When FITC reacts with antibody protein in alkaline solution, the r-amino group of lysine on the protein is mainly combined with the thiocarbmide bond of fluorescein to form FITC-protein conjugate, that is, fluorescent antibody or fluorescent conjugate . There are 86 lysine residues in an IgG molecule, which can generally bind to 15 to 20 at most. An IgG molecule can bind to 2 to 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 the conditions.
1. Marsshall method
(1) Materials Antibody globulin solution, 0.5mol/L (pH9.0) carbonate buffer, sterile saline, fluorescein isothiocyanate, 1% thimerosal aqueous solution, 50ml small beaker, 4°C refrigerator, Electromagnetic stirrer, dialysis bag, glass rod, 0.01mol/LPBS with pH 7.2 or 3.0, etc.
(2) Methods and steps
①Preparation of antibodies Take an appropriate amount of globulin solution of known concentration in a beaker, add human physiological saline and carbonate buffer, so that the final concentration of immunoglobulin is 20mg/ml, and the volume of carbonate buffer is 20% of the total volume. 1/10, mix evenly, and place the calcined on the electromagnetic stirrer (the speed is appropriate so that no foaming is appropriate) for 5-10 minutes.
② Preparation of fluorescein According to the total amount of protein to be labeled, add 0.01 mg of fluorescein per mg of immunoglobulin, and accurately weigh the required fluorescein isothiocyanate powder with an analytical balance. The following formulas can also be used to calculate the amount of immunoglobulin and fluorescein, and the amount of buffer to be added.
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 less 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. 0.5mol/L (pH9.5) carbonate buffer D/10=Fml.
f. PBS amount 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 the constant, mg; positive is the amount of fluorescein, mg; F is the volume of the carbonate buffer , ml; G is the volume of PBS, ml.
③ Combine (or label), gradually add the weighed fluorescent dye to the globulin solution while stirring, to avoid sticking the fluorescein to the wall of the flask (the addition will be completed within about 5-10 minutes). After adding, continue stirring for 12 hours in the dark about. The protein solution should be kept at about 4°C during the binding period, so the beaker and stirrer should be moved to a 4°C refrigerator.
④ After the dialysis is completed, centrifuge the labeled globulin solution (2500r/min) for 20min, remove a small amount of sediment, put it in a dialysis bag, put it in a beaker, and dialyze it with pH 8.0 buffered saline (0-4 ~C) overnight.
⑤ Passing the column Take the marker that has been dialyzed overnight, pass through Sephadex G-25 or G-50 column, separate free fluorescein, and collect the labeled fluorescent antibody for identification. Eluent: 0.01 mol/L phosphate buffer (pH 7.2); filtration volume: 12 ml of labeled global protein solution (without dialysis before filtration); collection volume: 20 ml (diluted about 1.7 times).
2. 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 pipette, sterile pipette and capillary dropper, beaker (500ml) dialysis bag, etc.
(2) Methods and steps
①Antibody preparation Dilute the globulin solution to a concentration of 30-40mg/ml with pH8.0 phosphate buffered saline at o-4-C, put it in a 25ml beaker, and place it in an ice bath.
② Fluorescent pigment preparation Calculated by adding 0.01 rug of fluorescein per mg of immunoglobulin, weigh the required fluorescein, and dissolve it with 3% sodium carbonate aqueous solution.
③ Mix the prepared antibody and the fluorescent dye solution in equal amounts, stir well, and combine in a refrigerator of o～4～C (preferably keep stirring on a magnetic stirrer) for 18～24h.
④ The methods of dialysis and column chromatography are the same as the Marsshall method.
3. Improvement method
(1) Reagents and materials
①In the preparation method of 0.01mol/L (pH7.2) PBS, calibrate the pH to 7.2. NaCi 18g, Na2HP04 1.15g, dissolved in 2000ml distilled water
② 0.5mol/L (pH9.0) carbonate buffer solution: Take 10ml of 0.5mol/LNazCOs (5.3%) and add 90ml of 0.5mol/LNaHC03 (4.2%), after mixing, calibrate the pH to 9.0.
③The preparation method of 3% sodium carbonate aqueous solution Weigh L 5g of anhydrous sodium carbonate and fully dissolve it in 50ml of sterilized distilled water.
④Other reagents and materials 1% sodium azide, centrifuge and centrifuge tube, beaker (25m1), stirrer, sterile pipette and capillary dropper, beaker (500m1) dialysis bag, etc.
(2) Methods and steps
①Take high titer anti-human globulin rabbit immune serum, separate the globulin, and dilute it with saline (0.15mol/LNaCl) and buffer (0.5mol/L NaHC03-Na2C03, pH9.0) so that each milliliter contains 10 mg of protein , the buffer is 10% of the total volume, and the temperature is lowered to 4 °C.
②Add isothiocyanate (FITC) fluorescein [protein: fluorescein = (50-80) mg’lmg], at 0 ~ 4 ~ C
Under electromagnetic stirring for 12 ~ 14h.
③ Then use half-saturated ammonium sulfate to precipitate and separate the labeled globulin to remove unbound fluorescein, and then dialyze with buffered saline to remove ammonium sulfate (test with Nessler reagent until the saline dialyzed overnight is free of ammonia ions and fluorescent pigments) .
④Add the prepared fluorescent antibody to sodium azide o. 01%, packed in 1ml ampoules, or freeze-dried, stored in the refrigerator (4°C) for more than half a year, and stored at -20°C for more than 2 years.
4. dialysis labeling
This method is suitable for fluorescein labeling of a small amount of antibody, which is simple and has less non-specific staining.
(1) Reagents and materials Reagents and materials are the same as the improved method.
(2) Methods and steps
① Use 0.025mol/L carbonate buffer pH 9.0 to dilute the immunoglobulin to be labeled to a concentration of 1% and put it into a dialysis bag.
② Use the same buffer to prepare a 0.1 mg/ml solution of FITC, put the FITC diluent in a cylindrical container at 10 times the volume of the 10 mg/ml globulin solution, and immerse the dialysis bag in the FITC solution.
③ The top of the container is tightly capped, and a stirring rod is placed at the bottom, and dialyzed for 24h under electromagnetic stirring at 4°C. Take out the labeling solution in the dialysis bag, immediately use Sephadex G50 gel filtration to remove free fluorescein, aliquot and store at 4°C.