The immunoferritin technique involves labeling antibodies with ferritin, allowing for the detection of specific antigens through electron microscopy. The process begins by conjugating ferritin to the antibody, which then binds to the target antigen. Under an electron microscope, the presence of ferritin indicates the location of the antigen.
**Materials and Reagents**
1. Horse spleen ferritin
2. Ammonium sulfate
3. Cadmium sulfate
4. Metaxylene (X)
5. Dl-isocyanate (XC)
6. 0.30 M pH 9.5 borate buffer
7. 0.1 M ammonium sulfate solution
8. 0.05 M pH 7.4 PBS
XC is prepared as a 1% solution in 0.30 M pH 9.5 borate buffer. It is crucial that the water and containers used are thoroughly cleaned and stored at 4°C for several days to prevent precipitation. If XC forms a polymer, it should be reconstituted before use.
**Procedure**
**1. Ferritin Extraction**
(1) Prepare a 2% ammonium sulfate solution and adjust the pH to 5.85 using 1 M NaOH or HCl. Dissolve 1 g of ferritin in 100 ml of this solution.
(2) Add 20% cadmium sulfate to achieve a final concentration of 5%, mix well, and incubate at 4°C overnight.
(3) Centrifuge at 4°C for 1 hour at 1,500 g. Remove the supernatant and add 2% ammonium sulfate to bring the volume back to 100 ml. Centrifuge again to remove impurities.
(4) Repeat step (2) with the supernatant, centrifuge, and discard the supernatant.
(5) Examine the precipitate under a microscope. It should appear as hexagonal, yellow-brown crystals with a double-four-point structure. If not, repeat steps (2)–(4).
(6) Dissolve the precipitate in a small amount of distilled water, add 50% saturated ammonium sulfate, precipitate, and centrifuge.
(7) Repeat step (6) once more.
(8) Redissolve the pellet in distilled water and dialyze against normal water for 24 hours, then against 0.05 M pH 7.5 PBS for another 24 hours.
(9) Centrifuge at 100,000 rpm for 2 hours, remove the upper supernatant (about 3/4), and store at 4°C overnight.
(10) Filter through a 0.45 μm membrane to obtain a concentration between 65–75 mg/ml. Store at 4°C without lyophilization to preserve the ferritin structure.
**2. Ferritin-Antibody Cross-Linking**
(1) Dilute ferritin to 20–25 mg/ml in 0.3 M pH 9.5 borate buffer.
(2) Add XC at a ratio of 1:1000 (w/w), stir at room temperature for 45 minutes, and centrifuge to remove any aggregates.
(3) Prepare purified IgG at 5 mg/ml in the same buffer. Mix IgG and ferritin-XC at a 1:4 (v/v) ratio and incubate at 4°C for 48 hours.
(4) Dialyze overnight against 0.1 M ammonium carbonate to remove excess isocyanate, then dialyze against 0.05 M pH 7.5 PBS to restore physiological pH.
(5) Centrifuge at 100,000 g for 5 hours. Remove the supernatant, resuspend the pellet in 0.05 M PBS, and centrifuge again to eliminate unbound IgG.
(6) Evaluate the conjugate’s specificity, immunological activity, and labeling efficiency using serological and immunological methods. With proper execution, the results should be reliable.
**3. Sample Treatment with Ferritin-Antibody Conjugate**
(1) Fix the specimen in 5% formalin in PBS (pH 7.2) at 4°C for 40–60 minutes.
(2) Wash with cold PBS and centrifuge.
(3) For tissue blocks, cut into smaller pieces under a dissecting microscope, place in a tube, and incubate with the conjugate at room temperature for 20 minutes, shaking occasionally.
(4) Wash three times with cold PBS and centrifuge.
(5) Fix the pellet in 2.5% glutaraldehyde for 20 minutes, then wash with PBS.
(6) Fix with citric acid and dehydrate. Proceed with ultrathin sectioning and staining for ferritin-antibody complexes.
For cultured cells:
(1) Fix in 1% formalin PBS at 4°C.
(2) Wash via centrifugation.
(3) Resuspend in 0.5 ml of 30% BSA in PBS inside a plastic dialysis bag. Place the bag on absorbent powder until the BSA gels. Transfer the bag to 2% pentane and fix with 0.1 M PBS (pH 7.5) for 3 hours.
(4) Cut into small pieces and wash with PBS.
(5) Dry in a desiccator with silica gel.
(6) Embed, section, and collect slices on a film coated with 4% BSA-PBS to reduce non-specific binding.
(7) Apply a drop of ferritin-antibody conjugate onto the grid.
(8) After 5 minutes, float the grid on PBS surface, face down, to remove excess conjugate.
(9) Stain with uranyl acetate or lead hydroxide after drying.
(10) Wash, dry, and observe under an electron microscope.
**Results Interpretation**
When a known control is available, any black particle visible under the electron microscope indicates the presence of the antigen. A positive result (+) is confirmed, while no such particle suggests a negative result (-).
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