The immunoferritin technique involves labeling antibodies with ferritin, which allows for the visualization of antigens through electron microscopy. The process relies on the binding between the ferritin-labeled antibody and the target antigen, making it possible to locate the antigen within the sample.
**Materials and Reagents**
1. Horse spleen ferritin
2. Ammonium sulfate
3. Cadmium sulfate
4. Metaxylene
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
The XC solution is prepared as a 1% concentration in 0.30 M pH 9.5 borate buffer. It's essential to use clean water and containers, and the solution should be stored at 4°C for several days before use. If precipitation occurs, the solution must be reconstituted.
**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 thoroughly, and incubate at 4°C overnight.
(3) Centrifuge at 1,500 g for 1 hour at 4°C, then discard the supernatant.
(4) Resuspend the pellet in 100 ml of 2% ammonium sulfate, mix, and centrifuge again to remove impurities.
(5) Repeat step (2), centrifuge, and discard the supernatant.
(6) Examine the sediment under a microscope. It should display hexagonal, yellow-brown crystals with a double-four-point structure. If not, repeat the steps.
(7) Dissolve the sediment in a small amount of distilled water, add 50% saturated ammonium sulfate to precipitate, centrifuge, and remove the supernatant.
(8) Repeat step (7) once more.
(9) Dissolve the pellet in distilled water and dialyze against 0.05 M pH 7.5 PBS for 24 hours.
(10) Centrifuge at 100,000 rpm for 2 hours, remove the upper supernatant, and store at 4°C.
(11) Filter the solution through a 0.45 μm membrane to obtain a concentration of 65–75 mg/ml. Store without lyophilization to preserve the structure of ferritin.
**2. Cross-linking of Ferritin with Antibody**
(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 precipitates.
(3) Prepare purified IgG at 5 mg/ml in the same buffer. Mix with ferritin-XC solution in a 1:4 (v/v) ratio and incubate at 4°C for 48 hours.
(4) Dialyze the mixture overnight in 0.1 M ammonium carbonate to remove excess XC, then dialyze in 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 PBS, and centrifuge again to eliminate unbound IgG.
(6) Assess the conjugate’s specificity, immunological activity, and labeling efficiency using serological and immunological methods. With proper execution, the results are typically reliable.
**3. Sample Treatment with Ferritin-Antibody Conjugate**
(1) Fix the specimen in 5% formalin (pH 7.2) in PBS 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 with 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 in PBS at 4°C.
(2) Wash by centrifugation.
(3) Resuspend in 0.5 ml of 30% BSA-PBS in a dialysis bag, place on desiccant powder until BSA gels, then transfer to 2% pentane dialdehyde 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 water surface 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.
(10) Wash, dry, and observe under an electron microscope.
**Results Interpretation**
Under controlled conditions, the presence of black iron particles indicates the presence of the antigen. A positive result is denoted as (+), while no visible particle signifies a negative result (-).
Water Flosser With Big Water Tank
Teeth flusher,Dental flusher,Oral irrigator,Water flosser,Teeth cleaning device
Shenzhen Shengkang Electronic Technology Co.,Ltd , https://www.shk-beauty.com