Journal of Immunological Methods, 17 (1977 ) 211--216 © Elsevier/North-Holland Biomedical Press
211
A SIMPLE TECHNIQUE FOR STUDYING IMMUNOGLOBULIN SYNTHESIS BY NORMAL AND MALIGNANT PLASMA CELLS IN VITRO
SUE PRITCHARD, J. WATKINS *, ANGELA MURPHY *, P.J. WYLD, F.E. PRESTON and G. HUDSON University Departments of Haematology and Immunology *, Hallamshire Hospital Medical School, Beech Hill Road, Sheffield $10 2RX, U.K.
(Received 9 March 1977, accepted 20 April 1977) Plasma cells from human marrows are saturated with C-14 labelled amino acids, harvested and recultured in unlabelled growth medium. The appearance of radioactivity in the growth medium then provides a simple and rapid measure of protein synthesis. The secreted radio-labelled material is characterized by isoelectric focusing and autoradiography in acrylamide gels, a technique which has advantages over established serological methods. INTRODUCTION T h e a p p e a r a n c e u p o n z o n a l e l e c t r o p h o r e s i s o f an intense i m m u n o g l o b u l i n b a n d of r e s t r i c t e d e l e c t r o p h o r e t i c m o b i l i t y in t h e s e r u m o f p a t i e n t s suffering f r o m p l a s m a cell t u m o u r s is a l m o s t diagnostic o f this t y p e o f m a l i g n a n c y . E x t r e m e l y high levels o f these ' m o n o c l o n a l ' i m m u n o g l o b u l i n s m a y be e n c o u n t e r e d in p a t i e n t s w i t h m u l t i p l e m y e l o m a usually w i t h s u p p r e s s i o n o f o t h e r i m m u n o g l o b u l i n classes. A l t h o u g h q u a n t i t a t i o n o f the s e r u m m o n o clonal p r o t e i n s p r o v i d e s a useful clinical a s s e s s m e n t o f r e s p o n s e to t h e r a p y , it s e e m s p r o b a b l e t h a t in vitro m e a s u r e m e n t o f the rate o f p r o d u c t i o n of t h e a b n o r m a l p r o t e i n f r o m m a r r o w cells g r o w n in c u l t u r e m i g h t yield i n f o r m a t i o n even m o r e useful f o r p r o g n o s i s and p a t i e n t m a n a g e m e n t . Q u a n t i t a t i o n o f s y n t h e s i z e d p r o t e i n in usually achieved b y a radioi m m u n o a s s a y t e c h n i q u e . This is r a t h e r u n s a t i s f a c t o r y since it m a k e s the f u n d a m e n t a l a s s u m p t i o n t h a t m o n o c l o n a l and p o l y c l o n a l i m m u n o g l o b u l i n s h a v e the s a m e specificity in r e a c t i o n w i t h class specific antisera. O u r o w n o b s e r v a t i o n s a n d t h o s e o f o t h e r w o r k e r s (Morell et al., 1 9 7 3 ) s h o w t h a t this is o f t e n n o t t h e case. In a d d i t i o n , i m m u n o c h e m i c a l assays are u n a b l e to distinguish b e t w e e n m o n o c l o n a l and a c c o m p a n y i n g p o l y c l o n a l i m m u n o globulins o f the s a m e class. In this r e p o r t we describe a s i m p l e t e c h n i q u e f o r evaluating i m m u n o globulin s y n t h e s i s by h u m a n m a r r o w cells in vitro w h i c h d o e s n o t d e p e n d on i m m u n o c h e m i c a l assay. S o m e p r e l i m i n a r y o b s e r v a t i o n s on d i f f e r e n c e s in s y n t h e s i s r a t e b e t w e e n n o r m a l a n d m a l i g n a n t cells are also p r e s e n t e d .
212 MATERIALS AND METHODS
Cell preparation The m e t h o d is similar to that described by Salmon and Smith (1970). Samples of bone marrow (1 ml) were obtained by needle aspiration and the leucocytes separated from red cells by sedimentation with Dextran 110 for 1 h at 37 °C. These nucleated cells were washed three times by centrifugation and resuspension in Hanks balanced salt solution (Gibco Bio-cult, Paisley) with 10% foetal calf serum (HBSS-FCS). After the final wash the cell b u t t o n was resuspended in the complete culture medium. This was Fischer's medium (Gibco Bio-Cult, Paisley) supplemented with 15% foetal calf serum and combined with 2.2 mg/ml sodium bicarbonate, 0.29 mg/ml L-glutamine, 50 U/ml penicillin, 50 pg/ml streptomycin and 2 U/ml heparin.
Measurement of immunoglobulin synthesis rate in cell culture The plasma cells were cultured in 1 ml volumes (0.4 × 106 cells) in sterile bijoux (Sterilin), each culture containing 2 pCi of a C-14 labelled amino acid mixture (Radiochemicals; CFB 152, Equimolar specific activity mixture, 10 mCi/mmol). Each culture was equilibrated with 5% CO2 in air and incubated for 24 h at 37°C. After this primary incubation the cells were pooled, washed six times with HBSS-FCS, resuspended in the supplemented Fischer's medium and incubated as before, but w i t h o u t labelled amino acids in the medium. At suitable intervals the cells and supernatants were harvested using Millipore filters (0.22 pm). The cells retained on the filters were washed with 2 ml of HBSS-FCS and washings combined with the corresponding supernatants. Radioactivity in the supernatants and in the cells on each filter was assessed by scintillation counting using Instagel (Packard) as the scintillator system.
Characterisation of the synthesized protein by isoelectric focusing Any supernatant remaining after scintillation counting (ca. 2 ml) was freeze dried, taken up in 0.1 ml of HBSS and 0.1 ml of the patient's serum added. This solution was dialyzed against phosphate buffered saline to remove excess salt and subjected to isoelectric focusing in a polyacrylamide gel using a pH 3--10 gradient (Multiphor system LKB-Producter AB, Stockholm, Sweden). Unlabelled samples of patient's serum were focused simultaneously as controls. The gels were stained, washed and dried in the conventional manner and set up for autoradiography against Kodak Industrex C film. Exposure times were o f the order of 2 months.
213 RESULTS AND DISCUSSION Any technique which depends on immunochemical quantitation of m onoclonal immunoglobulins is likely to lead to serious errors since monoclonal and polyclonal immunoglobulins do n o t always react similarly with class specific antisera. The conventional assay procedure established with the five Supra-regional Specific Protein Reference Units in the U.K. is a 'chemical' one based on zonal electrophoresis of the patients' serum on cellulose acetate, staining with Ponceau S and scanning with a microdensitometer. The 'density' of the m onoc l ona l protein zone is directly expressed by the d e n s i t o m e t e r in g/1 by reference to the total serum protein previously measured in the sample. A comparison of results with r a n d o m m y e l o m a sera received in this laborat o r y for routine m e a s u r e m e n t by both the chemical technique and by an i m m u n o c h e m i c a l pr oc e dur e (A.I.P -- Technicon A ut o Analyzer System II, Technicon I n s t r u m e n t Corporation, T a r r y t o w n , New York) is shown in table 1. Gross discrepancies are obvious and the immunochemical technique is incapable o f distinguishing mixtures o f monoclonal and polyclonal immunoglobulins o f the same class. U n f o r t u n a t e l y the relatively low yields o f protein available f r om the cell cultures do n o t lend themselves to chemical assay. Measurements based on release of radioactivity labelled materials can be readily made but require characterization of the labelled material. Isoelectric focusing, com bi ne d with autoradiography, combines specificity and a r e q u i r e m e n t for only very small quantities of protein ( A r b u t h n o t t and Beeley, 1975). The limited heterogeneity banding patterns of m y e l o m a pro-
TABLE 1 Comparison of results of monoclonal protein assays by chemical and immunochemical methods. Patient
Monoclonal protein class
Assay (g/l) Immunochemical
Chemical 23 21 6-monoclonal 6-polyclonal 7 18 6-monoclonal 5-polyclonal 22 30 2-monoclonal 2-polyclonal
EE BM BS
IgG IgG IgG
27 4 12
AR IS
IgA IgA
7 6
EH
IgA
11
KP NW GR
IgM IgM IgM
16 10 2
214
a
b
c
d
Fig. 1. Isoelectric focusing patterns of myeloma sera in acrylamide gel at pH 3--10. Samples (a), (b) (c) show various band arrangements for IgG monoclonal proteins. Sample (d) is from an IgA myeloma patient with almost total suppression of IgG protein.
teins are characteristic f o r a p a r t i c u l a r p a t i e n t b o t h in a r r a n g e m e n t and n u m bers o f bands (Cwynarski et al., 1 9 7 5 ) and their position in the isoelectric gradient (fig. 1). A u t o r a d i o g r a p h y o f tissue culture s u p e r n a t a n t s m i x e d with the p a t i e n t ' s serum m a y reveal n o t o n l y p r o d u c t i o n o f m o n o c l o n a l p r o t e i n b u t also t h e a m o u n t o f n o r m a l h e t e r o g e n e o u s i m m u n o g l o b u l i n which the individual is still able to p r o d u c e . The l a t t e r has clinical implications which we h o p e to e x p l o r e f u r t h e r . T h e necessary e x p o s u r e of the a u t o r a d i o g r a p h is at p r e s e n t very prol o n g e d , b u t in o u r e x p e r i e n c e released radioactivity is always associated with labelled p r o t e i n and n o t w i t h free a m i n o acids. It is t h e r e f o r e possible to apply the results of the c u l t u r e synthesis clinically w i t h o u t waiting for the c h a r a c t e r i s a t i o n o f the p r o t e i n s . F o r c o n v e n i e n c e we express m e a s u r e m e n t s as p e r c e n t a g e radioactivity p r e s e n t in the s u p e r n a t a n t at the t i m e o f cell harvesting c o m p a r e d with the t o t a l radioactivity o f the c u l t u r e . T y p i c a l 'synthesis' curves are shown in fig. 2. T h e r e are m a r k e d d i f f e r e n c e s in rate o f synthesis b e t w e e n cells f r o m
215 80
7O °
>, 6 0
>-
4O
306
~
&
~
~
~
2'~
Second incubatron ( h )
Fig. 2. R a d i o a c t i v i t y release i n t o g r o w t h m e d i u m by m a r r o w cells f r o m : o O, a p a t i e n t w i t h a d v a n c e d IgA m y e l o m a ; D, a p a t i e n t with a n u n d e f i n e d p l a s m a cell d i s t u r b a n c e ; • • , a n o r m a l individual.
m y e l o m a patients and those from normal marrow. The flattening of the m y e l o m a synthesis curves with increasing times reflects usage of the labelled amino acid pool within the cells and to a lesser degree, decreasing cell viability. Measurements of the relative rate of synthesis (RRS), are made on the portions of the curves for samples taken for up to 9 h of the second incubation period in 'cold' medium. Some preliminary results are tabulated (table 2). Obtaining normal marrow specimens poses both practical and ethical problems. The two 'controls' in table 2 are from patients and therefore by definition pathological. Since there was no indication of neoplastic growth
TABLE 2 Relative rates of s y n t h e s i s o f labelled p r o t e i n by n o r m a l a n d m y e l o m a m a r r o w s . Patient
Diagnosis
Period of therapy
Plasma m o n o c l o n a l p r o t e i n (g/l)
Degree of immune paresis
Relative rate of synthesis
CC * LB EW BS DL AM FC
Myeloma Myeloma Myeloma Myeloma CML Control Control
10 m o n t h s 4 years 16 m o n t h s 3 years 6 years N/A N/A
IgA 58 IgA 18 IgA 29 IgG 6 no monoclonal protein no monoclonal protein no monoclonal protein
v. m a r k e d moderate marginal none none none none
4.0 3.0 1.5 1.5 2.0 0.2 0.2
* P a t i e n t died s h o r t l y a f t e r t h e s e studies were made.
216 in these patients and we feel justified in considering t hem as representative of ground state synthesis. Their RRS values of 0.2 contrast markedly with those from other patients. The highest rate of synthesis was observed for IgA in a m y e l o m a patient with advanced disease. In contrast, two o t h e r patients with IgA m y e l o m a show significantly lower RRS values, apparently correlating with the less advanced nature of their disease (table 2). We have n o t y e t investigated correlations between m y e l o m a immunoglobulin class and synthesis rate but synthesis from cultures of BS cells, a therapy-controlled IgG m y e l o m a patient possessing a good level of heterogenous immunoglobulin is similar to bot h that observed in controlled IgA m y e l o m a patients and in DL, a patient with a plasma cell disturbance other than a m onocl onal g a m m o p a t h y (table 2). F r o m these preliminary observations this labelling technique appears to offer a simple and valid way of studying the products of cell culture. The highly specific technique of isoelectric focusing for charaeterisation of the products of cell synthesis has received little attention as a routine analytical tool. Its co mb inat i on with autoradiography offers considerable scope for quantitative as well as qualitative devel opm ent . REFERENCES Arbuthnott, J.P. and J.A. Beeley, 1975, eds. Isoelectric focusing (Butterworths). Cwynarski, M.T., J. Watkins and P.M. Johnson, 1975, in: Isoelectric focusing, eds. J.P. Arbuthnott and J.A. Beeley (Butterworths) p. 319. Morell, A., F. Skvaril and S. Barandum, 1973, Clin. Exp Immunol. 13,293. Salmon, S.E. and B.A. Smith, 1970, J. Immunol. 104,665.
211
A SIMPLE TECHNIQUE FOR STUDYING IMMUNOGLOBULIN SYNTHESIS BY NORMAL AND MALIGNANT PLASMA CELLS IN VITRO
SUE PRITCHARD, J. WATKINS *, ANGELA MURPHY *, P.J. WYLD, F.E. PRESTON and G. HUDSON University Departments of Haematology and Immunology *, Hallamshire Hospital Medical School, Beech Hill Road, Sheffield $10 2RX, U.K.
(Received 9 March 1977, accepted 20 April 1977) Plasma cells from human marrows are saturated with C-14 labelled amino acids, harvested and recultured in unlabelled growth medium. The appearance of radioactivity in the growth medium then provides a simple and rapid measure of protein synthesis. The secreted radio-labelled material is characterized by isoelectric focusing and autoradiography in acrylamide gels, a technique which has advantages over established serological methods. INTRODUCTION T h e a p p e a r a n c e u p o n z o n a l e l e c t r o p h o r e s i s o f an intense i m m u n o g l o b u l i n b a n d of r e s t r i c t e d e l e c t r o p h o r e t i c m o b i l i t y in t h e s e r u m o f p a t i e n t s suffering f r o m p l a s m a cell t u m o u r s is a l m o s t diagnostic o f this t y p e o f m a l i g n a n c y . E x t r e m e l y high levels o f these ' m o n o c l o n a l ' i m m u n o g l o b u l i n s m a y be e n c o u n t e r e d in p a t i e n t s w i t h m u l t i p l e m y e l o m a usually w i t h s u p p r e s s i o n o f o t h e r i m m u n o g l o b u l i n classes. A l t h o u g h q u a n t i t a t i o n o f the s e r u m m o n o clonal p r o t e i n s p r o v i d e s a useful clinical a s s e s s m e n t o f r e s p o n s e to t h e r a p y , it s e e m s p r o b a b l e t h a t in vitro m e a s u r e m e n t o f the rate o f p r o d u c t i o n of t h e a b n o r m a l p r o t e i n f r o m m a r r o w cells g r o w n in c u l t u r e m i g h t yield i n f o r m a t i o n even m o r e useful f o r p r o g n o s i s and p a t i e n t m a n a g e m e n t . Q u a n t i t a t i o n o f s y n t h e s i z e d p r o t e i n in usually achieved b y a radioi m m u n o a s s a y t e c h n i q u e . This is r a t h e r u n s a t i s f a c t o r y since it m a k e s the f u n d a m e n t a l a s s u m p t i o n t h a t m o n o c l o n a l and p o l y c l o n a l i m m u n o g l o b u l i n s h a v e the s a m e specificity in r e a c t i o n w i t h class specific antisera. O u r o w n o b s e r v a t i o n s a n d t h o s e o f o t h e r w o r k e r s (Morell et al., 1 9 7 3 ) s h o w t h a t this is o f t e n n o t t h e case. In a d d i t i o n , i m m u n o c h e m i c a l assays are u n a b l e to distinguish b e t w e e n m o n o c l o n a l and a c c o m p a n y i n g p o l y c l o n a l i m m u n o globulins o f the s a m e class. In this r e p o r t we describe a s i m p l e t e c h n i q u e f o r evaluating i m m u n o globulin s y n t h e s i s by h u m a n m a r r o w cells in vitro w h i c h d o e s n o t d e p e n d on i m m u n o c h e m i c a l assay. S o m e p r e l i m i n a r y o b s e r v a t i o n s on d i f f e r e n c e s in s y n t h e s i s r a t e b e t w e e n n o r m a l a n d m a l i g n a n t cells are also p r e s e n t e d .
212 MATERIALS AND METHODS
Cell preparation The m e t h o d is similar to that described by Salmon and Smith (1970). Samples of bone marrow (1 ml) were obtained by needle aspiration and the leucocytes separated from red cells by sedimentation with Dextran 110 for 1 h at 37 °C. These nucleated cells were washed three times by centrifugation and resuspension in Hanks balanced salt solution (Gibco Bio-cult, Paisley) with 10% foetal calf serum (HBSS-FCS). After the final wash the cell b u t t o n was resuspended in the complete culture medium. This was Fischer's medium (Gibco Bio-Cult, Paisley) supplemented with 15% foetal calf serum and combined with 2.2 mg/ml sodium bicarbonate, 0.29 mg/ml L-glutamine, 50 U/ml penicillin, 50 pg/ml streptomycin and 2 U/ml heparin.
Measurement of immunoglobulin synthesis rate in cell culture The plasma cells were cultured in 1 ml volumes (0.4 × 106 cells) in sterile bijoux (Sterilin), each culture containing 2 pCi of a C-14 labelled amino acid mixture (Radiochemicals; CFB 152, Equimolar specific activity mixture, 10 mCi/mmol). Each culture was equilibrated with 5% CO2 in air and incubated for 24 h at 37°C. After this primary incubation the cells were pooled, washed six times with HBSS-FCS, resuspended in the supplemented Fischer's medium and incubated as before, but w i t h o u t labelled amino acids in the medium. At suitable intervals the cells and supernatants were harvested using Millipore filters (0.22 pm). The cells retained on the filters were washed with 2 ml of HBSS-FCS and washings combined with the corresponding supernatants. Radioactivity in the supernatants and in the cells on each filter was assessed by scintillation counting using Instagel (Packard) as the scintillator system.
Characterisation of the synthesized protein by isoelectric focusing Any supernatant remaining after scintillation counting (ca. 2 ml) was freeze dried, taken up in 0.1 ml of HBSS and 0.1 ml of the patient's serum added. This solution was dialyzed against phosphate buffered saline to remove excess salt and subjected to isoelectric focusing in a polyacrylamide gel using a pH 3--10 gradient (Multiphor system LKB-Producter AB, Stockholm, Sweden). Unlabelled samples of patient's serum were focused simultaneously as controls. The gels were stained, washed and dried in the conventional manner and set up for autoradiography against Kodak Industrex C film. Exposure times were o f the order of 2 months.
213 RESULTS AND DISCUSSION Any technique which depends on immunochemical quantitation of m onoclonal immunoglobulins is likely to lead to serious errors since monoclonal and polyclonal immunoglobulins do n o t always react similarly with class specific antisera. The conventional assay procedure established with the five Supra-regional Specific Protein Reference Units in the U.K. is a 'chemical' one based on zonal electrophoresis of the patients' serum on cellulose acetate, staining with Ponceau S and scanning with a microdensitometer. The 'density' of the m onoc l ona l protein zone is directly expressed by the d e n s i t o m e t e r in g/1 by reference to the total serum protein previously measured in the sample. A comparison of results with r a n d o m m y e l o m a sera received in this laborat o r y for routine m e a s u r e m e n t by both the chemical technique and by an i m m u n o c h e m i c a l pr oc e dur e (A.I.P -- Technicon A ut o Analyzer System II, Technicon I n s t r u m e n t Corporation, T a r r y t o w n , New York) is shown in table 1. Gross discrepancies are obvious and the immunochemical technique is incapable o f distinguishing mixtures o f monoclonal and polyclonal immunoglobulins o f the same class. U n f o r t u n a t e l y the relatively low yields o f protein available f r om the cell cultures do n o t lend themselves to chemical assay. Measurements based on release of radioactivity labelled materials can be readily made but require characterization of the labelled material. Isoelectric focusing, com bi ne d with autoradiography, combines specificity and a r e q u i r e m e n t for only very small quantities of protein ( A r b u t h n o t t and Beeley, 1975). The limited heterogeneity banding patterns of m y e l o m a pro-
TABLE 1 Comparison of results of monoclonal protein assays by chemical and immunochemical methods. Patient
Monoclonal protein class
Assay (g/l) Immunochemical
Chemical 23 21 6-monoclonal 6-polyclonal 7 18 6-monoclonal 5-polyclonal 22 30 2-monoclonal 2-polyclonal
EE BM BS
IgG IgG IgG
27 4 12
AR IS
IgA IgA
7 6
EH
IgA
11
KP NW GR
IgM IgM IgM
16 10 2
214
a
b
c
d
Fig. 1. Isoelectric focusing patterns of myeloma sera in acrylamide gel at pH 3--10. Samples (a), (b) (c) show various band arrangements for IgG monoclonal proteins. Sample (d) is from an IgA myeloma patient with almost total suppression of IgG protein.
teins are characteristic f o r a p a r t i c u l a r p a t i e n t b o t h in a r r a n g e m e n t and n u m bers o f bands (Cwynarski et al., 1 9 7 5 ) and their position in the isoelectric gradient (fig. 1). A u t o r a d i o g r a p h y o f tissue culture s u p e r n a t a n t s m i x e d with the p a t i e n t ' s serum m a y reveal n o t o n l y p r o d u c t i o n o f m o n o c l o n a l p r o t e i n b u t also t h e a m o u n t o f n o r m a l h e t e r o g e n e o u s i m m u n o g l o b u l i n which the individual is still able to p r o d u c e . The l a t t e r has clinical implications which we h o p e to e x p l o r e f u r t h e r . T h e necessary e x p o s u r e of the a u t o r a d i o g r a p h is at p r e s e n t very prol o n g e d , b u t in o u r e x p e r i e n c e released radioactivity is always associated with labelled p r o t e i n and n o t w i t h free a m i n o acids. It is t h e r e f o r e possible to apply the results of the c u l t u r e synthesis clinically w i t h o u t waiting for the c h a r a c t e r i s a t i o n o f the p r o t e i n s . F o r c o n v e n i e n c e we express m e a s u r e m e n t s as p e r c e n t a g e radioactivity p r e s e n t in the s u p e r n a t a n t at the t i m e o f cell harvesting c o m p a r e d with the t o t a l radioactivity o f the c u l t u r e . T y p i c a l 'synthesis' curves are shown in fig. 2. T h e r e are m a r k e d d i f f e r e n c e s in rate o f synthesis b e t w e e n cells f r o m
215 80
7O °
>, 6 0
>-
4O
306
~
&
~
~
~
2'~
Second incubatron ( h )
Fig. 2. R a d i o a c t i v i t y release i n t o g r o w t h m e d i u m by m a r r o w cells f r o m : o O, a p a t i e n t w i t h a d v a n c e d IgA m y e l o m a ; D, a p a t i e n t with a n u n d e f i n e d p l a s m a cell d i s t u r b a n c e ; • • , a n o r m a l individual.
m y e l o m a patients and those from normal marrow. The flattening of the m y e l o m a synthesis curves with increasing times reflects usage of the labelled amino acid pool within the cells and to a lesser degree, decreasing cell viability. Measurements of the relative rate of synthesis (RRS), are made on the portions of the curves for samples taken for up to 9 h of the second incubation period in 'cold' medium. Some preliminary results are tabulated (table 2). Obtaining normal marrow specimens poses both practical and ethical problems. The two 'controls' in table 2 are from patients and therefore by definition pathological. Since there was no indication of neoplastic growth
TABLE 2 Relative rates of s y n t h e s i s o f labelled p r o t e i n by n o r m a l a n d m y e l o m a m a r r o w s . Patient
Diagnosis
Period of therapy
Plasma m o n o c l o n a l p r o t e i n (g/l)
Degree of immune paresis
Relative rate of synthesis
CC * LB EW BS DL AM FC
Myeloma Myeloma Myeloma Myeloma CML Control Control
10 m o n t h s 4 years 16 m o n t h s 3 years 6 years N/A N/A
IgA 58 IgA 18 IgA 29 IgG 6 no monoclonal protein no monoclonal protein no monoclonal protein
v. m a r k e d moderate marginal none none none none
4.0 3.0 1.5 1.5 2.0 0.2 0.2
* P a t i e n t died s h o r t l y a f t e r t h e s e studies were made.
216 in these patients and we feel justified in considering t hem as representative of ground state synthesis. Their RRS values of 0.2 contrast markedly with those from other patients. The highest rate of synthesis was observed for IgA in a m y e l o m a patient with advanced disease. In contrast, two o t h e r patients with IgA m y e l o m a show significantly lower RRS values, apparently correlating with the less advanced nature of their disease (table 2). We have n o t y e t investigated correlations between m y e l o m a immunoglobulin class and synthesis rate but synthesis from cultures of BS cells, a therapy-controlled IgG m y e l o m a patient possessing a good level of heterogenous immunoglobulin is similar to bot h that observed in controlled IgA m y e l o m a patients and in DL, a patient with a plasma cell disturbance other than a m onocl onal g a m m o p a t h y (table 2). F r o m these preliminary observations this labelling technique appears to offer a simple and valid way of studying the products of cell culture. The highly specific technique of isoelectric focusing for charaeterisation of the products of cell synthesis has received little attention as a routine analytical tool. Its co mb inat i on with autoradiography offers considerable scope for quantitative as well as qualitative devel opm ent . REFERENCES Arbuthnott, J.P. and J.A. Beeley, 1975, eds. Isoelectric focusing (Butterworths). Cwynarski, M.T., J. Watkins and P.M. Johnson, 1975, in: Isoelectric focusing, eds. J.P. Arbuthnott and J.A. Beeley (Butterworths) p. 319. Morell, A., F. Skvaril and S. Barandum, 1973, Clin. Exp Immunol. 13,293. Salmon, S.E. and B.A. Smith, 1970, J. Immunol. 104,665.
