Gel precipitation reaction

1. Single-phase agar diffusion test

principle

In a quantitative test, a certain amount of antibody is mixed in agar and poured onto a glass slide. After solidification, a hole is punched in the agar layer, and then the antigen is added to the well to diffuse it around. The diameter of the precipitation ring formed by the antigen-antibody complex is proportional to the concentration of the antigen. If a standard curve is prepared with different concentrations of standard antigens in advance, the antigen content in the unknown specimen can be obtained from the standard curve. This test is mainly used to check the content of various immunoglobulins and serum IgG in the specimen.

material

1. Antibody: goat anti-human IgG monovalent antiserum.

2. Antigen: serum to be tested.

3.3% saline agar, saline, glass slide, 3mm diameter punch, small triangular flask, capillary dropper, wet box.

method

1. Prepare agar plates containing antibodies

Take a bottle of goat anti-human IgG monovalent antiserum (potency 1:80), measure 0.3ml in a small triangular flask, add 11.7ml of physiological saline (40 times dilution and mix well, and place it in a 56 ° C water bath for 2 ~ 3 minute).

Take a tube of 3% saline agar, heat it to dissolve in water, and then put it in a 56 ° C water bath. When the agar temperature drops to 56 ° C, immediately add an equal amount of 1:40 diluted antiserum. Bubbles were generated, and quickly poured into a glass slide, 3.5ml each, and holes were punched after solidification. As shown below.

2. Dilute the serum to be tested

Dilute the serum to be tested with physiological saline 40 times,

3. Punch and add samples

Add two wells to each test sample, fill up (but do not overflow), and do not scratch the agar at the tip of the capillary dropper when loading.

4. Incubation

Place the agar plate in an enamel box, pad with wet gauze or sponge to prevent drying, and keep at 37 ℃ for 24h.

Results observation and analysis

Measure and calculate the average diameter of the precipitation ring of two wells of each sample, and calculate the IgG content according to the standard curve.

Standard antigen concentration (mg / 100ml)

Standard curve

2. Two-way agar diffusion test

principle

Commonly used for qualitative testing, but also for semi-quantitative testing. Add the antigen and antibody to the adjacent small holes on the agar gel plate to allow them to diffuse towards each other. When the two meet at the most appropriate ratio, a clear precipitation line is formed. Depending on whether there are precipitation lines, unknown antibodies can be identified with known antibodies, or unknown antibodies can be identified with known antigens. This method is commonly used clinically to check the alpha-fetoprotein in the serum of patients with primary liver cancer as an early auxiliary diagnosis of primary liver cancer.

A-Fetal protein (AFP) is a normal tissue component in fetal tissues and body fluids. This protein appears in the fetus only at the 9th week, peaks at 13-19 weeks, and gradually decreases after 21 weeks. The protein disappears or has little content after the fetus is born. It cannot be detected by ordinary test methods, and liver cancer patients and individual ovaries This protein has been detected in the serum of cancer or testicular cancer patients.

material

1. Anti-AFP serum, AFP positive serum of known liver cancer patients, and serum of patients to be tested.

2.1.2% agar (made up with physiological saline).

3. Slides, capillary straws, wet boxes.

method

1. Preparation of agar slides 3.5ml of 1.2% agar, known to be melted by heating, is quickly poured onto a slide.

2. Punching After solidifying, use a puncher to punch holes according to the pattern below. (Aperture 3mm, hole distance 4mm)

2. Serum to be tested in 3, 5 wells is positive for AFP

3. Loading

The above square hole is the first hole, which is called 2, 3, 4, 5 and 6 holes in the clockwise direction, and the central hole is 7 holes. Add anti-AFP serum in 7 wells, add well-known AFP positive serum (or AFP) in wells 1 and 4 as positive control wells; add well-known AFP negative serums in well 6 as negative control wells, and the remaining 2, 3, and 5 wells as the test Well, add the serum to be tested.

4. Incubation Incubate at room temperature or 37 ° C for 12-24 h.

Results observation

1. A white precipitation line should appear between holes 4 and 7 (as shown in Figures 1 and 7, 4 and 7 above), and no white precipitation line should appear between holes 6 and 7. If a white precipitation line appears between wells 2, 3, 5 and 7 and matches the precipitation line of the positive control as shown in wells 2, 3, 5 and 1, 4 in the figure, the experiment is positive, indicating that the serum to be tested contains AFP (Ie AFP positive). If there is a connection with the positive control, and it is spear-shaped as shown in the upper right picture between 4 and 5 holes, indicating that there is a common antigen component between them; if there is a cross precipitation line as shown in the upper right picture 3 and 4, it is a non-specific precipitation The reaction, which is a false positive reaction, is judged to be negative for the experiment, indicating that the serum to be tested is negative for AFP.

3. Convection immunoelectrophoresis

Principle Under certain conditions, colloidal particles are charged, and these charged colloidal particles move under the influence of electricity. If the colloidal particles are negatively charged, they move to the anode; otherwise, they move to the cathode. This phenomenon is called electrophoresis.

Convection immunoelectrophoresis is to put the patient's serum (antigen to be tested) in the cathode end hole of the agar plate, and the known antiserum (containing the known antibody) is placed in the anode end hole. At the same time, electrophoresis is performed under alkaline environmental conditions. The cathode moves to the anode fast, and the anti-gamma globulin has an isoelectric point between pH 6 and 7, so it has less negative charge and moves slowly. Due to the electroosmotic effect, it moves backward to the cathode, so the antigen and antibody meet in the electric field. When the ratio between the two is appropriate, the specific antigens and antibodies bind to each other, forming a white precipitation line visible to the naked eye.

material

1.1%, PH8.6, 0.075M barbiturate buffer.

2. Anti-AFP serum, known AFP positive serum, serum of patients to be tested.

3. Slides, pipettes, hole punches, capillary droppers, electrophoresis instruments, etc.

method

1. The 1.2% agar prepared with the buffer solution was heated to dissolve in water, and 3.5ml was added to the slide while hot, and the hole was drilled after condensation, the diameter of the hole was 3 mm, and the distance between the two holes was 3 mm.

2. Add anti-AFP sera to wells 1, 3, and 5 in the left column from top to bottom; add sera of the patient to be tested to 2 wells in the right column; add known AFP positive sera in 4 wells as a positive control; Know the negative control serum. Each hole is filled to the degree, don't make it overflow,

3. Place the agar plate in the electrophoresis tank, with the antigen end on the cathode side, the antibody on the anode side, and two layers of gauze strips impregnated with the electrophoresis buffer on both ends.

4. Power on, fixed voltage 5-6V / cm long, power on for 45 ~ 60 minutes.

Results observation

After electrophoresis, turn off the power and remove the agar plate. Above the black background, through the scattered light, first observe whether the white precipitation line between the 3 and 4 wells (positive control group) and between 5 and 6 wells (negative control group) appears; then look at the test well, if there is no between the wells With such a precipitation line, the serum to be tested is AFP negative serum.

4. Rocket immunoelectrophoresis

Principle: Rocket immunoelectrophoresis is an immunological technique that combines electrophoresis with unidirectional diffusion. After dissolving the antibody in the agar, the plate is made, a hole is punched on one side of the agar plate, and the sample to be tested and standard antigens of different concentrations are added. During electrophoresis, the antigen moves to the positive electrode in the agar containing the quantitative antibody, and forms a concentration gradient, forming a rocket-shaped precipitation peak at a suitable location. The height of the precipitation peak is proportional to the content of the antigen, so the antigen can be quantitatively determined.

method:

1. Preparation of agar plates

The antibody serum was mixed with the dissolved 1.5% agar in a 56 ° C water bath at a certain ratio, and quickly poured into a 2 mm thick agar plate.

2. Punch

3. Add sample: add standard antigen solution of known concentration and antigen to be tested respectively, 10 μl per well.

4. Electrophoresis: put the agar plate into the electrophoresis tank, the antigen is placed on the cathode side, the antibody is placed on the anode side, and four layers of gauze strips impregnated with the electrophoresis buffer are attached to both ends. The fixed voltage is 5 ~ 6V / cm long, and the energization time is about 45 ~ 60 minutes. Observe the results.

Section 4 Hemolytic reaction and complement binding test

In addition to agglutination and precipitation reactions, commonly used antigen-antibody reactions include complement binding test, neutralization test, and hemolytic plaque test. This experiment introduces the complement fixation test.

Experiment purpose and principle

Understand the complement complement test method and result analysis.

The complement fixation test is an anti-antigen system that involves complement and uses sheep red blood cells and hemolysin as indicator systems

体 反应。 Body reaction. If the corresponding antibody exists in the blood to be tested, add the corresponding antigen and complement. Since the antigen and antibody form a complex, the complement can be combined with it, and no free complement exists in the solution. However, since this combination is indistinguishable to the naked eye, an indicator system (sheep red blood cells and hemolysin) needs to be added. If the complement has been immobilized by the antigen-antibody complex of the test system, after the indicator system is added, hemolysis will not occur due to no complement binding (hemolysis or not, it is easy to distinguish with the naked eye). Corresponding. If hemolysis occurs. Negative for complement fixation test. It shows that the antigen and antibody in the test system do not correspond. The end of the complement is fixed, after joining the indication system. Complement complements it. There is a hemolytic reaction.

Experimental Materials

1. Antigen: type A (or type B) paratyphoid bacteria solution, 2% sheep red blood cell suspension.

2. Antibody: Serum of type A (or type B) paratyphoid bacteria diagnosis. Hemolysin (2 units / 0.25ml).

3. Complement: Rat serum (2 practical units / 0.25ml)

4. Others: normal saline, small test tube, pipette, etc.

experimental method:

1. Take 5 small test tubes, arrange them on the test tube rack and indicate the tube number.

2. Add 0.4ml of normal saline to the first tube, then use a pipette to take the inactivated (56 ℃ heating 30 minutes) antibody serum, lml (or test serum) into the first tube, and inhale three times to mix Then, aspirate 0.25ml into the second tube, and the serum in both tubes is diluted 1: 5.

3. Operate in the order shown in the table below.

Experimental results

First observe whether there is hemolysis and whether the results of each control tube are correct.

Hemolysis: red blood cells dissolve, the turbid liquid becomes red transparent liquid; non-hemolysis phenomenon: red blood cells do not dissolve, the turbid liquid remains unchanged.

Tubes 2, 3, and 4 should be completely hemolyzed due to the lack of corresponding antigen-antibody complexes to be tested, and tube 5 should not be hemolyzed because of only sheep red blood cells and saline. The above are control tubes.

The first tube without hemolysis was positive for complement binding reaction.

Experiment 5: Determination of complement by 50% hemolysis test (CH50)

The 50% hemolysis test is to obtain the minimum amount of serum that can cause 50% of sensitized sheep red blood cells to hemolyze, and then calculate the complement content per ml of serum.

Figure 12 Relationship between the degree of hemolysis of hemolysin-sensitized magenta cells and the increase in the amount of serum (complement) in intestinal mice

Using the amount of complement as the abscissa and the percentage of hemolysis as the ordinate, a clear "S" shaped curve can be obtained. Around 50% hemolysis, the line segment approximates a straight line. Therefore, when the amount of complement is slightly changed, it can have a great impact on the degree of hemolysis. Therefore, using 50% hemolysis as the end point is more sensitive than 100% hemolysis.

Materials and equipment

1. Barbiturate buffer (BB, pH7.4): diluted 1: 5 with distilled water when using

NaCl: 85g Barbiturate: 5.75g Barbiturate sodium: 3.75g Mgcl2: 1.017g Cacl2: 0.166g Distilled water 2000ml, filtered, stored at 4 ℃ When using this solution, add 1 part of this solution and 4 parts of distilled water.

2. 2% SRBC suspension: fresh defibrinated sheep blood, washed 3 times with 10 times NS. 2000rpm * 5min for the first two times and 2500rpm * 10min for the last time, take sheep red blood cells to make 2% SRBC suspension with BB solution

3. Hemolysin (2U), test serum (fresh guinea pig serum), horizontal centrifuge, water bath, 721 spectrophotometer,

4. Prepare 50% hemolysis standard tube: take 2% SRBC suspension 2ml and add distilled water 8ml, all SRBC will be dissolved, which is a 100% complete hemolysis tube. Take 2.5 ml of the supernatant of the complete dissolution tube and 2.5 ml of the BB solution, and mix them to obtain a 50% hemolytic standard tube.

method:

1. Preparation of 1:20 Serum. Venous blood was drawn into a test tube, allowed to stand at room temperature, serum was separated (within 2 hours), and diluted with BB solution to 1:20.

2. Prepare 50% hemolysis standard tube

3. Measurement of complement CH50 units: take 10 test tubes, numbered 1, 2, and 3 respectively. . . . . 10. Add samples according to the table below

â‘´Add samples according to the following table:

Results judgment:

Take a preliminary visual comparison of each tube with a 50% hemolytic standard tube, select two tubes close to the standard tube, use a 721 spectrophotometer to read the T value at a wavelength of 542 nm, and find the one that is closer to the standard tube transmittance One tube, according to the amount of diluted serum added to this tube, calculate the total complement value according to the following formula. Calculate the complement activity (U / ml) in each ml of serum:

Precautions:

1. The specimen to be tested should be free of hemolysis, pollution, and chylous blood. Laboratory equipment should be clean.

2. The buffer and sensitized sheep red blood cells should be freshly prepared.

3. The serum to be tested must be fresh. If left for more than 2 hours, the complement activity will decrease.

4. The hemolytic activity of complement is affected by many factors, such as sheep red blood cell concentration and the amount of hemolysin.

Clinical significance: This method determines the normal reference value of complement: 50 ～ 100 U / ml. In acute inflammation, infection, tissue damage (such as acute rheumatic fever, periarteritis nodosa, dermatomyositis, typhoid fever and multiple arthritis), tumors, myeloma, etc., often increase the complement content, making CH50 On the high side; hypocomplementemia is more common in diseases related to immunity, which is caused by the consumption of complement during the course of the disease, such as: acute glomerulonephritis, membrane proliferative glomerulonephritis,