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Quantification of β2-microglobulin by inhibition enzyme immunoassay
Journal o f Immunological Methods, 40 (1981) 231--238
231
© Elsevier/North-Holland Biomedical Press
Q U A N T I F I C A T I O N OF ~32-MICROGLOBULIN BY INHIBITION ENZYME IMMUNOASSAY
YVES CARLIER l, ALAIN COLLE 2, PIERRE TACHON 3, DANIEL BOUT 4 and ANDRI~ CAPRON 4 1 Instituto Boliviano de Biologia de Altura, La Paz, Bolivia, 2 Centre d'Immunologie, Marseille Luminy, 3 Laboratoire d'Immunochimie, Institut Pasteur, Lyon and 4 Cenlre d' Immunologie et de Biologie Parasitaire, Institut Pasteur, Lille, France
{Received 1 July 1980, accepted 14 September 1980) Inhibition enzyme immunoassay was applied to ~32-microglobulinfrom urine. The technical conditions of the assay were determined. The detection limits of the assay were 5-500 ng/ml. Correlation coefficient obtained between radioimmunoassay and inhibition enzyme immunoassay was 0.97. Inhibition enzyme immunoassay provides an alternative, low cost method for the quantification of/32-microglobulin. INTRODUCTION ~32-Microglobulin (/32mG) is a small protein (MW 11,800) containing a b o u t 100 amino acid residues, which is f o u n d in most b o d y fluids such as urine, serum, cerebrospinal fluid, saliva, colostrum, amniotic and seminal fluids (Berggard and Beam, 1968; Peterson et al., 1969; Evrin and Peterson, 1971; Jonasson et al., 1974; Colle et al., 1976a). It presents large structural homologies with some regions of the light or heavy chains of immunoglobulins (Smithies and Poulik, 1972; Peterson and Cunningham, 1972), and with t h e two HLA p o l y p e p t i d e chains ( N a ka m uro et al., 1973; Peterson et al., 1974). In the mouse, fl2-microglobulin is r e p o r t e d to be associated n o t only with the major histocompatibility antigen (H-2), but also with the structurally and genetically related T L (C)stberg et al., 1975; Vitetta et al., 1975) and Qa-2 (Michaelson et al., 1977) antigens, with the H-Y antigen {Fellous et al., 1978) and with a cell-cell interaction fact or (Amerding et al., 1975), b u t its f u n c t i o n in the i m m une response is still under investigation (Marx, 1974). In clinical use, fi2mG d e t e r m i n a t i o n can be helpful in m a n y fields, particularly in gynecology/obstetrics and in nephrology. The d e t e r m i n a t i o n of ~32mG in amniotic fluid can be used to de t e rm i ne foetal m a t u r i t y or to study some pregnancy disorders (Cejka et al., 1973; Hall and Roux, 1974; Jonasson et al., 1974). In nephr ol ogy the d e t e r m i n a t i o n of fl2mG in serum might be equivalent or superior to the d e t e r m i n a t i o n of creatinine in the estimation o f the glomerular filtration ratio and useful as a substitute for the more laborious inulin clearance d e t e r m i n a t i o n (Wibell et al., 1973). The deter-
232 mination of ~zmG in urine can be used to evaluate the kidney's ability to reabsorb proteins in the proximal tubules, since fl2mG is able to pass through the glomerular membrane relatively easily, but the normal tubular reabsorption of filtered fl2mG is closed to 100%. The increased /~=mG content in the urine appears to be an approximate measure of the decreased tubular reabsorption of such proteins (Wibell et al., 1973). The usual method for the determination of ~=mG level is a competitive solid-phase radioimmunoassay, from which a commercial kit was developed by Pharmacia (~2microtest, Uppsala, Sweden). This assay was shown to be sensitive and reproducible, but its use involves expensive and sophisticated counting equipment, radioisotope licensing, and a radiolabelled reagent with a short half-life requiring special protective measures for safe handling. Enzyme immunoassays, on the other hand, were shown to be equally sensitive and reproducible, do not have these inconveniences and are already applied in m a n y fields (Widson, 1976; Carlier et al., 1979a, 1980). In previous studies we showed the inhibition enzyme immunoassay (IEIA) particularly adapted for antigen quantification, in its application for apolipoprotein B (Carlier et al., 1978). The aim of this investigation was to apply an IEIA, which was shown to be simple, rapid, sensitive and reproducible, to quantify /~2mG in urine. MATERIALS AND METHODS
Antigens fi2mG was purified using urine from patients with severe tubulopathies, following the technique described previously by Colle et al. (1976b). The purity of the obtained batches was found to be around 97% using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Soluble antigen of Toxoplasma gondii was used as a non-related antigen to study the stability of the antigen coating. It was prepared as previously described (Carlier et al., 1980).
Anti-~zmG serum A rabbit anti-/32mG serum, commercially available, was obtained from Sebia (Issy les moulineaux, France). Its specificity was verified using immunoelectrophoresis and gel immunodiffusion. Only one precipitation band, showing an identity reaction with pure fl2mG, was obtained using human serum and concentrated urine from a patient with t u b u l o p a t h y as antigens.
Urine samples Nineteen urine samples adjusted to pH 7.0 were obtained from patients with clinically evident tubulopathies with a fl=mG level between 800 and 6700 pg/1 as determined by the Pharmacia radioimmunoassay (see below). Ten urine samples with a/3=mG level ~<240 pg/1 were used as controls.
233
Radioimrnunoassay (RIA ) for {J2mG evaluation A commercially available RIA kit (Phadebas-~2microtest; Pharmacia, Uppsala, Sweden) was used as reference method to quantify ~2mG in the urine samples. The test principle is based on antigenic competition between the standard 12SI-labelled ~2mG and the ~2mG of the studied samples, for an anti-~2mG antibody solid phase (Sephadex particles). The competitive capacity was then compared with that of standard ~2mG preparations of known concentration. A centrifugation step was necessary to separate bound and free ~2mG, and the radioactivity bound to the sedimented Sephadex particles was measured.
Inhibition enzyme-immunoassay (IEIA ) for fJ2rnG determination IEIA was performed in disposable polystyrene tubes as previously described for the quantification of apolipoprotein B (Carlier et al., 1978), with some modifications, particularly in the coating step (see below). Briefly, ~2mG-coated tubes were incubated with 0.5 ml of diluted urine samples or ~2mG preparations of known concentrations (for the reference inhibition curve), and 0.5 ml of anti-~2mG rabbit serum at a suitable dilution (see below) for 4 h at room temperature. The tubes were then emptied by suction, washed three times, and incubated overnight at 4°C with 1 ml of peroxidase-labelled sheep antibodies to rabbit immunoglobulins (conjugate: Institut Pasteur Production, Paris) at a suitable dilution (see below). Excess conjugate was then thoroughly washed and the a m o u n t of peroxidase fixed to the tubes determined using H202 as substrate and o-dianisidine as the hydrogen donor. After 1 h at r o o m temperature, the reaction was stopped by addition of a drop of 5 N HC1 and the yellow cotour development was measured in a spectrophotometer at 405 nm. All the dilutions were performed in phosphate-buffered saline (PBS) (0.01 M, pH 7.2) and the washing steps with the same PBS containing 0.05% Tween 20. RESULTS
Study of the coating conditions for fl2mG Preliminary assays using a coating procedure similar to that used for apolipoprotein B (Carlier et al., 1978) lead to weak inhibition curves (Fig. la). In order to stabilize ~2mG on the polystyrene surface, the tubes were previously incubated for 3 h at 56°C with 1 ml of a bifunctional reagent, glutaraldehyde (Merck) at a 0.1% concentration in 0.1 M carbonate/ bicarbonate buffer, pH 9.0. The tubes were washed three times and then incubated with 1 ml of fl2mG at a suitable concentration (see below) for 3 h at 37°C. After a further three washings, 1 ml of 0.5 M 4-aminobutyric acid (Merck) in 0.05 M carbonate/bicarbonate buffer, pH 10.0, was incubated in the tubes for 2 h at room temperature in order to block unused reactive groups of glutaraldehyde, to avoid non-specific binding of anti-/32mG antibodies or conjugate. After washing, such glutaraldehyde-coated tubes were
234
100
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60 I
~4o f©
20
O
O.01
0.1
I
Pg~2 m G
Fig. 1. ~2mG inhibition curves in IEIA using different conditions of coating: ¢, o, fi2mG ( l p g ) without glutaraldehyde; • A, fismG ( l p g ) with glutaraldehyde; [] . . . . . . D, T o x o p l a s m a g o n d i i (1 gg) with glutaraldehyde; anti-fl2mG serum at 1/250 dilution; 500 ng of peroxidase-labelled antibodies. B = absorption of the studied samples; B~ = absorption using 1 pg of free ]3~mG; B0 = absorption without free ~2mG; ~ = standard deviation.
further used for the inhibition reaction and gave an excellent inhibition curve with a 50% increase in sensitivity (Fig. lb). In order to verify the stability of such a coating, the following protocol was used: firstly, a non-related soluble antigen (Toxoplasma gondii) was coated using the procedure described above; secondly, the tubes were incubated for 3 h at 37°C with ~2mG; the inhibition reaction was then performed normally. Using these conditions, it was not possible to obtain any inhibition curve, thus showing that the antigen link to the plastic surface was strong and impossible to break (Fig. lc). The inverse protocol, using/~2mG-coated tubes, coated secondly with Toxoplasma antigen gave an excellent inhibition curve, identical to that obtained with tubes coated only with ~2mG.
Determination of optimal coating concentration of fl~mG The tubes were coated with different concentrations of ~2mG according to the procedure described above and then incubated with anti-fl2mG serum at a 1/250 dilution. After incubation with the conjugate and colorimetric quantitation of peroxidase fixed to the tubes, the absorbance values were plotted against concentration (gg/ml) of fl2mG used for coating (Fig. 2). The optimal coating concentration of 1 pg/ml was selected for further use.
Determination of the optimal dilution of anti-[JzmG serum Serial dilutions of the antiserum were incubated with a fixed a m o u n t of fizmG (1 pg/ml) coated on polystyrene tubes and the samples processed as described in Methods. A dilution curve is shown in Fig. 3. There was a
235
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Fig. 2. Binding of anti-~2mG serum to polystyrene tubes coated with various concentrations of /32mG: • v,, with anti-fl2mG serum at 1/250 dilution; • •, control without anti-fl2mG serum; arrow indicates the optimal coating antigen concentration; ~ = standard deviation.
maximal absorbance for antiserum dilutions smaller than 1/100. Without antiserum or coated fl2mG, non-specific binding was reduced to less than 1 and 7% respectively. Optimal sensitivity of IEIA was achieved by using A
1.2 1.o
/
< 0.6
/
0.4
0,2 O.O'
~
/~/ , 1/2000 1/1000 1/500 1/250
~ 1/100
,
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,-
A n t i - ~ 2 m G dilutions
Fig. 3. Binding of different anti-~2mG serum dilutions to fl2mG-coated polystyrene tubes: • ~,, with 1 pg/ml of coated ~2mG; A A, control without coated ~2mG; arrow indicates the optimal dilution to be used; ~ = standard deviation.
236
the antiserum dilution yielding 87% binding. This dilution could vary from one batch o f antiserum to another, but was generally 1/250 and this dilution was f u r t h e r used.
Determination o f the optimal dilution o f the conjugate Different conjugate concentrations were used with 1 pg/ml of ~2mG coated on p o l y s t y r e n e tubes and antiserum at 1/250 dilution (Fig. 4). The optimal c o n c e n t r a t i o n which gave a high range of extinction values and a saturation o f the antigenic sites of the anti-~2mG antibodies was 500 ng/ml. Inhibition reference curve Different co n c e nt r at i ons of ~2mG were used to obtain an inhibition reference curve (Fig. 5). Each sample was assayed in triplicate and the mean value was calculated. The results were expressed in percent as ( B - - B ~ ) / (B0 -- BI ) where B is the absorption of the studied sample, B, the absorption obtained with 1 pg of fl2mG, giving a maximal inhibition reaction and B0 the absorption obtained w i t h o u t free ~2mG, i.e. w i t h o u t inhibition reaction. Figure 5 shows a representative standard inhibition curve for fl2mG. As the a m o u n t of added soluble ~:mG was increased, the binding of anti-fl2mG
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250 ng C o n J u g a t e
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1
Fig. 4. B i n d i n g o f d i f f e r e n t a n t i b o d y c o n c e n t r a t i o n s o f t h e p e r o x i d a s e - l a b e l l e d c o n j u g a t e to ~ 2 m G - c o a t e d p o l y s t y r e n e t u b e s (1 ~tg), i n c u b a t e d w i t h a n t i - ~ : m G s e r u m ( 1 / 2 5 0 ) ; a r r o w i n d i c a t e s t h e o p t i m a l d i l u t i o n t o be used; ~ = s t a n d a r d deviation. Fig. 5. fl2mG i n h i b i t i o n r e f e r e n c e curve in I E I A . B = a b s o r p t i o n o f t h e s t u d i e d samples; B 1 = a b s o r p t i o n using 1/.tg of free ~ 2 m G ; B0 = a b s o r p t i o n w i t h o u t free ~ 2 m G ; ~ = s t a n d a r d deviation.
237 Y=O.B1X + 49.5 r = Q.97 p<(2OQ1 n= 29
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•
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°
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°
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Fig. 6. Correlation between IEIA and RIA for ~2mG determination.
antibodies to ~2mG solid phase was inhibited and the intensity of the color reaction decreased progressively. The d e t e c t i o n limits of the assay were 5--500 ng/ml, b u t the optimal working range in the linear part of the curve was f r o m 10 to 200 ng/ml.
Sensitivity and precision of assay The m i n i m u m detectable c o n c e n t r a t i o n (10 ng/ml in the linear part of the curve) is in excess o f t hat required to measure ~2mG in urine and urine dilutions were r o u t i ne l y used. Some urines f rom patients with severe tubulopathies must be diluted by as m uch as 1/ 100, but generally 1/10 and 1/100 dilutions in PBS (0.01 M, pH 7.2) were sufficient to study tubular urines. Within-batch precision was calculated f r o m the difference bet w een triplicate measurements of 29 urines. The coefficient of variation was found to be ~<2%.
Comparison between IEIA and RIA for [J2mG determination IEIA was mo r e accurate than Phadebas ~2micro-test since the within-assay coefficient of variation was 2% instead of 4.7--8.4% (Pharmacia h a n d b o o k information). Results obtained by the t w o m e t h o d s correlated well (r = 0.97; P < 0.001) (Fig. 6). The mean levels f o u n d by IEIA were sometimes lower than those obtained by RIA. DISCUSSION IEIA was shown to be a specific, precise and sensitive m e t h o d for the quantification of fi2mG and its results equaled those of the Phadebas
238 flzmicro test since a g o o d c o r r e l a t i o n was o b t a i n e d for the fl:mG u r i n a r y levels d e t e r m i n e d b y b o t h m e t h o d s . O n l y urine samples were studied in the p r e s e n t w o r k and f u r t h e r studies will be necessary to evaluate I E I A with serum or a m n i o t i c fluid. T h e main advantage o f I E I A over R I A , besides its higher precision and t h e e l i m i n a t i o n o f a c e n t r i f u g a t i o n step, is the a v o i d a n c e o f a r a d i o i s o t o p e labelled reagent, and h e n c e the a c c o m p a n y i n g sophisticated c o u n t i n g equipm e n t , r a d i o i s o t o p e licensing and special p r o t e c t i v e measures for i s o t o p e manipulations. I E I A provides an alternative, low cost m e t h o d , t h a t can easily be autom a t e d (Carlier et al., 1 9 7 9 b ) f o r q u a n t i f i c a t i o n o f flE-microglobulin in biological fluids. ACKNOWLEDGEMENTS T h e a u t h o r s wish to t h a n k Miss S. C a r t o n and Mr. L. H o r n e z f o r their excellent technical assistance and Miss M. del C a r m e n B u r g o a A. f o r her c o n t r i b u t i o n to the p r e p a r a t i o n o f the m a n u s c r i p t . REFERENCES Amerding, D., R.T. Kubo, H.M. Grey and D.H. Katz, 1975, Proc. Natl. Acad. Sci. U.S.A. 72, 4577. Berggard, I. and A.G. Beam, 1968, J. Biol. Chem. 243, 4095. Carlier, Y., D. Bout, J.C. Fruchart, C. Desreumaux, P. Dewailly, G. Sezille and J. Jaillard, 1978, J. Immunol. Methods 21,317. Carlier, Y., D. Bout and A. Capron, 1979a, Ann Inst. Pasteur (Lyon) 12, 25. Carlier, Y., D. Bout and A. Capron, 1979b, J. Immunol. Methods 31,237. Carlier, Y., D. Bout, J.P. Dessaint, A. Capron, F. Van Knapen, E.J. Ruitenberg, R. Bergquist and G. Huldt, 1980, Bull. WHO 58, 99. Cejka, J., F. Cohen and K. Kithier, 1973, Clin. Chim. Acta 47, 59. Colle, A., R. Guinet, M. Leclercq and Y. Manuel, 1976a, Clin. Chim. Acta 67, 93. Colle, A., C. Tonnelle, Y. Manuel, L. Rivat and C. Ropatz, 1976b, Eur. J. Immunol. 6, 660. Evrin, P.E. and P.A. Peterson, 1971, Scand, J. Clin. Lab. Invest. 28,439. Fellous, M., E. Giinther, R. Kemler, J. Wiels, R. Berger, J.L. Guente, H. Jacob and F. Jacob, 1978, J. Exp. Med. 148, 58. Hall, P.W. and J.F. Roux, 1974, Am. J. Obstet. Gynec. 120, 56. Jonasson, L.E., P.E. Evrin and L. Wibell, 1974, Acta Obstet. Gynec. Scand. 53, 49. Marx, J.L., 1974, Science 185,428. Michaelson, J., L. Flaherty, E. Vitetta and M.D. Poulik, 1977, J. Exp. Med. 145, 1066. Nakamuro, K., N. Tanigaki and D. Pressman, 1973, Proc. Natl. Acad. Sci. U.S.A. 70, 2863. Ostberg, L., L. Rask, H. Wigzell arid P.A. Peterson, 1975, Nature 253, 735. Peterson, P.A. and B.A. Cunningham, 1972, Proc. Natl. Acad. Sci. U.S.A. 69, 1967. Peterson, P.A., P.E. Evrin and I. Berggard, 1969, J. Clin. Invest. 48, 1189. Peterson, P.A., L. Rask and J.B. Lindbolm, 1974, Proc. Natl. Acad. U.S.A. 71, 35. Smithies, O. and M.D. Poulik, 1972, Science 175,187. Vitetta, E.S., J.W. Uhr and E.A. Boyse, 1975, J. Immunol. 114, 252. Wibell, L., P.E. Evrin and I. Berggard, 1973, Nephron 10, 320. Widson, G.B., 1976, Clin. Chem. 72, 1243.
231
© Elsevier/North-Holland Biomedical Press
Q U A N T I F I C A T I O N OF ~32-MICROGLOBULIN BY INHIBITION ENZYME IMMUNOASSAY
YVES CARLIER l, ALAIN COLLE 2, PIERRE TACHON 3, DANIEL BOUT 4 and ANDRI~ CAPRON 4 1 Instituto Boliviano de Biologia de Altura, La Paz, Bolivia, 2 Centre d'Immunologie, Marseille Luminy, 3 Laboratoire d'Immunochimie, Institut Pasteur, Lyon and 4 Cenlre d' Immunologie et de Biologie Parasitaire, Institut Pasteur, Lille, France
{Received 1 July 1980, accepted 14 September 1980) Inhibition enzyme immunoassay was applied to ~32-microglobulinfrom urine. The technical conditions of the assay were determined. The detection limits of the assay were 5-500 ng/ml. Correlation coefficient obtained between radioimmunoassay and inhibition enzyme immunoassay was 0.97. Inhibition enzyme immunoassay provides an alternative, low cost method for the quantification of/32-microglobulin. INTRODUCTION ~32-Microglobulin (/32mG) is a small protein (MW 11,800) containing a b o u t 100 amino acid residues, which is f o u n d in most b o d y fluids such as urine, serum, cerebrospinal fluid, saliva, colostrum, amniotic and seminal fluids (Berggard and Beam, 1968; Peterson et al., 1969; Evrin and Peterson, 1971; Jonasson et al., 1974; Colle et al., 1976a). It presents large structural homologies with some regions of the light or heavy chains of immunoglobulins (Smithies and Poulik, 1972; Peterson and Cunningham, 1972), and with t h e two HLA p o l y p e p t i d e chains ( N a ka m uro et al., 1973; Peterson et al., 1974). In the mouse, fl2-microglobulin is r e p o r t e d to be associated n o t only with the major histocompatibility antigen (H-2), but also with the structurally and genetically related T L (C)stberg et al., 1975; Vitetta et al., 1975) and Qa-2 (Michaelson et al., 1977) antigens, with the H-Y antigen {Fellous et al., 1978) and with a cell-cell interaction fact or (Amerding et al., 1975), b u t its f u n c t i o n in the i m m une response is still under investigation (Marx, 1974). In clinical use, fi2mG d e t e r m i n a t i o n can be helpful in m a n y fields, particularly in gynecology/obstetrics and in nephrology. The d e t e r m i n a t i o n of ~32mG in amniotic fluid can be used to de t e rm i ne foetal m a t u r i t y or to study some pregnancy disorders (Cejka et al., 1973; Hall and Roux, 1974; Jonasson et al., 1974). In nephr ol ogy the d e t e r m i n a t i o n of fl2mG in serum might be equivalent or superior to the d e t e r m i n a t i o n of creatinine in the estimation o f the glomerular filtration ratio and useful as a substitute for the more laborious inulin clearance d e t e r m i n a t i o n (Wibell et al., 1973). The deter-
232 mination of ~zmG in urine can be used to evaluate the kidney's ability to reabsorb proteins in the proximal tubules, since fl2mG is able to pass through the glomerular membrane relatively easily, but the normal tubular reabsorption of filtered fl2mG is closed to 100%. The increased /~=mG content in the urine appears to be an approximate measure of the decreased tubular reabsorption of such proteins (Wibell et al., 1973). The usual method for the determination of ~=mG level is a competitive solid-phase radioimmunoassay, from which a commercial kit was developed by Pharmacia (~2microtest, Uppsala, Sweden). This assay was shown to be sensitive and reproducible, but its use involves expensive and sophisticated counting equipment, radioisotope licensing, and a radiolabelled reagent with a short half-life requiring special protective measures for safe handling. Enzyme immunoassays, on the other hand, were shown to be equally sensitive and reproducible, do not have these inconveniences and are already applied in m a n y fields (Widson, 1976; Carlier et al., 1979a, 1980). In previous studies we showed the inhibition enzyme immunoassay (IEIA) particularly adapted for antigen quantification, in its application for apolipoprotein B (Carlier et al., 1978). The aim of this investigation was to apply an IEIA, which was shown to be simple, rapid, sensitive and reproducible, to quantify /~2mG in urine. MATERIALS AND METHODS
Antigens fi2mG was purified using urine from patients with severe tubulopathies, following the technique described previously by Colle et al. (1976b). The purity of the obtained batches was found to be around 97% using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Soluble antigen of Toxoplasma gondii was used as a non-related antigen to study the stability of the antigen coating. It was prepared as previously described (Carlier et al., 1980).
Anti-~zmG serum A rabbit anti-/32mG serum, commercially available, was obtained from Sebia (Issy les moulineaux, France). Its specificity was verified using immunoelectrophoresis and gel immunodiffusion. Only one precipitation band, showing an identity reaction with pure fl2mG, was obtained using human serum and concentrated urine from a patient with t u b u l o p a t h y as antigens.
Urine samples Nineteen urine samples adjusted to pH 7.0 were obtained from patients with clinically evident tubulopathies with a fl=mG level between 800 and 6700 pg/1 as determined by the Pharmacia radioimmunoassay (see below). Ten urine samples with a/3=mG level ~<240 pg/1 were used as controls.
233
Radioimrnunoassay (RIA ) for {J2mG evaluation A commercially available RIA kit (Phadebas-~2microtest; Pharmacia, Uppsala, Sweden) was used as reference method to quantify ~2mG in the urine samples. The test principle is based on antigenic competition between the standard 12SI-labelled ~2mG and the ~2mG of the studied samples, for an anti-~2mG antibody solid phase (Sephadex particles). The competitive capacity was then compared with that of standard ~2mG preparations of known concentration. A centrifugation step was necessary to separate bound and free ~2mG, and the radioactivity bound to the sedimented Sephadex particles was measured.
Inhibition enzyme-immunoassay (IEIA ) for fJ2rnG determination IEIA was performed in disposable polystyrene tubes as previously described for the quantification of apolipoprotein B (Carlier et al., 1978), with some modifications, particularly in the coating step (see below). Briefly, ~2mG-coated tubes were incubated with 0.5 ml of diluted urine samples or ~2mG preparations of known concentrations (for the reference inhibition curve), and 0.5 ml of anti-~2mG rabbit serum at a suitable dilution (see below) for 4 h at room temperature. The tubes were then emptied by suction, washed three times, and incubated overnight at 4°C with 1 ml of peroxidase-labelled sheep antibodies to rabbit immunoglobulins (conjugate: Institut Pasteur Production, Paris) at a suitable dilution (see below). Excess conjugate was then thoroughly washed and the a m o u n t of peroxidase fixed to the tubes determined using H202 as substrate and o-dianisidine as the hydrogen donor. After 1 h at r o o m temperature, the reaction was stopped by addition of a drop of 5 N HC1 and the yellow cotour development was measured in a spectrophotometer at 405 nm. All the dilutions were performed in phosphate-buffered saline (PBS) (0.01 M, pH 7.2) and the washing steps with the same PBS containing 0.05% Tween 20. RESULTS
Study of the coating conditions for fl2mG Preliminary assays using a coating procedure similar to that used for apolipoprotein B (Carlier et al., 1978) lead to weak inhibition curves (Fig. la). In order to stabilize ~2mG on the polystyrene surface, the tubes were previously incubated for 3 h at 56°C with 1 ml of a bifunctional reagent, glutaraldehyde (Merck) at a 0.1% concentration in 0.1 M carbonate/ bicarbonate buffer, pH 9.0. The tubes were washed three times and then incubated with 1 ml of fl2mG at a suitable concentration (see below) for 3 h at 37°C. After a further three washings, 1 ml of 0.5 M 4-aminobutyric acid (Merck) in 0.05 M carbonate/bicarbonate buffer, pH 10.0, was incubated in the tubes for 2 h at room temperature in order to block unused reactive groups of glutaraldehyde, to avoid non-specific binding of anti-/32mG antibodies or conjugate. After washing, such glutaraldehyde-coated tubes were
234
100
BO
60 I
~4o f©
20
O
O.01
0.1
I
Pg~2 m G
Fig. 1. ~2mG inhibition curves in IEIA using different conditions of coating: ¢, o, fi2mG ( l p g ) without glutaraldehyde; • A, fismG ( l p g ) with glutaraldehyde; [] . . . . . . D, T o x o p l a s m a g o n d i i (1 gg) with glutaraldehyde; anti-fl2mG serum at 1/250 dilution; 500 ng of peroxidase-labelled antibodies. B = absorption of the studied samples; B~ = absorption using 1 pg of free ]3~mG; B0 = absorption without free ~2mG; ~ = standard deviation.
further used for the inhibition reaction and gave an excellent inhibition curve with a 50% increase in sensitivity (Fig. lb). In order to verify the stability of such a coating, the following protocol was used: firstly, a non-related soluble antigen (Toxoplasma gondii) was coated using the procedure described above; secondly, the tubes were incubated for 3 h at 37°C with ~2mG; the inhibition reaction was then performed normally. Using these conditions, it was not possible to obtain any inhibition curve, thus showing that the antigen link to the plastic surface was strong and impossible to break (Fig. lc). The inverse protocol, using/~2mG-coated tubes, coated secondly with Toxoplasma antigen gave an excellent inhibition curve, identical to that obtained with tubes coated only with ~2mG.
Determination of optimal coating concentration of fl~mG The tubes were coated with different concentrations of ~2mG according to the procedure described above and then incubated with anti-fl2mG serum at a 1/250 dilution. After incubation with the conjugate and colorimetric quantitation of peroxidase fixed to the tubes, the absorbance values were plotted against concentration (gg/ml) of fl2mG used for coating (Fig. 2). The optimal coating concentration of 1 pg/ml was selected for further use.
Determination of the optimal dilution of anti-[JzmG serum Serial dilutions of the antiserum were incubated with a fixed a m o u n t of fizmG (1 pg/ml) coated on polystyrene tubes and the samples processed as described in Methods. A dilution curve is shown in Fig. 3. There was a
235
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i
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i
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i
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Fig. 2. Binding of anti-~2mG serum to polystyrene tubes coated with various concentrations of /32mG: • v,, with anti-fl2mG serum at 1/250 dilution; • •, control without anti-fl2mG serum; arrow indicates the optimal coating antigen concentration; ~ = standard deviation.
maximal absorbance for antiserum dilutions smaller than 1/100. Without antiserum or coated fl2mG, non-specific binding was reduced to less than 1 and 7% respectively. Optimal sensitivity of IEIA was achieved by using A
1.2 1.o
/
< 0.6
/
0.4
0,2 O.O'
~
/~/ , 1/2000 1/1000 1/500 1/250
~ 1/100
,
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,-
A n t i - ~ 2 m G dilutions
Fig. 3. Binding of different anti-~2mG serum dilutions to fl2mG-coated polystyrene tubes: • ~,, with 1 pg/ml of coated ~2mG; A A, control without coated ~2mG; arrow indicates the optimal dilution to be used; ~ = standard deviation.
236
the antiserum dilution yielding 87% binding. This dilution could vary from one batch o f antiserum to another, but was generally 1/250 and this dilution was f u r t h e r used.
Determination o f the optimal dilution o f the conjugate Different conjugate concentrations were used with 1 pg/ml of ~2mG coated on p o l y s t y r e n e tubes and antiserum at 1/250 dilution (Fig. 4). The optimal c o n c e n t r a t i o n which gave a high range of extinction values and a saturation o f the antigenic sites of the anti-~2mG antibodies was 500 ng/ml. Inhibition reference curve Different co n c e nt r at i ons of ~2mG were used to obtain an inhibition reference curve (Fig. 5). Each sample was assayed in triplicate and the mean value was calculated. The results were expressed in percent as ( B - - B ~ ) / (B0 -- BI ) where B is the absorption of the studied sample, B, the absorption obtained with 1 pg of fl2mG, giving a maximal inhibition reaction and B0 the absorption obtained w i t h o u t free ~2mG, i.e. w i t h o u t inhibition reaction. Figure 5 shows a representative standard inhibition curve for fl2mG. As the a m o u n t of added soluble ~:mG was increased, the binding of anti-fl2mG
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Fig. 4. B i n d i n g o f d i f f e r e n t a n t i b o d y c o n c e n t r a t i o n s o f t h e p e r o x i d a s e - l a b e l l e d c o n j u g a t e to ~ 2 m G - c o a t e d p o l y s t y r e n e t u b e s (1 ~tg), i n c u b a t e d w i t h a n t i - ~ : m G s e r u m ( 1 / 2 5 0 ) ; a r r o w i n d i c a t e s t h e o p t i m a l d i l u t i o n t o be used; ~ = s t a n d a r d deviation. Fig. 5. fl2mG i n h i b i t i o n r e f e r e n c e curve in I E I A . B = a b s o r p t i o n o f t h e s t u d i e d samples; B 1 = a b s o r p t i o n using 1/.tg of free ~ 2 m G ; B0 = a b s o r p t i o n w i t h o u t free ~ 2 m G ; ~ = s t a n d a r d deviation.
237 Y=O.B1X + 49.5 r = Q.97 p<(2OQ1 n= 29
? 4 <
•
W
°
X
°
R[A IO3ffg/l
Fig. 6. Correlation between IEIA and RIA for ~2mG determination.
antibodies to ~2mG solid phase was inhibited and the intensity of the color reaction decreased progressively. The d e t e c t i o n limits of the assay were 5--500 ng/ml, b u t the optimal working range in the linear part of the curve was f r o m 10 to 200 ng/ml.
Sensitivity and precision of assay The m i n i m u m detectable c o n c e n t r a t i o n (10 ng/ml in the linear part of the curve) is in excess o f t hat required to measure ~2mG in urine and urine dilutions were r o u t i ne l y used. Some urines f rom patients with severe tubulopathies must be diluted by as m uch as 1/ 100, but generally 1/10 and 1/100 dilutions in PBS (0.01 M, pH 7.2) were sufficient to study tubular urines. Within-batch precision was calculated f r o m the difference bet w een triplicate measurements of 29 urines. The coefficient of variation was found to be ~<2%.
Comparison between IEIA and RIA for [J2mG determination IEIA was mo r e accurate than Phadebas ~2micro-test since the within-assay coefficient of variation was 2% instead of 4.7--8.4% (Pharmacia h a n d b o o k information). Results obtained by the t w o m e t h o d s correlated well (r = 0.97; P < 0.001) (Fig. 6). The mean levels f o u n d by IEIA were sometimes lower than those obtained by RIA. DISCUSSION IEIA was shown to be a specific, precise and sensitive m e t h o d for the quantification of fi2mG and its results equaled those of the Phadebas
238 flzmicro test since a g o o d c o r r e l a t i o n was o b t a i n e d for the fl:mG u r i n a r y levels d e t e r m i n e d b y b o t h m e t h o d s . O n l y urine samples were studied in the p r e s e n t w o r k and f u r t h e r studies will be necessary to evaluate I E I A with serum or a m n i o t i c fluid. T h e main advantage o f I E I A over R I A , besides its higher precision and t h e e l i m i n a t i o n o f a c e n t r i f u g a t i o n step, is the a v o i d a n c e o f a r a d i o i s o t o p e labelled reagent, and h e n c e the a c c o m p a n y i n g sophisticated c o u n t i n g equipm e n t , r a d i o i s o t o p e licensing and special p r o t e c t i v e measures for i s o t o p e manipulations. I E I A provides an alternative, low cost m e t h o d , t h a t can easily be autom a t e d (Carlier et al., 1 9 7 9 b ) f o r q u a n t i f i c a t i o n o f flE-microglobulin in biological fluids. ACKNOWLEDGEMENTS T h e a u t h o r s wish to t h a n k Miss S. C a r t o n and Mr. L. H o r n e z f o r their excellent technical assistance and Miss M. del C a r m e n B u r g o a A. f o r her c o n t r i b u t i o n to the p r e p a r a t i o n o f the m a n u s c r i p t . REFERENCES Amerding, D., R.T. Kubo, H.M. Grey and D.H. Katz, 1975, Proc. Natl. Acad. Sci. U.S.A. 72, 4577. Berggard, I. and A.G. Beam, 1968, J. Biol. Chem. 243, 4095. Carlier, Y., D. Bout, J.C. Fruchart, C. Desreumaux, P. Dewailly, G. Sezille and J. Jaillard, 1978, J. Immunol. Methods 21,317. Carlier, Y., D. Bout and A. Capron, 1979a, Ann Inst. Pasteur (Lyon) 12, 25. Carlier, Y., D. Bout and A. Capron, 1979b, J. Immunol. Methods 31,237. Carlier, Y., D. Bout, J.P. Dessaint, A. Capron, F. Van Knapen, E.J. Ruitenberg, R. Bergquist and G. Huldt, 1980, Bull. WHO 58, 99. Cejka, J., F. Cohen and K. Kithier, 1973, Clin. Chim. Acta 47, 59. Colle, A., R. Guinet, M. Leclercq and Y. Manuel, 1976a, Clin. Chim. Acta 67, 93. Colle, A., C. Tonnelle, Y. Manuel, L. Rivat and C. Ropatz, 1976b, Eur. J. Immunol. 6, 660. Evrin, P.E. and P.A. Peterson, 1971, Scand, J. Clin. Lab. Invest. 28,439. Fellous, M., E. Giinther, R. Kemler, J. Wiels, R. Berger, J.L. Guente, H. Jacob and F. Jacob, 1978, J. Exp. Med. 148, 58. Hall, P.W. and J.F. Roux, 1974, Am. J. Obstet. Gynec. 120, 56. Jonasson, L.E., P.E. Evrin and L. Wibell, 1974, Acta Obstet. Gynec. Scand. 53, 49. Marx, J.L., 1974, Science 185,428. Michaelson, J., L. Flaherty, E. Vitetta and M.D. Poulik, 1977, J. Exp. Med. 145, 1066. Nakamuro, K., N. Tanigaki and D. Pressman, 1973, Proc. Natl. Acad. Sci. U.S.A. 70, 2863. Ostberg, L., L. Rask, H. Wigzell arid P.A. Peterson, 1975, Nature 253, 735. Peterson, P.A. and B.A. Cunningham, 1972, Proc. Natl. Acad. Sci. U.S.A. 69, 1967. Peterson, P.A., P.E. Evrin and I. Berggard, 1969, J. Clin. Invest. 48, 1189. Peterson, P.A., L. Rask and J.B. Lindbolm, 1974, Proc. Natl. Acad. U.S.A. 71, 35. Smithies, O. and M.D. Poulik, 1972, Science 175,187. Vitetta, E.S., J.W. Uhr and E.A. Boyse, 1975, J. Immunol. 114, 252. Wibell, L., P.E. Evrin and I. Berggard, 1973, Nephron 10, 320. Widson, G.B., 1976, Clin. Chem. 72, 1243.