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Rapid minichromatography for purification of samples prior to radioimmunoassay
ANALYTICAL
BIOCHEMISTRY
82, 69-75 (1977)
Rapid Minichromatography for Purification Prior to Radioimmunoassay KENNETH Endocrinology-Reproductive Neuroendocrinology Human Development,
of Samples
C. GORRAY AND W. B. QUAY’ Physiology Program, Section, Waisman Center University of Wisconsin,
Department of Zoology, and on Mental Retardation and Madison, Wisconsin 53706
Received February 28, 1977; accepted May 23, 1977 A method is presented for the treatment of serum or other biological fluids to remove interfering substances prior to radioimmunoassay. Studies with radioimmunoassay systems for measuring serum levels of insulin revealed the presence of such interfering substances, which could be removed by utilizing small columns of Sephadex G-25 prepared in disposable pipet tips. The columns were centrifuged at low, and finally, higher speeds, to recover the sample volume with essentially no alteration in insulin activity. Validity of the technique was evaluated by both parallelism and recovery studies. This system offers considerable advantages over previous extraction and chromatography procedures in terms of simplicity, reproducibility, and recovery.
The valid radioimmunoassay (RIA) of any one of a number of hormones is made difficult by the frequent presence in biological samples and fluids of endogenous interfering substances. It has been concluded that such heterogeneity of reacting species “may or may not require the fractionation of standards or unknowns in order to insure the validation of an assay” (1). We and other investigators have found this need especially true in the case of RIA of insulin. At least some of the factors and sources of the difficulties can be suggested: (A) high levels of nonspecific binding to various substances (2); (B) degradation of the insulin molecule in plasma or serum with the subsequent binding of insulin fragments to large serum proteins and the survival of their immunoreactivity in some RIA procedures (3,4); (C) the presence of insulin-like activity (ILA) in totally pancreatectomized animals (5) and including sometimes proinsulin (6). The extent of the difficulty posed by such factors is epitomized by a summary of the results of RIA of aliquots of insulin standards by 36 reputable Italian laboratories. Although within-assay variability was at an acceptable level (approximately IO%), betweenr Send reprint requests to K. C. Gorray, 481 Medical Sciences, Department of Medicine, University of Wisconsin Medical School, Madison, Wis. 53706. 69 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0003.2697
70
GORRAY
AND QUAY
laboratory variability was 0.5 to 1.5 times the median value of the unknowns (7). The presence of low-molecular-weight interfering substances in normal rat serum became apparent during our studies using RIA systems for insulin. One or both binding reactions were interfered with, and the factor(s) involved could not be removed with ethanol or polyethylene glycol extraction procedures. Nor could the effects of the interference be bypassed by utilization of “standard curves” containing low concenIOO-
60-
20-
(32)
RAT
INSULIN
ACTIVITY
(pU/ml)
FIG. 1. Standard curves or plots for percentage B0 (above) and the logit transformation (below) vs rat insulin concentrations. The data were pooled from 16 standard curves run in duplicate, with means plotted and 2x the standard error corresponding to each length of line above and below; sample sizes are noted within parentheses. % B, = counts in tubes containing rat insulin standard x 1OOkounts in tubes without.
PURIFICATION
BY
MINICHROMATOGRAPHY
71
trations of similarly prepared serum. This report presents a technique which we found rapid and successful in removing low-molecular-weight interfering substances from large numbers of small-volume samples prior to RIA. We believe that this technique has applicability to preparation of samples for RIA of diverse hormones in serum, other body fluids, tissue extracts, and incubation media. MATERIALS
AND METHODS
of serum. Blood was collected from male Holtzman rats, days of age, following decapitation. Blood in tubes on ice was to clot for 30 min and was then centrifuged (10,OOOg) for 10 supernatan’t fractions were pooled and stored in 0.5-ml aliquots until use. RIA protocol. The antibody preparations and double-antibody assay protocol of “Bio RIA” (Montreal, Canada) were used. lz51-Labeled porcine insulin (Cambridge Nuclear) prepared to contain 15-18,000 counts/O.1 ml was the test tracer. In addition, rat insulin, for use as standard, was kindly supplied by Eli Lilly and Co. Disposable polypropylene tubes (12 x 75 mm, Falcon No. 2053) were used as the assay tubes since they decreased insulin loss from nonspecific binding. A phosphate-buffered albumin solution (0.05 M phosphate, pH 7.4; 0.5% bovine serum albumin, Sigma Chemical Co.) was used for dilutions, and centrifugation at 14,OOOg was employed in the separation of bound from free insulin. The standard curves (Fig. 1) obtained demonstrated reproducible insulin measurements to be possible over a range of about 8.6 to 275 @J/ml of rat insulin standard activities. This illustration shows plots of both percentage of counts bound in the tubes containing no unlabeled insulin (%oB& (above), and the logit transformation (below) vs standard activities in microunits per milliliter for 16 assays performed over a period of several months. Column preparation and use. The essential idea of the minichromatographic procedure derives from “basket centrifugation” (Sephadex manual, Pharmacia Co.). Our adaptation is as follows: (i) A minicolumn is made by placing a very small plug of glass wool into a I-ml Eppendorf disposable pipet tip. (ii) A slurry of Sephadex G-25 (fine grade) is prepared by mixing 1 g of Sephadex/8 ml of assay buffer (containing bovine serum albumin) and preswelling the beads in solution for 3-4 hr at room temperature; 0.5 ml of the slurry is then added to each column. (iii) A rubber band is wrapped around the top of each column and each column is placed within a disposable tube (noted above) identical to that employed in the RIA. The rubber band is to support the column and to thus prevent its tip from resting on the bottom of the tube and retarding outflow. Preparation
48 to 60 allowed min. The at -20°C
72
GORRAY
AND
QUAY
(iv) Each tube containing a column is centrifuged at 1OOOg for 10 minutes to remove the buffer void volume, which is then discarded. (v) A sample, in OS-ml volume, is then added to each column. The assembled column-within-tube is immediately centrifuged at 48g for 10 min to force the progression of the sample through the column. (vi) The assembled unit is then centrifuged again for 10 min, but at lOOOg, to recover the remainder of the sample volume. Validation. Parallelism and recovery studies were done to assess the merits of this procedure for the removal of interfering substances from serum without altering true insulin content. In parallelism trials, three different dilutions of serum (1: 1, 1:3, and 1:7 v/v) were added to the columns and the eluant fractions were assayed for insulin by RIA. The slope of the logit dose-response curve for serum dilution was evaluated with respect to that of rat insulin standards run simultaneously. In the first set of recovery trials, three doses of rat insulin standard were added to a fixed serum dilution, chromatographed, and then assayed (RIA) to determine recovery of the added insulin. In the second set of recovery trials, 1251-labeled insulin was added to two concentrations of serum and the counts in the eluant were compared with the added counts to determine the number of counts lost in the column. The interassay variability of the technique was assessed by measuring the apparent insulin levels in two different concentrations of pooled serum, on different assay days, following chromatographic purification. RESULTS
AND DISCUSSION
The results of parallelism studies are summarized in Table 1. It is seen here that in four separate trials, b values, the slopes of the logit plots, were not significantly different in any case. These results support the contention that rat insulin in the serum was indeed the agent responsible for the inhibition or blocking of binding of the labeled hormone to the first TABLE RESULTS OF FOUR TRIALS OF PARALLELISM DILUTION (THREE TWOFOLD DILUTIONS
Trial No. 1 2 3 4
Slope standard
of rat logit line
-0.9589 -0.6648 -0.6648 -0.5902
1 BETWEEN RAT STANDARD PER CURVE) DOSE-RESPONSE Slope of logit line from three dilutions of serum -0.7040 -0.7569 -0.6561 -0.6340
AND SERUM CURVES
Student’s 0.9650 0.7936 0.0575 0.5160
t
PURIFICATION
BY
TABLE RECOVERY
OF THREE
73
MINICHROMATOGRAPHY 2
DOSES OF RAT INSULIN ADDED TO SERUM BY SEPHADEX MINICHROMATOGRAPHY
Rat insulin added (&J/ml) to I:3 (v/v) serum
No. of replications
68.8 34.4 17.2
AND TREATED
Percent recovered (mean _’ SE) 103.36 109.80 110.40
6 6 5
-+ 1.81 2 12.34 t 10.15
antibody. This conclusion is based on the unlikelihood of the dilution of any other interfering agent altering the dose-response plot in a manner identical to that of rat insulin standard. The results of recovery studies with nonlabeled rat insulin are summarized in Table 2. Rat insulin at three dosages in a 1: 1 (v/v) dilution of serum was essentially recovered fully. However, recoveries of labeled porcine insulin (Table 3) averaged only 76%, although they were highly consistent. The source of this difference remains unexplained, but may be related to species differences or to differences in the behavior of labeled and nonlabeled insulins in the columns. It is also possible that some of the labeled insulin underwent radiolysis due to the y emission of the 1251label. In such an instance the radioactively labeled fragments would have been trapped in the column rather than to have contributed to the counts in the eluate. Ail alternative interpretation meriting mention is that recovery of labeled insulin may have more accurately reflected the amount of insulin lost in the columns, under the assumption that the higher values for recovery TABLE RECOVERY
3
OF COUNTS FOR ?-LABELED INSULIN (PORCINE) ADDED CONCENTRATIONS OF SERUM AND TREATED BY SEPHADEX MINICHROMATOGRAPHY
Solution 0.4 ml of serum
Counts recovered
preparation
+ 0.1 ml of 12JI-labeled
0.2 ml of buffer, 0.2 ml of serum, lZSI-Iabeled insulin
TO Two
insulin
(%)
73.11 74.82
+ 0.1 ml of 76.54 80.68 76.36 76.30 2 1.26 (mean _’ SE)
74
GORRAY
AND QUAY
of nonlabeled insulin in the assays were due to incomplete removal of the interfering substance(s), leading to a 25% increase in apparent insulin levels. However, this interpretation seems untenable in view of the consistently close agreement between the observed mean values for three different doses of nonlabeled hormone and the expected value near 100%. Furthermore, the interassay variability of the system was low. Coefficients of variation from undiluted and 1:7 diluted serum samples run in duplicate were 5.72 and 23.13%, respectively. This finding also speaks against the possibility of only very incomplete removal of interfering substance(s), since considerably higher interassay variability would be expected if such substances remained. These results demonstrate the utility of employing minicolumns of Sephadex to cleanse from serum samples interfering substances which have a lower molecular weight than insulin. The eluates can then be taken directly to RIA without further difficulties in terms of interference or cross-reactivity. It may be noted that although Sephadex chromatography systems have been employed previously for the pretreatment of samples for RIA of both insulin (8) and other hormones (9), the “basket centrifugation” technique and other adaptations described here have not been presented for such purposes previously insofar as we are aware. Most importantly, however, the recoveries of nonlabeled insulin in our system (approximately 107%) averaged considerably higher than those (near 80%) generally reported in the literature for the extraction of insulin from antibody-containing serum, either by ethanol (10) or polyethylene glycol (11). The technique reported here is useful only in situations in which the compound of interest is notably higher in molecular weight than interfering contaminants or heterologous forms or derivatives. In the case of larger interfering molecules, other chromatographic or separation systems may be effective for this purpose. ACKNOWLEDGMENTS We are grateful to our colleagues in the Neuroendocrine Section, especially Drs. H. Karavolas and K. MacKenzie, for use of, and advice concerning, some RIA procedures. Appreciation is expressed to the University of Wisconsin, Madison, Graduate School for fellowship support (to K.C.G.), and to the National Institute of Mental Health (MN25091), the National Institutes of Health (HD-03352, HD-10263, 5-TOl-HD-00104-lo), USPHS, and the National Science Foundation (No. PCM 76-09953) for research grant support (to W.B.Q.).
REFERENCES 1. Yalow, R., and Berson, S. (1971) in Principles of Competitive Protein Binding Assays (W. Odell, and W. Daughaday, eds.), 1st ed.. p. 398, Lippincott, Philadelphia.
PURIFICATION
BY MINICHROMATOGRAPHY
75
2. Cuatrecasas, P., and Hollenberg, M. D. (1975) Biochem. Bioiphys. Res. Commun. 62, 31-41. 3. Piquet, C., Guidoux, R., and Peters, G. (1976) Experienria 32, 781. 4. Felix, J., Sutter-Dub, M., Legrele, C., Billandel, B., Sutter, B., and Jacquot, R. (1975) Pathol. Biol. 23, 847-848. 5. Schoeffling, K., Ditschumeit, H., Pitzouldt, R.. Beyer, J., Pfeiffer, E. F., Sirek, A,, Geerling, H., and Sirek, 0. V. (1965) Diabetes 14, 658-662. 6. Yalow, R., and Berson, S. (1971) in Principles of Competitive Protein Binding Assays (W. Odell, and W. Daughaday, eds.), 1st ed., pp. 388-390, Lippincott. Philadelphia. 7. Costantini, A., Lostia, O., Malvano, R., Rolleri, E.. Taggi, F.. and Zucchelli, G. (1975) J. Nucl. Biol. Med. 19, 164-176. 8. Davidson, P., and Dean, R. B. (1976) J. Lab. Clin. Med. 87, 1050- 1056. 9. Nuti, L. C.. McShan, W. H., and Meyer, R. K. (1974) Endocrinology 95, 682-689. 10. Heding, L. G. (1969) Horm. Metab. Res. 1, 14% 146. 11. Nakagawa, S., Nakayama, H., Sasaki, T., Yoshino, K., Yu, Y. Y., Shinozaki, K., Aok, S., and Mashino, K. (1973) Diabetes 22, 590-600.
BIOCHEMISTRY
82, 69-75 (1977)
Rapid Minichromatography for Purification Prior to Radioimmunoassay KENNETH Endocrinology-Reproductive Neuroendocrinology Human Development,
of Samples
C. GORRAY AND W. B. QUAY’ Physiology Program, Section, Waisman Center University of Wisconsin,
Department of Zoology, and on Mental Retardation and Madison, Wisconsin 53706
Received February 28, 1977; accepted May 23, 1977 A method is presented for the treatment of serum or other biological fluids to remove interfering substances prior to radioimmunoassay. Studies with radioimmunoassay systems for measuring serum levels of insulin revealed the presence of such interfering substances, which could be removed by utilizing small columns of Sephadex G-25 prepared in disposable pipet tips. The columns were centrifuged at low, and finally, higher speeds, to recover the sample volume with essentially no alteration in insulin activity. Validity of the technique was evaluated by both parallelism and recovery studies. This system offers considerable advantages over previous extraction and chromatography procedures in terms of simplicity, reproducibility, and recovery.
The valid radioimmunoassay (RIA) of any one of a number of hormones is made difficult by the frequent presence in biological samples and fluids of endogenous interfering substances. It has been concluded that such heterogeneity of reacting species “may or may not require the fractionation of standards or unknowns in order to insure the validation of an assay” (1). We and other investigators have found this need especially true in the case of RIA of insulin. At least some of the factors and sources of the difficulties can be suggested: (A) high levels of nonspecific binding to various substances (2); (B) degradation of the insulin molecule in plasma or serum with the subsequent binding of insulin fragments to large serum proteins and the survival of their immunoreactivity in some RIA procedures (3,4); (C) the presence of insulin-like activity (ILA) in totally pancreatectomized animals (5) and including sometimes proinsulin (6). The extent of the difficulty posed by such factors is epitomized by a summary of the results of RIA of aliquots of insulin standards by 36 reputable Italian laboratories. Although within-assay variability was at an acceptable level (approximately IO%), betweenr Send reprint requests to K. C. Gorray, 481 Medical Sciences, Department of Medicine, University of Wisconsin Medical School, Madison, Wis. 53706. 69 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0003.2697
70
GORRAY
AND QUAY
laboratory variability was 0.5 to 1.5 times the median value of the unknowns (7). The presence of low-molecular-weight interfering substances in normal rat serum became apparent during our studies using RIA systems for insulin. One or both binding reactions were interfered with, and the factor(s) involved could not be removed with ethanol or polyethylene glycol extraction procedures. Nor could the effects of the interference be bypassed by utilization of “standard curves” containing low concenIOO-
60-
20-
(32)
RAT
INSULIN
ACTIVITY
(pU/ml)
FIG. 1. Standard curves or plots for percentage B0 (above) and the logit transformation (below) vs rat insulin concentrations. The data were pooled from 16 standard curves run in duplicate, with means plotted and 2x the standard error corresponding to each length of line above and below; sample sizes are noted within parentheses. % B, = counts in tubes containing rat insulin standard x 1OOkounts in tubes without.
PURIFICATION
BY
MINICHROMATOGRAPHY
71
trations of similarly prepared serum. This report presents a technique which we found rapid and successful in removing low-molecular-weight interfering substances from large numbers of small-volume samples prior to RIA. We believe that this technique has applicability to preparation of samples for RIA of diverse hormones in serum, other body fluids, tissue extracts, and incubation media. MATERIALS
AND METHODS
of serum. Blood was collected from male Holtzman rats, days of age, following decapitation. Blood in tubes on ice was to clot for 30 min and was then centrifuged (10,OOOg) for 10 supernatan’t fractions were pooled and stored in 0.5-ml aliquots until use. RIA protocol. The antibody preparations and double-antibody assay protocol of “Bio RIA” (Montreal, Canada) were used. lz51-Labeled porcine insulin (Cambridge Nuclear) prepared to contain 15-18,000 counts/O.1 ml was the test tracer. In addition, rat insulin, for use as standard, was kindly supplied by Eli Lilly and Co. Disposable polypropylene tubes (12 x 75 mm, Falcon No. 2053) were used as the assay tubes since they decreased insulin loss from nonspecific binding. A phosphate-buffered albumin solution (0.05 M phosphate, pH 7.4; 0.5% bovine serum albumin, Sigma Chemical Co.) was used for dilutions, and centrifugation at 14,OOOg was employed in the separation of bound from free insulin. The standard curves (Fig. 1) obtained demonstrated reproducible insulin measurements to be possible over a range of about 8.6 to 275 @J/ml of rat insulin standard activities. This illustration shows plots of both percentage of counts bound in the tubes containing no unlabeled insulin (%oB& (above), and the logit transformation (below) vs standard activities in microunits per milliliter for 16 assays performed over a period of several months. Column preparation and use. The essential idea of the minichromatographic procedure derives from “basket centrifugation” (Sephadex manual, Pharmacia Co.). Our adaptation is as follows: (i) A minicolumn is made by placing a very small plug of glass wool into a I-ml Eppendorf disposable pipet tip. (ii) A slurry of Sephadex G-25 (fine grade) is prepared by mixing 1 g of Sephadex/8 ml of assay buffer (containing bovine serum albumin) and preswelling the beads in solution for 3-4 hr at room temperature; 0.5 ml of the slurry is then added to each column. (iii) A rubber band is wrapped around the top of each column and each column is placed within a disposable tube (noted above) identical to that employed in the RIA. The rubber band is to support the column and to thus prevent its tip from resting on the bottom of the tube and retarding outflow. Preparation
48 to 60 allowed min. The at -20°C
72
GORRAY
AND
QUAY
(iv) Each tube containing a column is centrifuged at 1OOOg for 10 minutes to remove the buffer void volume, which is then discarded. (v) A sample, in OS-ml volume, is then added to each column. The assembled column-within-tube is immediately centrifuged at 48g for 10 min to force the progression of the sample through the column. (vi) The assembled unit is then centrifuged again for 10 min, but at lOOOg, to recover the remainder of the sample volume. Validation. Parallelism and recovery studies were done to assess the merits of this procedure for the removal of interfering substances from serum without altering true insulin content. In parallelism trials, three different dilutions of serum (1: 1, 1:3, and 1:7 v/v) were added to the columns and the eluant fractions were assayed for insulin by RIA. The slope of the logit dose-response curve for serum dilution was evaluated with respect to that of rat insulin standards run simultaneously. In the first set of recovery trials, three doses of rat insulin standard were added to a fixed serum dilution, chromatographed, and then assayed (RIA) to determine recovery of the added insulin. In the second set of recovery trials, 1251-labeled insulin was added to two concentrations of serum and the counts in the eluant were compared with the added counts to determine the number of counts lost in the column. The interassay variability of the technique was assessed by measuring the apparent insulin levels in two different concentrations of pooled serum, on different assay days, following chromatographic purification. RESULTS
AND DISCUSSION
The results of parallelism studies are summarized in Table 1. It is seen here that in four separate trials, b values, the slopes of the logit plots, were not significantly different in any case. These results support the contention that rat insulin in the serum was indeed the agent responsible for the inhibition or blocking of binding of the labeled hormone to the first TABLE RESULTS OF FOUR TRIALS OF PARALLELISM DILUTION (THREE TWOFOLD DILUTIONS
Trial No. 1 2 3 4
Slope standard
of rat logit line
-0.9589 -0.6648 -0.6648 -0.5902
1 BETWEEN RAT STANDARD PER CURVE) DOSE-RESPONSE Slope of logit line from three dilutions of serum -0.7040 -0.7569 -0.6561 -0.6340
AND SERUM CURVES
Student’s 0.9650 0.7936 0.0575 0.5160
t
PURIFICATION
BY
TABLE RECOVERY
OF THREE
73
MINICHROMATOGRAPHY 2
DOSES OF RAT INSULIN ADDED TO SERUM BY SEPHADEX MINICHROMATOGRAPHY
Rat insulin added (&J/ml) to I:3 (v/v) serum
No. of replications
68.8 34.4 17.2
AND TREATED
Percent recovered (mean _’ SE) 103.36 109.80 110.40
6 6 5
-+ 1.81 2 12.34 t 10.15
antibody. This conclusion is based on the unlikelihood of the dilution of any other interfering agent altering the dose-response plot in a manner identical to that of rat insulin standard. The results of recovery studies with nonlabeled rat insulin are summarized in Table 2. Rat insulin at three dosages in a 1: 1 (v/v) dilution of serum was essentially recovered fully. However, recoveries of labeled porcine insulin (Table 3) averaged only 76%, although they were highly consistent. The source of this difference remains unexplained, but may be related to species differences or to differences in the behavior of labeled and nonlabeled insulins in the columns. It is also possible that some of the labeled insulin underwent radiolysis due to the y emission of the 1251label. In such an instance the radioactively labeled fragments would have been trapped in the column rather than to have contributed to the counts in the eluate. Ail alternative interpretation meriting mention is that recovery of labeled insulin may have more accurately reflected the amount of insulin lost in the columns, under the assumption that the higher values for recovery TABLE RECOVERY
3
OF COUNTS FOR ?-LABELED INSULIN (PORCINE) ADDED CONCENTRATIONS OF SERUM AND TREATED BY SEPHADEX MINICHROMATOGRAPHY
Solution 0.4 ml of serum
Counts recovered
preparation
+ 0.1 ml of 12JI-labeled
0.2 ml of buffer, 0.2 ml of serum, lZSI-Iabeled insulin
TO Two
insulin
(%)
73.11 74.82
+ 0.1 ml of 76.54 80.68 76.36 76.30 2 1.26 (mean _’ SE)
74
GORRAY
AND QUAY
of nonlabeled insulin in the assays were due to incomplete removal of the interfering substance(s), leading to a 25% increase in apparent insulin levels. However, this interpretation seems untenable in view of the consistently close agreement between the observed mean values for three different doses of nonlabeled hormone and the expected value near 100%. Furthermore, the interassay variability of the system was low. Coefficients of variation from undiluted and 1:7 diluted serum samples run in duplicate were 5.72 and 23.13%, respectively. This finding also speaks against the possibility of only very incomplete removal of interfering substance(s), since considerably higher interassay variability would be expected if such substances remained. These results demonstrate the utility of employing minicolumns of Sephadex to cleanse from serum samples interfering substances which have a lower molecular weight than insulin. The eluates can then be taken directly to RIA without further difficulties in terms of interference or cross-reactivity. It may be noted that although Sephadex chromatography systems have been employed previously for the pretreatment of samples for RIA of both insulin (8) and other hormones (9), the “basket centrifugation” technique and other adaptations described here have not been presented for such purposes previously insofar as we are aware. Most importantly, however, the recoveries of nonlabeled insulin in our system (approximately 107%) averaged considerably higher than those (near 80%) generally reported in the literature for the extraction of insulin from antibody-containing serum, either by ethanol (10) or polyethylene glycol (11). The technique reported here is useful only in situations in which the compound of interest is notably higher in molecular weight than interfering contaminants or heterologous forms or derivatives. In the case of larger interfering molecules, other chromatographic or separation systems may be effective for this purpose. ACKNOWLEDGMENTS We are grateful to our colleagues in the Neuroendocrine Section, especially Drs. H. Karavolas and K. MacKenzie, for use of, and advice concerning, some RIA procedures. Appreciation is expressed to the University of Wisconsin, Madison, Graduate School for fellowship support (to K.C.G.), and to the National Institute of Mental Health (MN25091), the National Institutes of Health (HD-03352, HD-10263, 5-TOl-HD-00104-lo), USPHS, and the National Science Foundation (No. PCM 76-09953) for research grant support (to W.B.Q.).
REFERENCES 1. Yalow, R., and Berson, S. (1971) in Principles of Competitive Protein Binding Assays (W. Odell, and W. Daughaday, eds.), 1st ed.. p. 398, Lippincott, Philadelphia.
PURIFICATION
BY MINICHROMATOGRAPHY
75
2. Cuatrecasas, P., and Hollenberg, M. D. (1975) Biochem. Bioiphys. Res. Commun. 62, 31-41. 3. Piquet, C., Guidoux, R., and Peters, G. (1976) Experienria 32, 781. 4. Felix, J., Sutter-Dub, M., Legrele, C., Billandel, B., Sutter, B., and Jacquot, R. (1975) Pathol. Biol. 23, 847-848. 5. Schoeffling, K., Ditschumeit, H., Pitzouldt, R.. Beyer, J., Pfeiffer, E. F., Sirek, A,, Geerling, H., and Sirek, 0. V. (1965) Diabetes 14, 658-662. 6. Yalow, R., and Berson, S. (1971) in Principles of Competitive Protein Binding Assays (W. Odell, and W. Daughaday, eds.), 1st ed., pp. 388-390, Lippincott. Philadelphia. 7. Costantini, A., Lostia, O., Malvano, R., Rolleri, E.. Taggi, F.. and Zucchelli, G. (1975) J. Nucl. Biol. Med. 19, 164-176. 8. Davidson, P., and Dean, R. B. (1976) J. Lab. Clin. Med. 87, 1050- 1056. 9. Nuti, L. C.. McShan, W. H., and Meyer, R. K. (1974) Endocrinology 95, 682-689. 10. Heding, L. G. (1969) Horm. Metab. Res. 1, 14% 146. 11. Nakagawa, S., Nakayama, H., Sasaki, T., Yoshino, K., Yu, Y. Y., Shinozaki, K., Aok, S., and Mashino, K. (1973) Diabetes 22, 590-600.