Production of FITC conjugate

Transcription

1 Production of FITC conjugate <Before labeling> # If you can purify IgG utilising Protein G column or by caprylic acid + ammonium sulfate method instead of the following precipitation method (Step A), the final purification by DEAE cellulose (Step E, Section II-1, 2 & 3) can be omitted!! The protein concentration of the purified IgG should be over 12 mg/ml. A. Purification of antibody by ammonium sulfate precipitation 1. Dilute serum (50 ml) with equal volume of PBS (50 ml). 2. Precipitation of antibodies (1/2 saturation): While the diluted serum (100 ml) being stirring gently, slowly add equal volume (100 ml) of saturated ammonium sulfate solution by dropwise. The saturated ammonium sulfate should be added slowly to ensure that the local concentration does not exceed the desired one. This step brings the final concentration to 1/2 saturation. # Saturated ammonium sulfate solution Dissoive excess amount of ammonium sulfate (76 g or more/1oo ml of distilled water) and boil or autoclave. Keep at room temperature until cooled and adjust the ph to 7.0 with ammonia solution. 3. Keep at room temperature for 1 hour. 4. Centrifugation at 5,000 rpm for 20 minutes or 8,000 rpm for 15 minutes. 5. Carefully discard the supernatant. Suspend the precipitate in PBS (half volume of the diluted serum, 50 ml) completely. If time is not enough to complete the next step, the suspension can be kept in 4 C until next day. 6. Add 25 ml (half volume of the suspension) saturated ammonium sulfate same as in step 3. This step brings the final concentration to 1/3 51

2 saturation. 7. Keep at room temperature for 1 hour. 8. Centrifugation at 5,OOO rpm for 2O minutes or 8,000 rpm for 15 minutes. 9. Carefully discard the supernatant and resuspend the precipitated antibody in 15 ml PBS completely. 10. Transfer the antibody solution to dialysis tube and dialyse in PBS at 4 C for overnight, PBS should be changed and dialyse for further 4 hrs. 11. After dialysis, the antibody solution should be kept at -80 C unless utilised soon. <DAY 1> B. Preparation of Sephadex G-25 Prior to the labeling of antibody with FITC, swelling, washing and equilibrating gels, and setting up the columns should be completed. Use Sephadex G-25: Pharmacia, Code No or Add Sephadex G-25 in distilled water, boil for 30 minutes and keep in the room temperature overnight (25 g dry gel swells to about 100 ml). 2. Discard supernatant, add distilled water and mix. Repeat this procedure 5 or 6 times to remove the small particles of gels. 3. Load Sephadex G-25 gels into a column at 4 C and equilibrate gels with 3-5 litre of PB (5 mm, ph 8.4). <DAY 2> C. Labeling (coupling) of antibodies with FITC FITC: Cappel, Code No Centrifuge the antibody solution at 8,000 rpm for 1O minutes at 4 C and apply the supernatant for FITC labeling. If the supernatant is turbid, filtrate the solution using a 0.45 µm membrane filter. 52

3 2. Adjustment of protein concentration a) Measure the volume of antibody solution (A ml). b) Assay protein concentration (B mg/ml) by Coomassie method. c) Calculate the amount of total protein (C mg) = Volume of antibody solution X protein concentration (A ml X B mg/ml = C mg) d) Calculate the volume of 1% (= 10 mg/ml) protein solution (D ml) = Protein concentration/10 X volume of antibody solution (B/10 X A ml = D ml) e) Calculate the amount of FITC (X mg) = Amount of total protein 70 (C mg 70 = X mg) f) Calculate the amount of 0.5 M carbonate buffer (E ml) = Volume of 1% protein solution X 0.1 (D ml X 0.1 = E ml) g) Calculate the volume of 0.85% NaCl (F ml). = Volume of 1% protein solution - volume of antibody solution -volume of 0.5 M carbonate buffer (D ml - A ml - E ml = F ml) h) Prepare 1% protein solution = Antibody solution M carbonate buffer % NaCl (A ml + E ml + F ml = 1% protein solution for labeling) * For example: The volume of antibody solution is 6 ml (= A). The protein concentration is 16 mg/ml (= B). C = A X B = 6 X 16 = 96 mg D = B/10 X A = 16/10 X 6 = 1.6 X 6 = 9.6 ml (= C/10) X = C 70 = = 1.37 mg E = D X 0.1 = 9.6 X 0.1 = 0.96 ml = 960 µl F = D - A - E = = 2.64 ml 3. Labeling of antibody with FITC Add FITC (X mg) to 1% protein solution and leave for 6 hours at 4 C with gentle stirring. The reaction bottle should be covered with aluminium foil to avoid light. NOTE: If it is difficult to weigh a small amount of FITC, follow the next procedure. Place an empty sealed container on a balance and tare. Then, transfer a small amount of FITC powder into the container and weigh the contents. Dissolve the FITC in 1 ml of 0.5 M carbonate buffer and use immediately. In case that the contents (FITC) weigh 5.2 mg, add G ml (= X mg 5.2) of this FITC solution to the 1% protein solution and substract 53

4 G ml from the volume of 0.5 M carbonate buffer (E' ml = E ml - G ml). In the case of above example; G = X 5.2 = = ml E' = E - G = = 697 µl Therefore, 1% protein solution + FITC is going to be (A + E' + F + G) ml. D. Preparation of DEAE-cellulose gels 1. Swell ion-exchanger DEAE-cellulose (DE52: Whatman No ) in 500 ml of 0.2 M PB (ph 8.4) and keep at room temp. for 2 to 3 hours. 2. Carefully discard the supernatant and equilibrate in 5 mm PB (ph ). Repeat this procedure more than 5 times. 3. Load DE52 gels into a column at 4 C and equilibrate with 5 mm PB (ph ). E. Purification of labeled antibody I. Separation of unbound FITC from the conjugate by gel filtration using Sephadex G Carefully overlay the conjugate mixture (FITC-antibody solution) on the top of the gel (column) and allow the mixture to flow into the column until it just enters the column bed (gels). 2. Carefully add 5 mm PB (ph 8.4) to the top of the gel and connect to a buffer supply. 3. The antibody labeled with FITC elutes first as described below. 4. Discard the first 5 ml of the eluting yellow solution and collect the conjugate until its yellow colour becomes weak. Towards the end of the collection, check the ph regularly with indicator paper or a meter. Stop collecting when the ph starts to increase. NOTE: Do not collect the mixture of carbonate buffer plus NaCl because the ionic strength of the mixture is high and it interferes purification of labeled antibody using DE52 (next step). When carbonate buffer comes out, the ph goes up to 9. 54

5 Unbound (free) FITC Carbonate buffer + NaCl FITC-labeled antibody II. Purification of labeled antibody (FA) by ion-exchange chromatography using DE52 gel The antibodies (FA) are allowed to bind to DE52 gels and eluted by changing the ionic strength and ph of the buffer. 1. Adsorb (bind) labeled antibody (FA) into the column for adsorption to DE52 gel. Wash out the unbound materials by applying about 5-column volumes of 5 mm PB (ph ). If time is not enough to complete the next step, stop here and start elution next day (avoid the column to dry and keep at 4 C). <Day 3> 2. Elute labeled antibody (FA) with 0.1 M PB (ph 6.3) and collect the yellow-coloured solution. 3. Concentrate the FA by ultrafiltration or dialysis with carboxymethylcellulose (CMC), Sephadex G-25 or 20% polyethylene glycol. For example, pour the FA in dialysis tubing, lay it on a tray and cover with CMC powder (or polyethylene glycol). Leave at 4 C for several hours. 4. Dialyse the FA against PBS (10 mm PB plus 0.1 M NaCl) overnight. <Day 4> F. Adsorption of FA with liver powder Add liver powders to FA at a concentration of 1% and incubate for 55

6 30 minutes at 4 C. Centrifuge the mixture at 8,000 rpm for 15 minutes at 4 C and filtrate the supernatant with 0.45 µm membrane. Make aliquots and store at -80 C. Keep one aliquot at 4 C for titration and daily use. # Prepare liver powders as follows: 1) Prepare a homogenate (fine suspension) of liver in saline. One gram of tissue should be suspended in approximately 1 ml of saline. 2) Transfer the homogenate onto ice for 5 minutes. 3) Add 8 ml of acetone (-20 C) per 2 ml of the homogenate and mix vigorously. Incubate at 0 C for 30 minutes with occasional vigorous mixing. 4) Collect the precipitate by centrifugation at 8,000 rpm for 15 minutes at 4 C. Discard the supernatant. 5) Resuspend the pellet in fresh acetone (-20 C) and mix vigorously. Allow to stand at 0 C for 1O minutes. 6) Centrifuge at 8,000 rpm for 1O minutes at 4 C. Transfer the pellet to a clean piece of filter paper. Spread the precipitate and allow to air-dry at room temperature. As it becomes dry, spread and disperse the pellet some more. 7) After the powder is dry, transfer it to an airtight container. Remove any large pieces that will not break into a fine powder. G. Titration of the prepared FITC conjugate Utilising the established indirect fluorescent antibody test (such as for Neospora), determine the working dilution of the conjugate. In case of the conjugates for direct fluorescent antibody test (such as for Campylobacter), examine the appropriate concentration with the antigens. H. Cleaning and storage the gel 1. Sephadex G-25 gels may be cleaned by treatment with; i) 0.2 M NaOH and ii) 2 M NaCl. After cleaning with NaOH and NaCl, wash with distilled water and equilibrate with 5 mm PB (ph 8.4). The gels can be stored in 5 mm PB (ph 8.4) with 0.1% NaN 3 or thimerosal in a cold room. 2. DE52 gels can be cleaned, washed and re-equilibrated by the methods 56

7 same as for Sephadex G-25. After equilibration, transfer the gels from the column into a bottle or flask, and store in 5 mm PB (ph 8.4) with 0.1% NaN 3 or thimerosal in a fridge. If DE52 gel is stored in a column, the particles of the gel packed tightly, which resulted in a slow elution speed. NOTE: NaN 3 should be removed from the columns before use. If it were not for fungus contamination, the gels could be used for several times. <SOLUTIONS> # PBS (10 mm, ph 7.0) NaH 2 P0 4 2H Na 2 HP0 4 12H 2 O... NaCl... Distilled water g 2.53 g 8.0 g 1 litre # PB (0.2 M, ph 8.4) Solution A: NaH 2 P0 4 2H g/litre (0.2 M) or NaH 2 PO 4 H 2 O g/litre (0.2 M) Solution B: Na 2 HP0 4 12H g/litre (0.2 M) or Na 2 HPO 4 anhydrous g/litre (0.2 M) or Na 2 HPO 4 2H 2 O g/litre (0.2 M) Add a small volume of solution A to solution B in order to bring the ph to 8.4. # PB (5 mm, ph 8.4) Dilute 0.2 M PB 40 times (25 ml in 975 ml of DW) with distilled water before use (0.2 M/40 = M = 5 mm). # PB (0.1 M, ph 6.3) Add solution B to solution A to bring the ph to 6.3 and dilute twice. # 0.5 M carbonate buffer Na 2 C g + NaHC g in 100 ml of distilled water The ph should become