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
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
72. Bacterial suspension of Staphylococcus (1×10cells/ml)
3. 6% sterile starch solution
4. Wright?s reagent
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 Wright′s reagent.
5. Wright′s reagent stain Add a little drop of wright′s 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
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 Hank′s 5 minutes at 1000r/min.
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65.Count cells and adjust concentration to 1×10 cells/ml with Hank′s.
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 wright′s.
9. Observe under microscope.
Part Two Specific Humoral Immunity
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.
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 examinee’s 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.
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
Anti-A serum + - + -
Anti-B serum - + + -
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
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.
81.Inactivated typhoid bacteria (7；10/ml)
2.Animal serum to be examined (Immunized by typhoid bacteria ‘H’ antigen)
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
Typhoid 0.5 0.5 0.5 0.5 0.5 0.5
1?20 1?40 1?80 1?160 1?320 Final serum control
3.shake rack gently to mix the fluid in test tubes. Put rack in 56? water bathe for
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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 (++).
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
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common carriers include RBC, polystyrene latex particle, SPA etc. According to the carrier, indirect agglutination reaction could be classified into different types.
Here we would introduce a latex agglutination test for pregnancy diagnosis.
Latex Agglutination Inhibition Test
Polystyrene latex particle could act as carrier. Antigen-sensitized latex could combine with corresponding antibody and then yield agglutination. If antibody is mixed with soluble antigen, and then antigen-sensitized latex is added, agglutination would not happen. So we call it latex agglutination inhibition test. Pregnant women urine contain human chorionic gonadotropin?HCG?hormone. Mix
the examinee’s urine and anti-HCG serum, the HCG in urine would combine and use up the anti-HCG antibody. Add HCG- sensitized latex particle afterwards, no anti-HCG antibody react with it and agglutination would not happen. Pregnancy test is positive. If the examinee’s urine contains no HCG, then sensitized latex combines with anti-HCG serum and agglutination occurs. Pregnancy test is negative.
1.HCG sensitized latex antigen
3.Urine of pregnancy women and control women
4.Pregnancy test plate, pipette
1.Add one drop of pregnant women urine and control women urine into adjacent wells on test plate, respectively.
2.Add one drop of anti-HCG serum into the two wells, respectively. Shake gently for 1-3min.
3.Add one drop of sensitized latex antigen into the two wells, respectively. Shake gently for 1-3min. Observe the result.
1.Add sample and reagents in turn.
2.Hang the bottle in the air when add anti-HCG serum and latex antigen. Do not stain the reagent.
3.Make sure to add same volume of reagents in different wells.
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
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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.
?Single agar diffusion test
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.
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
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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,
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.
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.
?Double Agar Diffusion Test
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.
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
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.
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