First, the principle
Although the labeled antibody method has many advantages, there are some problems that are difficult to overcome, such as increased molecular weight of the labeled antibody, weakening of the penetration of the cell membrane and tissue; chemical crosslinking reaction affects the activity of the antibody and the enzyme; The unlabeled antibody in the substance can compete with the antigen for binding, affecting the sensitivity of the detection method, and the like. The unlabeled antibody method can avoid the above-mentioned adverse factors, and the detection of the antigen in the tissue by the immunological reaction of a system of non-labeled antibodies. The unlabeled antibody method can be further divided into two methods of displaying with a marker and displaying with a different marker. The former utilizes a genetic engineering method to prepare F(ab')2 of a bispecific antibody, one Fab fragment binds to an antigen in a specimen, and one Fab is a binding fragment of an antibody protein. After the antibody interacts with the sample, ferritin is added to the antibody to display the site of the antigen in the tissue. The latter was negatively stained with tungsten phosphate and observed by direct electron microscopy.
Second, materials and reagents
1. The virus materials to be tested are feces, tracheal secretions, cerebrospinal fluid, tissue culture fluid, and the like.
2. High speed centrifuge
3. Known antibody
4. Phosphotungstic acid
5. Agarose
6. other
Third, the operation method
1. Classical method
1 Mix 0.9 ml of the tested virus material and add 0.1 ml of specific immune serum diluted 1..5 to 1..10.
2 At 37 ° C for 1 h or 37 ° C for 1 h and then at 4 ° C overnight.
3 Centrifuge at 17 000 r/min to 23 000 r/min for 90 min.
4 Aspirate the supernatant and place the centrifuge tube on the filter paper to remove any residual liquid.
5 Add a small amount of H 2 O in the precipitate, and dye 20Sec～30Sec with 3% phosphotungstic acid (pH6.0), add it to the 400 mesh copper mesh Fomnvar film, and gently suck it from the edge of the copper mesh with filter paper. Excess negative dye solution.
6 4×10 4 times observed under transmission electron microscope.
2. Fast method (agar diffusion method)
1 Preparation of immune complexes.
2 Prepare 1% agarose in normal saline or pH 7.2 barbital buffer, cast hot on a clean glass slide, 3ml each, and after cooling and solidification, place the 4mm diameter glass beads on the gel surface at the same distance. After granules, 2 ml to 3 ml of 1% agarose was poured on the gel surface, and after cooling and solidification, the glass beads were taken out to form a concave hole in the gel.
3 Cut the ordinary filter paper into 2×3cm size and 3-4 layers overlap. Use a knife to cut the agarose with a small hole into small pieces, each with a concave hole, placed flat on the filter paper.
4 Add the prepared virus-containing immune complex suspension to the well.
5 Place the Fomnvar membrane of the electron microscope carrier upside down on the droplets. Due to the water absorption effect of the agarose, after 20 minutes to 30 minutes, the water of the droplets can be sucked dry, and the concentrated immune complex adheres to the mesh film.
6 Add 3% phosphotungstic acid negative dye 20Sec to the net film, and excess negative dye solution is gently sucked on the edge of the net with filter paper.
7 Observed under a transmission electron microscope 4 × 10 4 times.
Fourth, the result is judged
Directly observe the morphology of the virions. Due to the centrifugation concentration treatment and the addition of specific antibodies, the sensitivity of the observation is greatly improved.
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