a rapid method for the purification of antibody–enzyme conjugates
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A rapid method for the purification of antibody-enzyme conjugates
Received October 3, 1978
Pa&. hf., Audette, M. %k Caron, .V. (1979) A rapid method for the purification of antibody- enzyme co~~jugates. Cmz. 9. BiocEtem. 57, 284-288
A method is described for a rapid and ef$lcient separation of enzy~ne-labelled antibodies from the free enzyme following the coupling reaction. A single passage sf the reaction mixture on a protein A - Sepharose CL-4B column gave a sharp separation of the free enzyme from the conjugate.
PagC, M., Audette, M. ebr Caron, bf. (1979) A rapid method for the purification of asatibody- enzyme conjugates. Cizim J. Biochem. 57, 284-2816
Nous proposons une mCthode rapide et efficace pour la separation des conjuguCs anticorps- enzyme de l'enzy me libre apr&s la rkaction de couglage. Un seul passage du mClange rkactionnel sur une colonlne de protein A - Sepharose CL-4B gerrnet une separation nette de l'enzyme libre et du corajuguk.
Labelled antibodies are widely used in a variety of insmunohistochemical and immunological techniques and in enzyme immunoassays. The separation of the enzymc-labelled antibodies from the free enzyme fol- lowing the coupling reaction has been accomplished by ammonium sealfate precipitation and gel filtration s n Bio-Gel P-300 ( 1 ) . These methods are long and very often yield conjugates which are contaminated with free enzyme s r enzyme psIyn~ers produced in the co~apling procedure. Passage of the reaction mixture on Sephadex C-25 separates proteins from tlae cross-Iinking agent. Tlne latter procedure which is widely used (see Ref. f for a revicw) yields a heterogeneous mixture of free enzyme, antibodies, and conjugates.
Protein A, a protein produced by Stophylocnccus terrra4s, specifically binds irnrnunoglobaalins through their Fc fragment (2 ) . The solid-phase derivative of this pro- tein. protein A - Sepharose CL-4B, has been used for the isolation of TgG-type antibodies and IgG subcIasses 6-31. We describe the use of protein A - Sepharose Ct- 4B for the paarification of enzyme antibody conjugates.
Experimental Procedure
Csizjzcgar~ Prepctratiore The gamma-globulin isolation and the labelling procedure
have already been reported by us (4). The same procedure was followed for tabelling various antibodies including antihuman carciraoembryonic antigen produced in rabbits. Briefly, 1 mL of gamma globulins ( 10 mg/rnL) was added
'Itfailing address: Centre de Recherclses, H6tel-Dieu de QuCbec, 11 C6te du Palais, Quktsec, (Qta6.1, Canada G l R 256.
to 0.6 naE of alkaline phssphatase (calf mkicosa type VIZ, 5 rng/rnL, Sigma chemical). The mixture was dialysed over- night at 4°C against 1 L of phosphate-bufered saline at pH 7.2 and 680 fib. of a I?& solution of glutaraldekyde in water was added. The mixture was left at room temperature for 2 h. The reaction mixture was then applied on a Sephadex G-50 column (0.9 x 12 cm) equilibrated with phosphate-buffered saline. This step eliminated free glu- taraldehyde and the protein mixture was obtained in the void volume of the column. Protein elution was monitored at 280 nrn and aha: enzyme activity was measured by the p-nitrophenyl phosphate method (5).
Chrsnzratc~grnphy on Prstc?iiz A -Sephaaose CE-41% The protein fraction obtained after gel chromatography
was applied (at 12 rnL/h) on a protein A - Sepharose CE-4B column (0.75 X 3.5 cm) (Pharmacia Fine Chemicals, Montreal) equilibrated with phssphate-buffered saline. The column was washed with 15 mE of buffer and followed by 3 ,%g potassium thiocyanate to elute the adsorbed proteins. El~ation was monitored as described above. Two sharp peaks were obtained (Fig. 11, the first containing Bee alkaline phosphatase and enzyme polymers and the second peak containing free antibodies and antibody-enzyme conjugates (Fig. 2 ) .
The apparent specific activity of the antibodies after pas- sage on Sephadex G-50 was 160 U/mg; after removal of the unconjugated enzyme by affinity chromatography, the specific activity was brought to 24.8 U/mg corresponding to a purification factor of 6.4.
The use of protein A - Sepharose CL-4B for the purifica- tion of antibody-enzyme conjugates offers many advantages. Owing to the high affinity and specificity of protein A, small columns may be used thus reducing the time required for ckromatsgraphy. Moreover, the reaction between protein A and the Fc fragment being rapid, relatively high flow rates may be used without altering the efficiency of the separation. The use of purified conjugates is desirable in both qualitative and quantitative techniques since it reduces the background
0008-4018/79/030286-03S01 .BB0/0 a ii) 979 National Research Council of Canada/Conseil national de recherches du Canada
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PERCY ET AIL.
FIG. 1. Separation of the enzyme-antibody conjugate from free enzyme on a protein A - Sepharose CHd-4B column. One-rnillilitre fractions were collected at 12 mL/h; the arrow indicates the application of a 3 174 KCNS solution to elute free and conjugated immunoglsbaalians.
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CAW. J. BIOCHBM. V8L. 57. 1979
level which interferes in highly sensitive techniques. This rapid afinity chromatography could be accomplished cn- tirely within 2 h. The method was used successfuUy in our laboratory for a variety of antibody-enzyme conjugates.
Acknowledgements
This work was supported by the National Cancer Institute of Canada and the Medical Research Council sf Canada.
I . Feteanu, A. ( 1978) Labelled Antibodies in Biology atatl Medicine, pp. 342-35 1 , McGraw-Hill, International. New York
2. Sj6quist, J., Meloun, B. 8r Hjelrn, H. (1972) EUP. I. Biocllcnz, 29, 572-5762
3. Hjelrn, H., Hjelm, M. & Sj~quist, S. (1972) FEBS Lett. 28,7P-76
4. ThCriault, L. & PagC, M. ( 1977) Clin. Clrenl. ( N . Y . ) 23 ,2 142-2 144
5. &%'olf, M., Dinwoodie, A. 8r Morgan, H. G. (1969) Cbin. C h h . Acga 24 , 13 1-134
FIL,. 2. Double irnmunodiffusion of iintihum:an IgG - enzyme conjugate prirified on protein A - Sepharose CL-4B. Well I[ contains the conj~egate (peak 2 of Fig. I ) , wells 2 and 4 contain fractions of peak 1, and well 6 contains goat rantirabbit HgG. The enzyme activity was determined with naphthol AS-MX phosphate after rinsing the plate 24 h in 0.9% sodiunl chloride.
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