Fluorescent antibody technology specific requirements Appreciation

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Appreciation of specific requirements for fluorescent antibody technology

Fluorescent antibody technology is a technique for antigen localization by fluorescently labeled antibodies. Compared with other serological methods for identifying bacteria, the technology has the characteristics of high speed, simple operation and high sensitivity. It has been used as a test for bacteria, viruses and parasites and for the diagnosis of autoimmune diseases in clinical tests.
(a) fluorescein labeling of antibodies

Antibodies for labeling are required to be highly specific and highly affinitive. The antisera used should not contain antibodies against normal tissues in the specimen. It is generally necessary to purify and extract the igg and then mark it. The fluorescein as a label should meet the following requirements:

1 should have a chemical group that can form a covalent bond with the protein molecule, and it is not easy to dissociate after binding with the protein, and the unbound pigment and its degradation product are easy to remove.

2 The fluorescence efficiency is high, and after binding with the protein, the fluorescence efficiency can still be maintained.

3 Fluorescent color contrasts with the color of the background tissue.

4 combined with protein does not affect the original biochemical and immunological properties of the protein.

5 marking method is simple, safe and non-toxic.

6 The combination with protein is stable and easy to store.

Common methods for labeling proteins include stirring and dialysis. Taking the fitc label as an example, the stirring labeling method is: first, the protein solution to be labeled is equilibrated with 0.5 ml/l ph 9.0 carbonate buffer, and then the fitc solution is added dropwise under magnetic stirring, and stirring is continued at room temperature for 4~. After 6 h, centrifugation, the supernatant is the marker. This method is suitable for labeling antibody solutions with large volume and high protein content. The advantage is that the labeling time is short and the amount of fluorescein is small. However, there are many influencing factors in this method. If improperly handled, it will cause more intense non-specific fluorescent staining.

The dialysis method is suitable for labeling antibody solutions with a small amount of sample and low protein content. This method is more uniform and non-specific staining is also lower. The method is as follows: firstly, the protein solution to be labeled is placed in a dialysis bag, and placed in a 0.01 mol/l ph 9.4 carbonate buffer solution containing fitc for overnight reaction, and then the free pigment is removed by pbs dialysis. Centrifuge at low speed and take the supernatant.

After labeling is complete, the labeled antibody should also be further purified to remove unbound free fluorescein and antibodies that bind too much to fluorescein. The purification method may be a dialysis method or a chromatographic separation method.

(II) Identification of fluorescent antibodies

Fluorescent antibodies should be identified prior to use. The identification finger includes the potency and the binding ratio of fluorescein to protein. The antibody titer can be titrated by the agar double diffusion method, and the titer is more than 1:16. The basic method for the determination and calculation of the ratio of fluorescein to protein (f/p) is to dilute the prepared fluorescent antibody to a2801≈1.0, and to measure the specific absorption peak of a280 (protein-specific absorption peak) and labeled fluorescein, respectively. Calculated according to the formula.

Fluorescent antibody staining

A suitably diluted fluorescent antibody is added to the fixed specimen. In the humid chamber, it is incubated at a certain temperature for a certain period of time, generally 25 to 37 ° C for 30 min, and the detection of thermolabile antigen is preferably 4 ° C overnight. Wash thoroughly with pbs and dry.

Main factors affecting the mark

Temperature, time, pH and labeling factors. The temperature is low, the marking time is long, the temperature is high, and the marking time should be short. FITC0-4 °C is preferably 6-12h, 20-25 °C is preferably 1-2h, and 37 °C is 30-45min. When the pH is low, the label is slow, the pH is high (greater than 10), the antibody is variability, and the pH is preferably 9.0-9.5. The antibody protein content is low and the labeling is slow, preferably 20-25 mg protein per ml. As for the labeling method, each has its own characteristics, but it cannot remove the non-specific fluorescent staining factors. Dialysis is suitable for small volume markings.