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Indirect immunofluorescence method for antibody detection

2021-02-20
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What is indirect immunofluorescence method for antibody detection?

1. Basic principles:

The staining procedure is divided into two steps. In the first step, an unknown unlabeled antibody (specimen to be tested) is added to the known antigen sample, and the sample is incubated at 37°C for 30 min in a humidified box to fully bind the antigen and antibody, and then washed to remove Unbound antibody. The second step is to add fluorescently labeled anti-globulin antibody or anti-IgG. IgM antibody. If an antigen-antibody reaction occurs in the first step, the labeled anti-globulin antibody will further bind to the antibody that has bound the antigen, so that the unknown antibody can be identified.

Indirect immunofluorescence method

2. Reagents and instruments:

1. Phosphate buffered saline (PBS): 0.01 mol/L, pH 7.4.

2. Buffered glycerol: 9 parts of analytically pure non-fluorescent glycerol + pH 9.2, 1 part of 0.2 M carbonate buffer.

3. Fluorescence-labeled anti-human globulin antibody: Dilute with 0.01 mol/L, pH7.4 PBS.

4. Three enamel buckets (with 0.01 mol/L, pH 7.4 PBS 1500 ml).

5. An enamel box with lid (with a layer of soaked gauze pad).

6. Fluorescence microscope.

7. Slide rack.

8. Filter paper.

9. 37℃ incubator, etc.

3. Experimental steps:

1. Add 0.01 mol/L, pH 7.4 PBS to the known antigen specimen sheet, and discard it after 10 minutes to keep the specimen sheet at a certain humidity.

2. Add 0.01 mol/L, pH 7.4 PBS appropriately diluted test antibody specimens to cover the known antigen specimen slides. Place the slides in an enamel box with a lid and keep them warm at 37°C for 30 min.

3. Take out the slides and place them on the slide rack. First rinse with 0.01 mol/L, pH 7.4 PBS for 1-2 times, and then sequentially immerse them in three cylinders of 0.01 mol/L, pH 7.4 PBS. The cylinder oscillates for 5 minutes from time to time.

4. Take out the glass slide and absorb the excess water with filter paper, but without drying the specimen, add a drop of fluorescently labeled anti-human globulin antibody with a certain dilution.

5. Place the slide flat in an enamel box with a lid, and keep it warm at 37°C for 30 min.

6. Repeat operation 3.

7. Take out the glass slide, absorb the excess water with filter paper, add a drop of buffer glycerin, and cover it with a cover glass.

8. Observe under the high power field of the fluorescence microscope, and the result judgment is the same as the direct method.

4. matters needing attention:

1. Observation is generally completed within 1 h after fluorescent staining, or stored at 4°C for 4 h. If the time is too long, the fluorescence will be weakened.

2. For each test, the following three types of controls need to be set:

(1) Positive control: positive serum + fluorescent marker.

(2) Negative control: negative serum + fluorescent marker.

(3) Fluorescent marker control: PBS + fluorescent marker.

3. The known antigen specimens should always be kept moist during each step of the operation to avoid drying.

4. The dripped antibody specimens or fluorescent markers to be tested should always be kept on the known antigen specimen slides to avoid fluid loss due to uneven placement, resulting in non-specific fluorescent staining

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