- Email: [email protected]
Automation of steroid radioimmunoassays for clinical and research purposes
1I. pp.
Jourwl of Steroid Biwhcmisrry. Vol. I25 10 I28 !‘Wgitmon Press Ltd 1979. Rinfed in Greaf Britain
AUTOMATION OF STEROID RADIOIMMUNOASSAYS CLINICAL AND RESEARCH PURPOSES ~pa~ment
FOR
R. VIHKO and G. L. HAMMOND of Clinical Chemistry, University of Oulu, SF-90220 Oulu 22, Finland SUMMARY
In recent years an exponential increase in the research and clinical application of radioimmunoassays has created a demand for manipulative aids, to increase the general efficiency of the technique as well as precision of the determinations. At present a number of systems exist for sample preparation and the automation of assays. However, due to inherent problems of carry-over. priming, cleaning and the necessity for chemically inert components most of these systems tend to be rather inflexible. The instrument of choice ought therefore to be extremely versatile and provide the operator with a wide spectrum of alternatives, in order to optimize the initial capital outlay. Moreover, in the light of technological developments in the field of simultaneous, multisample gamma-counting and data processing, it is anticipated that a new generation of multisample processors will emerge of sufficient flexibility to accommodate the wide variety of assay protocols in present use. in this paper, we have reviewed the problems encountered in the development of automated techniques for the radioimmunoassay of steroid hormones, and have presented a prefiminary description of a versatile modular discrete i~trument for the automation of radioimmuno~says, which is based on simuhaneous multisample preparation, and subsequent counting and data processing.
INTRODUCTION The increasing demand for steroid radioimmunoas-
says in clinical and research laboratories has stimulated the development of techniques designed to reduce the labour-intensive nature of the method, and to increase assay capacity, speed and reliability. The development of an i~trument for the complete automation of steroid r~io~muno~says has been limited by several factors inherent in the exploitation of immunochemical methods for the determination of small molecular weight compounds in biological samples, The structural similarity that exists between many of the physiologically important steroids has presented problems in the generation of antibodies of adequate specificity and, in addition, the presence of endogenous steroid binding proteins has necessitated the pre-purification of samples by solvent extraction and even chromatography. The separation of antibody bound from free r~io-labell~ ligand has caused serious problems in the development of instruments for the automation of radio~muno~says, and in overcoming these probfems the technological solution has often resulted in a severe restriction in the type of assay protocol which may be employed. Similarly, the wide use of [3H]-labelled steroids as labelled ligand, and the necessity for liquid scintillation counting, has also presented serious technical problems, but such problems have been largely overcome by the use of [1z51]-labelled steroid derivatives. In this paper, we endeavour to review the various attempts made to overcome these problems encountered in the automation of steroid radio~muno~say and to present a prel~ina~ description of a discrete multisample processor-counter unit.
ASSAY SPECIFICITY
The production of highly specific antisera for the radioimmunoassay of steroids has been limited by the structural similarity that exists between many physiologically important steroids. In recent years, however, various attempts have been made to select the site of conjugation of the steroid to the carrier molecule in such a way that the specific groups for the steroid in question may play a major role in determining the specificity of the anteriserum [l-3]. At present, the specificity of antisera used for the determination of clinically important steroids is in most cases adequate. Nevertheless, it is evident that under certain physiological and pathological conditions the influence of extremely high concentrations of other steroids necessitates the use of a purification procedure such as column chromatography. This is illustrated in Table 1, in which serum cortisol was determined with a highly specific antiserum (for details see [4]). These data show the sui~biIity of a nonchromato~aphic method for screening purposes, while it is essential to appreciate that during pregnancy and in infants with an adrenal steroid 21-hydroxylase deficiency grossly erroneous results may be obtained. For most research purposes, chromatographic purification of steroid extracts is essential. Although techniques have been described for partial automation of steroid extractions and chromatographies [S], these are generally cumbersome and so demanding that the benefit of this kind of automation is questionable. On the other hand, when several steroids are to be determined from a single extract, an automated technique capable of handling large numbers of different assays 125
R. VIHKO and G. L. HAMMOND
126 Table
1. Serum
cortisol
determined tography.
with and without Data from Ref. [4] Mean Serum Chromatography
Sample Normal serum (n = 5) Late pregnancy serum (n = 10) Infant serum (steroid 21-hydroxylase deficiency) Simple virilizing (n = 2) Salt losing (n = 2)
undoubtably increases the potential research capacity in an endocrine laboratory [63. In order to overcome the limitations to automation imposed by the necessity for extraction and/or chromatography in steroid radioimmunoassays for clinical purposes, additional techniques have been described which counteract the influence of endogenous serum binding proteins (albumin, corticosteroid binding globulin, sex hormone binding globulin). These include heat denaturation [7], lowering of pH [S]. and the addition of steroids or other agents which displace the steroid to be measured from serum binding proteins [9-l 11. LABELLED STEROIDS
An additional limitation in the development of automated techniques for steroid radioimmunoassays has been the widespread use of tritiated steroid as the labelled ligand. In recent years, the use of [‘251]-labelled steroid derivatives has attracted attention, especially in routine laboratories. The use of this label, as compared to tritium, is particularly advantageous in the development of automated systems, primarily because of the relatively simple counting procedures and shorter counting time required for gamma-emitting isotopes [12, 133. The higher specific activity of [ 12SI]-labelled steroids also plays a role in the increased sensitivity often associated with the use of this label. Moreover, [lZSI]-labelled steroid derivatives generally possess greater affinity towards the antibody and this is obviously due to the “bridge effect” exerted by the derivative side-chain [14]. This phenomenon may also contribute to the increased sensitivity and specificity often encountered when using [ ‘251]-labelled steroid ligands as compared to their tritiated counterparts [12, 131. SEPARATlON OF FREE AND BOUND RADIOACTIVITY
As in other radioimmunoassay procedures, the aration of bound and free radioactive steroids been accomplished by a wide range of techniques cluding dextran-coated charcoal adsorption of free ligand, polyethylene glycol and ammonium phate precipitation of the antibody-bound ligand,
sephas inthe sulgel
Lipidex-5000TM
Cortisol
chroma-
(ng/ml)
No chromatography
91.2 343
94.3 445
20.1 11.9
55.5 131.4
column chromatography, second antibody precipitation and filtration on glass filter discs, and a variety of solid-phase media. In view of the wide range of separation techniques in current use, severe limitations are imposed on instruments relying solely on one particular separation principle. At present, the use of dextran-coated charcoal is perhaps the most widely used technique for the separation of antibody bound and free steroids. This technique is particularly difficult to automate because of its time and temperature dependent nature [15, 163, and the difficulties encountered in dispensing a fine suspension of particles especially when using instruments relying on pumps. Recently, however, it has been reported that dextran-coated charcoal-magnetic particles in pellet form have been employed to overcome these problems, and also eliminate the necessity for centrifugation [17]. The major advantage of using solid-phase techniques such as antibody-coated tubes [18], glass beads [ll] and magnetic particles Cl73 is that centrifugation is not required. However, although the kinetics of the antibodysteroid interaction is thought to proceed rather rapidly in comparison with antibodyprotein interaction, it has been shown that assay incubations terminated at disequilibrium tend to be less sensitive and less specific than those conducted under of complete equilibrium [19]. This conditions phenomenon undoubtably imposes limitations on the use of short incubation continuous flow techniques.
INSTRUMENTATION
Recently, a comprehensive evaluation of the instruments currently available for the automation of radioimmunoassays has been published [20]. In many cases, the instrumentation in current use is applicable to only a limited number of procedures, often dictated by the availability of reagents specially prepared for the instrument in question. In addition, many of the instruments described are rather inflexible and require considerable cleaning and priming of components when. changing from one procedure to another. Nevertheless, they are generally capable of performing extremely large series of one particular assay with a high degree of precision. This motivates their use in
Automation of radioimmunoassays
121
improved by using Hamilton syringes of smaller in-
ternal diameter (4.5 mm) than previously used (7.5 mm)
Fig. 1. Multis~pIe
radio~muno~say
processor.
large screening projects or in a centralized laboratory service. During the last few years we have been engaged in developing a discrete modular approach for the automation of radio~muno~says. It has been our intention to develop a rapid and flexible instrument, which could be used universally for all radioimmunoassay protocols, and which would not suffer from the problem of carry-over. The original instrumentation was based on existing components of a clinical chemical analyzer {System Olli 3000), and has been described extensively for the automation of the radioimmunoassay of numerous steroids in biological samples [6,13]. The success of this preliminary work and the introduction of multisample gamma counters has led us to develop a new instrument, which combines sample prep~ation in units of 24 assay tubes and a simultaneous counting and evaluation of 12 tubes at one time. The development of the new instruments has proceeded in collaboration with the development teams at Kone Oy Instrument Division, Espoo, and Wallac Oy, Turku, Finland. MULTISAMPLE
RJA PROCESSOR/COUNTER
In many respects, the new multisample RIA processor (Fig. 1) resembles the original System Olli 3ooO dispenser 216 [13]. The samples are processed in duplicate units of 24 assay tubes, which are attached to pre-labelled code plates for convenient sample identification and to enable simultaneous transfer of tubes between the various modules in the system (sample processor and incubator and centrifuge, if required). The use of pre-arranged sets of 24 disposable pipette tips, which can be rapidly changed between the various steps in the procedures, ensures that carry-over is non-existent. In contrast to the earlier instrument, the new multisample RIA processor comprises a microprocessor which is capable of storing approximately 40 different assay protocols, which ensure that the correct sequence of the addition of samples and reagents is followed for any particular assay. The instrumentation also comprises facilities for continuous stirring of slurries and mixing of samples during pipetting. The precision of pipetting small volumes (down to lo& of serum has been S.B.I l/IA-J
and by filling most of the dead volume with a liquid After sample preparation, duplicate units of six assay tubes may be transferred to the multig~ma counter (Fig. 2), which comprises 12 wells arranged in two rows of six with a 50mm distance between the wells. The microprocessor is able to store information on particular assays, details of which may be recalled on a moving text display. The continuous monitoring of the performance of individual wells is provided, and counts in individual wells may also be displayed duting counting. The data processing relies on the use of spline function for the evaluation and opti~zation of standard curve parameters [21]. Unknown samples are interpreted, compiled and presented on a print-out in terms of concentration. Quality control data and identifi~tion of unreliable duplicates are provided. Due to the modular construction, the actual instrument time one operator requires to set up assays is minimal. Furthermore, due to the discrete nature of the sample processor, and the ease with which consecutively different samples and reagents can be dispensed, more than one operator and several different assay protocols may be accommodated simultaneously without problems due to carry-over. Although the instrument is directed principally for the counting of gamma-emmiting isotopes, the sample processor is applicable for the preparation of assays which rely on tritiated tracers. It is evident that the combination of multisample processing and counting will inevitably lead to a new generation of i~truments for the automation of radioimmunoassays, and it is anticipated that the Rexibility and discrete nature of the system described will open up possibilities for radioimmunoassay to assume a still more prominent position in both clinical and research laboratories.
Fig. 2. ~ultisample gamma counter.
R. VIHKO and G. L. HAMMOND
128 REFERENCES
1. Exley D.: The specificity and affinity of antibodies to steroids. In Radioimmunoassay in Clinical Biochemistry (Edited by C. A. Paster&). Heyden, London (1975) pp. 141-i51. 2. Bermtidez J. A., Coronado V., Mijares A., Le6n C., Veltiauez A.., Noble P. and Mateos J. L.: Stereochemi. cal approach to increase the specificity of steroid antibodies. J. Steroid Biochem. 6 (1975) 283290. 3. Grover P. K. and Ode11 W. D.: Specificity of antisera to sex steroids-l. The effect of substitution and stereochemistry. J. Steroid Biochem. 8 (1977) 121-126. 4. Apter D., JPnne 0. and Vihko R.: Lipidex chromat&aphy in the radioimmunoassay of serum and urinarv cortisol. Clin. chim. Acta 63 (1975) 139-148. 5. Sip&l W. G., Bidlingmaier F‘. aid Knorr D.: Mechanized Sephadex LH-20 multiple column chromatography as a prerequisite for automated multisteroid radioimmunoassay. In Radioimmunoassay and Related Procedures in Medicine, 1977, Vol. 1, Proceedings of a Symposium, Berlin, 31 October-4 November 1977. International Atomic Energy Agency, Vienna (1978) pp. 229-237. 6. Vihko R., Hammond G. L., Pakarinen A. and Viinikka L.: Multiple steroid radioimmunoassays and automation. Versatile techniques for reproductive endocrinology. In Radioimmunoassay and Related Procedures in Medicine 1977, Vol. 1, Proceedings of a Symposium, Berlin, 31 October-4 November 1977. International Atomic Energy Agency, Vienna (1978) pp. 221-228. 7. Foster L. B. and Dunn R. T.: Single-antibody technique for radioimmunoassay of cortisol in unextracted serum or plasma. Clin. Chm. 20 (1974) 365-368. 8. Seth J. and Brown L. M.: A simple radioimmunoassay for plasma cortisol. Clin. him. Acta 86 (1978) 10%120. 9. Pratt J. J., Wiegman T., Lappiihn R. E. and Woldring M. G.: Estimation of plasma testosterone without extraction and chromatography. Clin. chim. Acta 59 (1975) 337-346. 10. Jurgens H., Pratt J. J. and Woldring M. G.: Radioimmunoassay of plasma estradiol without extraction and chromatography. J. clin. Endocrinol. Metab. 40 (1975) 19-25. 11. Brock P., Eldred E. W., Woiszwillo J. E., Doran M. and Schoemaker H. J.: Direct solid-phase ‘251
radioimmunoassay of serum cortisol. C/in. Chem. 24 (1978) 1595-1598._ 12. Jeffcoate S. L.: Use of “‘iodine tracers in steroid radioimmunoassays. In Radioimmirnoassay of Steroid Hormones (Edited by D. Gupta). Verlag Chemie, Weinheim (1975) pp. 185-195. 13. Hammond G. L., Viinikka L. and Vihko R.: Automation of radioimmunoassays for some sex steroids with use of both iodinated and tritiated ligands. Clin. Chem. 23 (1977) 125&1257. 14. Collins W. P., Hennam J. F., Tyler J. P. P. and Barnard G. J. R.: Factors affecting the radioimmunoassay of gonadal steroids. In Radioimmunoassay in Clinical Biochemistry (Edited by C. A. Pasternak). Heyden, London (1975) pp. 15>169. 15. Binoux M. A. and Ode11W. D.: Use of dextran-coated charcoal to separate antibody-bound from free hormone: A critique. J. clin. Endocr. Metab. 36 (1973) 30>3 10. 16. de Hertogh R., van der Heyden I. and Ekka E.: “Unbound” ligand adsorption on dextran-coated charcoal: Practical considerations. J. Steroid Biochem. 6 (1975) 1333-1337. 17. Ithakissios D. S. and Kubiatowicz D. 0.: Use of protein containing magnetic microparticles in radioassays. Cfin. Chem. 23 (1977) 2072-2079.
18. Painter K. and Stonecypher T. E.: The development of a fully automated radioimmunoassay instrument based upon solid phase, antibody-coated tubes. In Radioimmunoassay and Related Procedures in Medicine, 1977, Vol. 1, Proceedings of a Symposium, Berlin, 31
October-4 November 1977. International Atomic Energy Agency, Vienna (1978) pp. 211-216. 19. Pratt J. J. and Woldring M. G.: Radioimmunoassay specificity and the “first-come first-served effect”. Clin. chim. Acta 68 (1976) 87-90.
20. Ingrand J.: Etude critique des equipements lourds destines a l’analyse radioimmunologique. In Radioimmunoassay and Related Procedures in Medicine, 1977, Vol. 1, Proceedings of a Symposium, Berlin, 31 October-4 November 1977. International Atomic Energy Agency, Vienna (1978) pp. 18s210. 21. Rawlins T. G. R. and Pietilii U.: Techniques in nuclear counting. Optimized precision counting and quality control. International Laboratory (1978) 2s 33.
Jourwl of Steroid Biwhcmisrry. Vol. I25 10 I28 !‘Wgitmon Press Ltd 1979. Rinfed in Greaf Britain
AUTOMATION OF STEROID RADIOIMMUNOASSAYS CLINICAL AND RESEARCH PURPOSES ~pa~ment
FOR
R. VIHKO and G. L. HAMMOND of Clinical Chemistry, University of Oulu, SF-90220 Oulu 22, Finland SUMMARY
In recent years an exponential increase in the research and clinical application of radioimmunoassays has created a demand for manipulative aids, to increase the general efficiency of the technique as well as precision of the determinations. At present a number of systems exist for sample preparation and the automation of assays. However, due to inherent problems of carry-over. priming, cleaning and the necessity for chemically inert components most of these systems tend to be rather inflexible. The instrument of choice ought therefore to be extremely versatile and provide the operator with a wide spectrum of alternatives, in order to optimize the initial capital outlay. Moreover, in the light of technological developments in the field of simultaneous, multisample gamma-counting and data processing, it is anticipated that a new generation of multisample processors will emerge of sufficient flexibility to accommodate the wide variety of assay protocols in present use. in this paper, we have reviewed the problems encountered in the development of automated techniques for the radioimmunoassay of steroid hormones, and have presented a prefiminary description of a versatile modular discrete i~trument for the automation of radioimmuno~says, which is based on simuhaneous multisample preparation, and subsequent counting and data processing.
INTRODUCTION The increasing demand for steroid radioimmunoas-
says in clinical and research laboratories has stimulated the development of techniques designed to reduce the labour-intensive nature of the method, and to increase assay capacity, speed and reliability. The development of an i~trument for the complete automation of steroid r~io~muno~says has been limited by several factors inherent in the exploitation of immunochemical methods for the determination of small molecular weight compounds in biological samples, The structural similarity that exists between many of the physiologically important steroids has presented problems in the generation of antibodies of adequate specificity and, in addition, the presence of endogenous steroid binding proteins has necessitated the pre-purification of samples by solvent extraction and even chromatography. The separation of antibody bound from free r~io-labell~ ligand has caused serious problems in the development of instruments for the automation of radio~muno~says, and in overcoming these probfems the technological solution has often resulted in a severe restriction in the type of assay protocol which may be employed. Similarly, the wide use of [3H]-labelled steroids as labelled ligand, and the necessity for liquid scintillation counting, has also presented serious technical problems, but such problems have been largely overcome by the use of [1z51]-labelled steroid derivatives. In this paper, we endeavour to review the various attempts made to overcome these problems encountered in the automation of steroid radio~muno~say and to present a prel~ina~ description of a discrete multisample processor-counter unit.
ASSAY SPECIFICITY
The production of highly specific antisera for the radioimmunoassay of steroids has been limited by the structural similarity that exists between many physiologically important steroids. In recent years, however, various attempts have been made to select the site of conjugation of the steroid to the carrier molecule in such a way that the specific groups for the steroid in question may play a major role in determining the specificity of the anteriserum [l-3]. At present, the specificity of antisera used for the determination of clinically important steroids is in most cases adequate. Nevertheless, it is evident that under certain physiological and pathological conditions the influence of extremely high concentrations of other steroids necessitates the use of a purification procedure such as column chromatography. This is illustrated in Table 1, in which serum cortisol was determined with a highly specific antiserum (for details see [4]). These data show the sui~biIity of a nonchromato~aphic method for screening purposes, while it is essential to appreciate that during pregnancy and in infants with an adrenal steroid 21-hydroxylase deficiency grossly erroneous results may be obtained. For most research purposes, chromatographic purification of steroid extracts is essential. Although techniques have been described for partial automation of steroid extractions and chromatographies [S], these are generally cumbersome and so demanding that the benefit of this kind of automation is questionable. On the other hand, when several steroids are to be determined from a single extract, an automated technique capable of handling large numbers of different assays 125
R. VIHKO and G. L. HAMMOND
126 Table
1. Serum
cortisol
determined tography.
with and without Data from Ref. [4] Mean Serum Chromatography
Sample Normal serum (n = 5) Late pregnancy serum (n = 10) Infant serum (steroid 21-hydroxylase deficiency) Simple virilizing (n = 2) Salt losing (n = 2)
undoubtably increases the potential research capacity in an endocrine laboratory [63. In order to overcome the limitations to automation imposed by the necessity for extraction and/or chromatography in steroid radioimmunoassays for clinical purposes, additional techniques have been described which counteract the influence of endogenous serum binding proteins (albumin, corticosteroid binding globulin, sex hormone binding globulin). These include heat denaturation [7], lowering of pH [S]. and the addition of steroids or other agents which displace the steroid to be measured from serum binding proteins [9-l 11. LABELLED STEROIDS
An additional limitation in the development of automated techniques for steroid radioimmunoassays has been the widespread use of tritiated steroid as the labelled ligand. In recent years, the use of [‘251]-labelled steroid derivatives has attracted attention, especially in routine laboratories. The use of this label, as compared to tritium, is particularly advantageous in the development of automated systems, primarily because of the relatively simple counting procedures and shorter counting time required for gamma-emitting isotopes [12, 133. The higher specific activity of [ 12SI]-labelled steroids also plays a role in the increased sensitivity often associated with the use of this label. Moreover, [lZSI]-labelled steroid derivatives generally possess greater affinity towards the antibody and this is obviously due to the “bridge effect” exerted by the derivative side-chain [14]. This phenomenon may also contribute to the increased sensitivity and specificity often encountered when using [ ‘251]-labelled steroid ligands as compared to their tritiated counterparts [12, 131. SEPARATlON OF FREE AND BOUND RADIOACTIVITY
As in other radioimmunoassay procedures, the aration of bound and free radioactive steroids been accomplished by a wide range of techniques cluding dextran-coated charcoal adsorption of free ligand, polyethylene glycol and ammonium phate precipitation of the antibody-bound ligand,
sephas inthe sulgel
Lipidex-5000TM
Cortisol
chroma-
(ng/ml)
No chromatography
91.2 343
94.3 445
20.1 11.9
55.5 131.4
column chromatography, second antibody precipitation and filtration on glass filter discs, and a variety of solid-phase media. In view of the wide range of separation techniques in current use, severe limitations are imposed on instruments relying solely on one particular separation principle. At present, the use of dextran-coated charcoal is perhaps the most widely used technique for the separation of antibody bound and free steroids. This technique is particularly difficult to automate because of its time and temperature dependent nature [15, 163, and the difficulties encountered in dispensing a fine suspension of particles especially when using instruments relying on pumps. Recently, however, it has been reported that dextran-coated charcoal-magnetic particles in pellet form have been employed to overcome these problems, and also eliminate the necessity for centrifugation [17]. The major advantage of using solid-phase techniques such as antibody-coated tubes [18], glass beads [ll] and magnetic particles Cl73 is that centrifugation is not required. However, although the kinetics of the antibodysteroid interaction is thought to proceed rather rapidly in comparison with antibodyprotein interaction, it has been shown that assay incubations terminated at disequilibrium tend to be less sensitive and less specific than those conducted under of complete equilibrium [19]. This conditions phenomenon undoubtably imposes limitations on the use of short incubation continuous flow techniques.
INSTRUMENTATION
Recently, a comprehensive evaluation of the instruments currently available for the automation of radioimmunoassays has been published [20]. In many cases, the instrumentation in current use is applicable to only a limited number of procedures, often dictated by the availability of reagents specially prepared for the instrument in question. In addition, many of the instruments described are rather inflexible and require considerable cleaning and priming of components when. changing from one procedure to another. Nevertheless, they are generally capable of performing extremely large series of one particular assay with a high degree of precision. This motivates their use in
Automation of radioimmunoassays
121
improved by using Hamilton syringes of smaller in-
ternal diameter (4.5 mm) than previously used (7.5 mm)
Fig. 1. Multis~pIe
radio~muno~say
processor.
large screening projects or in a centralized laboratory service. During the last few years we have been engaged in developing a discrete modular approach for the automation of radio~muno~says. It has been our intention to develop a rapid and flexible instrument, which could be used universally for all radioimmunoassay protocols, and which would not suffer from the problem of carry-over. The original instrumentation was based on existing components of a clinical chemical analyzer {System Olli 3000), and has been described extensively for the automation of the radioimmunoassay of numerous steroids in biological samples [6,13]. The success of this preliminary work and the introduction of multisample gamma counters has led us to develop a new instrument, which combines sample prep~ation in units of 24 assay tubes and a simultaneous counting and evaluation of 12 tubes at one time. The development of the new instruments has proceeded in collaboration with the development teams at Kone Oy Instrument Division, Espoo, and Wallac Oy, Turku, Finland. MULTISAMPLE
RJA PROCESSOR/COUNTER
In many respects, the new multisample RIA processor (Fig. 1) resembles the original System Olli 3ooO dispenser 216 [13]. The samples are processed in duplicate units of 24 assay tubes, which are attached to pre-labelled code plates for convenient sample identification and to enable simultaneous transfer of tubes between the various modules in the system (sample processor and incubator and centrifuge, if required). The use of pre-arranged sets of 24 disposable pipette tips, which can be rapidly changed between the various steps in the procedures, ensures that carry-over is non-existent. In contrast to the earlier instrument, the new multisample RIA processor comprises a microprocessor which is capable of storing approximately 40 different assay protocols, which ensure that the correct sequence of the addition of samples and reagents is followed for any particular assay. The instrumentation also comprises facilities for continuous stirring of slurries and mixing of samples during pipetting. The precision of pipetting small volumes (down to lo& of serum has been S.B.I l/IA-J
and by filling most of the dead volume with a liquid After sample preparation, duplicate units of six assay tubes may be transferred to the multig~ma counter (Fig. 2), which comprises 12 wells arranged in two rows of six with a 50mm distance between the wells. The microprocessor is able to store information on particular assays, details of which may be recalled on a moving text display. The continuous monitoring of the performance of individual wells is provided, and counts in individual wells may also be displayed duting counting. The data processing relies on the use of spline function for the evaluation and opti~zation of standard curve parameters [21]. Unknown samples are interpreted, compiled and presented on a print-out in terms of concentration. Quality control data and identifi~tion of unreliable duplicates are provided. Due to the modular construction, the actual instrument time one operator requires to set up assays is minimal. Furthermore, due to the discrete nature of the sample processor, and the ease with which consecutively different samples and reagents can be dispensed, more than one operator and several different assay protocols may be accommodated simultaneously without problems due to carry-over. Although the instrument is directed principally for the counting of gamma-emmiting isotopes, the sample processor is applicable for the preparation of assays which rely on tritiated tracers. It is evident that the combination of multisample processing and counting will inevitably lead to a new generation of i~truments for the automation of radioimmunoassays, and it is anticipated that the Rexibility and discrete nature of the system described will open up possibilities for radioimmunoassay to assume a still more prominent position in both clinical and research laboratories.
Fig. 2. ~ultisample gamma counter.
R. VIHKO and G. L. HAMMOND
128 REFERENCES
1. Exley D.: The specificity and affinity of antibodies to steroids. In Radioimmunoassay in Clinical Biochemistry (Edited by C. A. Paster&). Heyden, London (1975) pp. 141-i51. 2. Bermtidez J. A., Coronado V., Mijares A., Le6n C., Veltiauez A.., Noble P. and Mateos J. L.: Stereochemi. cal approach to increase the specificity of steroid antibodies. J. Steroid Biochem. 6 (1975) 283290. 3. Grover P. K. and Ode11 W. D.: Specificity of antisera to sex steroids-l. The effect of substitution and stereochemistry. J. Steroid Biochem. 8 (1977) 121-126. 4. Apter D., JPnne 0. and Vihko R.: Lipidex chromat&aphy in the radioimmunoassay of serum and urinarv cortisol. Clin. chim. Acta 63 (1975) 139-148. 5. Sip&l W. G., Bidlingmaier F‘. aid Knorr D.: Mechanized Sephadex LH-20 multiple column chromatography as a prerequisite for automated multisteroid radioimmunoassay. In Radioimmunoassay and Related Procedures in Medicine, 1977, Vol. 1, Proceedings of a Symposium, Berlin, 31 October-4 November 1977. International Atomic Energy Agency, Vienna (1978) pp. 229-237. 6. Vihko R., Hammond G. L., Pakarinen A. and Viinikka L.: Multiple steroid radioimmunoassays and automation. Versatile techniques for reproductive endocrinology. In Radioimmunoassay and Related Procedures in Medicine 1977, Vol. 1, Proceedings of a Symposium, Berlin, 31 October-4 November 1977. International Atomic Energy Agency, Vienna (1978) pp. 221-228. 7. Foster L. B. and Dunn R. T.: Single-antibody technique for radioimmunoassay of cortisol in unextracted serum or plasma. Clin. Chm. 20 (1974) 365-368. 8. Seth J. and Brown L. M.: A simple radioimmunoassay for plasma cortisol. Clin. him. Acta 86 (1978) 10%120. 9. Pratt J. J., Wiegman T., Lappiihn R. E. and Woldring M. G.: Estimation of plasma testosterone without extraction and chromatography. Clin. chim. Acta 59 (1975) 337-346. 10. Jurgens H., Pratt J. J. and Woldring M. G.: Radioimmunoassay of plasma estradiol without extraction and chromatography. J. clin. Endocrinol. Metab. 40 (1975) 19-25. 11. Brock P., Eldred E. W., Woiszwillo J. E., Doran M. and Schoemaker H. J.: Direct solid-phase ‘251
radioimmunoassay of serum cortisol. C/in. Chem. 24 (1978) 1595-1598._ 12. Jeffcoate S. L.: Use of “‘iodine tracers in steroid radioimmunoassays. In Radioimmirnoassay of Steroid Hormones (Edited by D. Gupta). Verlag Chemie, Weinheim (1975) pp. 185-195. 13. Hammond G. L., Viinikka L. and Vihko R.: Automation of radioimmunoassays for some sex steroids with use of both iodinated and tritiated ligands. Clin. Chem. 23 (1977) 125&1257. 14. Collins W. P., Hennam J. F., Tyler J. P. P. and Barnard G. J. R.: Factors affecting the radioimmunoassay of gonadal steroids. In Radioimmunoassay in Clinical Biochemistry (Edited by C. A. Pasternak). Heyden, London (1975) pp. 15>169. 15. Binoux M. A. and Ode11W. D.: Use of dextran-coated charcoal to separate antibody-bound from free hormone: A critique. J. clin. Endocr. Metab. 36 (1973) 30>3 10. 16. de Hertogh R., van der Heyden I. and Ekka E.: “Unbound” ligand adsorption on dextran-coated charcoal: Practical considerations. J. Steroid Biochem. 6 (1975) 1333-1337. 17. Ithakissios D. S. and Kubiatowicz D. 0.: Use of protein containing magnetic microparticles in radioassays. Cfin. Chem. 23 (1977) 2072-2079.
18. Painter K. and Stonecypher T. E.: The development of a fully automated radioimmunoassay instrument based upon solid phase, antibody-coated tubes. In Radioimmunoassay and Related Procedures in Medicine, 1977, Vol. 1, Proceedings of a Symposium, Berlin, 31
October-4 November 1977. International Atomic Energy Agency, Vienna (1978) pp. 211-216. 19. Pratt J. J. and Woldring M. G.: Radioimmunoassay specificity and the “first-come first-served effect”. Clin. chim. Acta 68 (1976) 87-90.
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