Journal of Steroid Biochemistry.Vol. 1I. pp.147 to 151 Wrgamon Press Ltd 1979. Printed in Great Britain
ENZYME-IMMUNOASSAY OF STEROIDS: POSSIBILITIES AND PITFALLS BAUKE K. VAN WEEMEN,ANNEKE M. G. BOSCH, EDWARD C. DAWSON and ANTON H. W. M. SCHUURS Organon Scientific Development Group, OS, The Netherlands SUMMARY
Enzyme-immunoassay for steroids can be based on either a combination of insolubilized steroid and labelled antibody or a combination of labelled steroid and antibody. Only the latter possibility has been worked out. Two types can be distinguish~: heterogeneous (in which a bound/free separation is required) and homogeneous. Heterogeneous assays have been developed for estrogens, progesterone, cortisot and testosterone. They can be equally sensitive, precise and accurate as radioimmunoas~y, and are therefore useful alternatives for the endocrinological laboratory.
The concentrations
at which steroid
hormones
occur
in human body fluids may vary from pmol/I. (e.g. serum oestradiol in the early luteal phase of the menstrual cycle) to as much as lOO~ol/l. (e.g. urinary estriol at the end of pregnancy). Of course, such huge variation has led to the development of different assay methods. We have already been able for many decades to measure the higher concentrations of steroid hormones, using either bioassay or chemical assay. Next to some other drawbacks-they may be lengthy, tedious and imprecise (bioassay), or relatively unspecific, requiring extensive purification before the assay proper can be performed-their main limitation is that they lack sensitivity for many an application. Therefore, the advent of radioreceptor- [l) and radioimmuno-assay [2] for steroid hormones was an important landmark in endocrinology. Not only could unpr~dent~ sensitivity (down to the pmol/I. level) be achieved, it was accompanied by high specificity, acceptable precision and a relatively easy performance. Hence, it is not surprising that these radioligand methods have become very widely used. The use of radioactively lahelled compound, however, has its drawbacks as well: specially equipped laboratories are required; counting equipment is often expensive; some radioactive isotopes have a very limited shelf-life; there is a waste-disposal problem; depending on local legislation, there may be license problems. With the use of non-isoto@ic labels, many of these drawbacks could be avoided, as was already suggested some 10 years ago[3]. Since then labelled ligand assays for steroid hormones using fluorophores 141, bacteriophages [SJ, coenzymes [6J and enzymes [7] have been described. The latter approach, which is mostly called enzyme-immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA), has attracted the most attention until now, and will be subject of this paper.
Assay principle
Two approaches have been described: (a) Enzyme-labelled antibodies are incubated with steroid-containing sample, and subsequently with excess insolubilized steroid. The amount of steroid in the sample is then inversely related to the amount of enzyme-~bell~ antibodies bound to the insolubilized steroid. This method has been mentioned only once in the literature, without any detail[lYJ. (b) Enzyme-labelled steroid and steroid-containing sample are incubated with a limited amount of steroid antibodies. The amount of steroid in the sample is inversely related to the amount of enzyme-labelled steroid bound to antibody. This, by ‘the way, is also the normal RIA principle. In homogeneous enzymeimmunoassuy, the non-bound enzyme-k&e&d steroid can easily be measured, because the enzyme activity of the ~z~~la~ll~ steroid is completely ~h~bit~ when it is bound to antibody. Although this method has not yet to our knowledge been used for steroid hormones, it works well for determination of the steroid glycoside digoxin, with an assay range of approx. 0.65-10 nmol/l. 191. In heterogeneous enzymeimmunoassay, antibody-bound and non-bound enzyme-labelled steroid are equally active, and must be separated before any measurement can be made. This separation can be obtained by double antibody or solid phase methods similar to those already used in RIA. Heterogeneous EIAs have been .reported for the following steroid hormones: estrogens [7, IO-14,173, prog~terone [B-17], testosterone [ 1t&20] and cortisol[21,22]. Which assay principle is preferable depends on several factors. Little is known about the. labelled antibody approach; its sensitivity potential and precision merit further investigation. Experience from similar RIA systems is encouraging. The choice between homogeneous and heterogeneous EIA should be based 0’1 the required sensitivity of the assay and the 147
148
B. K. VAN WEEMENet al.
org~ic/protein chemical ski11 of the investigator. It appears to be difficult to make homogeneous EIA sufficiently sensitive to detect less than 1nmol/l. of hapten, whereas heterogeneous EIAs can detect subpicomole amounts of progesterone [ 15,17] and estradiol [ 143. Also, preparation of enzyme-steroid conjugates that show the inhibition phenomenon required for homogeneous EIA requires sophisticated chemistry [24]. If, however, these drawbacks can be circumvented or overcome, the resulting homogeneous EIA is easy to perform and to automate. Heterogeneous EIA offers higher sensitivity and the conjugates are easier to prepare, but the assay is more complicated to perform, even if easy coated tube assay methods are used. Antiserum and conjugate
Add 0.1 mmol isobutyl~hloroformate. (3) Stir for 3 min at - 15°C. (4) Add dropwise to 0.005 mmol HRP in 3.5 ml dimethylformamid~water (4:3), precooled at 0°C. (5) Stir for L h at - 15”C, then 2 h at 0°C. (6) Add 20mg NaHCO,. (7) Dialyse against distilled water, then pass through Sephadex G-25. Conjugates of which up to 80% of the enzyme activity is bound by excess steroid antiserum can be prepared this way. These need no further purification. Recently, affinity chromatography has been used to purify conjugates, which resulted in highly sensitive assays [ 13,141. The number of steroid molecules introduced per enzyme molecule can be influenced by changing the molar ratio during the coupling reaction. This may be of importance for the ability of the conjugate to be bound by antiserum 1213 as well as for the sensitivity of the resulting assay[17]. The incorporation is most elegantly determined by coupling tritiated steroid to the enzyme[21]; the conjugate can also be digested by acid hydrolysis, and the liberated steroid derivative assayed by RIA or EIA [14]. Several enzymes have been used to produce steroid conjugates: hoqse-radish peroxidase [7,8,10-12, 16-191, p-u-galactosidase [ 13-15.211, glucoamylase [20] and alkaline phosphata~ [223. For homogeneous EIA, other enzymes must be used: glucose& phosphate dehydrogenase, malate dehydrogenase. Ali have their advantages and disadvantages, which mainly deal with inherent sensitivity, ease of assay, stability of substrate, presence of interfering factors. /I-D-Galactosidase has easily available chromogenic and fluorogenic substrates, which expands its sensitivity range considerably.
It is well known by now how antisera against steroid hormones can be produced and how their specificity can be engineered by selecting different sites of the steroid molecule for linkage to the carrier protein. In principle, any antiserum that can be used for RIA can be used for EIA as well. Enzyme-labelled steroids (conjugate) are in general prepared along the same lines as the immunogens, i.e. by linking a steroid derivative with a reactive group, mostly carboxyl, at the end of an introduced side chain to an appropriate group of the protein molecuk. Our early work [7,10] indicated that antiserum/ conjugate combinations for which the same steroid derivative had been used to prepare immunogen and conjugate (homologous systems) could lead to rather insensitive assay systems, presumably due to an important contribution of the bridge between steroid and protein to the antibody-conjugate binding. A way Boundflree separation method to overcome this problem was to use different steroid derivatives for antiserum and conjugate production As said before, in homogeneous EIA no bound/free (heterologous systems) [lo]. Similar results have been separation is required. This, of course, makes the found in RIA with iodinated labels. We also experi- assay very easy to perform. enced, however, that these phenomena are strongly In heterogeneous EIA, three bound/free separation antiserum dependent and that antisera can be found methods are in use: double antibody (DA) methods, that give highly sensitive homologous systems. It may solid phase (SP) methods and double antibody solid be advisable, therefore, to look for such antisera phase (DASP) methods. SP methods offer the easiest rather than to devise heterologous systems, because performance, if the coated tube variant is chosen. The the latter approach requires more chemistry. Moreother methods require centrifugation and wash steps. over, we have found a tendency towards a decrease which are more time-consuming, and sometimes (DA) in specificity of heterologous systems, although highly longer incubation periods. It has also been reported specific heterologous assays have been reported that the choice of bound/free separation may affect [IO, 141. On the other hand, we have found that the assay properties other than the ease of performance: dose response curves of heterologous systems may be a solid-phase method with Sephadex-coupled antisteeper than those of homologous systems [25] or body was considerably less sensitive than tbe corresponding DA method for progesterone [ 173, and a RIA [ 111. Preparation of steroid-enzyme cbnjugates is not DASP method was much less precise than the corredifficult, once the required ‘steroid derivatives are sponding DA method for cortisol[21]. This may. however, be very much dependent on the system and available. reagents under investigation. We have compared senWe have prepared conjugates of various carboxylated steroids and the enzyme horse-radish peroxidase sitivities of SP and DASP methods for testosterone and found’no large differences (see Table 1). Also, in good yields with the following procedure: with a DASP assay for total estrogens, we have found (1) Cool at - 15°C : 0.1 mm01 steroid and 0.1 mmol N-methylmorpholine in 2 ml dimethylformamide. (2) coefficients of variation of S&6.4% and 8.3-8.5% for
Enzyme-immunoassay Table 1. Sensitivities at midpoint (in pg/ml) of testosterone ElAs with various bound/free separation methods Midpoint (pg/ml) Relative conjugate concentration b/f Separation method Coated tube DASP Tube DASP Cellulose
1
1.67
180 250 500
190 200 490
within- and total-assay variation [ 113, which is cotnparable to data for RIA. Nearly always, the enzyme activity in the bound
fraction is measured. This offers the advantage that factors from the sample that might interfere with the enzyme reaction (e.g. endogeneous enzyme activity or substrate, inhibitors) can easily be removed by one or more washing steps. If a sample contains factors that irreversibly damage the enzyme, SP methods are preferable. Sample and insoluble antibodies can then be incubated in a first step, and the remainder of the sample removed before the conjugate is added, thus avoiding direct contact between sample and conjugate. In homogeneous EIA, these problems can be avoided only by pretreatment or predilution of the sample.
of steroids
149
Sensitivity
Depending on the choice of assay principle, bound/ free separation, enzyme and substrate, the sensitivity of steroid EIA can range from femtomoles to pmoles per tube. As usual, high. sensitivity is accompanied by long incubation times and laborious methodology, and vice versa. Direct comparisons have shown that steroid EIAs can be as sensitive as or even more sensitive than the corresponding RIAs [ll, 14,15,22]. As mentioned before, it has been claimed [14] that the theoretical sensitivity limits of immunoassay can be reached if the proper enzyme and substrate are chosen. Specificity
The antiserum is the most important factor which influences the specificity of steroid EIA. The general rule found for RIA, i.e. the highest specificity is obtained with antisera evoked with immunogens in which the steroid has been coupled to the carrier protein at a site which leaves the characteristic groups of that steroid exposed, applies equally well to EIA. Hence, by choosing the appropriate antiserum, we can design assay systems that react equally well with estrone, estradiol, estriol and all D-ring conjugated estrogens, based on antisera against a mixture of estriol 16- and estriol 17-monosuccinyl-albumin, but on the other hand we have also produced EIAs for Enzyme determination estradiol that show less than 1% cross-reaction with In homogeneous EIA, the enzyme activity is usually estrone, based on antisera against estradiol&carboxy and 1la-OH estradiol-1 l-succinyldetermined kinetically, although for more sensitive methyl-albumin assays endpoint methods have also been used. In albumin. Specificity patterns of RIA and EIA with the same heterogeneous EIA, only endpoint methods have been used. Often, kinetic methods cannot be used because antiserum are usually very similar, but sometimes of inhomogeneity of the test sample (SP or DASP higher specificity has been found foi EIA [16,19] or for RIA [15,20]. Both effects have been ascribed to methods). For all enzymes used various substrates have been the influence of the bridge ahtibody interaction. Heterologous assays may be somewhat less specific used, chromogenic as well as fluorogenic ones. The choice of substrate is also of influence on assay sensi- than homologous assays. For example, when we tested 14 antisera against testosterone+carboxymethyltivity. It has even been reported that the sensitivity oxime-albumin in homologous and heterologous potential of an estradiol-/?-D-galactosidase conjugate with a fluorogenic substrate could not be fully assay, the inean cross-reactions (range) with dihydroexploited, because the K-value of the antiserum testosterone were 54.5 (17-134)0/, and 61.5 (24-l 12)%, became the limiting factor [14]. respectively. For 5 antisera, the cross-reaction with dihydrotestosterone was more than 10% higher in the What to expect of a steroid enzyme-immunoassay heterologous assay than in the homologous one: for A satisfactory assay method should proide ade- 3 antisera, the reverse was found [25]. Clearly, the quate sensitivity, specificity, accuracy, precision and effect is not sufficiently serious to disqualify heterolopracticability. What can be expected of EIA in ‘these gous assay, if sensitivity gains can be obtained. A cautionary note should be made on ihe usual respects? Table 2. Correlation between EIA and RIA for various steroids Authors
Steroid
Number of samples
Correlation coefficient
Slope
Numazawa et al., 1977 Bosch et al., 1978 Joyce et al., 1977 Ogihara et al., 1977 Tateishi et al., 1977
Estradiol Total estrogens Progesterone Cortisol Testosterone
25 30 15 28 16
0.84 0.98 0.99 0.99 0.96
0.98 1.06 1.03-1.08 1.0 0.95
B. K.
1.50
VAN
WEEMENet al
Table 3. Variation coefficients of steroid EIAs
Authors
Steroid
Within-assay C.V. (%)
Numazawa et aI., 1977 Bosch et al.. 1978 Joyce et al.. 1977 Ogihara et al., 1977 Tateishi et al., 1977
Estradiol Total estrogens Progesterone Cortisol Testosterone
5.0-6.4 l&8.7 2.74.4 11.1
way of assessing immunoas~y specificity. We have that in estracliol assays where the cross-reactions of other estrogens look satisfactory when tested separately, cocktails of estrogens may be considerably overestimated, possibly because of some concerted action of the steroids[23]. We found this phenomenon to be more serious in EIA than in RIA. This aspect should be carefully evaluated. found
Precision
In our hands, steroid EIAs are often more precise than the corresponding RIAs using tritiated labels. The difference in steepness of the dose-response curve which we often observe [ 1l] will be an important factor in this, the lack of precision of &counting will be another. We can usually obtain coefficients of variation of less than 10yO for our steroid EIAs. Others report similar precision (see Table 3). Practicability
An important practical advantage of EIA over RIA is that the use of radioactivity is avoided. On the other hand, an extra incubation step (incubation with substrate) is added. Moreover, this incubation is more disturbance prone than radioactivity measurement; scrupulous washing is essential. In spite of the extra incubation step, total assay times for EIA and RIA are very similar. Usually, they can be completed within 24 h; some give results within one working day. The main practical problem for many users will be to obtain suitable reagents, i.e. antiserum and enzyme-conjugated steroid (or suitable steroid derivatives for coupling). It can, however, be expected that these will be commercially available in the future, as has been the case for RIA.
EIA provides a useful alternative to RIA for sensitive and precise measurement of steroids. Many methodological problems are encountered in both assay methods. Those spccificaliy connected to EIA can be
8.9-l 2.3 8.3-8.5 1lJ318.0 4.7-6.0 12.0
solved. The minimal equipment required and the stability of the reagents make the method especially attractive to the less affluent societies. Extensive reviews on enzyme-immunoassay have appeared recently [26,27].
REFERENCES
Accuracy
Provided that interfering factors in samples are removed by washing, EIA usually is as accurate as RIA. Coefficients of correlation between these two assays usually exceed 0.95, whereas the slope of the correlation line is very near to 1 (see Table 2). Recoveries are usually satisfactory [ll, 12,14,20,22].
Total-assay C.V. (%)
I. Murphy B. P., Engelberg W. and Pattee C. J.: Simple method for the determination of plasma corticoids. J. Cfin. Endocrinol. Metab. 23 (1963) 293-300. 2. Abraham G, E.: Solid-phase radioimmunoas~y of estradiol-178. ,f. C&n. ~~docri~o~. Metub. 29 (1969) 866870. 3. Miles, L. E. M. and Hales, C. N.: Labelled antibodies and immunological assay systems. Nature 219 (1968) 186-189. 4. Aalberse R. C.: Quantitative Ruoroimmunoassay. C/in. cltim. Aera (1973) lO%i 11. 5. Andrieu J. M., Mamas S. and Dray F.: Viroimmunoassay of Steroids. In Steroid Immunoassay (Edited by E. H D. Cameron, S. G. Hillier and K. Griffith@. Alpha Omega. Cardiff (1975) DD. 189-198. 6. Koh& F., Hollander’i.: Yaeger F. M., Carrico R. J. and Bogulaski R. C.: A homogeneous enzymeimmunoassay for estriol monitored by co-enzymic cycling reactions. In Enzyme &belled lrn~n~ss~y of hormones and lktqp (Edited by S. B. Pal). Walter de Gruyter, Berlin, New York (1978) pp. 67-80. 7. van Weemen, B. K. and Schuurs, A. H. W. M.: Immunoassay using haptenenzyme conjugates. FEB.5 Lett. 24 (1972) 77-81. 8. Yorde D. E., Sasse E. A., Wang T. Y.. Hussa R. 0. and Garancis J. C.: Competitive enzyme-linked immunoassay with use of sohtble ~zyme/antib~y immune complexes for labehng. C&t. Gem. 22 (1976) 1372-1377. 9. Chang J. J., Crow1 C. P. and Schneider R. S.: Homogeneous enzyme-immunoassay for digoxin. Clin. Chem. 21 (1975) 967. IO. van Weemen B. K. and Schuurs A. H. W. M.: The intluence of heterologous combinations of antiserum and enzym~la~lled estrogen on the characteristics of estrogen enzyme-immunoa~ay~ ~mmunoc~emjsfry 12 (1975) 667670. Il. Bosch A. M. G., Dijkhuizen D. M., Schuurs A. H. W. M. and van Weemen B. K.: Enzyme-immunoassay for total oestrogens in pregnancy plasma or serum. C/in. Chim. Acta 89 (1978) 59-70. 12. Numazawa M., Haryu A.. Kurosaka K. and Nambara T.: Picogram order enzyme-immunoa~y of oestradioL FEBS Left. 79 (I977) 396-398. 13. Exley D. and Abuknesha R.: The preparation and puri&cation of a /I-o-galactosidase-oestradiol-178 conjugate for enzyme-immunoassay. FEBS L&t. 79 (1977) 501-304.
-
14. Exley D. and Abuknesha R.: A highly sensitive and
Enzyme-immunoassay specific enzyme-immunoa~y method for oestradiol-17& FEBS L&r. 91 (1978) 162-165. IS. Dray F., Andrieu J. M. and Renaud F.: Enzymeimmunoassay of progesterone at the picogram level using #I-galactosidase as label. Rio&m. Biophys. Acta 403 (1975) 131-138. 16. Joyce B. G., Read G. F. and Fahmy D. R.: A specific enzyme-immunoassay for progesterone in human plasma. Steroids 29 (1977) 761-770. 17. Joyce B. G., Read G. F. and Riad-Fahmy D.: Enzymeimmunoassay for progesterone and estradiol. In Radioimmunoassay and Related Procedures in Medicine 1977. International Atomic Energy Agency, Vienna (1978) pp. 289-294. 18. Bosch A. M. G., Stevens W. H. J. M., van Wijngaarden C. J. and Schuurs A. H. W. M.: Solid-phase enzymeimmunoa~ay of testosterone. Z. AnaIyt. Chem. 2% (1978) 98. 19. Rajkowski K. M., Cittanova N., Urios P. and Jayle M. F.: An enzyme-tinked immunoassay of testosterone. Steroids 30 (1977) 129-137. 20. Tateishi K.. Yamamoto H.. Oaihara T. and Havashi C.: Enzyme-immunoassay fo; serum testosteione. Steroids
30 (1977) 25-32.
21. Comoglio
S. and Celada F.: An immuno-enzymatic
22.
23.
24.
25.
26. 27.
of steroids
151
assay of cortisol using E. cofi Bgalactosidase as label. J. Jmm~of. metals 10 (1976) 161-170. Ogihara T., Miyai K., Nishi K., Ishibashi K. and Kumahara Y.: Enzyme-ladled immunoassay for plasma cortisol. J. Cfin. Endocrinol. Metab. 4 (1977) 91-95. Dawson E. C., Bosch A. M. G. and van Weemen B. K.: Enzyme-immunoassay for steroids. In Recent Methods in Steroid Assay (Edited by C. Conti). In press. Rowley G. L., Rubenstein K. E., Huisjen J. and Ullman E. F.: Mechanism by which antibodies inhibit haptenmalate dehydrogenase conjugates. J. biol. Chem. 250 (1975) 3759-3766. Bosch A. M. G., van Hell H., Brands J. A. M. and Schuurs A. H. W. M.: Sensitivity, specificity and reprodu~bility of enzyme-immunoa~ys. In Proceedings of the International Sym~sium on Enzyme-la~lled Immunoassay of Hormones and Drugs, Uhn, July 1978. pp. 175-188. Schuurs A. H. W. M. and van Weemen B. K.: Enzymeimmunoassay. C/in. Chim. Acta 81 (1977) I-40. Engvall E. and Pesos A. J. (Editors): Quantitative enzyme-Immunoassay. Stand. J. Immunol. 8 (1978) Supplement 7.
ENZYME-IMMUNOASSAY OF STEROIDS: POSSIBILITIES AND PITFALLS BAUKE K. VAN WEEMEN,ANNEKE M. G. BOSCH, EDWARD C. DAWSON and ANTON H. W. M. SCHUURS Organon Scientific Development Group, OS, The Netherlands SUMMARY
Enzyme-immunoassay for steroids can be based on either a combination of insolubilized steroid and labelled antibody or a combination of labelled steroid and antibody. Only the latter possibility has been worked out. Two types can be distinguish~: heterogeneous (in which a bound/free separation is required) and homogeneous. Heterogeneous assays have been developed for estrogens, progesterone, cortisot and testosterone. They can be equally sensitive, precise and accurate as radioimmunoas~y, and are therefore useful alternatives for the endocrinological laboratory.
The concentrations
at which steroid
hormones
occur
in human body fluids may vary from pmol/I. (e.g. serum oestradiol in the early luteal phase of the menstrual cycle) to as much as lOO~ol/l. (e.g. urinary estriol at the end of pregnancy). Of course, such huge variation has led to the development of different assay methods. We have already been able for many decades to measure the higher concentrations of steroid hormones, using either bioassay or chemical assay. Next to some other drawbacks-they may be lengthy, tedious and imprecise (bioassay), or relatively unspecific, requiring extensive purification before the assay proper can be performed-their main limitation is that they lack sensitivity for many an application. Therefore, the advent of radioreceptor- [l) and radioimmuno-assay [2] for steroid hormones was an important landmark in endocrinology. Not only could unpr~dent~ sensitivity (down to the pmol/I. level) be achieved, it was accompanied by high specificity, acceptable precision and a relatively easy performance. Hence, it is not surprising that these radioligand methods have become very widely used. The use of radioactively lahelled compound, however, has its drawbacks as well: specially equipped laboratories are required; counting equipment is often expensive; some radioactive isotopes have a very limited shelf-life; there is a waste-disposal problem; depending on local legislation, there may be license problems. With the use of non-isoto@ic labels, many of these drawbacks could be avoided, as was already suggested some 10 years ago[3]. Since then labelled ligand assays for steroid hormones using fluorophores 141, bacteriophages [SJ, coenzymes [6J and enzymes [7] have been described. The latter approach, which is mostly called enzyme-immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA), has attracted the most attention until now, and will be subject of this paper.
Assay principle
Two approaches have been described: (a) Enzyme-labelled antibodies are incubated with steroid-containing sample, and subsequently with excess insolubilized steroid. The amount of steroid in the sample is then inversely related to the amount of enzyme-~bell~ antibodies bound to the insolubilized steroid. This method has been mentioned only once in the literature, without any detail[lYJ. (b) Enzyme-labelled steroid and steroid-containing sample are incubated with a limited amount of steroid antibodies. The amount of steroid in the sample is inversely related to the amount of enzyme-labelled steroid bound to antibody. This, by ‘the way, is also the normal RIA principle. In homogeneous enzymeimmunoassuy, the non-bound enzyme-k&e&d steroid can easily be measured, because the enzyme activity of the ~z~~la~ll~ steroid is completely ~h~bit~ when it is bound to antibody. Although this method has not yet to our knowledge been used for steroid hormones, it works well for determination of the steroid glycoside digoxin, with an assay range of approx. 0.65-10 nmol/l. 191. In heterogeneous enzymeimmunoassay, antibody-bound and non-bound enzyme-labelled steroid are equally active, and must be separated before any measurement can be made. This separation can be obtained by double antibody or solid phase methods similar to those already used in RIA. Heterogeneous EIAs have been .reported for the following steroid hormones: estrogens [7, IO-14,173, prog~terone [B-17], testosterone [ 1t&20] and cortisol[21,22]. Which assay principle is preferable depends on several factors. Little is known about the. labelled antibody approach; its sensitivity potential and precision merit further investigation. Experience from similar RIA systems is encouraging. The choice between homogeneous and heterogeneous EIA should be based 0’1 the required sensitivity of the assay and the 147
148
B. K. VAN WEEMENet al.
org~ic/protein chemical ski11 of the investigator. It appears to be difficult to make homogeneous EIA sufficiently sensitive to detect less than 1nmol/l. of hapten, whereas heterogeneous EIAs can detect subpicomole amounts of progesterone [ 15,17] and estradiol [ 143. Also, preparation of enzyme-steroid conjugates that show the inhibition phenomenon required for homogeneous EIA requires sophisticated chemistry [24]. If, however, these drawbacks can be circumvented or overcome, the resulting homogeneous EIA is easy to perform and to automate. Heterogeneous EIA offers higher sensitivity and the conjugates are easier to prepare, but the assay is more complicated to perform, even if easy coated tube assay methods are used. Antiserum and conjugate
Add 0.1 mmol isobutyl~hloroformate. (3) Stir for 3 min at - 15°C. (4) Add dropwise to 0.005 mmol HRP in 3.5 ml dimethylformamid~water (4:3), precooled at 0°C. (5) Stir for L h at - 15”C, then 2 h at 0°C. (6) Add 20mg NaHCO,. (7) Dialyse against distilled water, then pass through Sephadex G-25. Conjugates of which up to 80% of the enzyme activity is bound by excess steroid antiserum can be prepared this way. These need no further purification. Recently, affinity chromatography has been used to purify conjugates, which resulted in highly sensitive assays [ 13,141. The number of steroid molecules introduced per enzyme molecule can be influenced by changing the molar ratio during the coupling reaction. This may be of importance for the ability of the conjugate to be bound by antiserum 1213 as well as for the sensitivity of the resulting assay[17]. The incorporation is most elegantly determined by coupling tritiated steroid to the enzyme[21]; the conjugate can also be digested by acid hydrolysis, and the liberated steroid derivative assayed by RIA or EIA [14]. Several enzymes have been used to produce steroid conjugates: hoqse-radish peroxidase [7,8,10-12, 16-191, p-u-galactosidase [ 13-15.211, glucoamylase [20] and alkaline phosphata~ [223. For homogeneous EIA, other enzymes must be used: glucose& phosphate dehydrogenase, malate dehydrogenase. Ali have their advantages and disadvantages, which mainly deal with inherent sensitivity, ease of assay, stability of substrate, presence of interfering factors. /I-D-Galactosidase has easily available chromogenic and fluorogenic substrates, which expands its sensitivity range considerably.
It is well known by now how antisera against steroid hormones can be produced and how their specificity can be engineered by selecting different sites of the steroid molecule for linkage to the carrier protein. In principle, any antiserum that can be used for RIA can be used for EIA as well. Enzyme-labelled steroids (conjugate) are in general prepared along the same lines as the immunogens, i.e. by linking a steroid derivative with a reactive group, mostly carboxyl, at the end of an introduced side chain to an appropriate group of the protein molecuk. Our early work [7,10] indicated that antiserum/ conjugate combinations for which the same steroid derivative had been used to prepare immunogen and conjugate (homologous systems) could lead to rather insensitive assay systems, presumably due to an important contribution of the bridge between steroid and protein to the antibody-conjugate binding. A way Boundflree separation method to overcome this problem was to use different steroid derivatives for antiserum and conjugate production As said before, in homogeneous EIA no bound/free (heterologous systems) [lo]. Similar results have been separation is required. This, of course, makes the found in RIA with iodinated labels. We also experi- assay very easy to perform. enced, however, that these phenomena are strongly In heterogeneous EIA, three bound/free separation antiserum dependent and that antisera can be found methods are in use: double antibody (DA) methods, that give highly sensitive homologous systems. It may solid phase (SP) methods and double antibody solid be advisable, therefore, to look for such antisera phase (DASP) methods. SP methods offer the easiest rather than to devise heterologous systems, because performance, if the coated tube variant is chosen. The the latter approach requires more chemistry. Moreother methods require centrifugation and wash steps. over, we have found a tendency towards a decrease which are more time-consuming, and sometimes (DA) in specificity of heterologous systems, although highly longer incubation periods. It has also been reported specific heterologous assays have been reported that the choice of bound/free separation may affect [IO, 141. On the other hand, we have found that the assay properties other than the ease of performance: dose response curves of heterologous systems may be a solid-phase method with Sephadex-coupled antisteeper than those of homologous systems [25] or body was considerably less sensitive than tbe corresponding DA method for progesterone [ 173, and a RIA [ 111. Preparation of steroid-enzyme cbnjugates is not DASP method was much less precise than the corredifficult, once the required ‘steroid derivatives are sponding DA method for cortisol[21]. This may. however, be very much dependent on the system and available. reagents under investigation. We have compared senWe have prepared conjugates of various carboxylated steroids and the enzyme horse-radish peroxidase sitivities of SP and DASP methods for testosterone and found’no large differences (see Table 1). Also, in good yields with the following procedure: with a DASP assay for total estrogens, we have found (1) Cool at - 15°C : 0.1 mm01 steroid and 0.1 mmol N-methylmorpholine in 2 ml dimethylformamide. (2) coefficients of variation of S&6.4% and 8.3-8.5% for
Enzyme-immunoassay Table 1. Sensitivities at midpoint (in pg/ml) of testosterone ElAs with various bound/free separation methods Midpoint (pg/ml) Relative conjugate concentration b/f Separation method Coated tube DASP Tube DASP Cellulose
1
1.67
180 250 500
190 200 490
within- and total-assay variation [ 113, which is cotnparable to data for RIA. Nearly always, the enzyme activity in the bound
fraction is measured. This offers the advantage that factors from the sample that might interfere with the enzyme reaction (e.g. endogeneous enzyme activity or substrate, inhibitors) can easily be removed by one or more washing steps. If a sample contains factors that irreversibly damage the enzyme, SP methods are preferable. Sample and insoluble antibodies can then be incubated in a first step, and the remainder of the sample removed before the conjugate is added, thus avoiding direct contact between sample and conjugate. In homogeneous EIA, these problems can be avoided only by pretreatment or predilution of the sample.
of steroids
149
Sensitivity
Depending on the choice of assay principle, bound/ free separation, enzyme and substrate, the sensitivity of steroid EIA can range from femtomoles to pmoles per tube. As usual, high. sensitivity is accompanied by long incubation times and laborious methodology, and vice versa. Direct comparisons have shown that steroid EIAs can be as sensitive as or even more sensitive than the corresponding RIAs [ll, 14,15,22]. As mentioned before, it has been claimed [14] that the theoretical sensitivity limits of immunoassay can be reached if the proper enzyme and substrate are chosen. Specificity
The antiserum is the most important factor which influences the specificity of steroid EIA. The general rule found for RIA, i.e. the highest specificity is obtained with antisera evoked with immunogens in which the steroid has been coupled to the carrier protein at a site which leaves the characteristic groups of that steroid exposed, applies equally well to EIA. Hence, by choosing the appropriate antiserum, we can design assay systems that react equally well with estrone, estradiol, estriol and all D-ring conjugated estrogens, based on antisera against a mixture of estriol 16- and estriol 17-monosuccinyl-albumin, but on the other hand we have also produced EIAs for Enzyme determination estradiol that show less than 1% cross-reaction with In homogeneous EIA, the enzyme activity is usually estrone, based on antisera against estradiol&carboxy and 1la-OH estradiol-1 l-succinyldetermined kinetically, although for more sensitive methyl-albumin assays endpoint methods have also been used. In albumin. Specificity patterns of RIA and EIA with the same heterogeneous EIA, only endpoint methods have been used. Often, kinetic methods cannot be used because antiserum are usually very similar, but sometimes of inhomogeneity of the test sample (SP or DASP higher specificity has been found foi EIA [16,19] or for RIA [15,20]. Both effects have been ascribed to methods). For all enzymes used various substrates have been the influence of the bridge ahtibody interaction. Heterologous assays may be somewhat less specific used, chromogenic as well as fluorogenic ones. The choice of substrate is also of influence on assay sensi- than homologous assays. For example, when we tested 14 antisera against testosterone+carboxymethyltivity. It has even been reported that the sensitivity oxime-albumin in homologous and heterologous potential of an estradiol-/?-D-galactosidase conjugate with a fluorogenic substrate could not be fully assay, the inean cross-reactions (range) with dihydroexploited, because the K-value of the antiserum testosterone were 54.5 (17-134)0/, and 61.5 (24-l 12)%, became the limiting factor [14]. respectively. For 5 antisera, the cross-reaction with dihydrotestosterone was more than 10% higher in the What to expect of a steroid enzyme-immunoassay heterologous assay than in the homologous one: for A satisfactory assay method should proide ade- 3 antisera, the reverse was found [25]. Clearly, the quate sensitivity, specificity, accuracy, precision and effect is not sufficiently serious to disqualify heterolopracticability. What can be expected of EIA in ‘these gous assay, if sensitivity gains can be obtained. A cautionary note should be made on ihe usual respects? Table 2. Correlation between EIA and RIA for various steroids Authors
Steroid
Number of samples
Correlation coefficient
Slope
Numazawa et al., 1977 Bosch et al., 1978 Joyce et al., 1977 Ogihara et al., 1977 Tateishi et al., 1977
Estradiol Total estrogens Progesterone Cortisol Testosterone
25 30 15 28 16
0.84 0.98 0.99 0.99 0.96
0.98 1.06 1.03-1.08 1.0 0.95
B. K.
1.50
VAN
WEEMENet al
Table 3. Variation coefficients of steroid EIAs
Authors
Steroid
Within-assay C.V. (%)
Numazawa et aI., 1977 Bosch et al.. 1978 Joyce et al.. 1977 Ogihara et al., 1977 Tateishi et al., 1977
Estradiol Total estrogens Progesterone Cortisol Testosterone
5.0-6.4 l&8.7 2.74.4 11.1
way of assessing immunoas~y specificity. We have that in estracliol assays where the cross-reactions of other estrogens look satisfactory when tested separately, cocktails of estrogens may be considerably overestimated, possibly because of some concerted action of the steroids[23]. We found this phenomenon to be more serious in EIA than in RIA. This aspect should be carefully evaluated. found
Precision
In our hands, steroid EIAs are often more precise than the corresponding RIAs using tritiated labels. The difference in steepness of the dose-response curve which we often observe [ 1l] will be an important factor in this, the lack of precision of &counting will be another. We can usually obtain coefficients of variation of less than 10yO for our steroid EIAs. Others report similar precision (see Table 3). Practicability
An important practical advantage of EIA over RIA is that the use of radioactivity is avoided. On the other hand, an extra incubation step (incubation with substrate) is added. Moreover, this incubation is more disturbance prone than radioactivity measurement; scrupulous washing is essential. In spite of the extra incubation step, total assay times for EIA and RIA are very similar. Usually, they can be completed within 24 h; some give results within one working day. The main practical problem for many users will be to obtain suitable reagents, i.e. antiserum and enzyme-conjugated steroid (or suitable steroid derivatives for coupling). It can, however, be expected that these will be commercially available in the future, as has been the case for RIA.
EIA provides a useful alternative to RIA for sensitive and precise measurement of steroids. Many methodological problems are encountered in both assay methods. Those spccificaliy connected to EIA can be
8.9-l 2.3 8.3-8.5 1lJ318.0 4.7-6.0 12.0
solved. The minimal equipment required and the stability of the reagents make the method especially attractive to the less affluent societies. Extensive reviews on enzyme-immunoassay have appeared recently [26,27].
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Accuracy
Provided that interfering factors in samples are removed by washing, EIA usually is as accurate as RIA. Coefficients of correlation between these two assays usually exceed 0.95, whereas the slope of the correlation line is very near to 1 (see Table 2). Recoveries are usually satisfactory [ll, 12,14,20,22].
Total-assay C.V. (%)
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14. Exley D. and Abuknesha R.: A highly sensitive and
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151
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