7mm'nochemistry, 1976, Vol. 13, pp.871-872. Pergamon Press. Printed in Great Britain
THE QUESTION OF SPECIFICITY IN BINDING HUMAN IgG SUBCLASSES TO PROTEIN A-SEPHAROSE
P. Skvaril
Institute for Clinical and Experimental Cancer Research, University of Berne, Tiefenau-Hospital, 5004 Berne (Switzerland}
It was reported that Protein A isolated from Staphylococcus aureus reacts with the Fc part of human IgG1, IgG2 and IgG4 subclasses but not with the IgG5 subclass (Forsgren and SjSquist, 1966; Kronvall and Williams, 1969). Recently, IgG5 immunoglobulin was isolated from human normal and myeloma sera by using an immunosorbent prepared b7 coupling Protein A to Sepharose 4B (Hjelm et al., 1972; Hjelm, 1975). A similar immunosorbent is now available as a commercial product designated as Protein A-Sepharose CL-4B by Pharmacia (Uppsala, Sweden}. In the present study we confirmed that pure polyclonal IgG5 can be obtained with this immunosorbent. On the other hand, we found that a minor portion of IgG5 was always bound to the Protein A-Sepharose CL-4B at neutral pH and was eluted with other three subclasses,IgGl, IgG2 and IgG4 at acid pH. MATERIAL AND METHODS
The immunosorbent column (10 x 75 mm) was prepared according to the manufacturer's instructions using 1.5 g of dry Protein A-Sepharose CL-4B. Phosphate buffer, 0.1M, pH 7.5, was used for the elution of non-bound proteins (starting buffer); glycine-HC1 buffer, 0.1M, pH 2.8, for the desorption of bound proteins. In consecutive experiments the column was loaded (i) with 50 mg, 25 mg and 5 mg of Cohn Fraction II dissolved in 2 ml of starting buffer, (2} with 25 mg of Cohn Fraction II dissolved in 10 ml of the same buffer (5) with 2 ml of normal human serum pool (SRK), (4) with 2 ml of a individual serum with a high concentration of a polyclonal IgG5 immunoglobulin and (5} with Z5 mg of a monomeric IgG preparation isolated from the Cohn Fraction II by DEAE Sephadex chromatography followed by Sephadex G200 gel filtration. The same column was used throughout ali experiments. The flow rate was adjusted by a peristaltic pump to 2 ml/h and fractions at 1 h intervals were collected in the Ultro-Rac (LKB, Stockholm). The absorbance of the effluent was registered by LKB Uvicord II absorptiometer. Protein fractions eluted at neutral pH were pooled and concentrated under vacuum; fractions eluted with glycine pH 2.8 buffer were neutralized with 0.SM NaOH before concentration. All procedures were carried out at 4 ° C. IgG subclasses were detected in the immunoelectrophoresis as described previously (Skvaril, 1972). RESULTS A typical elution profile o f a c h r o m a t o g r a p h y r u n o f Cohn F r a c t i o n II on P r o t e i n A - S e p h a r o s e CL-4B i s shown i n F i g . 1. I n t h e f i r s t peak, only the IgG3 p r o t e i n was e l u t e d , i n t h e s e c o n d p e a k I g G 1 , IgG2 a n d IgG4 w e r e d e t e c t e d with a small amount of IgGS. Identical results were obtained in all experiments carried out under conditions described in Material and Methods: in addition to other three subclasses a detectable a m o u n t o f IgG3 was a l w a y s e l u t e d a t a c i d pH. In one experiment, IgG3 was m e a s u r e d i n s i n g l e r a d i a l immunodiffusion in both effluent fractions:about 55~ o f t h e t o t a l IgG3 was f o u n d t o b e e l u t e d in peak If. In hemagglutination inhibition tests, m o l e c u l e s b e a r i n g Gm(b) a n d Gm(g) markers were distributed in both fractions in about the same ratio as in the starting material.
871
OO2so pH 7.0buffer
pH2,8 buffer
1,0 05 025
I
10
210
3~}
Fraction No.
Fig. i. Left side: Chromatography of 25 mg Cohn Fraction II on a column of Protein A-Sepharose CL-4B (I0x75 mm) in 0.1M phosphate buffer pH 7.0 {flow rate 2 ml/h; fraction volume 2 ml). Bound proteins were eluted with 0.1M glycine HCI buffer, pH 2.8. Right side:Immunoelectrophoresis of normal human serum {NS) and peak I and II proteins developed with IgG subclass specific antisera. DISCUSSION At present the binding of a portion of the IgG3 protein to the Protein A-Sepharose CL-4B in 0.1M phosphate buffer, pH 7,0, cannot be explained. It is difficult to believe that this is due to technical factors. The elution rate of 2 ml/h is adequate for the size of column and gel volume. A sufficient amount of neutral buffer was always used for washing the column prior to the elution with acid buffer. Binding of IgG3 could be detected even with the lowest amount of IgG applied to the column, i.e. with 1 mg per ml gel: the capacity should be as high as 25 mg per ml gel {Pharmacia leaflet: Protein A-Sepharose CL-4B, Nov. 1975). Spontaneous fragmentation of IgG (Skvaril,1960) was never observed in starting material or in chromatographically isolated fractions I and II. In two experiments a normal human serum and a monomeric IgG preparation were separated with the same effect as the Cohn Fraction II. Therefore the possibility was excluded that the aggregated portion of IgG3 may react unspecifically with the Protein A immunosorbent. Another explanation could be a nonspecific binding of a part of IgG3 to protein{s) of other subclasses by protein-protein interactions. However, such interactions among poly. clonal IgG subclass proteins in neutral 0.1M phosphate buffers have never been observed until now.- A possibility that a sub-subclass of the IgG3 may exist cannot be completely excluded and a screening of a collection of monoclonal IgG3 proteins is envisaged. Acknowledgements. This investigation was supported by the Swiss National Foundation for Scientific Research. Determinations of Gm markers were kindly done by Mr. Scherz, Central Laboratory of the Swiss Red Cross Blood Transfusion Service in Berne. The skilful technical assistance of Mrs. Marianne Probst is greatly acknowledged.
REFERENCES Hjelm H. {1975) Scand. J . Immunol. 4, 633. Hjelm H., Hjelm K and S j 0 q u i s t J . {1972) FEBS L e t t e r s 288, 73. K r o n v a l l G. and W i l l i a m s R.C. {1969) J . Immunol. 103, 828. F o r s g r e n A. and S j S q u i s t J . {1966} J . Immunol. 9_~7, 822. S k v a r i l F. {1960) N a t u r e {London) 185, 475. S k v a r i l F . , M o r e l l A. and Barandun S. {1972) Vox Sang. 233, 546. 872
THE QUESTION OF SPECIFICITY IN BINDING HUMAN IgG SUBCLASSES TO PROTEIN A-SEPHAROSE
P. Skvaril
Institute for Clinical and Experimental Cancer Research, University of Berne, Tiefenau-Hospital, 5004 Berne (Switzerland}
It was reported that Protein A isolated from Staphylococcus aureus reacts with the Fc part of human IgG1, IgG2 and IgG4 subclasses but not with the IgG5 subclass (Forsgren and SjSquist, 1966; Kronvall and Williams, 1969). Recently, IgG5 immunoglobulin was isolated from human normal and myeloma sera by using an immunosorbent prepared b7 coupling Protein A to Sepharose 4B (Hjelm et al., 1972; Hjelm, 1975). A similar immunosorbent is now available as a commercial product designated as Protein A-Sepharose CL-4B by Pharmacia (Uppsala, Sweden}. In the present study we confirmed that pure polyclonal IgG5 can be obtained with this immunosorbent. On the other hand, we found that a minor portion of IgG5 was always bound to the Protein A-Sepharose CL-4B at neutral pH and was eluted with other three subclasses,IgGl, IgG2 and IgG4 at acid pH. MATERIAL AND METHODS
The immunosorbent column (10 x 75 mm) was prepared according to the manufacturer's instructions using 1.5 g of dry Protein A-Sepharose CL-4B. Phosphate buffer, 0.1M, pH 7.5, was used for the elution of non-bound proteins (starting buffer); glycine-HC1 buffer, 0.1M, pH 2.8, for the desorption of bound proteins. In consecutive experiments the column was loaded (i) with 50 mg, 25 mg and 5 mg of Cohn Fraction II dissolved in 2 ml of starting buffer, (2} with 25 mg of Cohn Fraction II dissolved in 10 ml of the same buffer (5) with 2 ml of normal human serum pool (SRK), (4) with 2 ml of a individual serum with a high concentration of a polyclonal IgG5 immunoglobulin and (5} with Z5 mg of a monomeric IgG preparation isolated from the Cohn Fraction II by DEAE Sephadex chromatography followed by Sephadex G200 gel filtration. The same column was used throughout ali experiments. The flow rate was adjusted by a peristaltic pump to 2 ml/h and fractions at 1 h intervals were collected in the Ultro-Rac (LKB, Stockholm). The absorbance of the effluent was registered by LKB Uvicord II absorptiometer. Protein fractions eluted at neutral pH were pooled and concentrated under vacuum; fractions eluted with glycine pH 2.8 buffer were neutralized with 0.SM NaOH before concentration. All procedures were carried out at 4 ° C. IgG subclasses were detected in the immunoelectrophoresis as described previously (Skvaril, 1972). RESULTS A typical elution profile o f a c h r o m a t o g r a p h y r u n o f Cohn F r a c t i o n II on P r o t e i n A - S e p h a r o s e CL-4B i s shown i n F i g . 1. I n t h e f i r s t peak, only the IgG3 p r o t e i n was e l u t e d , i n t h e s e c o n d p e a k I g G 1 , IgG2 a n d IgG4 w e r e d e t e c t e d with a small amount of IgGS. Identical results were obtained in all experiments carried out under conditions described in Material and Methods: in addition to other three subclasses a detectable a m o u n t o f IgG3 was a l w a y s e l u t e d a t a c i d pH. In one experiment, IgG3 was m e a s u r e d i n s i n g l e r a d i a l immunodiffusion in both effluent fractions:about 55~ o f t h e t o t a l IgG3 was f o u n d t o b e e l u t e d in peak If. In hemagglutination inhibition tests, m o l e c u l e s b e a r i n g Gm(b) a n d Gm(g) markers were distributed in both fractions in about the same ratio as in the starting material.
871
OO2so pH 7.0buffer
pH2,8 buffer
1,0 05 025
I
10
210
3~}
Fraction No.
Fig. i. Left side: Chromatography of 25 mg Cohn Fraction II on a column of Protein A-Sepharose CL-4B (I0x75 mm) in 0.1M phosphate buffer pH 7.0 {flow rate 2 ml/h; fraction volume 2 ml). Bound proteins were eluted with 0.1M glycine HCI buffer, pH 2.8. Right side:Immunoelectrophoresis of normal human serum {NS) and peak I and II proteins developed with IgG subclass specific antisera. DISCUSSION At present the binding of a portion of the IgG3 protein to the Protein A-Sepharose CL-4B in 0.1M phosphate buffer, pH 7,0, cannot be explained. It is difficult to believe that this is due to technical factors. The elution rate of 2 ml/h is adequate for the size of column and gel volume. A sufficient amount of neutral buffer was always used for washing the column prior to the elution with acid buffer. Binding of IgG3 could be detected even with the lowest amount of IgG applied to the column, i.e. with 1 mg per ml gel: the capacity should be as high as 25 mg per ml gel {Pharmacia leaflet: Protein A-Sepharose CL-4B, Nov. 1975). Spontaneous fragmentation of IgG (Skvaril,1960) was never observed in starting material or in chromatographically isolated fractions I and II. In two experiments a normal human serum and a monomeric IgG preparation were separated with the same effect as the Cohn Fraction II. Therefore the possibility was excluded that the aggregated portion of IgG3 may react unspecifically with the Protein A immunosorbent. Another explanation could be a nonspecific binding of a part of IgG3 to protein{s) of other subclasses by protein-protein interactions. However, such interactions among poly. clonal IgG subclass proteins in neutral 0.1M phosphate buffers have never been observed until now.- A possibility that a sub-subclass of the IgG3 may exist cannot be completely excluded and a screening of a collection of monoclonal IgG3 proteins is envisaged. Acknowledgements. This investigation was supported by the Swiss National Foundation for Scientific Research. Determinations of Gm markers were kindly done by Mr. Scherz, Central Laboratory of the Swiss Red Cross Blood Transfusion Service in Berne. The skilful technical assistance of Mrs. Marianne Probst is greatly acknowledged.
REFERENCES Hjelm H. {1975) Scand. J . Immunol. 4, 633. Hjelm H., Hjelm K and S j 0 q u i s t J . {1972) FEBS L e t t e r s 288, 73. K r o n v a l l G. and W i l l i a m s R.C. {1969) J . Immunol. 103, 828. F o r s g r e n A. and S j S q u i s t J . {1966} J . Immunol. 9_~7, 822. S k v a r i l F. {1960) N a t u r e {London) 185, 475. S k v a r i l F . , M o r e l l A. and Barandun S. {1972) Vox Sang. 233, 546. 872
