Convective immunoelectrophoresis is a straightforward and quick technique that combines agar diffusion with electrophoresis. This method allows for rapid results, making it ideal for fast diagnosis. Compared to the two-way diffusion method, it offers a sensitivity that is 10 to 15 times higher.
**Purpose**
By conducting this experiment, students will reinforce their understanding of immunology basics, gain insight into the principles and applications of convective immunoelectrophoresis, and develop practical skills in performing the procedure.
**Principle of the Experiment**
In a solution with a specific pH, antigens and antibodies carry electrical charges and can 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, so it moves slowly. Moreover, the antibody is significantly affected by electroosmosis, which is stronger than its own movement. As a result, during electrophoresis, the antibody may even move toward the negative electrode instead of the positive one. Antigens, on the other hand, are typically negatively charged and are placed near the negative electrode, while antibodies are placed near the positive electrode. After electrophoresis, when the two substances meet in the right proportion, a visible precipitation line forms.
**Reagents and Equipment**
1. Antigen and corresponding immune serum.
2. Barbiturate buffer (pH 8.6, 0.05 M):
- Sodium barbital: 10.3 g
- Barbital: 1.84 g
- Distilled water: 1000 ml
3. Agar plate prepared with barbiturate buffer (pH 8.6, 0.025 M) diluted twice, resulting in a 1.5% agar solution. Add 0.01%–0.02% merthiolate as a preservative and store in the refrigerator.
4. Additional materials: electrophoresis equipment, saline, punchers, micro-injectors, and glass slides.
**Experimental Procedure**
1. Prepare a 1% to 1.5% agar gel plate using a barbiturate buffer (pH 8.6, 0.05 M), with a thickness of 2–3 mm.
2. Once the agar is cooled, create two rows of small holes, each with a diameter of 0.3–0.6 cm and spaced 0.4–1.0 cm apart. Remove the agar from the center of each hole to 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 plate under a light source against a dark background. A clear, dense white precipitate line indicates a positive reaction. If the line is not clear, place the agar plate in a humid chamber at 37°C for several hours or overnight in the electrophoresis tank.
**Precautions**
1. The concentration ratio of antigen to antibody must be appropriate. If the ratio is incorrect, no visible precipitate line may form. To ensure accuracy, especially when using expensive sera, test different dilutions of each sample.
2. To avoid false positives, include a control antigen next to the test antigen. If the sample's antigen merges with the precipitation line formed by the control antigen, it confirms the specificity of the antigen.
3. Electroosmotic action is essential in convective immunoelectrophoresis. Poor-quality agar may cause excessive electroosmosis, leading to non-specific reactions. In some cases, agarose cannot be used because it lacks sufficient electroosmotic properties.
4. If the antigen and antibody have similar charges or migration patterns, they may move in the same direction during electrophoresis, making it impossible to detect them using this method.
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