1.agglutination reaction

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Experiment of Medical Immunology

The Department of Immunology

September, 2014


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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 someunexpected thing is taken place.

4.Every one should not waste any material and abuse

the apparatus.


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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.


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Experiment report

1. The nameof the experiment.

2. Materials: some important reagent.

3. Techniques and Procedures.

4. Results.

5. Conclusion.

6. Discussion.

Note: Scores=30% report + 70% examination


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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).


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[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.


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[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


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(1)Specificity

Lock and Key Concept: Ag+Ab=Ag-Abcomplex


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(2)Reversible

Ag-Ab reactions occur by non-covalent bonds, so

they are by their nature reversible.


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(3)Ag:Ab ratioNot all Ag/Ab reaction

can be observed by eyes.The ratiobetween the

antigen and antibody

influences the sizes of

Ag/Ab complexes.Ab excess Ag excess

EquivalenceLattice formation


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2. Factors affecting Ag/Ab reactions

(1)Electrolytes: normal saline

(2)pH level: 7.0

(3)Temperature 370C


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3. Categories of antigen-antibody reaction

*Agglutination reaction

*Precipitation reaction

*Complement fixation reaction

*Neutralization reaction

*Immunolabeling technique

Classical Ag-Abreaction


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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.


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Agglutination tests

Direct agglutination reactionIndirect agglutination reaction


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Direct agglutination reaction

Particle antigen + antibody = agglutinate (directly )

Slide agglutination test

Tube agglutination test


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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.


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2)Tube agglutination test

The direct agglutination is carried out in tube.

Directly mix known particle Agwith 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 )


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(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)


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Application:

Used to detect specific antibody for the soluble antigen.

*Diagnosis some virus infection

*Detect the Rheumatiod factors (RF)

*Diagnosis early pregnant


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.Today Experiment

1. Slide agglutination test----Blood group examination

(every one do it)

ABO blood type


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[Materials]

(1)Known antibody: Anti-type A, Anti-type B.

(2)Unkown antigen: You blood type antigen on your

own RBC.


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[Procedures]

(1)Preparation of 2-5% RBC suspension.

(2)Add 1drop of anti-type Aand anti-type B

respectively on the clean slide.

(3)Add 1drop 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.


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[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 aresome RBC precipitate on the slide. When shake it, it

will became clouding.


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Judge you blood type:

Type Ablood: Anti-A Agglutination;

Type Bblood: Anti-B Agglutination;Type ABblood: Anti-A and Anti-B Agglutination;

Type Oblood: Have no agglutination in Anti-A

and Anti-B.


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[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.


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2. Indirect agglutination reaction----Latex agglutination test for detection of RF

Rheumatoid factors (RF) are a group of abnormal

immunoglobulinsin 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.


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[Materials]

(1)Human IgG sensitized latex particle;

(2)RF positive control, RF negative control.


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[Procedures]

(1)Add one drop of RFpositive controland RF

negative controlon the slide (black paper) respectively.

(2)Add one drop of IgG sensitized latex particlesinto

the above sera respectively. Shake the slide gently to

mix well.

(3)Leave it for 5 minutes at room temperature andobserve the results.


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[Results]

(1)Positive results:

In the suspension, there are some white agglutination

particles. When shake slide, the supernatant willbecame more and more transparent.

(2)Negative results:

In the solution, there is no agglutination particles.

When shake slide, the whole solution still keep turbid.


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[Attentions]

(1)Sera should be mixed very well with latex reagent.

(2)The white agglutination particles show very well

against the black background.

1.agglutination reaction

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