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Solid-phase radioimmunoassay of immunoglobulins G, A and M: Applicability in analysis of sucrose gradients
Journal of Immunological Methods, 71 (1984) 127-132
127
Elsevier JIM 03110
Solid-Phase Radioimmunoassay of Immunoglobulins G, A and M: Applicability in Analysis of Sucrose Gradients Erik F. Eriksen
1,*
Henning Danielsen 1, Anders S. Johansen 2 and Lars I. Larsson 3
I Medical Department C, A arhus Kommunehospital, 2 Institute of Medical Biochemistry, University of A arhus, A arhus, and ~ Unit of Histochemistry, University Institute of Pathology, Copenhagen, Denmark
(Received 2 November 1983, accepted 2 March 1984)
A simple and sensitive solid-phase radioimmunoassay for the detection of immunoglobulins G, A and M in sucrose gradients is described. The solid-phase consisted of immunoglobulins adsorbed to polystyrene tubes. Using buffers without detergent and 1251-labeled sheep anti-rabbit igG as radioligand, the assay was able to detect 0.8 ng per tube in the IgG assay and 1.6 ng per tube in the IgA and lgM assays. Standard curves with antigen dissolved in 10% and 32% sucrose were superimposable and did not deviate from standard curves with antigen dissolved in buffer without sucrose. Using these techniques on ultracentrifugation samples from patients with systemic lupus erythematosus, Sch6nlein-Henoch nephritis and lgA glorulonephritis it was possible to detect both immunoglobulin fragments and immunoglobulin aggregates at the same time without prior dialysis of the samples. Key words: solid-phase radioimmunoassay - immunoglobulins - sucrose gradient centrifugation
Introduction Since the a p p e a r a n c e o f solid-phase techniques for r a d i o i m m u n o a s s a y ( Cat t and Tregear, 1967) these m e t h o d s h a v e been used extensively for the d e t e c t i o n of small a m o u n t s of a great variety of substances. T h e m e t h o d s a p p l y i n g r a d i o l a b e l e d ligands m a i n l y use p r o t ei n A ( L a n g o n e , 1978, 1980; L a n g o n e et al., 1979; G e e and L a n g o n e , 1981) or I g G (Blankstein et al., 1980). T h e aim of this study was to d e v e l o p a sensitive solid-phase r a d i o i m m u n o a s s a y to m a k e possible the d e t e c t i o n of small a m o u n t s o f i m m u n o g l o b u l i n s G, A a n d M in u l t r a c e n t r i f u g a t i o n samples. * Address for correspondence and reprints: Erik Fink Eriksen, Medical Department III, Aarhus Amtssygehus, Tage Hansensgade 2, DK-8000 Aarhus C., Denmark. 0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.
128 Materials and Methods
Polystyrene tubes (70 mm × 11 mm) were obtained from Nunc (Copenhagen). Human IgG and IgM were obtained from Behringwerke (Marburg), and colostrum IgA was obtained from Calbiochem (San Diego, CA). Rabbit antibodies against human IgG (-/-chain) and IgM (tt-chain) were obtained from Dako (Copenhagen). Rabbit antibody against IgA (a-chain) was generously supplied by Dako. Sheep anti-rabbit IgG was purchased from Statens Bakteriologiska Laboratorium (Stockholm), bovine serum albumin (BSA) from Sigma (St. Louis, MO), human serum albumin (HSA) from Novo (Copenhagen) and normal sheep serum (NSS) from Statens Serum Institut (Copenhagen). The crossreactivity of the IgG, IgA and IgM antibodies were tested in our solid-phase system by adsorbing the respective antigens to polystyrene tubes and comparing the displacing ability of the different antigens towards the different antibodies used. The IgG and IgM antibodies exhibited less than 1% crossreactivity towards the 2 other antigens, while the IgA antibody revealed 5% crossreactivity with IgM and less than 1% with IgG.
Iodination of sheep anti-rabbit IgG One hundred microliters of sheep anti-rabbit IgG (8 m g / m l in demineralized water) were mixed with 40 ~1 chloramine-T (0.4 m g / m l in 0.5 m o l / l phosphate buffer, pH 7.4) and 1.2 mCi 125I. The mixture was shaken for 20 s and allowed to stand for 1 min before the addition of 40 ~1 bisulphite (0.4 m g / m l in 0.5 mol/1 phosphate buffer, pH 7.4). Before gel filtration on a Sephadex G-25 fine column the mixture was diluted to a final volume of 1.3 ml with demineralized water.
Coating of polystyrene tubes with immunoglobulins Immunoglobulins G, A and M were dissolved in phosphate buffer (0.1 mol/l, pH 7.4) to a concentration of 2 ttg/ml. Five hundred microliters of these solutions were pipetted into each polystyrene tube and the tubes were placed in a water bath (37 o C) overnight. After incubation, the tubes were used directly or stored for up to 1 month at 4°C. Before use the tubes were washed 3 times with phosphate buffer.
Assay procedure One hundred microliters of antigen solution (IgG, IgA and IgM standard or sample) were added to each tube followed by 200 ttl antibody (rabbit anti-human IgG, IgA or IgM). Oi0timal dilution for anti-IgG was 1 : 50,000 and 1 : 40,000 for anti-IgA and IgM. The mixture of antigen and antibody was incubated for 24 h at 4°C. Standards, samples and antibody were diluted in phosphate-buffered saline (PBS) + 0.1% BSA + 0.1% NaN 3 (pH 7.4). After incubation the tubes were washed 3 times with PBS. Between each washing the tubes were allowed to stand 5 min with washing buffer. After the last wash the tubes were emptied by suction and 100 ttl of 125I-sheep anti-rabbit IgG (105 cpm) were added with 200 ttl of incubation buffer. Again the tubes were incubated for 24 h at 4°C, followed by 3 washes with PBS. After the last wash the tubes were counted in a Packard Gamma spectrometer.
129
Sucrose gradient ultracentrifugation Sucrose gradients (10-32% w / w ) were prepared in a linear gradient former and pumped directly into the centrifuge tubes. The sera were diluted 1 : 4 in PBS and 100 ~1 of the sample were put on top of the gradient. Centrifugation was carried out at 35,000 rpm for 16 h (rotor temperature 8°C). A Beckman SW 40 rotor was used in a Beckman ultracentrifuge. After centrifugation the gradient was pumped through a flow cuvette and the protein concentration monitored in a Beckman DB spectrophotometer. The extinction at 280 nm was plotted with a Philips PM 8000 plotter. Sixteen fractions from each centrifuge tube were collected on an LKB fraction collector. The effluent profiles were read into a Tektronix 4051 computer as data pairs and subsequently converted to sedimentation coefficients according to the principle of Martin and Ames (1961). The program used was a Fortran program made as described by Steensgaard et al. (1978).
Results
Influence of high sucrose concentrations The suitability of the assay procedures in the analysis of sucrose gradient centrifugation samples of sera depended on the assay exhibiting constant performance in the concentration range of sucrose used in the gradient. Our gradients ranged from 10 to 32% and we therefore tested the standard curves with standards % of
IgG
maximal binding
IgM
IgA
qol-
BC :
60
n
o
I 1
1o
I
I
I
1oQ
1o 3
lo 4
I l o ~ 't
I
I
I
lO
,to I!
io 2
/ I
log cone.
lo 4
I o Is I
I
I
I
I
I
1o
1o i
1o 3
lo 4
,io B
ng/ml
Fig. 1. Standard curves obtained for all 3 immunoglobulins (solid line). Superimposed are the results obtained when standards were dissolved in 10% (e) and 32% (D) sucrose.
130 Ig JJglml A
I
I 10
I ;~0
3o
I0~
~0
3o
II 3O
t
C -~
III
, g~ .
,..'"'\'-
ol
i "Io
Ro
Sed. Coeff
3o
S
Fig. 2. Distributions of IgG ( ), IgA (...) and lgM (. . . . . . ) in relation to sedimentation coefficients obtained on sucrose gradient ultracentrifugation. A: normal individual; B: patient with Sch6nlein-Henoch nephritis; and C: patient with SLE. Arrow I indicates the presence of IgA, lgG and IgM aggregates at 25-30 S. Arrow I1 indicates the presence of 7 S lgM fragments and arrow 111 the presence of IgA and IgM aggregates at 20-30 S. m a d e up in 10% and 32% sucrose, giving a final concentration of 3.3% and 10.6% respectively in the incubation mixture. Fig. 1 shows the results obtained. The standard curves were superimposable in the range 10 n g / m l to 10 ~ g / m l for all 3 immunoglobulins tested.
Analysis of sera from patients with immune complex disease Fig. 2 shows the distribution of IgA, I g G and IgM in sucrose gradient centrifugations samples of sera from a normal individual (A), a patient with Sch6nlein-Henoch glomerulonephritis (B) and a patient with systemic lupus erythematosus (SLE) (C).
131 In the normal individual the 3 peaks for I g G at 7 S, IgA at 10 S and IgM at 19 S are clearly visible. The Serum of the patient with Sch6nlein-Henoch glomerulonephritis revealed aggregates of IgG, A and M at 25-30 S. The most profound changes in immunoglobulin distribution patterns were Seen in the SLE serum. A large peak of IgM immunoreactive substances was demonstrated at 7 S, and high molecular weight aggregates containing IgM and IgA were seen at 25-30 S.
Discussion This simple assay procedure using commercial available antisera and antigens is comparable with other immunoglobulin assays described previously (Gee and Langone, 1981; Ischiguru et al., 1981; Nerenberg and Prasad, 1981) with respect to sensitivity. Further, it requires iodination of only one tracer substance to measure 3 different antigens. The avoidance of the separation step necessary in conventional radioimmunoassay techniques makes it a very productive assay. In 6 h work one technician can analyze more than 300 samples. The assay covers a wide range of concentrations, which diminishes the dilution problems encountered in other systems with smaller ranges. The analysis of ultracentrifugation samples in this way gives information which otherwise could only be obtained by the use of several different techniques. The system is not influenced by sucrose concentrations up to 32%, which makes it an ideal method for the analysis of samples subjected to sucrose gradient centrifugation. It may even be of importance in relation to other media, where high solute concentrations might interfere with other techniques. The small volume needed in this assay makes the analysis of several different substances in the same gradient fraction possible. Without prior dialysis of the sucrose gradient samples, it is possible to determine the quantitative distribution of immunoglobulins in relation to sedimentation coefficients. At the same time the presence of immunoglobulin fragments and aggregates is detected, giving a detailed picture of disturbances in immunoglobulin synthesis and possible immune complex formation. Furthermore, C l q fixation techniques can only detect complexes containing IgG and IgM, while our technique also detects IgA complexes.
References Blankstein, L.K., B.D. Stollar and S.B. Levy, 1980, Anal. Biochem. 104, 168. Catt, K. and G.W. Tregear, 1967, Science 158, 1570. Gee, A.P. and J.J. Langone, 1981, Anal. Biochem. 116, 524. lschiguru, Y, K. Kato and T. Ito, 1981, Clin. Claim. Acta 116, 237. Langone, J.J., 1978, J. Immunol. Methods 24, 269. Langone, J.J., 1980, J. Immunol. Methods 34, 93. Langone, J.J., M.D. Boyle and T. Borsos, 1979, Anal. Biochem. 93, 207.
132 Martin, R.G. and B.N. Ames, 1961, J. Biol. Chem. 236, 1372. Nerenberg, S.T. and R. Prasad, 1981, in: Methods of Enzymology, eds. J.J. Langone and H. Van Vunakis (Academic Press, New York) p. 666. Steensgaard, J., N.P.H. Moller and L. Funding, 1978, in: Centrifugal Separation in Molecular and Cell Biology, eds. G.D.B. Birnie and D. Richwood (Butterworth, London) p. 115.
127
Elsevier JIM 03110
Solid-Phase Radioimmunoassay of Immunoglobulins G, A and M: Applicability in Analysis of Sucrose Gradients Erik F. Eriksen
1,*
Henning Danielsen 1, Anders S. Johansen 2 and Lars I. Larsson 3
I Medical Department C, A arhus Kommunehospital, 2 Institute of Medical Biochemistry, University of A arhus, A arhus, and ~ Unit of Histochemistry, University Institute of Pathology, Copenhagen, Denmark
(Received 2 November 1983, accepted 2 March 1984)
A simple and sensitive solid-phase radioimmunoassay for the detection of immunoglobulins G, A and M in sucrose gradients is described. The solid-phase consisted of immunoglobulins adsorbed to polystyrene tubes. Using buffers without detergent and 1251-labeled sheep anti-rabbit igG as radioligand, the assay was able to detect 0.8 ng per tube in the IgG assay and 1.6 ng per tube in the IgA and lgM assays. Standard curves with antigen dissolved in 10% and 32% sucrose were superimposable and did not deviate from standard curves with antigen dissolved in buffer without sucrose. Using these techniques on ultracentrifugation samples from patients with systemic lupus erythematosus, Sch6nlein-Henoch nephritis and lgA glorulonephritis it was possible to detect both immunoglobulin fragments and immunoglobulin aggregates at the same time without prior dialysis of the samples. Key words: solid-phase radioimmunoassay - immunoglobulins - sucrose gradient centrifugation
Introduction Since the a p p e a r a n c e o f solid-phase techniques for r a d i o i m m u n o a s s a y ( Cat t and Tregear, 1967) these m e t h o d s h a v e been used extensively for the d e t e c t i o n of small a m o u n t s of a great variety of substances. T h e m e t h o d s a p p l y i n g r a d i o l a b e l e d ligands m a i n l y use p r o t ei n A ( L a n g o n e , 1978, 1980; L a n g o n e et al., 1979; G e e and L a n g o n e , 1981) or I g G (Blankstein et al., 1980). T h e aim of this study was to d e v e l o p a sensitive solid-phase r a d i o i m m u n o a s s a y to m a k e possible the d e t e c t i o n of small a m o u n t s o f i m m u n o g l o b u l i n s G, A a n d M in u l t r a c e n t r i f u g a t i o n samples. * Address for correspondence and reprints: Erik Fink Eriksen, Medical Department III, Aarhus Amtssygehus, Tage Hansensgade 2, DK-8000 Aarhus C., Denmark. 0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.
128 Materials and Methods
Polystyrene tubes (70 mm × 11 mm) were obtained from Nunc (Copenhagen). Human IgG and IgM were obtained from Behringwerke (Marburg), and colostrum IgA was obtained from Calbiochem (San Diego, CA). Rabbit antibodies against human IgG (-/-chain) and IgM (tt-chain) were obtained from Dako (Copenhagen). Rabbit antibody against IgA (a-chain) was generously supplied by Dako. Sheep anti-rabbit IgG was purchased from Statens Bakteriologiska Laboratorium (Stockholm), bovine serum albumin (BSA) from Sigma (St. Louis, MO), human serum albumin (HSA) from Novo (Copenhagen) and normal sheep serum (NSS) from Statens Serum Institut (Copenhagen). The crossreactivity of the IgG, IgA and IgM antibodies were tested in our solid-phase system by adsorbing the respective antigens to polystyrene tubes and comparing the displacing ability of the different antigens towards the different antibodies used. The IgG and IgM antibodies exhibited less than 1% crossreactivity towards the 2 other antigens, while the IgA antibody revealed 5% crossreactivity with IgM and less than 1% with IgG.
Iodination of sheep anti-rabbit IgG One hundred microliters of sheep anti-rabbit IgG (8 m g / m l in demineralized water) were mixed with 40 ~1 chloramine-T (0.4 m g / m l in 0.5 m o l / l phosphate buffer, pH 7.4) and 1.2 mCi 125I. The mixture was shaken for 20 s and allowed to stand for 1 min before the addition of 40 ~1 bisulphite (0.4 m g / m l in 0.5 mol/1 phosphate buffer, pH 7.4). Before gel filtration on a Sephadex G-25 fine column the mixture was diluted to a final volume of 1.3 ml with demineralized water.
Coating of polystyrene tubes with immunoglobulins Immunoglobulins G, A and M were dissolved in phosphate buffer (0.1 mol/l, pH 7.4) to a concentration of 2 ttg/ml. Five hundred microliters of these solutions were pipetted into each polystyrene tube and the tubes were placed in a water bath (37 o C) overnight. After incubation, the tubes were used directly or stored for up to 1 month at 4°C. Before use the tubes were washed 3 times with phosphate buffer.
Assay procedure One hundred microliters of antigen solution (IgG, IgA and IgM standard or sample) were added to each tube followed by 200 ttl antibody (rabbit anti-human IgG, IgA or IgM). Oi0timal dilution for anti-IgG was 1 : 50,000 and 1 : 40,000 for anti-IgA and IgM. The mixture of antigen and antibody was incubated for 24 h at 4°C. Standards, samples and antibody were diluted in phosphate-buffered saline (PBS) + 0.1% BSA + 0.1% NaN 3 (pH 7.4). After incubation the tubes were washed 3 times with PBS. Between each washing the tubes were allowed to stand 5 min with washing buffer. After the last wash the tubes were emptied by suction and 100 ttl of 125I-sheep anti-rabbit IgG (105 cpm) were added with 200 ttl of incubation buffer. Again the tubes were incubated for 24 h at 4°C, followed by 3 washes with PBS. After the last wash the tubes were counted in a Packard Gamma spectrometer.
129
Sucrose gradient ultracentrifugation Sucrose gradients (10-32% w / w ) were prepared in a linear gradient former and pumped directly into the centrifuge tubes. The sera were diluted 1 : 4 in PBS and 100 ~1 of the sample were put on top of the gradient. Centrifugation was carried out at 35,000 rpm for 16 h (rotor temperature 8°C). A Beckman SW 40 rotor was used in a Beckman ultracentrifuge. After centrifugation the gradient was pumped through a flow cuvette and the protein concentration monitored in a Beckman DB spectrophotometer. The extinction at 280 nm was plotted with a Philips PM 8000 plotter. Sixteen fractions from each centrifuge tube were collected on an LKB fraction collector. The effluent profiles were read into a Tektronix 4051 computer as data pairs and subsequently converted to sedimentation coefficients according to the principle of Martin and Ames (1961). The program used was a Fortran program made as described by Steensgaard et al. (1978).
Results
Influence of high sucrose concentrations The suitability of the assay procedures in the analysis of sucrose gradient centrifugation samples of sera depended on the assay exhibiting constant performance in the concentration range of sucrose used in the gradient. Our gradients ranged from 10 to 32% and we therefore tested the standard curves with standards % of
IgG
maximal binding
IgM
IgA
qol-
BC :
60
n
o
I 1
1o
I
I
I
1oQ
1o 3
lo 4
I l o ~ 't
I
I
I
lO
,to I!
io 2
/ I
log cone.
lo 4
I o Is I
I
I
I
I
I
1o
1o i
1o 3
lo 4
,io B
ng/ml
Fig. 1. Standard curves obtained for all 3 immunoglobulins (solid line). Superimposed are the results obtained when standards were dissolved in 10% (e) and 32% (D) sucrose.
130 Ig JJglml A
I
I 10
I ;~0
3o
I0~
~0
3o
II 3O
t
C -~
III
, g~ .
,..'"'\'-
ol
i "Io
Ro
Sed. Coeff
3o
S
Fig. 2. Distributions of IgG ( ), IgA (...) and lgM (. . . . . . ) in relation to sedimentation coefficients obtained on sucrose gradient ultracentrifugation. A: normal individual; B: patient with Sch6nlein-Henoch nephritis; and C: patient with SLE. Arrow I indicates the presence of IgA, lgG and IgM aggregates at 25-30 S. Arrow I1 indicates the presence of 7 S lgM fragments and arrow 111 the presence of IgA and IgM aggregates at 20-30 S. m a d e up in 10% and 32% sucrose, giving a final concentration of 3.3% and 10.6% respectively in the incubation mixture. Fig. 1 shows the results obtained. The standard curves were superimposable in the range 10 n g / m l to 10 ~ g / m l for all 3 immunoglobulins tested.
Analysis of sera from patients with immune complex disease Fig. 2 shows the distribution of IgA, I g G and IgM in sucrose gradient centrifugations samples of sera from a normal individual (A), a patient with Sch6nlein-Henoch glomerulonephritis (B) and a patient with systemic lupus erythematosus (SLE) (C).
131 In the normal individual the 3 peaks for I g G at 7 S, IgA at 10 S and IgM at 19 S are clearly visible. The Serum of the patient with Sch6nlein-Henoch glomerulonephritis revealed aggregates of IgG, A and M at 25-30 S. The most profound changes in immunoglobulin distribution patterns were Seen in the SLE serum. A large peak of IgM immunoreactive substances was demonstrated at 7 S, and high molecular weight aggregates containing IgM and IgA were seen at 25-30 S.
Discussion This simple assay procedure using commercial available antisera and antigens is comparable with other immunoglobulin assays described previously (Gee and Langone, 1981; Ischiguru et al., 1981; Nerenberg and Prasad, 1981) with respect to sensitivity. Further, it requires iodination of only one tracer substance to measure 3 different antigens. The avoidance of the separation step necessary in conventional radioimmunoassay techniques makes it a very productive assay. In 6 h work one technician can analyze more than 300 samples. The assay covers a wide range of concentrations, which diminishes the dilution problems encountered in other systems with smaller ranges. The analysis of ultracentrifugation samples in this way gives information which otherwise could only be obtained by the use of several different techniques. The system is not influenced by sucrose concentrations up to 32%, which makes it an ideal method for the analysis of samples subjected to sucrose gradient centrifugation. It may even be of importance in relation to other media, where high solute concentrations might interfere with other techniques. The small volume needed in this assay makes the analysis of several different substances in the same gradient fraction possible. Without prior dialysis of the sucrose gradient samples, it is possible to determine the quantitative distribution of immunoglobulins in relation to sedimentation coefficients. At the same time the presence of immunoglobulin fragments and aggregates is detected, giving a detailed picture of disturbances in immunoglobulin synthesis and possible immune complex formation. Furthermore, C l q fixation techniques can only detect complexes containing IgG and IgM, while our technique also detects IgA complexes.
References Blankstein, L.K., B.D. Stollar and S.B. Levy, 1980, Anal. Biochem. 104, 168. Catt, K. and G.W. Tregear, 1967, Science 158, 1570. Gee, A.P. and J.J. Langone, 1981, Anal. Biochem. 116, 524. lschiguru, Y, K. Kato and T. Ito, 1981, Clin. Claim. Acta 116, 237. Langone, J.J., 1978, J. Immunol. Methods 24, 269. Langone, J.J., 1980, J. Immunol. Methods 34, 93. Langone, J.J., M.D. Boyle and T. Borsos, 1979, Anal. Biochem. 93, 207.
132 Martin, R.G. and B.N. Ames, 1961, J. Biol. Chem. 236, 1372. Nerenberg, S.T. and R. Prasad, 1981, in: Methods of Enzymology, eds. J.J. Langone and H. Van Vunakis (Academic Press, New York) p. 666. Steensgaard, J., N.P.H. Moller and L. Funding, 1978, in: Centrifugal Separation in Molecular and Cell Biology, eds. G.D.B. Birnie and D. Richwood (Butterworth, London) p. 115.