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Direct time-resolved fluorescence immunoassay for serum oestradiol based on the idiotypic anti-idiotypic approach
95
Journal of Immunological Methods, 138 (1991) 95-101 © 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 ADONIS 002217599100130H JIM05879
Direct time-resolved fluorescence immunoassay for serum oestradiol based on the idiotypic anti-idiotypic approach A. Altamirano-Bustamante 1, G. Barnard 2 and F. K o h e n 1 1 Department of Hormone Research, The Weizmann Institute of Science, Rehovot, Israel, and 2 Department of Chemistry, City University, Northampton Square, London, U.K. (Received 4 September 1990, revised received 12 November 1990, accepted 17 December 1990)
We report a novel competitive type immunoassay for oestradiol based on the idiotypic anti-idiotypic approach. This has been achieved by the production of an anti-idiotypic antibody (anti-Id) which is directed against the oestradiol binding site of the primary idiotypic antibody (Abl). In this format the primary Ab I was captured onto the surface of microtitre wells and oestradiol standards or serum samples were then allowed to compete with europium labelled anti-Id for the binding sites of Ab 1. Fluorescence was proportional to the concentration of oestradiol over the range 0-8 ng/ml. The sensitivity of the assay was 80 + 20 pg/ml, whilst the intra-assay variation ranged from 3 to 10%, and the inter-assay variation from 7.3 to 15%. The results obtained by the fluorescence immunoassay correlated well with those obtained by an extraction radioimmunoassay using tritiated antigen and dextran-coated charcoal for separation of bound and free ligand (n = 60, r = 0.98). The idiotypic anti-idiotypic approach in hapten immunoassays enables antibodies to be labelled instead of haptens, and thus permits the development of robust and sensitive immunoassays. Key words: Serum estradiol; Idiotypic antibody; Anti-idiotypic antibody; Time-resolved fluorescence
Introduction
Most hapten assays developed to date, involve tagging a hapten with a marker, such as a radioactive isotype, an enzyme, a chemiluminescent marker or a fluorescent label, etc. Although reliable systems are possible using haptens labelled with radioactive markers, it has been difficult to achieve the same reliability with non-isotopic labels. In general, it is easier to prepare and to purify non-radioactive labels conjugated to antibodies than to label haptens with non-radioactive tags. Correspondence to." F. Kohen, Department of Hormone Research, The Weizmann Institute of Science, Rehovot 76100, Israel.
In order to label anti-hapten antibodies with non-radioactive markers recourse can be made to the anti-idiotypic procedure since it has been reported that anti-idiotypic antibodies of the betatype, Ab2fl, have the capacity to mimic the original antigen used to generate the primary antibody (Ab 0 (Strasberg, 1989). A large number of anti-Id classified as Ab2a, Ab2fl and Ab2y has been described in the literature (Farid and Lo, 1985; Bona et al., 1986). These anti-Id have proved to be useful tools in identifying by immunocytochemistry various cell surface receptors (Knigge et al., 1987; Sege and Peterson, 1987; Lombes et al., 1990), in mimicking the mode of action of various hormones (Schechter et al., 1982) and as putative vaccines against infectious agents (Sacks et al., 1982). To our knowledge, monoclonal anti-
96
nb2J3
Antigen
Fig. 1. Principles of a separation assay for haptens based on the idiotypic anti-idiotypic approach. The anti-idiotype, Ab2/~, bears on its Fab region a structural counterpart of the hormone binding site which is shared by the idiotype Ab1 and by the hormone. Thus AbzB can completewith the antigen (hormone) for the binding sites of Ab1. In this paper the solid phase Ab1 is represented by the idiotypic oestradiol antibody clone 2F9 and the europium labelled Ab2t~by the anti-idiotypicantibody against anti-oestradiol,clone 1D5. idiotypic steroid specific antibodies of the beta type that can compete with the original steroid for the binding sites of Ab I in immunoassay procedures have not yet been reported. In a previous report (Barnard and Kohen, 1990), we have described the production and characterization of monoclonal anti-idiotypic antibodies of the alpha (Ab2a) and the beta type (Ab2fl) against monoclonal anti-oestradiol. We now report the development of a direct assay for serum oestradiol based on the idiotypic-anti-idiotypic approach. In this format, the primary monoclonal idiotypic antibody to oestradiol (Abl, clone no. 2F9) served as the solid phase and europium labelled monoclonal anti-idiotypic antibody (clone no. 1D 5, Ab2fl type) directed against the paratopic region of the primary anti-oestradiol antibody as the marker (see Fig. 1). The end-point was measured by timeresolved fluorescence.
Materials and methods
Reagent and solutions Steroids, Tris-(hydroxymethyl)-aminomethane, bovine serum albumin (BSA, RIA grade), Tween 20, diethylenetriamine pentaacetic acid (DTPA), bovine-~/-globulin, incomplete and complete Freund's adjuvant were purchased from Sigma
Chemical Co., St. Louis, MO. Affinity purified rabbit anti-mouse immunoglobulin was obtained from Dakopatts, Glostrup, Denmark. SepharoseProtein A and Sephadex G-25 were purchased from Pharmacia, Uppsala, Sweden. Anti-mouse immunoglobulins for the Ouchterlony immunodiffusion technique were from Serotec, Oxford, U.K. The labelling reagent (N-l(p-isothiocyanatophenyl)-di-ethylene-triamine-N 1, N 2, N 3-tetraacetate, DTTA) chelated with Eu 3+ was kindly provided by Dr. IIkka Hemmila, Wallac Oy, Turku, Finland. Danazol (17c~-pregna-2,4-dien-20-yno[2,3d]isoxazol-17-ol) was a gift from Winthrop Labs, Brussels, Belgium. Time resolved fluorescence was measured using the enhancement solution obtained from Wallac, Oy, Turku, Finland. The assay buffer used throughout was Tris-HC1 buffer, pH 7.75, 50 mmol/1, containing bovine serum albumin (5 g/l), bovine-y-globulin (0.5 g/l), 20 /zmol of DTPA, sodium chloride (9 g/l), Tween 20 (0.5 ml/1) and sodium azide (0.5 g/l). The wash solution was Tris-HC1 buffer, p H 7.75, 50 mmol/1, containing Tween 20 (0.05 ml/1), sodium chloride (0.9 g/l) and sodium azide (0.5 g/l).
Procedures The preparation of rabbit anti-mouse IgG coated plates Rabbit anti-mouse immunoglobulin (2.9 r a g / ml) was diluted in 6 vols of diluted HC1 (1/400 v/v). After 5 min, the acid-treated IgG was diluted to 5 /~g/ml in acidic coating buffer (0.2 M, p H 3-4, prepared by dissolving 27.2 g dihydrogen phosphate monohydrate in 1 litre of doubly distilled water). 200 ~1 of IgG were added to the wells of polystyrene microtiter strips (1 x 12 well, from Labsystems, Helsinki, Finland). After an overnight incubation at 37 ° C in a humid atmosphere, the coating buffer was aspirated to waste and the strips washed and aspirated twice with wash solution. The strips were then blocked overnight with 50 mM sodium hydrogen phosphate, containing sodium chloride 9 g/l, sodium azide 0.1 g/1 and bovine serum albumin, 1 g/1. The blocking solution was then aspirated, and the strips sealed with sealing tape (Nunc, Kanstrup, Denmark).
97
Purification of monoclonal antibodies with specificity for oestradiol Ascitic fluid from monoclonal anti-oestradiol antibodies derived from a rat-mouse hybridoma cell line (clone no. 2F9) (Kohen and Lichter, 1986) was purified by affinity chromatography on Immunobind Avid Gel A x (Bioprobe International, Tustin, CA). Briefly, ascitic fluid (1 ml) was diluted with 9 ml of sodium phosphate buffer (PBS), 10 mM, pH 7.4, containing sodium chloride 9 g / l and sodium azide 0.1 g/1 and applied to a small column containing 3 ml gel. The IgG fraction was eluted from the matrix by lowering the pH to 3.0 with 0.1 M sodium acetate. The pooled IgG containing peak was then dialysed against PBS and stored at - 20 ° C until use.
Preparation of europium labelled antibodies The following procedure was used for labelling antibodies with europium. As an example, the labelling of anti-oestradiol is described in detail. Anti-oestradiol IgG (1 m g / m l PBS) was dialysed against carbonate buffer (50 mM, pH 9.8) for 2 h. At the same time a vial containing 1 mg of europium chelate was reconstituted in 200 /~1 of double distilled water. An 80-fold excess of the labelling reagent (140 /tl; 3.83 n m o l / / d ) was added to the IgG solution. The reaction mixture was stirred overnight at 4 ° C and subsequently purified by gel filtration on Sephadex G-25. The labelled IgG was eluted with Tris-HC1, 50 mM, pH 7.75 containing 0.2% sodium chloride and 0.05% sodium azide. In general 10-15 mol of europium were incorporated per mol of protein. The labelled IgG was stored at 4 °C until use. The same labelling procedure was used for tagging anti-idiotype antibodies with europium.
Preparation of monoclonal anti-idiotypic antibodies against anti-oestradiol Female CD 2 mice (age:2 months) were immunized with purified monoclonal anti-oestradiol IgG (clone 2F9, 50 /~g/mouse) in complete Freund's adjuvant. Subsequently, two booster injections were given using anti-oestradiol IgG in incomplete Freund's adjuvant. After 2 months of immunization, the anti-idiotypic response was checked as described (Barnard and Kohen, 1990). 3 months after the initial immunization the spleen
cells of the mouse showing the highest serum titre of antibodies which recognized europium labelled anti-oestradiol IgG were fused with a mouse myeloma cell line (NSO, kindly donated by Dr. Milstein, Cambridge) using the hybridoma technique of K/Shler and Milstein (1976). The culture supernatants of growing hybridomas were screened for antibody activity as described by Barnard and Kohen (1990).
Screening of anti-idiotypes of the betatype Preparation of probes. The screening procedures involved first the identification of anti-idiotypic antibodies that recognized europium-labelled anti-oestradiol IgG followed by identification of anti-idiotypic antibodies of the beta type which were classified as antigen-sensitive and could compete with oestradiol for the binding sites of antioestradiol (Barnard and Kohen, 1990). Briefly, in the first screening, hybridoma culture supernatants (5 /~l/well) were added to the anti-mouse IgG coated microstrips containing 200 ~1 of assay buffer/well. The plates were incubated for 1 h. The reaction mixture was aspirated and the plates were washed three times. The plates were then blocked for 10 min with 25/~1 of mouse serum, and subsequently 200 /zl of europium labelled anti-oestradiol IgG (approximately 107 cps/ml) were added to each well. The plates were incubated for 1 h at room temperature and subsequently the strips were washed six times. Enhancement solution (200/~1) was added to each well and the strips were agitated on the shaker for 15 min. Fluorescence was then measured using an Arcus time-resolved fluorimeter (Wallac Oy, Turku, Finland). The wells that gave positive results in this assay were classified as anti-allotypes and anti-idiotypes. In the second screening procedure, the hybridomas that gave positive results in the first screening assay were added (5 #l/well) in quadruplicate to anti-mouse IgG coated microtiter wells containing 200/~1 of assay buffer. The plates were incubated, washed and blocked as described in the first screening procedure (see above). Subsequently to one set of duplicates, 200 /xl of assay buffer containing europium labelled antioestradiol IgG (107 cps/ml) were added and, to the other set of duplicates, 200/~1 of assay buffer
98 containing an excess amount of oestradiol (1 /~g/ml) and europium-labelled anti-oestradiol (10 7 cps/ml) were added. The plates were processed as described in the first screening procedure. Immobilized hybridoma culture supernatants that failed to bind europium labelled anti-oestradiol in the presence of oestradiol were classified as paratypic antibodies (beta type). From these screening procedures one clone, no. 1D5, was selected as a beta type anti-idiotype and was used in the development of a competitive immunoassay for oestradiol. This clone belonged to the IgG1 heavy chain class. Ascites of this clone were prepared in pristane primed C D 2 mice as described previously (Strasburger et al., 1989). An immunoglobulin fraction (IgG) of clone no 1D 5 was isolated from ascitic fluid by chromatography on Sepharose-protein A as described by Strasburger and Kohen (1990). Purified IgG was then labelled with europium using the procedure described in this paper.
Assay Procedure Preparation of monoclonal anti-oestradiol coated plates A stock solution of monoclonal anti-oestradiol IgG (clone 2F9) was diluted with PBS to 2.5 /xg/ml, and 200 /~1 of this solution were pipetted into each well of the microtitre strips. After an overnight incubation at room temperature the coating buffer was aspirated to waste and the strips were washed three times. The plates were then ready for use. Time-resolved fluorescence immunoassay Preparation of the matrix for the assay. The displacing agents, danazol and 5a-dihydrotestosterone were prepared at a concentration of 5 n g / m l in assay buffer. To 2 ml of horse serum 1 ml of each of the displacing agents was added and the mixture was left for 1 h at room temperature. A solution of 40 ng oestradiol/ml was prepared by directly mixing 100/~1 of an ethanolic solution of 1 t~g/ml solution of oestradiol with 2.4 ml of 1 : 1 mixture of horse serum with the displacing agents, and by incubating the mixture for 15 min at 37 ° C. This stock solution of oestradiol in horse serum and displacing agents and the 1 : 1 mixture
of horse-serum and displacing agents were used to prepare oestradiol standards, ranging from 0 to 200 pg/well. 25 /~1 of standards, quality control samples or unknowns were pipetted into the wells of the antibody coated plates. In addition, the quality control samples and unknowns received 25 /~1 of assay buffer containing displacing agents (5 ng danazol and 5 ng 5a-dihydrotestosterone/ml assay buffer). Europium labelled anti-idiotypic antibody (clone 1D 5, 20 ng, 1 × 10 6 cps, 100/~1) was added to each well and the total volume in each well was adjusted with assay buffer to 200/xl. The plates were then incubated at room temperature for 1 h in an automatic plate shaker. The reaction mixture was aspirated to waste, and the strips were washed six times. Enhancement solution (200 /~1) was then added to each well, and the strips were agitated on the shaker for 10 min. Fluorescence was then measured with an Arcus time-resolved fluorometer.
Sample collection Blood samples were obtained by venipuncture from patients undergoing ovulation induction at King's College Medical School, London, U.K. We thank Prof. W.P. Collins for providing these sampies. Calculation of results Non-specific blank values were subtracted from the fluorimeter readings and a logit-log transformation was used for plotting the calibration curve and the calculation of results of oestradiol concentration from unknowns.
Results
Calibration curves The effects of the addition of danazol, 5a-dihydrotestosterone and horse serum to the standards are shown in Fig. 2. Accurate results could be obtained using the calibration curve established in the presence of danazol (5 ng/ml), 5a-dihydrotestosterone (5 n g / m l ) and horse serum (25 /xl/well). This curve was used in the direct fluorescence immunoassay. Sensitivity, defined as the minimum detectable dose of oestradiol that could be distinguished from zero (mean + SD), was
TABLE I
\-E 4000
PRECISION OF OESTRADIOL M E A S U R E M E N T BY DIRECT FIA
7_o 3000
99
,~n~
"o t_
Pool
Mean and SD
Intra-assay
Interassay
no.
oestradiol concen-
CV a, %
CV a, %
7.34 6.24 9.98 3.37 3.14
11.34 8.62 14.67 7.34 10.62
"~ 2000 oJ
tration ( p g / m l ) 1 2 3 4 5
82.5 332.7 436.5 1,652 2,276
+ 9.35 + 28.69 + 61.61 + 121.2 +242
~1000.
a CV = coefficient of variation, defined as
SD
mean
x
LL
W~m e I
1000
I
I
I
2000
3000
4000
RIA, pg esfradiot/rnl Fig. 3. Comparison of serum oestradiol concentrations determined by the extraction RIA (x) and FIA (y) methods. The
100.
regression equation summarizing the results (n = 60) was y = 1.0x + 4.31 with a correlation coefficient r = 0.98.
c a l c u l a t e d f r o m f o u r consecutive c a l i b r a t i o n curves p r e p a r e d in d u p l i c a t e (see Fig. 2). T h e m e a n value d e r i v e d f r o m the curve used in the direct fluorescence i m m u n o a s s a y was 80 + 20 p g / m l , a n d the reference logit %. B / T value r e a d f r o m the curve was 91%.
Recovery I n c r e a s i n g a m o u n t s of u n l a b e l l e d o e s t r a d i o l ( 3 . 2 - 1 0 0 p g / w e l l ) were a d d e d to horse s e r u m a n d the s a m p l e s were a n a l y s e d several times (n = 17)
b y direct F I A . T h e m e a n r e c o v e r y was 103.7 + 4.15%.
Precision I n t r a - a s s a y a n d i n t e r - a s s a y v a r i a t i o n were e s t i m a t e d b y m e a s u r i n g o e s t r a d i o l levels in duplic a t e in five s e r u m p o o l s used for i n t e r n a l q u a l i t y c o n t r o l in seven consecutive assays. T h e results are s h o w n in T a b l e I.
Parallelism S e r u m f r o m a p r e g n a n t w o m e n was d i l u t e d 10-, 20-, 40- a n d 80-fold w i t h the 1 : 1 m i x t u r e o f horse s e r u m a n d d i s p l a c i n g agents, a n d the o e s t r a d i o l c o n t e n t was a s s a y e d in the d i l u t e d s a m p l e s b y direct F I A ( y ) . T h e l i n e a r regression e q u a t i o n s u m m a r i z i n g the results was: y = 0.977 x - 1.051, with a c o r r e l a t i o n coefficient of r = 0.99.
,~7.0 90
80
=~
60
~' 30
Correlation with radioimmunoassay
20
10
1=0 100 Estradiol (pg/wel[)
1000
Fig. 2. Effect of displacing agents and horse serum on oestradiol calibration curves in the FlA. Assays buffer only (A. . . . . . zx); danazol, (50 n g / m l ) , 5a-dihydrotestosterone (50 ng/ml), horse serum, 50 #l/well ( O - - - - - O ) ; danazol (5 ng/ml), 5a-dihydrotestosterone (5 n g / m l ) , horse serum 50 ~ l / w e l l (o o); danazol (5 ng/ml), 5a-dihydrotestosterone (5 n g / m l ) , horse serum 25 ~l/well (e e), m e a n + S D of four consecutive determinations - the curve used in the direct FIA. B = specific counts that were bound; T = total counts.
T h e c o n c e n t r a t i o n of o e s t r a d i o l in 60 s e r u m s a m p l e s as d e t e r m i n e d b y d i r e c t F I A ( y ) a n d as m e a s u r e d b y R I A ( x ) after e x t r a c t i o n o f the sera s h o w e d excellent c o r r e l a t i o n characteristics. T h e l i n e a r regression e q u a t i o n was y = 1.0 x + 4.31 w i t h a c o r r e l a t i o n r = 0.98. T h e results are s h o w n in Fig. 3.
Discussion I n recent y e a r s several n o n - i s o t o p i c i m m u n o a s say techniques have b e e n d e s c r i b e d for the m e a -
100 surement of serum oestradiol from plasma. These methods rely on the use of a derivative of oestradiol conjugated to an enzyme (Marcus and Durnford, 1988) or to a chemiluminescent marker (De Boever et al., 1986). The use of these labels in immunoassay methods have certain disadvantages. For instance, the purification of oestradiol-enzyme conjugates is tedious and difficult and the endpoint measurement of steroid-chemiluminescent marker conjugates cannot be repeated (De Boever et al., 1990). On the other hand, labelling of antibodies with an enzyme is rather easy and the antibody-enzyme conjugate can be separated easily from unreacted enzyme and antibody by gel filtration. In addition, antibodies can be labelled with universal reagents, such as biotin, for use in avidin-biotin mediated i m m u n o a s s a y systems (Strasburger et al., 1988) or with fluorescent labels for use in time-resolved fluorescence (Barnard, 1988). The availability in our hands of an anti-idiotypic antibody (clone 1D 5) (Barnard and Kohen, 1990) capable of mimicking oestradiol at the binding site of the primary anti-oestradiol antibody (clone no. 2F9) permitted the use of labelled antibody in the immunoassay method. In this paper we describe the development of an immunoassay for serum oestradiol which is based on the idiotypic anti-idiotypic approach. In this format the primary anti-oestradiol antibody (clone 2F9) serves as the solid-phase, and the anti-idiotypic antibody (clone 1Ds) labelled with europium as the marker (see Fig. 1). The direct fluorescent immunoassay (FIA) proved satisfactory with respect to sensitivity, accuracy and precision. A good correlation was observed between the FIA and the R I A using an extraction step (see Fig. 3). This direct FIA has the advantage of a short assay time, requiring in total about 2 h including counting and calculation of results. The direct assay for oestradiol can be performed with 25 t~l of serum per well (see Fig. 2) and the sensitivity (80 p g / m l ) of the present method is adequate for measuring oestradiol during the human ovarian cycle as well as during ovulation induction. In summary, this paper reports the use of an anti-idiotypic antibody and an idiotypic antibody in hapten immunoassay as exemplified by the direct measurement of oestradiol from serum. This
technique offers interesting possibilities for the development of competitive-type non-isotopic immunoassays for haptens.
Acknowledgements This work has been supported in part by a grant (BR-IS-001) from the Institute for International Studies in Natural Family Planning, Department of Obstetrics and Gynaecology, Georgetown University Medical School, Washington DC, U.S.A. (G.B. and F.K.) and from the Special Programme of Research, Development and Research Training in H u m a n Reproduction, World Health Organization (to F.K.). We thank Prof. W.P. Collins of the Department of Obstetrics and Gynaecology, King's College School of Medicine and Dentistry, London, for the provision of samples and the results of the extraction RIA. We are grateful to Wallac Oy, Turku, Finland, for the provision of equipment and reagents, to Mrs. M. Kopelowitz for secretarial assistance, and to Dr. T. Ngo, of Bio-Probe Int. Tustin Co., for providing us the I m m u n o b i n d Avid Gel Ax. We thank the Special Programme of Research, Development and Research Training in H u m a n Reproduction, World Health Organization, for a training grant to A. A-B.
References Barnard, G. (1988) The development of fluorescence immunoassays. In: Non-Radiometric Assays: Technology and Application in Polypeptide and Steroid Hormone Detection. Alan R. Liss, New York, p. 15. Barnard, G. and Kohen, F. (1990) Idiometric assay: A noncompetitive immunoassay for haptens typified by the measurement of serum oestradiol. Clin. Chem. 36, 1945. Bona, C.A., Kang, C.-Y., Kohler, H. and Monestier, M. (1986) Epibody: the image of the network created by a single antibody. Immunol. Rev. 90, 116. De Boever, J., Kohen, F., Usanachitt, C., Vandekerckhove, D., Leyseele, D. and Vandewalle, L. (1986) Direct chemiluminescence immunoassay for oestradiol in serum. Clin. Chem. 32, 1895. De Boever, J., Kohen, F., Leyseele, D. and Vandekerckhove, D. (1990) Isoluminol as a marker in direct chemiluminescence immunoassays for steroid hormones. J. Biolum. Chemilum. 5, 5.
101 Farid, N. and Lo, T. (1985) Antidiotypic antibodies as probes for receptor structure and function. Endocr. Rev. 6, 1. Knigge, K.M., Plekut, D.T., Berlove, D.J., Junig, J.T. and Melrose, P.A. (1987) Staining of magnocellular neurons of the supraoptic and paraventricular nuclei with vasopresin anti-idiotype antibody: a potential method for receptor immunocytochemistry. Mol. Brian Res. 2, 69. Kohen, F. and Lichter, S. (1986) Monoclonal antibodies to steroid hormones. In: G. Forti, M.B. Lipsett and M. Serio (Eds.), Monoclonal Antibodies: Basic Principles, Experimental and Clinical Applications in Endocrinology, Raven Press, New York, p. 87. KShler, G. and Milstein, C. (1976) Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur. J. Immunol., 6, 511. Lombes, M., Farman, N., Oblin, M., Baulieu, E., Bonvalet, J. and Erlanger, B. (1990) Immunohistochemical localization of renal mineralocorticoid receptor by using an anti-idiotypic antibody that is an internal image of aldesterone. Proc. Natl. Acad. Sci. U.S.A. 87, 1086. Marcus, G.J. and Durnford, R. (1988) Oestradiol assay by microtitre plate enzyme immunoassay. J. Steroid Biochem. 29, 207. Sacks, D., Esser, K. and Sher, A. (1982) Immunization of mice against African trypanosomiasis using anti-idiotypic antibodies. J. Exp. Med. 115, 1108.
Sege, K. and Peterson, P. (1978) Use of anti-idiotypic antibodies as cell-surface receptor probes. Proc. Natl. Acad. Sci. U.S.A. 73, 2443. Shechter, Y., Maron, R., Elias, D. and Cohen, I. (1982) Autoantibodies to insulin receptor spontaneously develop as anti-idiotypes in mouse immunized with insulin. Science 216, 542. Strasberg, A.D. (1989) Interaction of anti-idiotypic antibodies with membrane receptors: Practical considerations. In: J.J. Langone (Ed.), Methods in Enzymology, Vol. 178. Academic Press, New York, p. 179. Strasburger, C.J. and Kohen, F. (1990) Biotinylated probes in immunoassay. In: M. Wilchek and E. Bayer (Eds.), Methods in Enzymology, Vol. 181, Academic Press, New York, p. 481. Strasburger, C.J., Arnir-Zaltzman, Y. and Kohen, F. (1988) The avidin-biotin reaction as a universal amplification system in irnmunoassays. In: Non-Radiometric Assays: Technology and Application in Polypeptide and Steroid Hormone Detection. Ala R. Liss, New York, p. 79. Strasburger, C., Barnard, G., Toldo, L., Zarmi, B., Zadik, Z., Kowarski, A. and Kohen, F. (1989) Somatotropin as measured by a two-site time-resolved immunofluorometric assay. Clin. Chem. 35, 55.
Journal of Immunological Methods, 138 (1991) 95-101 © 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 ADONIS 002217599100130H JIM05879
Direct time-resolved fluorescence immunoassay for serum oestradiol based on the idiotypic anti-idiotypic approach A. Altamirano-Bustamante 1, G. Barnard 2 and F. K o h e n 1 1 Department of Hormone Research, The Weizmann Institute of Science, Rehovot, Israel, and 2 Department of Chemistry, City University, Northampton Square, London, U.K. (Received 4 September 1990, revised received 12 November 1990, accepted 17 December 1990)
We report a novel competitive type immunoassay for oestradiol based on the idiotypic anti-idiotypic approach. This has been achieved by the production of an anti-idiotypic antibody (anti-Id) which is directed against the oestradiol binding site of the primary idiotypic antibody (Abl). In this format the primary Ab I was captured onto the surface of microtitre wells and oestradiol standards or serum samples were then allowed to compete with europium labelled anti-Id for the binding sites of Ab 1. Fluorescence was proportional to the concentration of oestradiol over the range 0-8 ng/ml. The sensitivity of the assay was 80 + 20 pg/ml, whilst the intra-assay variation ranged from 3 to 10%, and the inter-assay variation from 7.3 to 15%. The results obtained by the fluorescence immunoassay correlated well with those obtained by an extraction radioimmunoassay using tritiated antigen and dextran-coated charcoal for separation of bound and free ligand (n = 60, r = 0.98). The idiotypic anti-idiotypic approach in hapten immunoassays enables antibodies to be labelled instead of haptens, and thus permits the development of robust and sensitive immunoassays. Key words: Serum estradiol; Idiotypic antibody; Anti-idiotypic antibody; Time-resolved fluorescence
Introduction
Most hapten assays developed to date, involve tagging a hapten with a marker, such as a radioactive isotype, an enzyme, a chemiluminescent marker or a fluorescent label, etc. Although reliable systems are possible using haptens labelled with radioactive markers, it has been difficult to achieve the same reliability with non-isotopic labels. In general, it is easier to prepare and to purify non-radioactive labels conjugated to antibodies than to label haptens with non-radioactive tags. Correspondence to." F. Kohen, Department of Hormone Research, The Weizmann Institute of Science, Rehovot 76100, Israel.
In order to label anti-hapten antibodies with non-radioactive markers recourse can be made to the anti-idiotypic procedure since it has been reported that anti-idiotypic antibodies of the betatype, Ab2fl, have the capacity to mimic the original antigen used to generate the primary antibody (Ab 0 (Strasberg, 1989). A large number of anti-Id classified as Ab2a, Ab2fl and Ab2y has been described in the literature (Farid and Lo, 1985; Bona et al., 1986). These anti-Id have proved to be useful tools in identifying by immunocytochemistry various cell surface receptors (Knigge et al., 1987; Sege and Peterson, 1987; Lombes et al., 1990), in mimicking the mode of action of various hormones (Schechter et al., 1982) and as putative vaccines against infectious agents (Sacks et al., 1982). To our knowledge, monoclonal anti-
96
nb2J3
Antigen
Fig. 1. Principles of a separation assay for haptens based on the idiotypic anti-idiotypic approach. The anti-idiotype, Ab2/~, bears on its Fab region a structural counterpart of the hormone binding site which is shared by the idiotype Ab1 and by the hormone. Thus AbzB can completewith the antigen (hormone) for the binding sites of Ab1. In this paper the solid phase Ab1 is represented by the idiotypic oestradiol antibody clone 2F9 and the europium labelled Ab2t~by the anti-idiotypicantibody against anti-oestradiol,clone 1D5. idiotypic steroid specific antibodies of the beta type that can compete with the original steroid for the binding sites of Ab I in immunoassay procedures have not yet been reported. In a previous report (Barnard and Kohen, 1990), we have described the production and characterization of monoclonal anti-idiotypic antibodies of the alpha (Ab2a) and the beta type (Ab2fl) against monoclonal anti-oestradiol. We now report the development of a direct assay for serum oestradiol based on the idiotypic-anti-idiotypic approach. In this format, the primary monoclonal idiotypic antibody to oestradiol (Abl, clone no. 2F9) served as the solid phase and europium labelled monoclonal anti-idiotypic antibody (clone no. 1D 5, Ab2fl type) directed against the paratopic region of the primary anti-oestradiol antibody as the marker (see Fig. 1). The end-point was measured by timeresolved fluorescence.
Materials and methods
Reagent and solutions Steroids, Tris-(hydroxymethyl)-aminomethane, bovine serum albumin (BSA, RIA grade), Tween 20, diethylenetriamine pentaacetic acid (DTPA), bovine-~/-globulin, incomplete and complete Freund's adjuvant were purchased from Sigma
Chemical Co., St. Louis, MO. Affinity purified rabbit anti-mouse immunoglobulin was obtained from Dakopatts, Glostrup, Denmark. SepharoseProtein A and Sephadex G-25 were purchased from Pharmacia, Uppsala, Sweden. Anti-mouse immunoglobulins for the Ouchterlony immunodiffusion technique were from Serotec, Oxford, U.K. The labelling reagent (N-l(p-isothiocyanatophenyl)-di-ethylene-triamine-N 1, N 2, N 3-tetraacetate, DTTA) chelated with Eu 3+ was kindly provided by Dr. IIkka Hemmila, Wallac Oy, Turku, Finland. Danazol (17c~-pregna-2,4-dien-20-yno[2,3d]isoxazol-17-ol) was a gift from Winthrop Labs, Brussels, Belgium. Time resolved fluorescence was measured using the enhancement solution obtained from Wallac, Oy, Turku, Finland. The assay buffer used throughout was Tris-HC1 buffer, pH 7.75, 50 mmol/1, containing bovine serum albumin (5 g/l), bovine-y-globulin (0.5 g/l), 20 /zmol of DTPA, sodium chloride (9 g/l), Tween 20 (0.5 ml/1) and sodium azide (0.5 g/l). The wash solution was Tris-HC1 buffer, p H 7.75, 50 mmol/1, containing Tween 20 (0.05 ml/1), sodium chloride (0.9 g/l) and sodium azide (0.5 g/l).
Procedures The preparation of rabbit anti-mouse IgG coated plates Rabbit anti-mouse immunoglobulin (2.9 r a g / ml) was diluted in 6 vols of diluted HC1 (1/400 v/v). After 5 min, the acid-treated IgG was diluted to 5 /~g/ml in acidic coating buffer (0.2 M, p H 3-4, prepared by dissolving 27.2 g dihydrogen phosphate monohydrate in 1 litre of doubly distilled water). 200 ~1 of IgG were added to the wells of polystyrene microtiter strips (1 x 12 well, from Labsystems, Helsinki, Finland). After an overnight incubation at 37 ° C in a humid atmosphere, the coating buffer was aspirated to waste and the strips washed and aspirated twice with wash solution. The strips were then blocked overnight with 50 mM sodium hydrogen phosphate, containing sodium chloride 9 g/l, sodium azide 0.1 g/1 and bovine serum albumin, 1 g/1. The blocking solution was then aspirated, and the strips sealed with sealing tape (Nunc, Kanstrup, Denmark).
97
Purification of monoclonal antibodies with specificity for oestradiol Ascitic fluid from monoclonal anti-oestradiol antibodies derived from a rat-mouse hybridoma cell line (clone no. 2F9) (Kohen and Lichter, 1986) was purified by affinity chromatography on Immunobind Avid Gel A x (Bioprobe International, Tustin, CA). Briefly, ascitic fluid (1 ml) was diluted with 9 ml of sodium phosphate buffer (PBS), 10 mM, pH 7.4, containing sodium chloride 9 g / l and sodium azide 0.1 g/1 and applied to a small column containing 3 ml gel. The IgG fraction was eluted from the matrix by lowering the pH to 3.0 with 0.1 M sodium acetate. The pooled IgG containing peak was then dialysed against PBS and stored at - 20 ° C until use.
Preparation of europium labelled antibodies The following procedure was used for labelling antibodies with europium. As an example, the labelling of anti-oestradiol is described in detail. Anti-oestradiol IgG (1 m g / m l PBS) was dialysed against carbonate buffer (50 mM, pH 9.8) for 2 h. At the same time a vial containing 1 mg of europium chelate was reconstituted in 200 /~1 of double distilled water. An 80-fold excess of the labelling reagent (140 /tl; 3.83 n m o l / / d ) was added to the IgG solution. The reaction mixture was stirred overnight at 4 ° C and subsequently purified by gel filtration on Sephadex G-25. The labelled IgG was eluted with Tris-HC1, 50 mM, pH 7.75 containing 0.2% sodium chloride and 0.05% sodium azide. In general 10-15 mol of europium were incorporated per mol of protein. The labelled IgG was stored at 4 °C until use. The same labelling procedure was used for tagging anti-idiotype antibodies with europium.
Preparation of monoclonal anti-idiotypic antibodies against anti-oestradiol Female CD 2 mice (age:2 months) were immunized with purified monoclonal anti-oestradiol IgG (clone 2F9, 50 /~g/mouse) in complete Freund's adjuvant. Subsequently, two booster injections were given using anti-oestradiol IgG in incomplete Freund's adjuvant. After 2 months of immunization, the anti-idiotypic response was checked as described (Barnard and Kohen, 1990). 3 months after the initial immunization the spleen
cells of the mouse showing the highest serum titre of antibodies which recognized europium labelled anti-oestradiol IgG were fused with a mouse myeloma cell line (NSO, kindly donated by Dr. Milstein, Cambridge) using the hybridoma technique of K/Shler and Milstein (1976). The culture supernatants of growing hybridomas were screened for antibody activity as described by Barnard and Kohen (1990).
Screening of anti-idiotypes of the betatype Preparation of probes. The screening procedures involved first the identification of anti-idiotypic antibodies that recognized europium-labelled anti-oestradiol IgG followed by identification of anti-idiotypic antibodies of the beta type which were classified as antigen-sensitive and could compete with oestradiol for the binding sites of antioestradiol (Barnard and Kohen, 1990). Briefly, in the first screening, hybridoma culture supernatants (5 /~l/well) were added to the anti-mouse IgG coated microstrips containing 200 ~1 of assay buffer/well. The plates were incubated for 1 h. The reaction mixture was aspirated and the plates were washed three times. The plates were then blocked for 10 min with 25/~1 of mouse serum, and subsequently 200 /zl of europium labelled anti-oestradiol IgG (approximately 107 cps/ml) were added to each well. The plates were incubated for 1 h at room temperature and subsequently the strips were washed six times. Enhancement solution (200/~1) was added to each well and the strips were agitated on the shaker for 15 min. Fluorescence was then measured using an Arcus time-resolved fluorimeter (Wallac Oy, Turku, Finland). The wells that gave positive results in this assay were classified as anti-allotypes and anti-idiotypes. In the second screening procedure, the hybridomas that gave positive results in the first screening assay were added (5 #l/well) in quadruplicate to anti-mouse IgG coated microtiter wells containing 200/~1 of assay buffer. The plates were incubated, washed and blocked as described in the first screening procedure (see above). Subsequently to one set of duplicates, 200 /xl of assay buffer containing europium labelled antioestradiol IgG (107 cps/ml) were added and, to the other set of duplicates, 200/~1 of assay buffer
98 containing an excess amount of oestradiol (1 /~g/ml) and europium-labelled anti-oestradiol (10 7 cps/ml) were added. The plates were processed as described in the first screening procedure. Immobilized hybridoma culture supernatants that failed to bind europium labelled anti-oestradiol in the presence of oestradiol were classified as paratypic antibodies (beta type). From these screening procedures one clone, no. 1D5, was selected as a beta type anti-idiotype and was used in the development of a competitive immunoassay for oestradiol. This clone belonged to the IgG1 heavy chain class. Ascites of this clone were prepared in pristane primed C D 2 mice as described previously (Strasburger et al., 1989). An immunoglobulin fraction (IgG) of clone no 1D 5 was isolated from ascitic fluid by chromatography on Sepharose-protein A as described by Strasburger and Kohen (1990). Purified IgG was then labelled with europium using the procedure described in this paper.
Assay Procedure Preparation of monoclonal anti-oestradiol coated plates A stock solution of monoclonal anti-oestradiol IgG (clone 2F9) was diluted with PBS to 2.5 /xg/ml, and 200 /~1 of this solution were pipetted into each well of the microtitre strips. After an overnight incubation at room temperature the coating buffer was aspirated to waste and the strips were washed three times. The plates were then ready for use. Time-resolved fluorescence immunoassay Preparation of the matrix for the assay. The displacing agents, danazol and 5a-dihydrotestosterone were prepared at a concentration of 5 n g / m l in assay buffer. To 2 ml of horse serum 1 ml of each of the displacing agents was added and the mixture was left for 1 h at room temperature. A solution of 40 ng oestradiol/ml was prepared by directly mixing 100/~1 of an ethanolic solution of 1 t~g/ml solution of oestradiol with 2.4 ml of 1 : 1 mixture of horse serum with the displacing agents, and by incubating the mixture for 15 min at 37 ° C. This stock solution of oestradiol in horse serum and displacing agents and the 1 : 1 mixture
of horse-serum and displacing agents were used to prepare oestradiol standards, ranging from 0 to 200 pg/well. 25 /~1 of standards, quality control samples or unknowns were pipetted into the wells of the antibody coated plates. In addition, the quality control samples and unknowns received 25 /~1 of assay buffer containing displacing agents (5 ng danazol and 5 ng 5a-dihydrotestosterone/ml assay buffer). Europium labelled anti-idiotypic antibody (clone 1D 5, 20 ng, 1 × 10 6 cps, 100/~1) was added to each well and the total volume in each well was adjusted with assay buffer to 200/xl. The plates were then incubated at room temperature for 1 h in an automatic plate shaker. The reaction mixture was aspirated to waste, and the strips were washed six times. Enhancement solution (200 /~1) was then added to each well, and the strips were agitated on the shaker for 10 min. Fluorescence was then measured with an Arcus time-resolved fluorometer.
Sample collection Blood samples were obtained by venipuncture from patients undergoing ovulation induction at King's College Medical School, London, U.K. We thank Prof. W.P. Collins for providing these sampies. Calculation of results Non-specific blank values were subtracted from the fluorimeter readings and a logit-log transformation was used for plotting the calibration curve and the calculation of results of oestradiol concentration from unknowns.
Results
Calibration curves The effects of the addition of danazol, 5a-dihydrotestosterone and horse serum to the standards are shown in Fig. 2. Accurate results could be obtained using the calibration curve established in the presence of danazol (5 ng/ml), 5a-dihydrotestosterone (5 n g / m l ) and horse serum (25 /xl/well). This curve was used in the direct fluorescence immunoassay. Sensitivity, defined as the minimum detectable dose of oestradiol that could be distinguished from zero (mean + SD), was
TABLE I
\-E 4000
PRECISION OF OESTRADIOL M E A S U R E M E N T BY DIRECT FIA
7_o 3000
99
,~n~
"o t_
Pool
Mean and SD
Intra-assay
Interassay
no.
oestradiol concen-
CV a, %
CV a, %
7.34 6.24 9.98 3.37 3.14
11.34 8.62 14.67 7.34 10.62
"~ 2000 oJ
tration ( p g / m l ) 1 2 3 4 5
82.5 332.7 436.5 1,652 2,276
+ 9.35 + 28.69 + 61.61 + 121.2 +242
~1000.
a CV = coefficient of variation, defined as
SD
mean
x
LL
W~m e I
1000
I
I
I
2000
3000
4000
RIA, pg esfradiot/rnl Fig. 3. Comparison of serum oestradiol concentrations determined by the extraction RIA (x) and FIA (y) methods. The
100.
regression equation summarizing the results (n = 60) was y = 1.0x + 4.31 with a correlation coefficient r = 0.98.
c a l c u l a t e d f r o m f o u r consecutive c a l i b r a t i o n curves p r e p a r e d in d u p l i c a t e (see Fig. 2). T h e m e a n value d e r i v e d f r o m the curve used in the direct fluorescence i m m u n o a s s a y was 80 + 20 p g / m l , a n d the reference logit %. B / T value r e a d f r o m the curve was 91%.
Recovery I n c r e a s i n g a m o u n t s of u n l a b e l l e d o e s t r a d i o l ( 3 . 2 - 1 0 0 p g / w e l l ) were a d d e d to horse s e r u m a n d the s a m p l e s were a n a l y s e d several times (n = 17)
b y direct F I A . T h e m e a n r e c o v e r y was 103.7 + 4.15%.
Precision I n t r a - a s s a y a n d i n t e r - a s s a y v a r i a t i o n were e s t i m a t e d b y m e a s u r i n g o e s t r a d i o l levels in duplic a t e in five s e r u m p o o l s used for i n t e r n a l q u a l i t y c o n t r o l in seven consecutive assays. T h e results are s h o w n in T a b l e I.
Parallelism S e r u m f r o m a p r e g n a n t w o m e n was d i l u t e d 10-, 20-, 40- a n d 80-fold w i t h the 1 : 1 m i x t u r e o f horse s e r u m a n d d i s p l a c i n g agents, a n d the o e s t r a d i o l c o n t e n t was a s s a y e d in the d i l u t e d s a m p l e s b y direct F I A ( y ) . T h e l i n e a r regression e q u a t i o n s u m m a r i z i n g the results was: y = 0.977 x - 1.051, with a c o r r e l a t i o n coefficient of r = 0.99.
,~7.0 90
80
=~
60
~' 30
Correlation with radioimmunoassay
20
10
1=0 100 Estradiol (pg/wel[)
1000
Fig. 2. Effect of displacing agents and horse serum on oestradiol calibration curves in the FlA. Assays buffer only (A. . . . . . zx); danazol, (50 n g / m l ) , 5a-dihydrotestosterone (50 ng/ml), horse serum, 50 #l/well ( O - - - - - O ) ; danazol (5 ng/ml), 5a-dihydrotestosterone (5 n g / m l ) , horse serum 50 ~ l / w e l l (o o); danazol (5 ng/ml), 5a-dihydrotestosterone (5 n g / m l ) , horse serum 25 ~l/well (e e), m e a n + S D of four consecutive determinations - the curve used in the direct FIA. B = specific counts that were bound; T = total counts.
T h e c o n c e n t r a t i o n of o e s t r a d i o l in 60 s e r u m s a m p l e s as d e t e r m i n e d b y d i r e c t F I A ( y ) a n d as m e a s u r e d b y R I A ( x ) after e x t r a c t i o n o f the sera s h o w e d excellent c o r r e l a t i o n characteristics. T h e l i n e a r regression e q u a t i o n was y = 1.0 x + 4.31 w i t h a c o r r e l a t i o n r = 0.98. T h e results are s h o w n in Fig. 3.
Discussion I n recent y e a r s several n o n - i s o t o p i c i m m u n o a s say techniques have b e e n d e s c r i b e d for the m e a -
100 surement of serum oestradiol from plasma. These methods rely on the use of a derivative of oestradiol conjugated to an enzyme (Marcus and Durnford, 1988) or to a chemiluminescent marker (De Boever et al., 1986). The use of these labels in immunoassay methods have certain disadvantages. For instance, the purification of oestradiol-enzyme conjugates is tedious and difficult and the endpoint measurement of steroid-chemiluminescent marker conjugates cannot be repeated (De Boever et al., 1990). On the other hand, labelling of antibodies with an enzyme is rather easy and the antibody-enzyme conjugate can be separated easily from unreacted enzyme and antibody by gel filtration. In addition, antibodies can be labelled with universal reagents, such as biotin, for use in avidin-biotin mediated i m m u n o a s s a y systems (Strasburger et al., 1988) or with fluorescent labels for use in time-resolved fluorescence (Barnard, 1988). The availability in our hands of an anti-idiotypic antibody (clone 1D 5) (Barnard and Kohen, 1990) capable of mimicking oestradiol at the binding site of the primary anti-oestradiol antibody (clone no. 2F9) permitted the use of labelled antibody in the immunoassay method. In this paper we describe the development of an immunoassay for serum oestradiol which is based on the idiotypic anti-idiotypic approach. In this format the primary anti-oestradiol antibody (clone 2F9) serves as the solid-phase, and the anti-idiotypic antibody (clone 1Ds) labelled with europium as the marker (see Fig. 1). The direct fluorescent immunoassay (FIA) proved satisfactory with respect to sensitivity, accuracy and precision. A good correlation was observed between the FIA and the R I A using an extraction step (see Fig. 3). This direct FIA has the advantage of a short assay time, requiring in total about 2 h including counting and calculation of results. The direct assay for oestradiol can be performed with 25 t~l of serum per well (see Fig. 2) and the sensitivity (80 p g / m l ) of the present method is adequate for measuring oestradiol during the human ovarian cycle as well as during ovulation induction. In summary, this paper reports the use of an anti-idiotypic antibody and an idiotypic antibody in hapten immunoassay as exemplified by the direct measurement of oestradiol from serum. This
technique offers interesting possibilities for the development of competitive-type non-isotopic immunoassays for haptens.
Acknowledgements This work has been supported in part by a grant (BR-IS-001) from the Institute for International Studies in Natural Family Planning, Department of Obstetrics and Gynaecology, Georgetown University Medical School, Washington DC, U.S.A. (G.B. and F.K.) and from the Special Programme of Research, Development and Research Training in H u m a n Reproduction, World Health Organization (to F.K.). We thank Prof. W.P. Collins of the Department of Obstetrics and Gynaecology, King's College School of Medicine and Dentistry, London, for the provision of samples and the results of the extraction RIA. We are grateful to Wallac Oy, Turku, Finland, for the provision of equipment and reagents, to Mrs. M. Kopelowitz for secretarial assistance, and to Dr. T. Ngo, of Bio-Probe Int. Tustin Co., for providing us the I m m u n o b i n d Avid Gel Ax. We thank the Special Programme of Research, Development and Research Training in H u m a n Reproduction, World Health Organization, for a training grant to A. A-B.
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Sege, K. and Peterson, P. (1978) Use of anti-idiotypic antibodies as cell-surface receptor probes. Proc. Natl. Acad. Sci. U.S.A. 73, 2443. Shechter, Y., Maron, R., Elias, D. and Cohen, I. (1982) Autoantibodies to insulin receptor spontaneously develop as anti-idiotypes in mouse immunized with insulin. Science 216, 542. Strasberg, A.D. (1989) Interaction of anti-idiotypic antibodies with membrane receptors: Practical considerations. In: J.J. Langone (Ed.), Methods in Enzymology, Vol. 178. Academic Press, New York, p. 179. Strasburger, C.J. and Kohen, F. (1990) Biotinylated probes in immunoassay. In: M. Wilchek and E. Bayer (Eds.), Methods in Enzymology, Vol. 181, Academic Press, New York, p. 481. Strasburger, C.J., Arnir-Zaltzman, Y. and Kohen, F. (1988) The avidin-biotin reaction as a universal amplification system in irnmunoassays. In: Non-Radiometric Assays: Technology and Application in Polypeptide and Steroid Hormone Detection. Ala R. Liss, New York, p. 79. Strasburger, C., Barnard, G., Toldo, L., Zarmi, B., Zadik, Z., Kowarski, A. and Kohen, F. (1989) Somatotropin as measured by a two-site time-resolved immunofluorometric assay. Clin. Chem. 35, 55.