Journal of Immunological Methods, 147 (1992) 173-179 @
173
1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00
JIM 06202
Sandwich immunoassay of small molecules I. Investigation with testosterone as model hapten Esahak Ali, Judhajit Sengupta and Tarun K. Dhar Indian Institute of Chemical Biology, Calcutta-700032, India
(Received 12 August 1991, accepted 29 October 1991)
A new strategy has been developed for enzyme immunoassay of small molecules. The method is based on the formation and estimation of the complex Ab.H-H.Ab* where Ab and Ab* are immobilised and labeled antibodies respectively and H-H is a synthetic bis-analogue of the hapten H. The amount of the complex formed is inversely related to the concentration of free hapten present. The estimation of the sandwich complex increases the sensitivity and the specificity of the assay compared to conventional assays. Using the N,N'-diamide of testosterone-3-(O-carboxymethyl)oxime with ethylenediamine and a polyclonal rabbit anti-testosterone antibody (for both labeling and immobilisation) a sensitivity of 100 fg/well (3.5 x 10- 16 mol) was obtained for testosterone. The cross-reactivity of 5a-dihydrotestosterone was 16% compared to 27% obtained by conventional competitive assays. Key
words: Sandwich immunoassay; Testosterone dimer; Testosterone; Testosterone-3-(O-carboxymethyl)oxime
Introduction Enzyme Immunoassay (Engvall and Perlmann, 1971; Van Weemen and Schuurs, 1971) is now a widely used technique for estimation of antigens, antibodies, drugs and hormones. Most of these assays are of the competitive type with the antigen or the antibody immobilised to a solid phase (Tijssen, 1985). For macromolecular antigens with more than one epitope the two-site or sandwich immunoassay (Engvall and Perlmann, 1972) gives better specificity and sensitivity than the competi-
Cormpondence to: E. Ali, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Calcutta-700032, India. Abbreviations: s, singlet; d, doublet; t, triplet; br s, broad singlet;DASP, double-antibody solid phase; BSA, bovine serum albumin.
tive assays. However, this type of assay is not applicable to small molecules like steroid hormones. Although sensitivities of enzyme immunoassay is now comparable or sometimes even superior to radioimmunoassay there is an increasing demand for assays with still higher sensitivities. Strategies for increasing sensitivity have so far been based on elaborate purification of and manipulation of antibodies and enzyme conjugates (Ishikawa et aI., 1989) and/or the use of some form of enzyme amplification system such as avidin-biotin (Kendall et aI., 1983) or enzymeanti-enzyme complex (Koertge et aI., 1985). In this paper we describe a new strategy for enzyme immunoassay of haptens which have considerably higher specificity and sensitivity compared to conventional assays. The principle of the assay is based on the following equilibrium (eq. 1), where Ab is the immobilised antibody directed
174
--0
rAD
0 0
-
Immobili zed
alS
antlOody
0
+
- an alogue hap ten
~
Free hapten In
~
')
~ Ant lb ody-
samp le
enzy me conJuQote
IncubO Ie
2) Wash
3) Add substro te
S uOS l rO le
....
,I,
-- ' I '
I mmobil i zed Immune
PrOduct
co mplex
Fig. I. Principle of sandwich enzyme immunoassay of haptens using bis-analogue of the hapten.
against the hapten H, Ab * is the same antibody labeled with enzyme and H-H is a synthetic bisanalogue of the hapten.
Ab+H-H+H+Ab* ... Ab.H-H.Ab* +Ab.H+Ab.*H +Ab"'.H-H.Ab* +Ab.H-H+Ab*.H-H
+
I. CH 2 CI 2
J
Stirred ot RT for 2 hours.
2. Separation of dicyclohexyl urea. 3. Chromatography over Silica gel.
2 Fig. 2. Synthesis of the bis-testosterone analogue 2.
(1)
175
The sandwich complex Ab.H-H.Ab* is the species estimated, the production of which is inversely related to the hapten concentration. A diagrammatic representation of the methodology is given in Fig. 1. The antibody used in the present study was anti-testosterone antibody raised against testosterone-3-{ O-carboxymethyl) oxime-BSA conjugate and the same antibody was labeled with horseradish peroxidase. The testosterone dimer 2 was synthesized by condensation of testosterone-3-{ O-carboxymethyl)oxime 1 with ethylenediamine (Fig. 2). Materials and Methods Materials
Flat bottomed 96 well polystyrene microtitre plates were from Costar, Cambridge, MA, USA. The automatic microtitre plate reader was from Bio-Rad laboratories, Richmond, USA. Testosterone, dicyclohexylcarbodiimide, N-hydroxysuccinimide, (aminooxy)acetic acid, pyrrolidine, ethylenediamine, Sephadex G-50, horseradish peroxidase type VI (EC 1.11.7), polyoxyethylenesorbitan monolaurate (Tween 20), bovine serum albumin, casein, 3,3',5,5'-tetramethylbenzidine were purchased from Sigma, St. Louis, USA. All other chemicals and buffer salts were of analytical grade. Testosterone-3-(O-carboxymethyl) oxime was synthesized according to a standard procedure (Janoski et aI., 1974; Samanta and Ali, 1990).
Buffers
Coating buffer was sodium bicarbonate / sodium carbonate (50 mmoljl, pH 9.6); washing buffer was Na zHP04/KH 2 P04 (50 mmoljl, pH 7.6) containing 0.5 ml of Tween 20/1; post-coating buffer was Na2HP04/KH2P04 (50 mmoljl, pH 7.6) containing 4.0 g of casein and 0.1 g of thiomersaljl; incubation buffer was borate buffer (50 mmoljl, pH 8.0) containing per litre, 20 mg of, KCl, 20 mg Ca02' 20 mg Mg0 2, 2.0 g BSA and 0.1 g of thiomersal; enzyme assay buffer was sodium acetate/ citric acid (100 mmoljl, pH 3.95). Chromogen
Tetramethylbenzidine was dissolved in dimethylsulphoxide to give a final concentration of 42
mmoljl. 8 ml of this solution was added to 1 litre of enzyme assay buffer. Before addition, 1.5 ml of 30 mljl aqueous hydrogen peroxide was added. Standards
Testosterone stock standard solution (1 mg/ml in ethanol) was kept at - 20°C and seven working standards (0.1-50 pg/weII) were prepared by dilution in incubation buffer. Preparation of the his-testosterone analogue 2
To a stirred suspension of testosterone-3-(Ocarboxymethyl)oxime (180 mg, 0.5 mmon in 5 ml dry methylene chloride was added 210 mg dicyclohexylcarbodiimide. The mixture was stirred till the turbidity disappeared and ethylenediamine (28 mg, 0.46 mmol) was added slowly in portions. The reaction mixture was stirred continuously for 2 h at room temperature and then filtered to remove the separated urea. Evaporation of the filtrate yielded a white solid residue which was redissolved in 5 ml warm methylene chloride. The solution was allowed to stand at 4°C for 30 min when additional dicyclohexylurea separated which was removed by filtration. The process was repeated once more to remove a further crop of dicyclohexylurea. The final filtrate was concentrated to a viscous oil and chromatographed on a silica gel column (1.5 cm x 20 em). Elution with 2% methanol in chloroform gave 40 mg (23% yield) of the bis analogue 2. It was crystallised from chloroform-petroleum ether and the product dried in vacuo. m.p. 200°C; [a]D + 165.6° (MeOH); IR(KBr) v!:3600-3420 (NH,OH), 1660 (e--o), 1635 (C=N) em-I; IHNMR(CDCI 3)5 :0.80 s(18-Me), 1.09 s(19-Me), 3.45 br s(NH-C!iz), 3.62 t(17-H, J = 7 Hz), 4.46 s(-CH 2CO), 5.72 s(4H,anti), 6.40 s(4H,syn), 6.8 br s(N!!,OH); (found C 70.53%; H, 8.86%; C 44 H 66N40 6 requires C, 70.75%; H, 8.89%). Preparation of antibody and enzyme conjugate
The preparation of anti-testosterone antibody was described in the preceding paper (Dhar and Ali, 1992). Anti-testosterone antibody was conjugated to peroxidase by periodate method according to Wilson and Nakane, (1978). The resulting conjugate was purified by dialysis against phosphate buffer (10 mmoljl, pH 7.4) containing 0.9%
176
NaCI, followed by chromatography on Sephadex G-50. After the addition of BSA, 20g/1, the conjugate was stored in aliquots at - 20°C (final concentration 1 gil). Working solutions were made in incubation buffer. Coating of microtitre plate
Each well of the microtitre plate was coated with 200 JLl of diluted anti-testosterone antibody in coating buffer. After 1 h incubation at room temperature it was kept at 4°C for 16 h. The wells were then emptied and washed with washing buffer three times. The unoccupied binding sites were then blocked by incubation for 2 h at 37°C with 200 JLl of post coating buffer. The plates were washed again and stored at 4°C until used. No reduction in assay sensitivity was recorded after storage for at least 2 weeks. Immunoassay procedures One step method. Standards (50 JL I) were
added into the wells of a microtitre plate in duplicate. This was followed by the addition of 100 JLl of immunoassay reagent containing the testosterone dimer 2 and the peroxidase conjugate at 500-fold dilution. Plates were covered and incubated at 37°C for 1 h and washed three times with 300 JLl portions of washing buffer. The chromogen solution was then added (150 ~ljwell) and incubated in dark for 30 min. The reaction was stopped by adding 100 JLl of sulphuric acid (4 N) to each well in the same sequence as the chromogen solution was added. The solution was mixed for 30 s and absorbance measured at 450 om. Two step method. Standards were added (SO JLl) into each well of a microtitre plate in duplicate followed by 100 JLl of testosterone dimer in incubation buffer. Plates were covered and incubated at 3TC for 1 h and washed three times with 300 JLl portions of washing buffer. The enzyme conjugate (150 JLl) was added at 750-fold dilution and incubated at room temperature for 30 min. The rest of the procedure was same as described for the one step procedure. Competitive assay
For comparison, testosterone was also assayed
by the double antibody solid phase method de-
scribed in the preceding paper (Dhar and Ali, 1992) except that the testosterone standards were made in the incubation buffer instead of charcoal stripped serum.
Results and discussion
Condensation of testosterone-3-{O-carbo"Ymethyl)oxime with ethylenediamine gave the bistestosterone derivative 2 in 23% yield. The compound was fully characterised by its IR and NMR spectra. Like the starting material 1 the oximino group of the dimer exists as an equilibrium mixture of syn and anti isomers, ratio (1: 2), as evident from two signals for the vinyl hydrogens at C-4 (Shoppee et aI., 1964). The conditions used for immobilisation of the antibody, e.g., the pH and composition of the coating and post-coating buffers, the incubation periods and the post-coating protein was optimised by standard checker board titration. Thereafter, the effects of other variables on the assay performance were systematically studied. Effect of bis-analogue concentration
Incubation of increasing concentrations of the bis-analogue (hereafter called the 'dimer') with a fixed concentration of the labeled antibody in absence of testosterone yielded a bell shaped curve in the one step procedure (Fig. 3). With the coating antibody dilution at 1/4000 a broad maximum was obtained centred around the dimer concentration of 60 pg/well. Change of the antibody dilution to 1/3000 shifted the maximum to 100 pg/well. The curve obtained in the two step procedure was however quite different. No decrease in the absorbance was observed upto a dimer concentration of 1 ng/well. The shape of the curves seem to be sigmoidal becoming an asymptot at the higher concentration (and possibly at a lower concentration also). In absence of free testosterone the following equilibrium (eq. 2) is eJ[pected in the one step procedure, with the ternary immune-complex a being responsible for color development.
E c: o
10
173
1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00
JIM 06202
Sandwich immunoassay of small molecules I. Investigation with testosterone as model hapten Esahak Ali, Judhajit Sengupta and Tarun K. Dhar Indian Institute of Chemical Biology, Calcutta-700032, India
(Received 12 August 1991, accepted 29 October 1991)
A new strategy has been developed for enzyme immunoassay of small molecules. The method is based on the formation and estimation of the complex Ab.H-H.Ab* where Ab and Ab* are immobilised and labeled antibodies respectively and H-H is a synthetic bis-analogue of the hapten H. The amount of the complex formed is inversely related to the concentration of free hapten present. The estimation of the sandwich complex increases the sensitivity and the specificity of the assay compared to conventional assays. Using the N,N'-diamide of testosterone-3-(O-carboxymethyl)oxime with ethylenediamine and a polyclonal rabbit anti-testosterone antibody (for both labeling and immobilisation) a sensitivity of 100 fg/well (3.5 x 10- 16 mol) was obtained for testosterone. The cross-reactivity of 5a-dihydrotestosterone was 16% compared to 27% obtained by conventional competitive assays. Key
words: Sandwich immunoassay; Testosterone dimer; Testosterone; Testosterone-3-(O-carboxymethyl)oxime
Introduction Enzyme Immunoassay (Engvall and Perlmann, 1971; Van Weemen and Schuurs, 1971) is now a widely used technique for estimation of antigens, antibodies, drugs and hormones. Most of these assays are of the competitive type with the antigen or the antibody immobilised to a solid phase (Tijssen, 1985). For macromolecular antigens with more than one epitope the two-site or sandwich immunoassay (Engvall and Perlmann, 1972) gives better specificity and sensitivity than the competi-
Cormpondence to: E. Ali, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Calcutta-700032, India. Abbreviations: s, singlet; d, doublet; t, triplet; br s, broad singlet;DASP, double-antibody solid phase; BSA, bovine serum albumin.
tive assays. However, this type of assay is not applicable to small molecules like steroid hormones. Although sensitivities of enzyme immunoassay is now comparable or sometimes even superior to radioimmunoassay there is an increasing demand for assays with still higher sensitivities. Strategies for increasing sensitivity have so far been based on elaborate purification of and manipulation of antibodies and enzyme conjugates (Ishikawa et aI., 1989) and/or the use of some form of enzyme amplification system such as avidin-biotin (Kendall et aI., 1983) or enzymeanti-enzyme complex (Koertge et aI., 1985). In this paper we describe a new strategy for enzyme immunoassay of haptens which have considerably higher specificity and sensitivity compared to conventional assays. The principle of the assay is based on the following equilibrium (eq. 1), where Ab is the immobilised antibody directed
174
--0
rAD
0 0
-
Immobili zed
alS
antlOody
0
+
- an alogue hap ten
~
Free hapten In
~
')
~ Ant lb ody-
samp le
enzy me conJuQote
IncubO Ie
2) Wash
3) Add substro te
S uOS l rO le
....
,I,
-- ' I '
I mmobil i zed Immune
PrOduct
co mplex
Fig. I. Principle of sandwich enzyme immunoassay of haptens using bis-analogue of the hapten.
against the hapten H, Ab * is the same antibody labeled with enzyme and H-H is a synthetic bisanalogue of the hapten.
Ab+H-H+H+Ab* ... Ab.H-H.Ab* +Ab.H+Ab.*H +Ab"'.H-H.Ab* +Ab.H-H+Ab*.H-H
+
I. CH 2 CI 2
J
Stirred ot RT for 2 hours.
2. Separation of dicyclohexyl urea. 3. Chromatography over Silica gel.
2 Fig. 2. Synthesis of the bis-testosterone analogue 2.
(1)
175
The sandwich complex Ab.H-H.Ab* is the species estimated, the production of which is inversely related to the hapten concentration. A diagrammatic representation of the methodology is given in Fig. 1. The antibody used in the present study was anti-testosterone antibody raised against testosterone-3-{ O-carboxymethyl) oxime-BSA conjugate and the same antibody was labeled with horseradish peroxidase. The testosterone dimer 2 was synthesized by condensation of testosterone-3-{ O-carboxymethyl)oxime 1 with ethylenediamine (Fig. 2). Materials and Methods Materials
Flat bottomed 96 well polystyrene microtitre plates were from Costar, Cambridge, MA, USA. The automatic microtitre plate reader was from Bio-Rad laboratories, Richmond, USA. Testosterone, dicyclohexylcarbodiimide, N-hydroxysuccinimide, (aminooxy)acetic acid, pyrrolidine, ethylenediamine, Sephadex G-50, horseradish peroxidase type VI (EC 1.11.7), polyoxyethylenesorbitan monolaurate (Tween 20), bovine serum albumin, casein, 3,3',5,5'-tetramethylbenzidine were purchased from Sigma, St. Louis, USA. All other chemicals and buffer salts were of analytical grade. Testosterone-3-(O-carboxymethyl) oxime was synthesized according to a standard procedure (Janoski et aI., 1974; Samanta and Ali, 1990).
Buffers
Coating buffer was sodium bicarbonate / sodium carbonate (50 mmoljl, pH 9.6); washing buffer was Na zHP04/KH 2 P04 (50 mmoljl, pH 7.6) containing 0.5 ml of Tween 20/1; post-coating buffer was Na2HP04/KH2P04 (50 mmoljl, pH 7.6) containing 4.0 g of casein and 0.1 g of thiomersaljl; incubation buffer was borate buffer (50 mmoljl, pH 8.0) containing per litre, 20 mg of, KCl, 20 mg Ca02' 20 mg Mg0 2, 2.0 g BSA and 0.1 g of thiomersal; enzyme assay buffer was sodium acetate/ citric acid (100 mmoljl, pH 3.95). Chromogen
Tetramethylbenzidine was dissolved in dimethylsulphoxide to give a final concentration of 42
mmoljl. 8 ml of this solution was added to 1 litre of enzyme assay buffer. Before addition, 1.5 ml of 30 mljl aqueous hydrogen peroxide was added. Standards
Testosterone stock standard solution (1 mg/ml in ethanol) was kept at - 20°C and seven working standards (0.1-50 pg/weII) were prepared by dilution in incubation buffer. Preparation of the his-testosterone analogue 2
To a stirred suspension of testosterone-3-(Ocarboxymethyl)oxime (180 mg, 0.5 mmon in 5 ml dry methylene chloride was added 210 mg dicyclohexylcarbodiimide. The mixture was stirred till the turbidity disappeared and ethylenediamine (28 mg, 0.46 mmol) was added slowly in portions. The reaction mixture was stirred continuously for 2 h at room temperature and then filtered to remove the separated urea. Evaporation of the filtrate yielded a white solid residue which was redissolved in 5 ml warm methylene chloride. The solution was allowed to stand at 4°C for 30 min when additional dicyclohexylurea separated which was removed by filtration. The process was repeated once more to remove a further crop of dicyclohexylurea. The final filtrate was concentrated to a viscous oil and chromatographed on a silica gel column (1.5 cm x 20 em). Elution with 2% methanol in chloroform gave 40 mg (23% yield) of the bis analogue 2. It was crystallised from chloroform-petroleum ether and the product dried in vacuo. m.p. 200°C; [a]D + 165.6° (MeOH); IR(KBr) v!:3600-3420 (NH,OH), 1660 (e--o), 1635 (C=N) em-I; IHNMR(CDCI 3)5 :0.80 s(18-Me), 1.09 s(19-Me), 3.45 br s(NH-C!iz), 3.62 t(17-H, J = 7 Hz), 4.46 s(-CH 2CO), 5.72 s(4H,anti), 6.40 s(4H,syn), 6.8 br s(N!!,OH); (found C 70.53%; H, 8.86%; C 44 H 66N40 6 requires C, 70.75%; H, 8.89%). Preparation of antibody and enzyme conjugate
The preparation of anti-testosterone antibody was described in the preceding paper (Dhar and Ali, 1992). Anti-testosterone antibody was conjugated to peroxidase by periodate method according to Wilson and Nakane, (1978). The resulting conjugate was purified by dialysis against phosphate buffer (10 mmoljl, pH 7.4) containing 0.9%
176
NaCI, followed by chromatography on Sephadex G-50. After the addition of BSA, 20g/1, the conjugate was stored in aliquots at - 20°C (final concentration 1 gil). Working solutions were made in incubation buffer. Coating of microtitre plate
Each well of the microtitre plate was coated with 200 JLl of diluted anti-testosterone antibody in coating buffer. After 1 h incubation at room temperature it was kept at 4°C for 16 h. The wells were then emptied and washed with washing buffer three times. The unoccupied binding sites were then blocked by incubation for 2 h at 37°C with 200 JLl of post coating buffer. The plates were washed again and stored at 4°C until used. No reduction in assay sensitivity was recorded after storage for at least 2 weeks. Immunoassay procedures One step method. Standards (50 JL I) were
added into the wells of a microtitre plate in duplicate. This was followed by the addition of 100 JLl of immunoassay reagent containing the testosterone dimer 2 and the peroxidase conjugate at 500-fold dilution. Plates were covered and incubated at 37°C for 1 h and washed three times with 300 JLl portions of washing buffer. The chromogen solution was then added (150 ~ljwell) and incubated in dark for 30 min. The reaction was stopped by adding 100 JLl of sulphuric acid (4 N) to each well in the same sequence as the chromogen solution was added. The solution was mixed for 30 s and absorbance measured at 450 om. Two step method. Standards were added (SO JLl) into each well of a microtitre plate in duplicate followed by 100 JLl of testosterone dimer in incubation buffer. Plates were covered and incubated at 3TC for 1 h and washed three times with 300 JLl portions of washing buffer. The enzyme conjugate (150 JLl) was added at 750-fold dilution and incubated at room temperature for 30 min. The rest of the procedure was same as described for the one step procedure. Competitive assay
For comparison, testosterone was also assayed
by the double antibody solid phase method de-
scribed in the preceding paper (Dhar and Ali, 1992) except that the testosterone standards were made in the incubation buffer instead of charcoal stripped serum.
Results and discussion
Condensation of testosterone-3-{O-carbo"Ymethyl)oxime with ethylenediamine gave the bistestosterone derivative 2 in 23% yield. The compound was fully characterised by its IR and NMR spectra. Like the starting material 1 the oximino group of the dimer exists as an equilibrium mixture of syn and anti isomers, ratio (1: 2), as evident from two signals for the vinyl hydrogens at C-4 (Shoppee et aI., 1964). The conditions used for immobilisation of the antibody, e.g., the pH and composition of the coating and post-coating buffers, the incubation periods and the post-coating protein was optimised by standard checker board titration. Thereafter, the effects of other variables on the assay performance were systematically studied. Effect of bis-analogue concentration
Incubation of increasing concentrations of the bis-analogue (hereafter called the 'dimer') with a fixed concentration of the labeled antibody in absence of testosterone yielded a bell shaped curve in the one step procedure (Fig. 3). With the coating antibody dilution at 1/4000 a broad maximum was obtained centred around the dimer concentration of 60 pg/well. Change of the antibody dilution to 1/3000 shifted the maximum to 100 pg/well. The curve obtained in the two step procedure was however quite different. No decrease in the absorbance was observed upto a dimer concentration of 1 ng/well. The shape of the curves seem to be sigmoidal becoming an asymptot at the higher concentration (and possibly at a lower concentration also). In absence of free testosterone the following equilibrium (eq. 2) is eJ[pected in the one step procedure, with the ternary immune-complex a being responsible for color development.
E c: o
10
