Reversible
(3)Ag:Ab ratio
(1)Specificity
Lock and Key Concept: Ag+Ab=Ag-Ab complex
(2)Reversible
Ag-Ab reactions occur by non-covalent bonds, so
they are by their nature reversible.
(3)Ag:Ab ratio
Not all Ag/Ab reaction
can be observed by eyes.
The ratio between the
antigen and antibody
influences the sizes of
Ag/Ab complexes.
Ab excess Ag excess
Equivalence Lattice formation
2. Factors affecting Ag/Ab reactions
(1)Electrolytes: normal saline
(2)pH level: 7.0
(3)Temperature
37
0
C
3. Categories of antigen-antibody reaction
*Agglutination reaction
*Precipitation reaction
*Complement fixation reaction
*Neutralization reaction
*Immunolabeling technique
Classical Ag-Ab
reaction
4. Agglutination reaction
Insoluble particle antigen + specific antibodies
---- agglutinates
Particle antigen:
Such as RBC, and bacteria.
Can be observed under microscope , cloudiness (eye).
Take part in agglutination reaction.
Soluble antigen:
Such as protein and polysaccharides.
Can not be observed under microscope , clear(eye)
Take part in precipitation reaction.
Agglutination tests
Direct agglutination reaction
Indirect agglutination reaction
Direct agglutination reaction
Particle antigen + antibody = agglutinate (directly )
Slide agglutination test
Tube agglutination test
1)Slide agglutination test
The direct agglutination is carried out on the slides.
Application
*It is qualitative test
*Use the known Ab to identify the unknown Ag.
Determination of blood type (ABO types);
Identification of bacteria and its serotype.
2)Tube agglutination test
The direct agglutination is carried out in tube.
Directly mix known particle Ag with serial dilution
of diagnostic serum (unknown Ab) in the tubes.
Application:
*It is quantitative test.
*Use the known antigen to identify the unknown
antibody.
Widal test (Salmonella infections )
(3)Indirect agglutination reaction
If the soluble antigen was adsorbed on some particle,
the agglutination reaction will occur.
Particle:
*Red blood cell (hemagglutination)
*Latex particle(latex agglutination)
Application:
Used to detect specific antibody for the soluble antigen.
*Diagnosis some virus infection
*Detect the Rheumatiod factors (RF)
*Diagnosis early pregnant
.Today Experiment
1. Slide agglutination test----Blood group examination
(every one do it)
ABO blood type
[Materials]
(1)Known antibody: Anti-type A, Anti-type B.
(2)Unkown antigen: You blood type antigen on your
own RBC.
[Procedures]
(1) Preparation of 2-5% RBC suspension.
(2) Add 1 drop of anti-type A and anti-type B
respectively on the clean slide.
(3) Add 1 drop of you RBC in the anti-typeA
and anti-typeB on the slide.
(4) Mix it by tooth pick.
(5) Wait for 5-10 minutes and observe the result.
[Results]
Positive result: has some agglutination particles in
the solutions. When shake it, it keep transparent
and not cloudling.
Negative result: has no agglutination, but there are
some RBC precipitate on the slide. When shake it, it
will became clouding.
Judge you blood type:
Type A blood: Anti-A Agglutination;
Type B blood: Anti-B Agglutination;
Type AB blood: Anti-A and Anti-B Agglutination;
Type O blood: Have no agglutination in Anti-A
and Anti-B.
[Attentions]
(1)When you take the RBC, please do it each other,
and pay attention to sterilize carefully.
(2)When we observe the result, we observe it on the
white background, then we will get good effect.
2. Indirect agglutination reaction
----Latex agglutination test for detection of RF
Rheumatoid factors (RF) are a group of abnormal
immunoglobulins in the sera from patients of
rheumatoid arthritis. These antibody activity is directed
against Fc of human IgG.
The RF is antibody, the IgG is antigen
Adsorb the IgG
on the surface
of latex.
[Materials]
(1)Human IgG sensitized latex particle;
(2)RF positive control, RF negative control.
[Procedures]
(1)Add one drop of RF positive control and RF
negative control on the slide (black paper) respectively.
(2)Add one drop of IgG sensitized latex particles into
the above sera respectively. Shake the slide gently to
mix well.
(3)Leave it for 5 minutes at room temperature and
observe the results.
[Results]
(1)Positive results:
In the suspension, there are some white agglutination
particles. When shake slide, the supernatant will
became more and more transparent.
(2)Negative results:
In the solution, there is no agglutination particles.
When shake slide, the whole solution still keep turbid.
[Attentions]
(1)Sera should be mixed very well with latex reagent.
(2)The white agglutination particles show very well
against the black background.