Convective immunoelectrophoresis is a straightforward and quick technique that combines agar diffusion with electrophoresis. It allows for rapid results, making it suitable for fast diagnostic purposes. Compared to the two-way diffusion method, this technique offers a sensitivity that is 10 to 15 times higher.
**Purpose**
This experiment aims to reinforce fundamental knowledge in immunology, deepen understanding of the principles and applications of convective immunoelectrophoresis, and develop hands-on skills in performing the procedure.
**Principle**
In a solution with a specific pH, antigens and antibodies carry electrical charges and move directionally under an electric field. When antigen and antibody are placed in an agar gel at pH 8.6, the antibody has a weak negative charge and a large molecular weight, which causes it to migrate slowly. Moreover, the antibody is significantly affected by electroosmosis, meaning the electroosmotic flow exceeds its own movement. As a result, during electrophoresis, the antibody moves toward the negative electrode instead of the positive one. Antigens, typically negatively charged, are placed on the negative side, while antibodies are placed on the positive side. After electrophoresis, when the two meet, a visible precipitate line forms at the correct ratio.
**Reagents and Equipment**
1. Antigen and corresponding immune serum.
2. Barbital buffer (pH 8.6, 0.05 M):
- Barbital sodium: 10.3 g
- Barbital: 1.84 g
- Distilled water: 1000 ml
3. Buffer agar plate: Prepare 1.5% agar using barbital buffer at pH 8.6 and 0.025 mol/L (diluted twice), add 0.01%–0.02% mercury chloride for preservation, and store in the refrigerator.
4. Other materials: Electrophoresis apparatus, saline, punchers, micro-injectors, glass slides.
**Procedure**
1. Prepare a 1% to 1.5% agar gel plate using a 0.05 M barbital buffer at pH 8.6, with a thickness of 2–3 mm.
2. Once the agar solidifies, punch two small holes (diameter: 0.3–0.6 cm, spacing: 0.4–1.0 cm). Remove the agar inside the holes and seal the bottom.
3. Load one well with the known (or tested) antigen and the other with the test (or known) antibody.
4. Place the agar plate in the electrophoresis chamber with the antigen well on the negative side. Apply a voltage of 2.5–6 V/cm or a current of 3–5 mA/cm for 30–90 minutes.
After electrophoresis, observe the gel under a light source against a dark background. A clear, dense white precipitate line between the wells indicates a positive result. If the line is not distinct, incubate the plate in a moist box at 37°C for several hours or overnight in the electrophoresis tank.
**Precautions**
1. The concentration ratio of antigen and antibody is critical. An improper ratio may prevent a visible precipitate line from forming. To ensure accuracy, test samples should be diluted and tested at multiple concentrations.
2. To avoid false positives, include a control antigen near the sample well. If the sample antigen fuses with the precipitation line formed by the control antigen, it confirms the specificity of the sample.
3. Electroosmotic effect is essential for proper convective immunoelectrophoresis. Poor-quality agar can cause excessive electroosmosis, leading to non-specific reactions. Some types of agarose may not be suitable due to lack of electroosmotic activity.
4. If the antigen and antibody have similar charges or migration patterns, they will move in the same direction during electrophoresis, making it impossible to detect a precipitate line using this method.
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