Convection immunoelectrophoresis assay
Convection immunoelectrophoresis assay
Convection immunoelectrophoresis is essentially an immunodiffusion technique with directional acceleration, the basic principle of which is: two rows of holes are punched on an agar plate, the left holes are filled with the antigen to be tested, and the right holes are filled with the corresponding antibody, which is on the cathode side and the antibody is on the anode side. After electrification, the negatively charged antigen swims to the anode side of the antibody, while the antibody flows to the cathode side of the antigen by electroosmosis, forming a precipitation line between the two or on the other side of the antibody.
Operation method
convection immunoelectrophoresis
Principle
Convection immunoelectrophoresis is a simple and rapid method based on agar diffusion combined with electrophoresis. This method can produce results in a short period of time, so it can be used for rapid diagnosis, and the sensitivity is 10-15 times higher than that of the bidirectional diffusion technique.
Materials and Instruments
Serum to be tested Move 1. The preparation of agar plates is based on the need to use two types of large slides (6 cm × 9 cm) and (small slides). About 10 ml of agar is required for the large slide and 3.5 ml for the small slide. After solidification, the holes are punched according to the diagram in the same way as in the agar diffusion test. For more product details, please visit Aladdin Scientific website.
Agar Barbiturate Buffer Saline
Electrophoresis machine Centrifuge Perforator Microsampler
2. Addition of samples: patient serum (original serum and 10-fold diluted serum each occupy one hole) is added to the left hole, and antiserum is added to the right side, and there should be a positive control for each slide. 
Figure 5 Schematic diagram of the location of antigen and antibody wells for convection immunoelectrophoresis3. ElectrophoresisUse a domestic ordinary electrophoresis apparatus. Add 0.05 m PH8.6 barbiturate buffer to two-thirds of the height of the electrophoresis tank, and pay attention to the liquid level in the two tanks to be as horizontal as possible. Place the glass plate with the added sample on the electrophoresis tank, with the antigen end connected to the negative pole and the antibody end connected to the positive pole, and use 2~4 layers of filter paper moistened as a salt bridge, with the filter paper connected to the agar plate at 0.5 cm. The current was calculated by the width of the plate and the voltage by the length of the plate. The required current was 2 to 3 mA/cm, i.e., 20 mA for large plates and 10 mA for small plates. The voltage is 4~6 volts/cm. The results were observed after 45 minutes to 2 hours of energization.
4. Observation of the results: above the black background, observed with scattered light from multiple angles, there is a white precipitation line between the pair of holes, that is, the positive control should appear obvious white precipitation line. If the antigen, the two poles of the micro-precipitation streak is not clear, at 37 ℃ insulation for several hours can enhance the clarity of the precipitation streak.
5. Factors affecting the outcome
(1) Antigen-Antibody Ratio: Precipitation bands tend to appear when the antigen-antibody ratio is adapted, and vice versa. When the antibody concentration is constant and the serum being examined contains a high concentration of alpha-fetoprotein, making a 10-fold, 20-fold or higher dilution can increase the positive rate. With the increase of dilution, the ratio of antigen and antibody changes, the precipitation line from near the antiserum hole to gradually move to the middle of the two holes, and there may be atypical precipitation line such as curved, eight whiskers, diagonal, which are also positive and should be noted.
(2) Several groups of electrophoresis buffers showed the highest sensitivity for electrophoresis with sodium barbital-hydrochloric acid buffer. Sodium barbital-barbitone was the next most sensitive buffer. tris buffer was worse.
(3) Electrophoresis time needs to be longer when voltage and current: electrophoresis time can be shorter when voltage and current increase. However, if the voltage is too high, the aperture will be deformed, and if the current is too high, the antigen and antibody proteins will be easily denatured, which will interfere with the experimental results. Generally choose 5 ml per cm, electrophoresis time changed to 1.5 hours.