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Direct Fluorescent Antibody Test

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Fluorescent Antibody Test is based on the principle of antigen-antibody reaction. Firstly, the known antigen or antibody is labeled with fluorescein to make fluorescent antibody, and then this fluorescent antibody (or antigen) is used as a probe to detect the corresponding antigen in the tissue or cell (Or antibody). The antigen-antibody complex formed in the tissue or cell contains labeled fluorescein. Observe the specimen with a fluorescence microscope. The fluorescein is irradiated by external excitation light to produce bright fluorescence (yellow-green or orange-red), and you can see where the fluorescence is. Organize cells to determine the nature and location of the antigen or antibody, and use quantitative techniques to determine the content.

There are two types of immunofluorescence: Direct Fluorescent Antibody Test and Indirect Fluorescent Antibody Test.

(1) Direct Fluorescent Antibody Test: Add the labeled specific fluorescent antibody directly to the antigen sample, stain it for a certain temperature and time, wash off the excess fluorescent antibody that has not participated in the reaction, and dry it at room temperature before mounting and microscopic inspection .

(2) Indirect Fluorescent Antibody Test: If checking for unknown antigens, first use a known unlabeled specific antibody (antibody) to react with the antigen sample, wash off the unreacted antibody with water, and then use the labeled anti-antibody (second antibody) to react with the antigen The specimen is reacted to form an antigen-antibody-antibody complex, and then washed with water to remove unreacted labeled antibodies, dried, mounted and then examined under a microscope. If the unknown antibody is checked, it indicates that the antigen sample is known, the serum to be tested is antibody, and the antigen check in other steps is the same. The labeled anti-antibody is an anti-globulin antibody, which has the same specificity as serum globulin.

Direct Fluorescent Antibody Test combines immunological methods (antigen-antibody specific binding) with fluorescent labeling technology to study the distribution of specific protein antigens in cells. Because the fluorescence emitted by fluorescein can be detected under a fluorescence microscope, the antigen can be located on the cell. The method of using fluorescent antibody to trace or check the corresponding antigen is called the fluorescent antibody method; the method of using known fluorescent antigen markers to trace or check the corresponding antibody is called the fluorescent antigen method. These two methods are collectively referred to as immunofluorescence technology, and the fluorescent antibody method More commonly used.

Direct Fluorescent Antibody Test

1. Direct Fluorescent Antibody Test Basic Principles

Fluorescein is labeled on the corresponding antibody and reacts directly with the corresponding antigen. Its advantages are simple method, high specificity, less non-specific fluorescent staining, and relatively large amount of labeled antibody.

2. Direct Fluorescent Antibody Test Reagents and Instruments

  1, phosphate buffered saline (PBS): 0.01mol/L, pH7.4;

  2, fluorescently labeled antibody solution: diluted with 0.01mol/L, pH7.4 PBS;

  3. Buffered glycerol: 9 parts of analytically pure non-fluorescent glycerol + 1 part of pH9.2 0.2M carbonate buffer solution;

  4. Three enamel buckets (with 0.01mol/L, pH7.4 PBS1500ml);

   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. Direct Fluorescent Antibody Test experimental steps

1. Add 0.01 mol/L, pH 7.4 PBS to the specimens to be tested, and discard them after 10 minutes to keep the specimens at a certain humidity;

2. Add appropriately diluted fluorescently labeled antibody solution dropwise to completely cover the specimen, place it in an enamel box with a lid, and keep it warm for a certain period of time (reference: 30min);

3. Take out the slides and place them on the slide rack, rinse them with 0.01mol/L, pH7.4 PBS, and then sequentially soak them in 0.01mol/L, pH7.4 PBS three-cylinder, 3-5min per cylinder , Oscillate from time to time;

4. Take out the glass slide, absorb excess water with filter paper, but do not dry the specimen, add a drop of buffer glycerin and cover it with a cover glass;   

5. Immediately observe with a fluorescence microscope. Observe the specific fluorescence intensity of the specimen. Generally, “+” can be used to indicate: (-) no fluorescence; (±) extremely weak and suspicious fluorescence; (+) fluorescence is weak but clearly visible; (++) fluorescence is bright; (+) ++–++++) Fluorescence shines. The specific fluorescence staining intensity of the specimen to be tested is above “++”, and various controls show (±) or (-), it can be judged as positive.   

4. Attention

  1. For the dilution of fluorescently labeled antibodies, ensure that the protein of the antibody has a certain concentration, generally between 1:20-100. It is necessary to find the best gradient and establish the best dilution ratio. The antibody concentration is too low. It will cause the fluorescence to be too weak and affect the observation of the results;

  2. The temperature and time of staining need to be changed according to various specimens and antigens. The staining time can range from 10 minutes to several hours, and 30 minutes is generally sufficient. The staining temperature is usually room temperature (about 25°C), higher than 37°C can enhance the staining effect, but for heat-labile antigens (such as Japanese encephalitis virus), a low temperature of 0-2°C can be used to extend the staining time. Staining at low temperature overnight is much better than 30min at 37℃;   

  3. In order to ensure the correctness of fluorescent staining, the following controls should be set during the first experiment to eliminate the interference of some non-specific fluorescent staining:    (1) Specimen autofluorescence control: add 1-2 drops of 0.01mol/L, pH7.4 PBS to the specimen;  (2) Specificity control (inhibition test): Specimen plus unlabeled specific antibody, plus fluorescently labeled specific antibody.   

  4. Generally, if specimens are irradiated under a high-pressure lamp for more than 3 minutes, the fluorescence will weaken. Specimens stained with fluorescence should be observed on the same day. As time goes by, the fluorescence intensity will gradually decrease.

Related Articles:

Indirect Fluorescent Antibody Test

Fluorescent Antibody Technique

Fluorescent Antibody Technique (Direct, Indirect)

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