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Experiment of Immunology

By Kenneth Campbell,2014-10-24 00:30
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Experiment of Immunology

Experiment of Immunology

Institute of Immunology

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     Part One Nonspecific Immunity Function Assay Exp. 1 Phagocytosis of Macrophage……………………………… 27

    Part Two Specific Humoral Immunity Function Assay Exp.2 Measurement of Antibody………………………………… 28

Exp.3 Enzyme-Linked Immunosorbent Assay (ELISA)…………39

Exp.4 Analysis of antibody-forming cells in vitro……………… 44

    Part Three Specific Cellular Immune Function Assay Exp.5 Separation of Mononuclear Cells from Peripheral Human

    Blood………………………………………………………… 46

Exp.6 Erythrocyte Rosette Forming Cell Assay………………‥ 48

Exp.7 Lymphocyte Proliferation Assay………………………… 50

    Part Four Immune Pathology

    Exp.8 Hypersensitivity Test of Guinea Pig………………‥…‥ 54

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    Part One Nonspecific Immunity Function Assay

    Exp. 1 Phagocytosis of Macrophage

    Macrophage is primary cell of the mononuclear phagocyte system. The main function of Macrophage is to dispose of microbes and dead cells through the process of phagocytosis. It is important in the innate immune system and adaptive immune system. It can be stimulated by antigen, and then activate. Here, we introduce a method to measure the ability of macrophage.

    ?. Phagocytosis of Macrophage

    in vivo

    Materials:

    1. Mouse

    72. Bacterial suspension of Staphylococcus (1×10cells/ml)

    3. 6% sterile starch solution

    4. Wright?s reagent

    Methods:

    1. Inject 1ml of 6% sterile starch solution intraperitoneally to mouse.

    2.72 hours later, inject 1ml of bacterial suspension of Staphylococcus, massage softly the abdomen to ensure spread of bacterial.

    3.After 30-40 minutes, draw neck to kill mouse, cut open the skin and peritoneum to expose the peritoneal cavity.

    4.Draw a little of fluid and place on a slide, smear symmetrically and stain with Wrights reagent.

    5. Wrights reagent stain Add a little drop of wrights reagent, stain for 1

    minute and then add the same amount of 0.9% NaCl solution on it. Shake it, gently, stain 5 mins.

    6. Dry with bibulous paper, observe under microscope to see

    ?. Phagocytosis of Macrophage

    in vitro

    1. Inject 1ml of 6% sterile starch solution intraperitoneally to mouse.

    2. After 72 hurs, draw neck to kill mouse, cut open the skin and peritoneum to expose the peritoneal cavity.

    3. Draw the fluid from peritoneal cavity, centrifuge 5 min at 1000r/min, and discard supernatant.

    4.Wash macrophage with Hanks 5 minutes at 1000r/min.

    Repeat once.

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    65.Count cells and adjust concentration to 1×10 cells/ml with Hanks.

    6.Add equal volume macrophage suspension and bacterial suspension of Staphylococcus to tube.

    7. Incubate at 37? for 30 min (shake two times), centrifuge 5 min at 500r/min, and discard supernatant.

    8.Shake tube slightly, place a drop of supernatant on a slide and mix with a drop of wrights.

    9. Observe under microscope.

    Part Two Specific Humoral Immunity

    Function Assay

    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.

    Agglutination Reaction

    I. Direct Agglutination Reaction

    Direct agglutination reaction refers to the antigen particle (e.g., complete bacteria or cell) combine with corresponding antibody in vitro and yield the visible agglutination reaction.

    Direct agglutination could be classified into two types, slide agglutination and tube agglutination.

    Slide Agglutination Test (human ABO blood type assay)

    This test is designed to observe the direct agglutination reaction of known antibody and unknown particle antigen on slide. Here, we take the human blood type assay as an example.

    The reaction is simple and rapid. Usually it is used to detect qualitatively the antigen or antibody. Such as bacteria identify, type and ABO blood type assay. Principle

    ABO blood type assay is nominated according to the antigen molecule on RBC. Human ABO blood type has two antigens: antigen A and antigen B. RBC surface of A type blood have antigen A. RBC surface of B type blood have antigen B. RBC surface of AB type blood have antigen A and B. RBC surface of O type blood have neither of

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the two type antigens.

    If we use the known anti-A serum and anti-B serum to combine with examinees RBC

    antigen, we can judge his or her blood type by observing the agglutination of RBC. Materials

    1.wtandrd andard anti-A serum, anti-B serum

    2.Slide, ethanol, iodine, aseptic needle, wooden stick, tampon stick Methods

    1.Take a clean slide and divide it into two sides with a marker pencil. Write A and B on different sides.

    2.Cnvert the standard serum reagent bottle and squeeze one drop of anti-A serum on A side. Add anti-B serum on B side.

    3.Dsinfect the finger skin. Stab the skin quickly with aseptic needle after the skin is dry.

    4.Take blood with two sides of the stick. Mix with anti-A and anti-B serum respectively.

    5.Press finger to stop bleeding with aseptic tampon stick.

    6.Keep the slide quietly for a few minutes. Observe the result on white background.

    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 A B AB O

    Serum

    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.

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    Tube Agglutination Test

    Principle

    Dilute the examinees 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

    81.Inactivated typhoid bacteria (710/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 1 2 3 4 5 6

    Normal saline 0.9 0.5 0.5 0.5 0.5 0.5

    Typhoid 0.1 0.5 0.5 0.5 0.5 Discard

    immunized 0.5

    serum

    Typhoid 0.5 0.5 0.5 0.5 0.5 0.5

    bacteria

    suspension

    1?20 1?40 1?80 1?160 1?320 Fina