Rocket immunoelectrophoresis experiment principles, materials and operating procedures

1. Experimental principle

Rocket immunoelectrophoresis (RIEP) is a quantitative detection technique that combines unidirectional immunodiffusion and electrophoresis. During electrophoresis, the antibody contained in the agar gel does not move, but the antigen in the sample is caused to swim toward the positive electrode under the action of the electric field. When the antigen and antibody molecules reach the appropriate ratio, a peak of insoluble immune complex shaped like a rocket is formed, and the height of the peak is positively correlated with the antigen concentration in the sample collection. Therefore, when the antibody concentration in the agar is fixed, the standard curve is drawn with the precipitation peak formed after the standard antigens of different dilutions swimming and the antigen concentration as the abscissa. The content of the antigen to be tested can be calculated according to the length of the precipitation peak of the sample; conversely, when the concentration of the antigen in the agar is fixed, the content of the antibody to be tested can be determined (ie reverse rocket immunoelectrophoresis).

2. Experimental materials

(1) Materials and reagents

1. PH8.6, 0.1M barbiturate-barbiturate sodium buffer

Barbital sodium 10.3 g

Barbitur 1.84 g

Thimerosal 100 mg (preservative)

Distilled water is heated to dissolve and set the volume to 500ml

2. 1% pre-recovered agar (or agarose)

1g agar (or agarose) plus 100ml of distilled water can be dissolved.

3. 1.5% agarose gel

1.5g agarose plus 50ml of distilled water, dissolve in water and boil to dissolve or heat and dissolve in a wave oven (be careful not to overflow and pay attention to adding evaporated water), then add 50ml of the above barbiturate buffer to mix, store at 4 ℃ for use.

4. Antigen and corresponding immune serum.

(2) Equipment and equipment

Glass plates, molds, hole punches and picks, droppers, covered enamel boxes (with wet filter paper inside), thermostats (25 ° C), triangular beakers, glass agitators, micro-samplers, other commonly used equipment.

3. Operation steps

1. Selection of antigen and antibody concentration

With the method of fixing the antigen content and antibody concentration separately, the minimum antigen amount and the lowest antibody concentration are selected. After immunoelectrophoresis, the sharpest and sharp conical precipitation peaks appear at the clearest term, up to 2 to 5 cm. Generally, the dosage of antigen is 0.5 to 5 micrograms, the dosage of antiserum is 5 to 10 microliters, and the dilution is selected from 1:10 to 1: 200.

2. Preparation of pre-recovered agar glass plate

Add the dissolved 1% pre-recombined agar to the glass plate with a dropper so that it can cover the surface, put it in the incubator to dry (or naturally dry), and then it can be used to prepare the gel plate.

3. Preparation of antiserum agar plate

According to the above selection results, a certain amount of antiserum is mixed with a certain amount of 1.5% barbiturate buffer agar melted and cooled to 56 ° C, so that the concentration of antiserum is the optimal concentration selected by the above method, and the antiserum agar is poured board. The thickness of the agar is 1-2mm, and the size of the glass plate can be determined according to the needs of the experiment. If using a 5 × 7.5cm glass plate, about 6ml of antiserum agar is required.

4. Punch and add samples

After the agar solidifies, punch a row with a 3mm hole punch at a distance of 10mm from the long side of the glass plate, and the hole distance is 5mm. Add antigen solution to each well, 10μl per well. Several standard antigens of different concentrations should be added for each experiment as a control. The dilution range includes the highest and lowest levels of specimen content.

5. Electrophoresis

Place the agar plate on the beam of the electrophoresis tank, the sample end is at the negative pole, and connect the agar plate and the buffer in the electrophoresis tank with double-layer filter paper (the buffer in the tank is 0.06M pH8.6 barbiturate buffer). Turn on the power, the voltage at the end of the board is 3v / cm, the current intensity is 3mA / cm, and the electrophoresis is for 5 hours.

6. Judgment of results (click to expand the schematic diagram of electrophoresis results)

After electrophoresis, remove the agar plate, soak it with physiological saline and distilled water in sequence, add 1% tannic acid to rinse the agar plate, and dry it to observe and determine the results. The results can also be observed after staining by conventional protein staining methods. There are two methods for determining the results: one is to measure the height of the precipitation peak (from the center of the hole to the peak tip) in mm; the other is to measure the area of ​​the precipitation peak with a multimeter, in mm2. The former is simpler and the latter is more accurate. According to the measurement results, the antigen content in the specimen to be tested is calculated from the standard curve.

7. Formulation of standard curve

Prepare the agar plate with the optimal concentration of antiserum selected above. Drill holes, add 5-10 standard antigen concentrations in appropriate proportions, and repeat the test 10 times. Take the average value of the height (or area) of the precipitation peak measured after each electrophoresis and draw a standard curve.

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