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Immunofixation on cellulose acetate: An improved screening method for monoclonal immunoglobulins
Journal of Immunological Methods, 26 (1979) 365--368
365
Q Elsevier/North-Holland Biomedical Press
IMMUNOFIXATION ON CELLULOSE ACETATE: AN IMPROVED SCREENING METHOD FOR MONOCLONAL IMMUNOGLOBULINS
MARCO A. PIZZOLATO, F E R N A N D O R. GO/~I and RO BERTO C. S A L V A R E Z Z A
Department of Clinical Chemistry, Faculty of Biochemistry, University of Buenos Aires, Jun ~'n 956, 1113 Buenos Aires, Argentina (Received 20 July 1978, accepted 10 October 1978)
A rapid, simple and economical method is described for typing monoclonal immunoglobulins. It is a modification of the immunofixation electrophoresis method and cellulose acetate has been used as a supporting medium. It involves an initial electrophoretic separation followed by an antigen (Ag)-antibody (Ab) reaction 'in situ'. Eight samples can be typed on each 5.7 cm × 10.5 cm strip and only 20 pl of commercial antiserum are required (about 2.5 /~l per sample). The method permits detection of monoclonal proteins at concentrations as low as 100 ng[pl in only 60 min.
INTRODUCTION
Immunofixation was initially described by Alper and Johnson (1969}. Although these authors suggest the use of alternative media, most workers have systematically used agarose gel (Cawley et al., 1976; Cejka and Kithier, 1976), polyacrylamide gel and acrylamide and isoelectric focusing for high resolution (Carrel et al., 1969; Makomkawkeyoon and Haque, 1970; Arnaud et al., 1977), and cellulose acetate has only been mentioned perfunctorily (Chang and Inglis, 1975; Grunbaum and Zajac, 1977). In our method cellulose acetate serves as a supporting medium for immunofixation. After conventional electrophoresis on cellulose acetate the antiserum is applied with a volumetric distributor as previously described (Pizzolato, 1973). The technique has several advantages over previous methods and has proved suitable for rapid screening of monoclonal immunoglobulins. MA TER I ALS AND METHODS
Serum, plasma, urine, cerebrospinal fluid, isolated fractions and others biological fluids have been used. To obtain an adequate antigen : antibody ratio, serum was diluted to give * Presented in part to the Xth International Congress of Clinical Chemistry, Mexico City, February 1978.
366
a monoclonal protein c onc e nt r at i on between 300 and 1000 mg/1, depending on the antiserum titer. Electrophoresis was p e r f o r m e d by the usual procedure using veronal buffer (pH 8.6, I = 0.05). Cellulose acetate strips of different brands have buen used: b o th dry (Beckman, Gelman, Helena, Sartorius) and gelatinized (Cellogel). Beckman's m i cr oz one system was used to apply samples to the strips and electrophoresis p e r f o r m e d at r o o m temperature, using 250 V for 15 min. After electrophoresis, 20 ~1 of undiluted m o n o v a l e n t antiserum (Nephelometric grade, Dako, Kallestad, Behringwerke) were spread on the dry strips, and 40 ~1 on the gelatinized ones, by means of a volumetric distribut o r (Pizzolato, 1973) from the cathodic end to the albumin front. The strips were then incubated in a moist chamber for 5 rain at r o o m temperature. Non-precipitated proteins were removed from the strips by continuous shaking in saline for at least 15 min. The strips were then placed for 10 min in a staining solution of Coomassie Brilliant Blue R250 (2.5 g/1 in m e t h a n o l - water--acetic acid, 5 : 5 : 1, by vol.). Methanol--water--acetic acid (5 : 5 : 1 by vol.) was used for destaining. For a p e r m a n e n t record the strips were dipped in eth an ol - - w at er (95 : 5 by vol.) for 30 sec, in ethanol--acetic acid (75 : 25 by vol.) for 1 min and were then placed on glass slides and warmed at 50°C to 60°C until t he y become com pl e tel y transparent. R E S U L T S AND D I S C U S S I O N
Fig. I demonstrates the effectiveness of i m m u n o f i x a t i o n with various dilutions of an IgG-kappa m onocl onal protein, in which the optimal Ag-Ab ratio is shown in Fig. 1B~ (400 mg/1 of m o n o c l o n a l protein). It also shows the kind o f precipitation bands obtained at different times and incubation temperatures. At 37°C (Fig. 1C and F) there is accelerated dissolution of the complexes because of faster diffusion. Using different temperatures and times o f incubation, we have det er m i ned that the best conditions are 5 min at r o o m t e m p e r a t u r e (between 20 and 25 ° C). The use of a goat antiserum is also r e c o m m e n d e d to minimize Ag-Ab dissolution. Correct dilution, ternperature and incubation time are i m p o r t a n t , particularly in the presence of oligoclonal proteins which can make interpretation difficult, because of their apparent restricted electrophoretic heterogeneity on i m m u n o f i x a t i o n . I m m u n o f i x a t i o n on cellulose acetate allows detection of monoclonal proteins at co n cen tr a t i ons as low as 100 ng/pl (20 ng in approxi m at el y 0.2 pl of sample, see Fig. 1B2). The technique is particularly suitable for characterization of double or multiple c o m p o n e n t s , mainly if t h e y have similar electrophoretic mobilities or low concentrations such as shown in Fig. 2A, where a sharp resolution of the double band is shown. This c a n n o t be satisfactorily resolved by converttional immunoelectrophoresis, when the use of two-dimensional immunoelectrophoresis (Laurell, 1965) or transfer immunoelectrophoresis (Kohn,
367
®
I
II +
I
i
I
++
+
D
II~
A .1
t
2
oO o
o
I
I
++ +
E
+
F
B
C
Fig. 1. A a n d D : e l e c t r o p h e r o g r a m s o f several d i l u t i o n s o f a s e r u m I g G - k a p p a m o n o c l o n a l g a m m o p a t h y , o, n o r m a l s e r u m ; ~o, p o l y c l o n a l g a m m o p a t h y . B, C a n d E, F s h o w i r n m u n o f i x a t i o n o n cellulose a c e t a t e o f t h e s a m e s a m p l e s as A a n d D r e s p e c t i v e l y w i t h m o n o v a l e n t IgG a n t i s e r u m . B: 5 m i n i n c u b a t i o n at r o o m t e m p e r a t u r e . C: 5 rain i n c u b a t i o n at 3 7 ° C . E: 15 rain i n c u b a t i o n at r o o m t e m p e r a t u r e . F : 15 rain i n c u b a t i o n at 3 7 ° C .
0 A
B
B
C ~!i+F :++ i:!++++ i++ :i++ i+ :
19(5 ® Fig. 2. I m m u n o f i x a t i o n of: ( A ) a d o u b l e I g G - k a p p a m o n o c l o n a l g a m m o p a t h y ; (B) an Fc c o m p o n e n t f r o m a p a t i e n t w i t h a g a m m a h e a v y c h a i n disease; (C) an I g G - l a m b d a ' M ' corn+ ponent.
368
1971) is required. Both of the latter procedures are laborious, time-consuming and require large amounts of material. With reference to the original m e t h o d using agarose gel, the present technique gives similar resolution and has the following advantages. Firstly, smaller quantities of reactants are used (2.5 pl of antiserum per sample), and secondly the application of the antiserum b y cellulose acetate or filter paper strips is avoided, thus reducing the risk of losing immunocomplexes (Arnaud et al., 1977). Furthermore the time required for the different steps -- electrophoresis, incubation, washing, staining and destaining -- is much reduced. Cellulose acetate is thinner than agarose gel, so smaller quantities of antiserum are required, and the Ag-Ab reaction is practically instantaneous because Ab diffusion is negligible. One disadvantage with this m e t h o d is that less immune complexes can be washed in the soaking phase, especially when the critical parameters ('M' c o m p o n e n t concentration, temperature and incubation time) are n o t in the correct range. In our experience immunofixation on cellulose acetate -- preferably the dry t y p e -- is the m e t h o d of choice for routine screening of proteins with restricted electrophoretic heterogeneity such as those found in monoclonal gammopathies and has also proved satisfactory for studying the polymorphism of alpha-1 antitrypsin. REFERENCES Alper, C.A. and A.M. Johnson, 1969, Vox. Sang. 17,445. Arnaud, P., G.B. Wilson, J. Koistinen and H.H. Fudenberg, 1977, J. Immunol. Methods 16, 221. Carrel, S., L. Theilkaes, S. Skvaril and S. Barandun, 1969, J. Chromatogr. 4 5 , 4 8 3 . Cawley, L.P., B.J. Minard, W.W. Tourtellote, B.I. Ma and C. Chelle, 1976, Clin. Chem. 22, 1262. Cejka, J. and K. Kithier, 1976, Immunochemistry 13,629. Chang, C.H. and N.R. Inglis, 1975, Clin. Chim. Acta 65, 91. Grunbaum, B.W. and P.L. Zajac, 1977, J. Forensic Sci. 22, 586. Kohn, J., 1971, in: Methods in Immunology and Immunochemistry, eds. C.A. Williams and M.W. Chase (Academic Press, New York) p. 275. Laurel], C.B., 1965, Anal. Biochem. 10, 358. Makomkawkeyoon, S. and R. Haque, 1970, Anal. Biochem. 3 6 , 4 4 2 . Pizzolato, M.A., 1973, Clin. Chim. Acta 4 5 , 2 0 7 .
365
Q Elsevier/North-Holland Biomedical Press
IMMUNOFIXATION ON CELLULOSE ACETATE: AN IMPROVED SCREENING METHOD FOR MONOCLONAL IMMUNOGLOBULINS
MARCO A. PIZZOLATO, F E R N A N D O R. GO/~I and RO BERTO C. S A L V A R E Z Z A
Department of Clinical Chemistry, Faculty of Biochemistry, University of Buenos Aires, Jun ~'n 956, 1113 Buenos Aires, Argentina (Received 20 July 1978, accepted 10 October 1978)
A rapid, simple and economical method is described for typing monoclonal immunoglobulins. It is a modification of the immunofixation electrophoresis method and cellulose acetate has been used as a supporting medium. It involves an initial electrophoretic separation followed by an antigen (Ag)-antibody (Ab) reaction 'in situ'. Eight samples can be typed on each 5.7 cm × 10.5 cm strip and only 20 pl of commercial antiserum are required (about 2.5 /~l per sample). The method permits detection of monoclonal proteins at concentrations as low as 100 ng[pl in only 60 min.
INTRODUCTION
Immunofixation was initially described by Alper and Johnson (1969}. Although these authors suggest the use of alternative media, most workers have systematically used agarose gel (Cawley et al., 1976; Cejka and Kithier, 1976), polyacrylamide gel and acrylamide and isoelectric focusing for high resolution (Carrel et al., 1969; Makomkawkeyoon and Haque, 1970; Arnaud et al., 1977), and cellulose acetate has only been mentioned perfunctorily (Chang and Inglis, 1975; Grunbaum and Zajac, 1977). In our method cellulose acetate serves as a supporting medium for immunofixation. After conventional electrophoresis on cellulose acetate the antiserum is applied with a volumetric distributor as previously described (Pizzolato, 1973). The technique has several advantages over previous methods and has proved suitable for rapid screening of monoclonal immunoglobulins. MA TER I ALS AND METHODS
Serum, plasma, urine, cerebrospinal fluid, isolated fractions and others biological fluids have been used. To obtain an adequate antigen : antibody ratio, serum was diluted to give * Presented in part to the Xth International Congress of Clinical Chemistry, Mexico City, February 1978.
366
a monoclonal protein c onc e nt r at i on between 300 and 1000 mg/1, depending on the antiserum titer. Electrophoresis was p e r f o r m e d by the usual procedure using veronal buffer (pH 8.6, I = 0.05). Cellulose acetate strips of different brands have buen used: b o th dry (Beckman, Gelman, Helena, Sartorius) and gelatinized (Cellogel). Beckman's m i cr oz one system was used to apply samples to the strips and electrophoresis p e r f o r m e d at r o o m temperature, using 250 V for 15 min. After electrophoresis, 20 ~1 of undiluted m o n o v a l e n t antiserum (Nephelometric grade, Dako, Kallestad, Behringwerke) were spread on the dry strips, and 40 ~1 on the gelatinized ones, by means of a volumetric distribut o r (Pizzolato, 1973) from the cathodic end to the albumin front. The strips were then incubated in a moist chamber for 5 rain at r o o m temperature. Non-precipitated proteins were removed from the strips by continuous shaking in saline for at least 15 min. The strips were then placed for 10 min in a staining solution of Coomassie Brilliant Blue R250 (2.5 g/1 in m e t h a n o l - water--acetic acid, 5 : 5 : 1, by vol.). Methanol--water--acetic acid (5 : 5 : 1 by vol.) was used for destaining. For a p e r m a n e n t record the strips were dipped in eth an ol - - w at er (95 : 5 by vol.) for 30 sec, in ethanol--acetic acid (75 : 25 by vol.) for 1 min and were then placed on glass slides and warmed at 50°C to 60°C until t he y become com pl e tel y transparent. R E S U L T S AND D I S C U S S I O N
Fig. I demonstrates the effectiveness of i m m u n o f i x a t i o n with various dilutions of an IgG-kappa m onocl onal protein, in which the optimal Ag-Ab ratio is shown in Fig. 1B~ (400 mg/1 of m o n o c l o n a l protein). It also shows the kind o f precipitation bands obtained at different times and incubation temperatures. At 37°C (Fig. 1C and F) there is accelerated dissolution of the complexes because of faster diffusion. Using different temperatures and times o f incubation, we have det er m i ned that the best conditions are 5 min at r o o m t e m p e r a t u r e (between 20 and 25 ° C). The use of a goat antiserum is also r e c o m m e n d e d to minimize Ag-Ab dissolution. Correct dilution, ternperature and incubation time are i m p o r t a n t , particularly in the presence of oligoclonal proteins which can make interpretation difficult, because of their apparent restricted electrophoretic heterogeneity on i m m u n o f i x a t i o n . I m m u n o f i x a t i o n on cellulose acetate allows detection of monoclonal proteins at co n cen tr a t i ons as low as 100 ng/pl (20 ng in approxi m at el y 0.2 pl of sample, see Fig. 1B2). The technique is particularly suitable for characterization of double or multiple c o m p o n e n t s , mainly if t h e y have similar electrophoretic mobilities or low concentrations such as shown in Fig. 2A, where a sharp resolution of the double band is shown. This c a n n o t be satisfactorily resolved by converttional immunoelectrophoresis, when the use of two-dimensional immunoelectrophoresis (Laurell, 1965) or transfer immunoelectrophoresis (Kohn,
367
®
I
II +
I
i
I
++
+
D
II~
A .1
t
2
oO o
o
I
I
++ +
E
+
F
B
C
Fig. 1. A a n d D : e l e c t r o p h e r o g r a m s o f several d i l u t i o n s o f a s e r u m I g G - k a p p a m o n o c l o n a l g a m m o p a t h y , o, n o r m a l s e r u m ; ~o, p o l y c l o n a l g a m m o p a t h y . B, C a n d E, F s h o w i r n m u n o f i x a t i o n o n cellulose a c e t a t e o f t h e s a m e s a m p l e s as A a n d D r e s p e c t i v e l y w i t h m o n o v a l e n t IgG a n t i s e r u m . B: 5 m i n i n c u b a t i o n at r o o m t e m p e r a t u r e . C: 5 rain i n c u b a t i o n at 3 7 ° C . E: 15 rain i n c u b a t i o n at r o o m t e m p e r a t u r e . F : 15 rain i n c u b a t i o n at 3 7 ° C .
0 A
B
B
C ~!i+F :++ i:!++++ i++ :i++ i+ :
19(5 ® Fig. 2. I m m u n o f i x a t i o n of: ( A ) a d o u b l e I g G - k a p p a m o n o c l o n a l g a m m o p a t h y ; (B) an Fc c o m p o n e n t f r o m a p a t i e n t w i t h a g a m m a h e a v y c h a i n disease; (C) an I g G - l a m b d a ' M ' corn+ ponent.
368
1971) is required. Both of the latter procedures are laborious, time-consuming and require large amounts of material. With reference to the original m e t h o d using agarose gel, the present technique gives similar resolution and has the following advantages. Firstly, smaller quantities of reactants are used (2.5 pl of antiserum per sample), and secondly the application of the antiserum b y cellulose acetate or filter paper strips is avoided, thus reducing the risk of losing immunocomplexes (Arnaud et al., 1977). Furthermore the time required for the different steps -- electrophoresis, incubation, washing, staining and destaining -- is much reduced. Cellulose acetate is thinner than agarose gel, so smaller quantities of antiserum are required, and the Ag-Ab reaction is practically instantaneous because Ab diffusion is negligible. One disadvantage with this m e t h o d is that less immune complexes can be washed in the soaking phase, especially when the critical parameters ('M' c o m p o n e n t concentration, temperature and incubation time) are n o t in the correct range. In our experience immunofixation on cellulose acetate -- preferably the dry t y p e -- is the m e t h o d of choice for routine screening of proteins with restricted electrophoretic heterogeneity such as those found in monoclonal gammopathies and has also proved satisfactory for studying the polymorphism of alpha-1 antitrypsin. REFERENCES Alper, C.A. and A.M. Johnson, 1969, Vox. Sang. 17,445. Arnaud, P., G.B. Wilson, J. Koistinen and H.H. Fudenberg, 1977, J. Immunol. Methods 16, 221. Carrel, S., L. Theilkaes, S. Skvaril and S. Barandun, 1969, J. Chromatogr. 4 5 , 4 8 3 . Cawley, L.P., B.J. Minard, W.W. Tourtellote, B.I. Ma and C. Chelle, 1976, Clin. Chem. 22, 1262. Cejka, J. and K. Kithier, 1976, Immunochemistry 13,629. Chang, C.H. and N.R. Inglis, 1975, Clin. Chim. Acta 65, 91. Grunbaum, B.W. and P.L. Zajac, 1977, J. Forensic Sci. 22, 586. Kohn, J., 1971, in: Methods in Immunology and Immunochemistry, eds. C.A. Williams and M.W. Chase (Academic Press, New York) p. 275. Laurel], C.B., 1965, Anal. Biochem. 10, 358. Makomkawkeyoon, S. and R. Haque, 1970, Anal. Biochem. 3 6 , 4 4 2 . Pizzolato, M.A., 1973, Clin. Chim. Acta 4 5 , 2 0 7 .