- Email: [email protected]
Ultrastructure-immunoreactivity interference in the radioimmunoassay of the Tamm-Horsfall urinary mucoprotein
Journal of Immunological Methods,
54 (1982) 343- 353
343
Elsevier Biomedical Press
Ultrastructure-Immunoreactivity Interference in the Radioimmunoassay of the Tamm-Horsfall Urinary Mucoprotein Lucien H a r t m a n n *, A n n i e - F r a n c e Bringuier * and Etienne Delain ** • Laboratoire de Chimie Clinique et Biologie Molbculaire, lnstitut Biomkdical des Cordeliers, 15, rue de l'Ecole de Mbdecine, 75270 Paris Cbdex 06, and ** Laboratoire de Microscopie Cellulaire et Mol~culaire, Institut Gustave Roussy, 94800 Villejuif, France
(Received 26 January 1982, accepted 3 May 1982)
We report the systematic study of various factors (pH, SDS, NaC1, urea, albumin, temperature, glycoprotein concentration) that could interfere in the radioimmunoassay of the Tamm-Horsfall urinary mucoprotein. The proposed procedure relates antigenicity of the molecule with a defined ultrastructure, enabling systematic physio-pathological studies in the future and valid comparisons of results obtained. Key words: Tamm-Horsfall urinary protein - - protein ultrastructure - - factors affecting radioimmunoassay
Introduction Until now, there has been no standard procedure for r a d i o i m m u n o a s s a y ( R I A ) of the T a m m - H o r s f a l l (TH) urinary mucoprotein, which explains the variety of procedures and the disparity of results reported ( G r a n t and Neuberger, 1973; Avis, 1977; Meberg et al., 1979). The work described here was undertaken to eliminate this inconvenience b y considering relationships between the antigenicity of the molecule and its different ultrastructural states.
Materials and Methods Materials Reagents. Products used to prepare buffers were obtained f r o m Merck: phosphoric, boric and acetic acids, Tris-(hydroxymethyl)-aminomethane, N a O H , m o n o and dibasic phosphates, NaC1 and polyethylene glycol (PEG). Bovine g a m m a globulin (Cohn fraction II), bovine serum albumin (BSA), sodium dodecyl sulfate
0022-1759/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
344 (SDS), sodium azide, sodium metabisulfite and chloramine T were purchased from Sigma. Chromatography. Sephadex G-50 (20--80 /~m) and G-200 (40-120 ktm) were obtained from Pharmacia. Radiochemicals. NaJ25I was obtained from New England Nuclear. Instrumentation. Disposable polystyrene tubes were used for the RIA. All centrifugations were performed in a Jouan E 96 centrifuge at 4°C. An Intertechnique CG 4000 gammacounter was used for determinations of radioactivity. Universal Britton-Robinson buffer. 0.035 M phosphoric acid, 0.04 M boric acid, 0.04 M acetic acid, pH 1.8. This buffer was diluted 1/8, adjusted to pH 7.4 with 0.05 N NaOH and added with 0.25% (w/v) BSA, 0.4% (w/v) bovine gamma globulins and 0.1% (w/v) SDS for the RIA. Biological material 24 h urines from 13 healthy adults between 21 and 30 years of age were collected over 0.1% Na azide and stored at - 2 0 ° C . Bacterial sterility was verified before the assay with Pasteur D G U kits, as described by Mackey and Sandys (1966), and Guttmann and Naylor (1967). One milliliter of urine was dialyzed at constant volume against 100 ml of double distilled water for 4 h at laboratory temperature, and was then diluted for the RIA.
Radioimmunoassay of the TH protein Reference glycoprotein. This protein was prepared by NaCl 0.58 M salting out as described by Tamm and Horsfall (1952) and Hartmann et al. (1981). A qualitative and quantitative analysis of impurities (albumin, a2M, ceruloplasmin, haptoglobin, transferrin) was systematically performed with specific antisera. Protein identity was determined by 4 criteria: a single well circumscribed spot with c~2/~ relative mobility after cellulose acetate electrophoresis, a single precipitin line on immunoelectrophoresis, a single peak after 2-dimensional electrophoresis (Oilier and Hartmann, 1974) and by amino acid composition and percentages of neutral and aminomonosaccharides (Delain et al., 1980). Protein concentration was determined by amido black assay (Hartmann, 1971). The reference glycoprotein was stored at a minimal concentration of 100/~g/ml in 0.05 M sodium phosphate buffer (pH 7.4) containing 0.2% (w/v) SDS (maximal concentration required to preserve the glycoprotein immunological integrity). Iodination. The glycoprotein was labeled with Na12SI by the chloramine T method (Hunter and Greenwood, 1962), using 0.05 M phosphate buffer (pH 7.4). Two micrograms of the T H protein (10 /~1 at 0.5% SDS, a concentration which allows sub-unit formation), followed by 50/~g of chloramine T, were added to 1 mCi of Na1251 dissolved in 10 /~1 of 0.5 M phosphate buffer (pH 7.4). The reaction was stopped after 15 sec by adding 125 #g sodium metabisulfite. Labeled T H protein was separated from free iodide by filtering through a 20 cm X 9 mm column of Sephadex G-50 (fine), and eluting with 0.05 M Tris-HC1 (pH 7.4) containing 0.5% SDS. Elution fractions of ~25I-labeled T H giving the best binding percentages with antiserum were stored for one month at - 2 0 ° C in approximately 150 × 103 dpm (70 nCi) aliquots. Antiserum. The purified glycoprotein preparation was used to raise antiserum in
345 'Fauve de Bourgogne' rabbits as described by Ollier et al. (1979). Antisera were stored in 1 ml aliquots in 0.1% Na azide in liquid nitrogen ( - 170°C). The specificity of anti-TH g a m m a globulins was verified by 2-dimensional electrophoresis and micro-immunodiffusion ( H a r t m a n n and Toilliez, 1957). Procedure. RIA was performed in duplicate in a final volume of 0.3 ml with the Britton-Robinson buffer (pH 7.4), containing 0.25% BSA, 0.4% bovine gamma globulin and 0.1% SDS. 2500 d p m of J25I-labeled T H were used per tube. The reaction mixture contained 0.1 ml of antiserum (diluted to obtain 30-40% binding of ~25I-labeled TH) to which were successively added 0.1 ml of the reference solution or the sample to be assayed and, after a 1 h preincubation at 37°C, 0.1 ml of 125I-labeled TH. After overnight incubation at laboratory temperature and stabilization for 1 h at 4°C, the antigen-antibody complex was precipitated by adding 0.3 ml of 10% PEG 6000 ( w / v ) in distilled water. Tubes were immediately centrifuged for 30 rain at 2000 × g at 4°C. After aspiration of the supernatant, the radioactivity of the bound antigen fraction was determined in the precipitate. VaBdity of the assay. This was controlled both by the overload test and by a parallelism between reference and various urinary assay curves. Increasing quantities of glycoprotein, 10, 20, and 40 ~g, were added to 1 ml of urine. After dialysis, the samples were assayed as described above. The identity of the product assayed in 2-fold serial dilutions of urine was also verified by comparing its behaviour with that of the reference protein towards antiserum (parallelism of curves). The precision of the assay was determined by establishing a standard curve of 10 determinations per point and by 10 assays of the same urine sample. Reproducibility was verified by measuring 8 samples of the same urine analyzed in 8 different assay series. Statistics. The coefficient of variation was determined by the study of precision and reproducibility. The significance of the coefficient of correlation of the linear regression curves established for the overload test was evaluated with the Student's t test.
Factors participating in glycoprotein aggregation Using the RIA, the immunoreactivity of T H towards the antiserum was studied with respect to the following parameters: pH, NaC1, SDS, BSA, urea, temperature. A glycoprotein solution at 2000 n g / m l was prepared in Britton-Robinson buffer (pH 7.4). It was diluted 1/10 in the same buffer, in which were separately and successively varied the p H (1.8-10), and the concentrations of NaCI (0.15-0.45 M), SDS (0.1-0.6%), BSA (10-4-100 m g / m l ) and urea (0.1-0.3 M). Finally, the mixtures were exposed to temperatures ranging from 20°C to 100°C for 30 rain. All the reaction mixtures then underwent RIA. Concentration and SDS. A T H solution at 1 m g / m l in 0.05 M phosphate buffer containing 0.2% SDS was diluted 1/2 and 1 / 4 in the same buffer to obtain solutions at 500 and 250 ~ g / m l . Each solution was then brought to a final concentration of 10 ~ g / m l when diluted in Britton-Robinson buffer in order to establish a sensitivity curve.
346
Aliquots of 4 reference solutions at 10, 40, 50 and 60 # g / m l were treated with increasing quantities of SDS from 0 to 0.4% and then diluted for RIA. An identical quantity of protein was iodinated in the presence of 0.2 and 0.5% SDS. Specific radioactivities were then determined after chromatography and elimination of free iodide.
Results
Validity of the radioimmunoassay Under our experimental conditions, the mean specific radioactivity (calculated for n - - 7 ) of the ~25I-labeled T H was 70 #Ci//~g. The antiserum titer, defined by the final dilution which fixed 30-40% of the t25I-labeled T H (2500 dpm) was 1/15,000. The sensitivity of the assay, as defined by the quantity of non-radioactive glycoprotein required to displace 50% of bound 125I-labeled TH, was 20 n g / m l . The reference curve was established with 10 points in the range of 5-100 n g / m l , The system adopted for separating the antigen-antibody complex gave non-specific precipitation of the free protein no greater than 10%. Overload test. The equation of the regression line comparing theoretical and
100!
Bloc %)
90B07060 5O 4-0 30
T.H.
(ng/ml )
-/
iO
15
~'o ~o
5o -~o a'oo
Fig. 1. Superposition of the standard curve (O O) and values obtained in assay of dialyzed and diluted urine (, • ). The percent inhibition is shown on the y-axis and the TH concentration on the logarithmic x-axis.
347
experimental values was, for n = 9, y =- 0.99 × - 6 . 5 , with r = 0.95 and P < 0.001. (Experimental measurements are on the x-axis, theoretical calculation on the y-axis.) Parallelism (Fig. 1). The inhibition percentages of 125I-labeled T H binding to the antiserum in the presence of antigen in urine at different dilutions were identical with those obtained with the reference glycoprotein. Thus, the antigenic identity of the material assayed and the T H protein is proved.
~ng/m[ ng/m[
2@
100
250
,
)
pH
2 4- 6
~-~
- - ~
8
'
10
~
200
4~
~ng/m t , ng/m[ 300'
20(
?
/I I
250
100 v
I /
A v
Noel
(m)
/ urea
200
(m) i
0.1 02
0~3 0.4- 0.'5
0.1
ng/mt
0.2
0.3
ng/m[
201
~g.~;rn
20c
100
100
[) 1~'*0 lo"1o"1o-' J ' '
i 1;
Temp
20
io
60
) 80
100
Fig. 2. Influence of various factors on glycoprotein immunoreactivity. The concentrations of the T H solution (read from a standard curve) are shown on the y-axes. The increasing variations of each factor are on the x-axes.
348
\\\
4o-
i~
30211IH__
2
4-
6 8 10
20
40
60 80100
Fig. 3. Variations of reference curve sensitivities: 50% inhibitions of binding of antiserum with 125l-labeled TH in the presence of variable quantities of non-radioactive TH according to the reference TH solutions: 1000 • , , 500 • • and 250/~g/ml • • . Percent inhibition is shown on the y-axis and concentrations on non-radioactive TH on the logarithmic x-axis.
I( T.H. laq/ml )
I I00~ 8O 6O 4O 2O
0
0'.1
0.'2
0.3
0.4
S.DS. ('i'.) 0]5 -"
Fig. 4. TH glycoprotein immunoreactivity according to concentration, in the presence of SDS. Initial TH concentrations at 0% SDS: 10 C) ©, 40 • • , 50 • • and 60 ~tg/ml • •. The quantities of SDS used for the protein preparation are shown on the x-axis. Variations in immunoreactivity, in # g / m l , after RIA are shown on the y-axis.
349
Precision. The coefficient of variation (CV) calculated for the 10 points of the calibration range was always less than 4.5% (n -- 10). The CV for urine assays was 3% (n -- 10, m -- 5 . 7 / t g / m l ) . Reproducibi#ty. The CV for all points in 8 different calibration ranges was less than 7% and was 6.5% for the urine assays (n = 8, m = 6.3/~g/ml). Glycoprotein immunoreactivity The results obtained on RIA of T H protein with respect to the various factors studied are shown in Fig. 2. From the standard curve, the apparent concentration of the glycoprotein solution decreased with pH, SDS concentration and increasing NaC1 molarity. It remained stable with physiological concentrations of urea and with BSA up to l0 m g / m l (beyond this, BSA increased non-specific precipitation of the tracer). The protein retained complete antigenicity up to 80°C. The sensitivities of the reference curves (Fig. 3) obtained from 1000, 500 and 250 /~g/ml solutions treated with 0.2% SDS were respectively, at 50% B / B 0, 58, 47 and 36 n g / m l . This demonstrates increasing sensitivity with decreasing protein concentrations. The effect of increasing SDS concentrations on glycoprotein immunoreactivity at different concentrations is shown in Fig. 4. For l0 and 40 f f g / m l solutions, maximal immunoreactivity was obtained with 0.2% SDS, while it was not yet reached with 0.4% SDS, for 50 and 6 0 / l g / m l solutions.
6 i ,c.p.m
Bo/T
3x10 -
(%)
2 ~106
loo -80 6O
~o6 40
°/
.7 k m
225
2ss
20
2-;0
e[ufion(m()
Fig. 5. Gel exclusion chromatography of 1251-labeled T H on Sephadex G-200. The elution volume is shown on the x-axis, the radioactivities of eluted fractions on the left-hand y-axis and the binding percentage of these fractions with diluted ( l / 1 5 , 0 0 0 final) anti-TH serum on the right-hand y-axis, cpm: • O, B o / T O O. Sephadex G-200 was equilibrated with 0.05 M Tris-HCl, pH 7.4, containing 0.5% SDS. H = 9 4 cm, D = 2 . 6 cm. Elution rate: l0 m l / h ; 2.5 ml fractions.
350 The specific radioactivities obtained after iodination of identical quantities of T H protein in the presence of 0.2 or 0.5% SDS were 13 /~Ci//~g and 31 /~Ci//tg. In addition, Sephadex G-200 chromatography of T H protein iodinated in the presence of 0.5% SDS (Fig. 5) showed homogeneity of immunoreactivity of the labeled protein in the initial and maximal portions of the elution peak. Decreased tracer binding was observed only in the trailing portion of the peak. A small peak of radioactivity appearing after 125I-labeled T H and well before free iodide, did not bind anti-TH, anti-a2M, anti-haptoglobin or anti-albumin sera. These proteins are present in small quantities when T H is prepared.
Discussion
Ultrastructural study has shown that the T H protein molecule is composed of an axial filament 2 nm in diameter with fine lateral spicules 14-16 nm long and 2 nm thick. The spicules can fold to take on the aspect of spherules. The attachment of the spicules on the central axis would occur with a helicoidal arrangement, explaining the alternating spiked and zig-zag aspects along the polymer length. Chemically, the central axis is probably a polypeptide chain. The lateral spicules correspond to glycan chains terminating either by mannose/glucose or by sialic acid. This study also showed that the macromolecule had a high tendency to form parallel appositions, forming fibers of variable thicknesses (Delain et al., 1980). As reported here, potential interfering factors in RIA tended to prevent chain joining, and to dissociate and reduce chains to sub-units, while retaining total antigenicity (Figs, 6 and 7). Glycoprotein dissociation into sub-units and improved assay sensitivity were both favored by the use of Britton-Robinson buffer of low ionic strength and lacking NaC1. Once the glycoprotein was reduced to subunits, the quantity required to displace 50% of antiserum-tracer binding decreased. Similarly, the presence of 0.1% SDS and buffer p H at 7.4 contributed to glycoprotein solubility and thus assay sensitivity without affecting (at the chosen concentrations) the formation of the antigen-antibody complex. Finally, the separation of the complex from the free antigen fraction was favored by the use of BSA and bovine gamma globulins as carriers. When used at the respective concentrations of 0.25 and 0.4%, they had no effect on the non-specific precipitation of the T H protein. The effect of glycoprotein concentration on its physico-chemical behavior is also known (McQueen and Engel, 1966). On the basis of ultrastructural knowledge of the protein, in small chains or filaments (Delain et al., 1980), and of results obtained under various experimental conditions, we can formulate certain hypotheses on the relationships between structure and immunoreactivity. Sensitivity is undeniably increased by use of reference stock solutions at low concentrations, in which there is a greater probability of sub-unit formation. The quantity of the glycoprotein required to saturate a given number of antibody sites seems to decrease in this case. The presence of 0.5% SDS during iodination favors maximal sub-unit formation,
351
Fig. 6. Native TH glycoprotein Dark-field electron microscopy.
air-dried and tungsten X 70,000.
shadowed
as described
by Delain
et al. (1980).
thus rendering the tyrosyl groups of the protein more accessible to iodine, which results in an increased specific radioactivity. The lack of binding of the second eluted peak of radioactivity (Fig. 5) with the antiserum suggests protein degradation due to the relatively high SDS concentration and to the iodination. The latter modifies both the molecular weight and the immunoreactivity of the TH protein. Except for the detectable decrease of binding in the last elution fractions of the ‘251-labeled TH peak, certainly due to partial degradation of the glycoprotein, G-200 chromatography showed good homogeneity of the solution. Thus, step elution on Sephadex G-50 (fine) and verification of the binding of each elution fraction with the antiserum are sufficient to eliminate the iodinated but degraded fraction of the glycoprotein. The SDS concentration required for maximal sub-unit formation depends on TH
352
Fig. 7. TH glycoprotein treated with 0.1% SDS. Electron microscopy as in Fig. 6. The 2-dimensional arrangement is completely destroyed. × 70,000.
c o n c e n t r a t i o n . F o r the same T H c o n c e n t r a t i o n , m a x i m a l sub-unit f o r m a t i o n increases i m m u n o r e a c t i v i t y a n d the results of the R I A . The strict r e l a t i o n s h i p s b e t w e e n s t r u c t u r e - c o n c e n t r a t i o n and i m m u n o r e a c t i v i t y b e c o m e a limiting factor for the a p p l i c a t i o n of R I A of T H p r o t e i n to biological samples. T h e m e a n s ( ± 1 S.D.) of 13 values o b t a i n e d after assays in the same i n c u b a t i o n a n d in reference to s t a n d a r d curves p r e p a r e d from stock solutions of 200 a n d 9 0 0 / L g / m l were 11 ± 3.5 a n d 18 ± 6.5 ~ g / m l , or 12 + 5.5 a n d 19 ± 8 m g / 2 4 h. T h e 60% increase o b t a i n e d in the second case is due to the different c o n c e n t r a t i o n , structure a n d b e h a v i o r of the reference protein. Q u a n t i t a t i v e R I A of the T H p r o t e i n is r e n d e r e d a p p r o x i m a t e b y the difficulty of o b t a i n i n g the reference p r o t e i n a n d the u r i n a r y p r o t e i n in the s a m e structural state. This r e p o r t confirms that the d i s p a r i t y of u r i n a r y T H p r o t e i n assays r e p o r t e d in the literature is at least p a r t i a l l y due to the
353
diversity of procedures used, which gives rise to different ultrastructural states of the protein.
Acknowledgements This work was supported by the C.N.R.S. (E.R.A. no. 696), the I.N.S.E.R.M. (U 202) and the Claude Bernard Association (C 21). The authors thank P. Marcon for typing the manuscript.
References Avis, P.J.G., 1977, Clin. Sci. Mol. Med. 52, 183. Oelain, E., J.P. Thiery, D. Coulaud, A. Jolivi6re and L. Hartmann, 1980. Biol. Cell. 39, 31. Fletcher, A.P., A. Neuberger and W.A. Ratcliffe, 1970, Biochem, J. 120, 425. Grant, A.M.S. and A. Neuberger, 1973, Clin. Sci. 44, 163. G u t t m a n n , D. and G.R.E. Naylor, 1967, Brit. Med. J. 3, 343. Hartmann, L., 1971, in: Techniques Modernes du Laboratoire et Exploration Fonctionelle. ed. L. Hartmann (Masson, Paris) p. 276. Hartmann, L. and M. Toilliez, 1957, Rev. Fr. Etud. Clin. Biol. II, 197. Hartmann, L., J.P. Ambert, M.P. Ollier-Hartmann, G. Richet and C. Raynaud, 1981, Biomedicine 35, 30. Hunter, W.M. and F.C. Greenwood, 1962, Nature (London) 194, 495. Mackey, J.P. and G.H. Sandys, 1966, Brit. Med. J. 1, 1173. McQueen, E.G. and G.B. Engel, 1966, J. Clin. Path. 19, 392. Meberg, A., H. Haugen, I. Akesson and H. Sande, 1979, Nephron 23, 28. Ollier, M.P. and L. Hartmann, 1974, Biomedicine 21, 444. Ollier, M.P., M.A. Auger-Buendia and L. Hartmann, 1979, C.R. Acad. Sci. Paris, D. 289, 189. T a m m , I. and F.L. Horsfall, 1952, J. Exp. Med. 95, 71.
54 (1982) 343- 353
343
Elsevier Biomedical Press
Ultrastructure-Immunoreactivity Interference in the Radioimmunoassay of the Tamm-Horsfall Urinary Mucoprotein Lucien H a r t m a n n *, A n n i e - F r a n c e Bringuier * and Etienne Delain ** • Laboratoire de Chimie Clinique et Biologie Molbculaire, lnstitut Biomkdical des Cordeliers, 15, rue de l'Ecole de Mbdecine, 75270 Paris Cbdex 06, and ** Laboratoire de Microscopie Cellulaire et Mol~culaire, Institut Gustave Roussy, 94800 Villejuif, France
(Received 26 January 1982, accepted 3 May 1982)
We report the systematic study of various factors (pH, SDS, NaC1, urea, albumin, temperature, glycoprotein concentration) that could interfere in the radioimmunoassay of the Tamm-Horsfall urinary mucoprotein. The proposed procedure relates antigenicity of the molecule with a defined ultrastructure, enabling systematic physio-pathological studies in the future and valid comparisons of results obtained. Key words: Tamm-Horsfall urinary protein - - protein ultrastructure - - factors affecting radioimmunoassay
Introduction Until now, there has been no standard procedure for r a d i o i m m u n o a s s a y ( R I A ) of the T a m m - H o r s f a l l (TH) urinary mucoprotein, which explains the variety of procedures and the disparity of results reported ( G r a n t and Neuberger, 1973; Avis, 1977; Meberg et al., 1979). The work described here was undertaken to eliminate this inconvenience b y considering relationships between the antigenicity of the molecule and its different ultrastructural states.
Materials and Methods Materials Reagents. Products used to prepare buffers were obtained f r o m Merck: phosphoric, boric and acetic acids, Tris-(hydroxymethyl)-aminomethane, N a O H , m o n o and dibasic phosphates, NaC1 and polyethylene glycol (PEG). Bovine g a m m a globulin (Cohn fraction II), bovine serum albumin (BSA), sodium dodecyl sulfate
0022-1759/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
344 (SDS), sodium azide, sodium metabisulfite and chloramine T were purchased from Sigma. Chromatography. Sephadex G-50 (20--80 /~m) and G-200 (40-120 ktm) were obtained from Pharmacia. Radiochemicals. NaJ25I was obtained from New England Nuclear. Instrumentation. Disposable polystyrene tubes were used for the RIA. All centrifugations were performed in a Jouan E 96 centrifuge at 4°C. An Intertechnique CG 4000 gammacounter was used for determinations of radioactivity. Universal Britton-Robinson buffer. 0.035 M phosphoric acid, 0.04 M boric acid, 0.04 M acetic acid, pH 1.8. This buffer was diluted 1/8, adjusted to pH 7.4 with 0.05 N NaOH and added with 0.25% (w/v) BSA, 0.4% (w/v) bovine gamma globulins and 0.1% (w/v) SDS for the RIA. Biological material 24 h urines from 13 healthy adults between 21 and 30 years of age were collected over 0.1% Na azide and stored at - 2 0 ° C . Bacterial sterility was verified before the assay with Pasteur D G U kits, as described by Mackey and Sandys (1966), and Guttmann and Naylor (1967). One milliliter of urine was dialyzed at constant volume against 100 ml of double distilled water for 4 h at laboratory temperature, and was then diluted for the RIA.
Radioimmunoassay of the TH protein Reference glycoprotein. This protein was prepared by NaCl 0.58 M salting out as described by Tamm and Horsfall (1952) and Hartmann et al. (1981). A qualitative and quantitative analysis of impurities (albumin, a2M, ceruloplasmin, haptoglobin, transferrin) was systematically performed with specific antisera. Protein identity was determined by 4 criteria: a single well circumscribed spot with c~2/~ relative mobility after cellulose acetate electrophoresis, a single precipitin line on immunoelectrophoresis, a single peak after 2-dimensional electrophoresis (Oilier and Hartmann, 1974) and by amino acid composition and percentages of neutral and aminomonosaccharides (Delain et al., 1980). Protein concentration was determined by amido black assay (Hartmann, 1971). The reference glycoprotein was stored at a minimal concentration of 100/~g/ml in 0.05 M sodium phosphate buffer (pH 7.4) containing 0.2% (w/v) SDS (maximal concentration required to preserve the glycoprotein immunological integrity). Iodination. The glycoprotein was labeled with Na12SI by the chloramine T method (Hunter and Greenwood, 1962), using 0.05 M phosphate buffer (pH 7.4). Two micrograms of the T H protein (10 /~1 at 0.5% SDS, a concentration which allows sub-unit formation), followed by 50/~g of chloramine T, were added to 1 mCi of Na1251 dissolved in 10 /~1 of 0.5 M phosphate buffer (pH 7.4). The reaction was stopped after 15 sec by adding 125 #g sodium metabisulfite. Labeled T H protein was separated from free iodide by filtering through a 20 cm X 9 mm column of Sephadex G-50 (fine), and eluting with 0.05 M Tris-HC1 (pH 7.4) containing 0.5% SDS. Elution fractions of ~25I-labeled T H giving the best binding percentages with antiserum were stored for one month at - 2 0 ° C in approximately 150 × 103 dpm (70 nCi) aliquots. Antiserum. The purified glycoprotein preparation was used to raise antiserum in
345 'Fauve de Bourgogne' rabbits as described by Ollier et al. (1979). Antisera were stored in 1 ml aliquots in 0.1% Na azide in liquid nitrogen ( - 170°C). The specificity of anti-TH g a m m a globulins was verified by 2-dimensional electrophoresis and micro-immunodiffusion ( H a r t m a n n and Toilliez, 1957). Procedure. RIA was performed in duplicate in a final volume of 0.3 ml with the Britton-Robinson buffer (pH 7.4), containing 0.25% BSA, 0.4% bovine gamma globulin and 0.1% SDS. 2500 d p m of J25I-labeled T H were used per tube. The reaction mixture contained 0.1 ml of antiserum (diluted to obtain 30-40% binding of ~25I-labeled TH) to which were successively added 0.1 ml of the reference solution or the sample to be assayed and, after a 1 h preincubation at 37°C, 0.1 ml of 125I-labeled TH. After overnight incubation at laboratory temperature and stabilization for 1 h at 4°C, the antigen-antibody complex was precipitated by adding 0.3 ml of 10% PEG 6000 ( w / v ) in distilled water. Tubes were immediately centrifuged for 30 rain at 2000 × g at 4°C. After aspiration of the supernatant, the radioactivity of the bound antigen fraction was determined in the precipitate. VaBdity of the assay. This was controlled both by the overload test and by a parallelism between reference and various urinary assay curves. Increasing quantities of glycoprotein, 10, 20, and 40 ~g, were added to 1 ml of urine. After dialysis, the samples were assayed as described above. The identity of the product assayed in 2-fold serial dilutions of urine was also verified by comparing its behaviour with that of the reference protein towards antiserum (parallelism of curves). The precision of the assay was determined by establishing a standard curve of 10 determinations per point and by 10 assays of the same urine sample. Reproducibility was verified by measuring 8 samples of the same urine analyzed in 8 different assay series. Statistics. The coefficient of variation was determined by the study of precision and reproducibility. The significance of the coefficient of correlation of the linear regression curves established for the overload test was evaluated with the Student's t test.
Factors participating in glycoprotein aggregation Using the RIA, the immunoreactivity of T H towards the antiserum was studied with respect to the following parameters: pH, NaC1, SDS, BSA, urea, temperature. A glycoprotein solution at 2000 n g / m l was prepared in Britton-Robinson buffer (pH 7.4). It was diluted 1/10 in the same buffer, in which were separately and successively varied the p H (1.8-10), and the concentrations of NaCI (0.15-0.45 M), SDS (0.1-0.6%), BSA (10-4-100 m g / m l ) and urea (0.1-0.3 M). Finally, the mixtures were exposed to temperatures ranging from 20°C to 100°C for 30 rain. All the reaction mixtures then underwent RIA. Concentration and SDS. A T H solution at 1 m g / m l in 0.05 M phosphate buffer containing 0.2% SDS was diluted 1/2 and 1 / 4 in the same buffer to obtain solutions at 500 and 250 ~ g / m l . Each solution was then brought to a final concentration of 10 ~ g / m l when diluted in Britton-Robinson buffer in order to establish a sensitivity curve.
346
Aliquots of 4 reference solutions at 10, 40, 50 and 60 # g / m l were treated with increasing quantities of SDS from 0 to 0.4% and then diluted for RIA. An identical quantity of protein was iodinated in the presence of 0.2 and 0.5% SDS. Specific radioactivities were then determined after chromatography and elimination of free iodide.
Results
Validity of the radioimmunoassay Under our experimental conditions, the mean specific radioactivity (calculated for n - - 7 ) of the ~25I-labeled T H was 70 #Ci//~g. The antiserum titer, defined by the final dilution which fixed 30-40% of the t25I-labeled T H (2500 dpm) was 1/15,000. The sensitivity of the assay, as defined by the quantity of non-radioactive glycoprotein required to displace 50% of bound 125I-labeled TH, was 20 n g / m l . The reference curve was established with 10 points in the range of 5-100 n g / m l , The system adopted for separating the antigen-antibody complex gave non-specific precipitation of the free protein no greater than 10%. Overload test. The equation of the regression line comparing theoretical and
100!
Bloc %)
90B07060 5O 4-0 30
T.H.
(ng/ml )
-/
iO
15
~'o ~o
5o -~o a'oo
Fig. 1. Superposition of the standard curve (O O) and values obtained in assay of dialyzed and diluted urine (, • ). The percent inhibition is shown on the y-axis and the TH concentration on the logarithmic x-axis.
347
experimental values was, for n = 9, y =- 0.99 × - 6 . 5 , with r = 0.95 and P < 0.001. (Experimental measurements are on the x-axis, theoretical calculation on the y-axis.) Parallelism (Fig. 1). The inhibition percentages of 125I-labeled T H binding to the antiserum in the presence of antigen in urine at different dilutions were identical with those obtained with the reference glycoprotein. Thus, the antigenic identity of the material assayed and the T H protein is proved.
~ng/m[ ng/m[
2@
100
250
,
)
pH
2 4- 6
~-~
- - ~
8
'
10
~
200
4~
~ng/m t , ng/m[ 300'
20(
?
/I I
250
100 v
I /
A v
Noel
(m)
/ urea
200
(m) i
0.1 02
0~3 0.4- 0.'5
0.1
ng/mt
0.2
0.3
ng/m[
201
~g.~;rn
20c
100
100
[) 1~'*0 lo"1o"1o-' J ' '
i 1;
Temp
20
io
60
) 80
100
Fig. 2. Influence of various factors on glycoprotein immunoreactivity. The concentrations of the T H solution (read from a standard curve) are shown on the y-axes. The increasing variations of each factor are on the x-axes.
348
\\\
4o-
i~
30211IH__
2
4-
6 8 10
20
40
60 80100
Fig. 3. Variations of reference curve sensitivities: 50% inhibitions of binding of antiserum with 125l-labeled TH in the presence of variable quantities of non-radioactive TH according to the reference TH solutions: 1000 • , , 500 • • and 250/~g/ml • • . Percent inhibition is shown on the y-axis and concentrations on non-radioactive TH on the logarithmic x-axis.
I( T.H. laq/ml )
I I00~ 8O 6O 4O 2O
0
0'.1
0.'2
0.3
0.4
S.DS. ('i'.) 0]5 -"
Fig. 4. TH glycoprotein immunoreactivity according to concentration, in the presence of SDS. Initial TH concentrations at 0% SDS: 10 C) ©, 40 • • , 50 • • and 60 ~tg/ml • •. The quantities of SDS used for the protein preparation are shown on the x-axis. Variations in immunoreactivity, in # g / m l , after RIA are shown on the y-axis.
349
Precision. The coefficient of variation (CV) calculated for the 10 points of the calibration range was always less than 4.5% (n -- 10). The CV for urine assays was 3% (n -- 10, m -- 5 . 7 / t g / m l ) . Reproducibi#ty. The CV for all points in 8 different calibration ranges was less than 7% and was 6.5% for the urine assays (n = 8, m = 6.3/~g/ml). Glycoprotein immunoreactivity The results obtained on RIA of T H protein with respect to the various factors studied are shown in Fig. 2. From the standard curve, the apparent concentration of the glycoprotein solution decreased with pH, SDS concentration and increasing NaC1 molarity. It remained stable with physiological concentrations of urea and with BSA up to l0 m g / m l (beyond this, BSA increased non-specific precipitation of the tracer). The protein retained complete antigenicity up to 80°C. The sensitivities of the reference curves (Fig. 3) obtained from 1000, 500 and 250 /~g/ml solutions treated with 0.2% SDS were respectively, at 50% B / B 0, 58, 47 and 36 n g / m l . This demonstrates increasing sensitivity with decreasing protein concentrations. The effect of increasing SDS concentrations on glycoprotein immunoreactivity at different concentrations is shown in Fig. 4. For l0 and 40 f f g / m l solutions, maximal immunoreactivity was obtained with 0.2% SDS, while it was not yet reached with 0.4% SDS, for 50 and 6 0 / l g / m l solutions.
6 i ,c.p.m
Bo/T
3x10 -
(%)
2 ~106
loo -80 6O
~o6 40
°/
.7 k m
225
2ss
20
2-;0
e[ufion(m()
Fig. 5. Gel exclusion chromatography of 1251-labeled T H on Sephadex G-200. The elution volume is shown on the x-axis, the radioactivities of eluted fractions on the left-hand y-axis and the binding percentage of these fractions with diluted ( l / 1 5 , 0 0 0 final) anti-TH serum on the right-hand y-axis, cpm: • O, B o / T O O. Sephadex G-200 was equilibrated with 0.05 M Tris-HCl, pH 7.4, containing 0.5% SDS. H = 9 4 cm, D = 2 . 6 cm. Elution rate: l0 m l / h ; 2.5 ml fractions.
350 The specific radioactivities obtained after iodination of identical quantities of T H protein in the presence of 0.2 or 0.5% SDS were 13 /~Ci//~g and 31 /~Ci//tg. In addition, Sephadex G-200 chromatography of T H protein iodinated in the presence of 0.5% SDS (Fig. 5) showed homogeneity of immunoreactivity of the labeled protein in the initial and maximal portions of the elution peak. Decreased tracer binding was observed only in the trailing portion of the peak. A small peak of radioactivity appearing after 125I-labeled T H and well before free iodide, did not bind anti-TH, anti-a2M, anti-haptoglobin or anti-albumin sera. These proteins are present in small quantities when T H is prepared.
Discussion
Ultrastructural study has shown that the T H protein molecule is composed of an axial filament 2 nm in diameter with fine lateral spicules 14-16 nm long and 2 nm thick. The spicules can fold to take on the aspect of spherules. The attachment of the spicules on the central axis would occur with a helicoidal arrangement, explaining the alternating spiked and zig-zag aspects along the polymer length. Chemically, the central axis is probably a polypeptide chain. The lateral spicules correspond to glycan chains terminating either by mannose/glucose or by sialic acid. This study also showed that the macromolecule had a high tendency to form parallel appositions, forming fibers of variable thicknesses (Delain et al., 1980). As reported here, potential interfering factors in RIA tended to prevent chain joining, and to dissociate and reduce chains to sub-units, while retaining total antigenicity (Figs, 6 and 7). Glycoprotein dissociation into sub-units and improved assay sensitivity were both favored by the use of Britton-Robinson buffer of low ionic strength and lacking NaC1. Once the glycoprotein was reduced to subunits, the quantity required to displace 50% of antiserum-tracer binding decreased. Similarly, the presence of 0.1% SDS and buffer p H at 7.4 contributed to glycoprotein solubility and thus assay sensitivity without affecting (at the chosen concentrations) the formation of the antigen-antibody complex. Finally, the separation of the complex from the free antigen fraction was favored by the use of BSA and bovine gamma globulins as carriers. When used at the respective concentrations of 0.25 and 0.4%, they had no effect on the non-specific precipitation of the T H protein. The effect of glycoprotein concentration on its physico-chemical behavior is also known (McQueen and Engel, 1966). On the basis of ultrastructural knowledge of the protein, in small chains or filaments (Delain et al., 1980), and of results obtained under various experimental conditions, we can formulate certain hypotheses on the relationships between structure and immunoreactivity. Sensitivity is undeniably increased by use of reference stock solutions at low concentrations, in which there is a greater probability of sub-unit formation. The quantity of the glycoprotein required to saturate a given number of antibody sites seems to decrease in this case. The presence of 0.5% SDS during iodination favors maximal sub-unit formation,
351
Fig. 6. Native TH glycoprotein Dark-field electron microscopy.
air-dried and tungsten X 70,000.
shadowed
as described
by Delain
et al. (1980).
thus rendering the tyrosyl groups of the protein more accessible to iodine, which results in an increased specific radioactivity. The lack of binding of the second eluted peak of radioactivity (Fig. 5) with the antiserum suggests protein degradation due to the relatively high SDS concentration and to the iodination. The latter modifies both the molecular weight and the immunoreactivity of the TH protein. Except for the detectable decrease of binding in the last elution fractions of the ‘251-labeled TH peak, certainly due to partial degradation of the glycoprotein, G-200 chromatography showed good homogeneity of the solution. Thus, step elution on Sephadex G-50 (fine) and verification of the binding of each elution fraction with the antiserum are sufficient to eliminate the iodinated but degraded fraction of the glycoprotein. The SDS concentration required for maximal sub-unit formation depends on TH
352
Fig. 7. TH glycoprotein treated with 0.1% SDS. Electron microscopy as in Fig. 6. The 2-dimensional arrangement is completely destroyed. × 70,000.
c o n c e n t r a t i o n . F o r the same T H c o n c e n t r a t i o n , m a x i m a l sub-unit f o r m a t i o n increases i m m u n o r e a c t i v i t y a n d the results of the R I A . The strict r e l a t i o n s h i p s b e t w e e n s t r u c t u r e - c o n c e n t r a t i o n and i m m u n o r e a c t i v i t y b e c o m e a limiting factor for the a p p l i c a t i o n of R I A of T H p r o t e i n to biological samples. T h e m e a n s ( ± 1 S.D.) of 13 values o b t a i n e d after assays in the same i n c u b a t i o n a n d in reference to s t a n d a r d curves p r e p a r e d from stock solutions of 200 a n d 9 0 0 / L g / m l were 11 ± 3.5 a n d 18 ± 6.5 ~ g / m l , or 12 + 5.5 a n d 19 ± 8 m g / 2 4 h. T h e 60% increase o b t a i n e d in the second case is due to the different c o n c e n t r a t i o n , structure a n d b e h a v i o r of the reference protein. Q u a n t i t a t i v e R I A of the T H p r o t e i n is r e n d e r e d a p p r o x i m a t e b y the difficulty of o b t a i n i n g the reference p r o t e i n a n d the u r i n a r y p r o t e i n in the s a m e structural state. This r e p o r t confirms that the d i s p a r i t y of u r i n a r y T H p r o t e i n assays r e p o r t e d in the literature is at least p a r t i a l l y due to the
353
diversity of procedures used, which gives rise to different ultrastructural states of the protein.
Acknowledgements This work was supported by the C.N.R.S. (E.R.A. no. 696), the I.N.S.E.R.M. (U 202) and the Claude Bernard Association (C 21). The authors thank P. Marcon for typing the manuscript.
References Avis, P.J.G., 1977, Clin. Sci. Mol. Med. 52, 183. Oelain, E., J.P. Thiery, D. Coulaud, A. Jolivi6re and L. Hartmann, 1980. Biol. Cell. 39, 31. Fletcher, A.P., A. Neuberger and W.A. Ratcliffe, 1970, Biochem, J. 120, 425. Grant, A.M.S. and A. Neuberger, 1973, Clin. Sci. 44, 163. G u t t m a n n , D. and G.R.E. Naylor, 1967, Brit. Med. J. 3, 343. Hartmann, L., 1971, in: Techniques Modernes du Laboratoire et Exploration Fonctionelle. ed. L. Hartmann (Masson, Paris) p. 276. Hartmann, L. and M. Toilliez, 1957, Rev. Fr. Etud. Clin. Biol. II, 197. Hartmann, L., J.P. Ambert, M.P. Ollier-Hartmann, G. Richet and C. Raynaud, 1981, Biomedicine 35, 30. Hunter, W.M. and F.C. Greenwood, 1962, Nature (London) 194, 495. Mackey, J.P. and G.H. Sandys, 1966, Brit. Med. J. 1, 1173. McQueen, E.G. and G.B. Engel, 1966, J. Clin. Path. 19, 392. Meberg, A., H. Haugen, I. Akesson and H. Sande, 1979, Nephron 23, 28. Ollier, M.P. and L. Hartmann, 1974, Biomedicine 21, 444. Ollier, M.P., M.A. Auger-Buendia and L. Hartmann, 1979, C.R. Acad. Sci. Paris, D. 289, 189. T a m m , I. and F.L. Horsfall, 1952, J. Exp. Med. 95, 71.