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Fluorescein FITC-labeled antibody method

2021-02-19
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Immunofluorescence technology combines fluorescein such as fluorescein isothiocyanate (FITC) with the corresponding antibody (or antigen) in a chemical way, and combines the fluorescent-labeled antibody (or antigen) with the corresponding antigen in the specimen to form a fluorescent-labeled antibody -Antigen complex, observe with a fluorescence microscope. The presence of fluorescence under the microscope indicates the presence of an antigen-antibody complex, and the presence of another unknown antigen (or antibody) can be inferred based on the known antibody (or antigen).

Fluorescein FITC-labeled antibody method
Fluorescein FITC-labeled antibody method

Fluorescein FITC-labeled antibody technology principle

  1. Direct immunofluorescence method “Direct immunofluorescence method uses specific antibodies labeled with fluorescent pigments to directly bind to corresponding antigens to identify unknown antigens. This method has the advantages of simple operation, short duration, and high specificity, but it also has disadvantages such as inability to detect antibodies and poor sensitivity.

  2. Indirect immunofluorescence method “Indirect immunofluorescence method uses fluorescein-labeled anti-globulin antibody. After the antibody binds to the corresponding antigen, the fluorescent-labeled anti-globulin antibody interacts with the bound antibody, thereby inferring the presence of the antigen or antibody. Indirect fluorescence technology can detect unknown antibodies with known antigens, and can also detect unknown antigens with known antibodies.

Now take the detection of virus antibodies (IgG) as an example

When FITC reacts with an antibody protein in an alkaline solution, the r amino group of lysine on the protein is combined with the thiocarbmide bond of fluorescein to form FITC-protein conjugates, namely fluorescent antibodies or fluorescent conjugates . There are 86 lysine residues in an IgG molecule, and generally can bind 15-20 at most, and an IgG molecule can bind 2-8 molecules of FITC. The reaction formula is as follows:

FITC-N=C=S + N-H2-Protein → FITC-NS-C-N-H2-Protein

The Marsshall (1958) method is commonly used to label fluorescent antibodies, and the labeling method of Chadwick et al. or the dialysis labeling method of Clark et al. (1963) can also be used according to conditions.

Marsshall method

(1) Material

Antibody globulin solution, 0.5mol/L (pH9.0) carbonate buffer, sterile physiological saline, fluorescein isothiocyanate, 1% thimerosal aqueous solution, 50ml small beaker, 4℃ refrigerator, electromagnetic stirrer, Dialysis bag, glass rod, 0.01mol/LPBS with pH 7.2 or 3.0, etc.

(2) Methods and steps

①Preparation of antibody Take a proper amount of globulin solution of known concentration in a beaker, and add human saline and carbonate buffer to make the final immunoglobulin concentration 20mg/ml, and the carbonate buffer capacity is the total amount 1/10, mix well, put the burning ball on the electromagnetic stirrer (the speed is appropriate to avoid foam) for 5-10min.

② Preparation of fluorescein According to the total amount of protein to be labeled, add 0.01 mg of fluorescent pigment per mg of immunoglobulin, and accurately weigh the required fluorescein isothiocyanate powder with an analytical balance. The following formula can also be used to calculate the amount of immunoglobulin and fluorescein, and the amount of buffer to be added can also be calculated.

a. Protein solution: content Amg/m1; volume Bml.

b. Total protein amount (AXB)=Crag.

c. C/20~C/10=Dmg (if the protein content is lower than 20mg/ml, use C/10; if it is higher than 20mg/ml, use C/20).

d. The amount of fluorescein FITC: (1/50~2/100) XC=Emg.

e. It is 0.5 mol/L (pH 9.5) carbonate buffer solution D/10=Fml.

f. The amount of PBS D-(B+F)=Gml.

Note: A is the protein content, mg/ml; B is the volume of the protein solution; C is the total protein, mg; D is a constant, mg; positive is the amount of fluorescein, mg; F is the volume of carbonate buffer , Ml; G is the volume of PBS, ml.

③Combine (or label) Gradually add the weighed fluorescent pigment into the globulin solution while stirring, avoid sticking the fluorescein to the wall of the flask (approximately within 5-10 minutes), after adding, continue to stir for 12h in the dark about. During the binding period, the protein solution should be kept at about 4°C, so move the beaker and stirrer into a 4°C refrigerator.

④After dialysis and binding, centrifuge the labeled globulin solution (2500r/min) for 20rains to remove a small amount of sediment, put it into a dialysis bag, and then place it in a beaker, dialyzed with pH 8.0 buffered saline (0~4 ~C) Overnight.

⑤Passing the column Take the dialysis overnight marker, pass it through Sephadex G-25 or G-50 column, separate free fluorescein, and collect the labeled fluorescent antibody for identification. Eluent: 0.01mol/L phosphate buffer (pH 7.2); filtration volume: 12ml labeled global protein solution (not dialyzed before filtration); collection volume: 20ml (diluted by about 1.7 times).

Chadwick method

(1) Reagents and materials

Antibody globulin solution, fluorescein isothiocyanate, 3% sodium carbonate aqueous solution, 0.01mol/L pH8.0PBS, 1% thimerosal, centrifuge and centrifuge tube, beaker (25ml) stirrer, sterile straw, sterile Pipette, capillary dropper, beaker (500ml) dialysis bag, etc.

(2) Methods and steps

① Antibody preparation Dilute the globulin solution to a concentration of 30-40 mg/ml with pH 8.0 phosphate buffered saline at o~4~C, put it in a 25ml beaker, and put it in an ice tank.

② Preparation of Fluorescent Pigment Calculated by adding fluorescein 0.01rug per mg of immunoglobulin, weigh the required fluorescein and dissolve it with 3% sodium carbonate aqueous solution.

③Mix the prepared antibody and the fluorescent pigment solution in equal amounts, stir thoroughly, and combine in a refrigerator at o~4~C (preferably keep stirring on a magnetic stirrer) for 18~24h.

④The dialysis and column chromatography methods are the same as the Marsshall method.

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