Biochem. J.:(1975) 145, 607-616 Printed in Great Britain
607
Inmunoassay of Serum Polypeptide Hormones by Using '25I-Labelled Anti-(Immunoglobulin G) Antibodies By PETER BECK and C. NICHOLAS HALES Department ofMedical Biochemistry, The Welsh National School ofMedicine, Heath Park, Cardiff CF4 4XN, U.K. (Received 20 August 1974) 1. A technique for indirectly labelling antibodies to polypeptide hormones, by combining them with radioactively labelled anti-(immunoglobulin G) is described. (a) 125I-labelled anti-(rabbit immunoglobulin G) and anti-(guinea-pig immunoglobulin G) antibodies with high specific radioactivity were prepared after purification of the antibodies on immunoadsorbents containing the respective antigens. (b) Rabbit immunoglobulin G antibodies to human growth hormone, porcine glucagon and guinea-pig immunoglobulin G antibodies to bovine insulin and bovine parathyroid hormone were combined with immunoadsorbents containing the respective polypeptide hormone antigen. (c) The immunoglubulin G antibodies to the polypeptide hormones were reacted with 1251-labelled anti-(immunoglobulin G) antibodies directed against the appropriate species of immunoglobulin G, and the anti-hormone antibodies were combined with the hormone-containing immunoadsorbent. (d) 1251-labelledanti-(immunoglobulinG)antibodiesandanti-hormone antibodies were simultaneously eluted from the hormone-containing immunoadsorbent by dilute HCI, pH 2.0. After elution the anti-(immunoglobulin G) antibodies and antihormone antibodies were allowed to recombine at pH 8.0 and 4°C. 2. The resultant immunoglobulin G-anti-immunoglobulin G complex was used in immunoradiometric (labelled antibody) and two-site assays of the respective polypeptide hormone. 3. By using these immunoassays, concentrations down to 90pg of human growth hormone/ml, lOOpg of bovine insulin/ml, 80pg of bovine parathyroid hormone/ml and 15Opg of glucagon/ml were readily detected. Assays of human plasma for growth hormone and insulin by these methods showed good agreement with results obtained by using a directly '251-labelled anti-hormone antibody in an immunoradiometric assay of human growth hormone or by radioimmunoassay of human insulin. 4. The method described allows immunoradiometric or two-site assays to be performed starting with as little as 450ng of polypeptide hormone-antibody protein. An additional advantage of the method is that a single iodination of the readily available antibodies to immunoglubulin G allows the establishment of several polypeptide hormone assays. The preparation and properties of purified antibodies labelled to high specific radioactivity with radioactive iodine have been described (Miles & Hales, 1968b). The theoretical considerations leading to the use of labelled antibodies in sensitive assay systems have been discussed (Miles & Hales, 1968a; Rodbard & Weiss, 1973) and assays using such labelled antibodies have now been described for many polypeptide hormones as well as for other proteins of both low and high molecular weight (Addison et al., 1971). An important factor limiting the general application of the technique is the requirement for approximately 10g of purified antibody to achieve a satisfactory iodination. Antisera suitable for sensitive and specific immunoassays are often available in only small amounts because of Vol. 145
difficulties in raising specific high-affinity antibodies to certain polypeptides. This paper describes
a
technique for indirectly labelling antibodies to polypeptide hormones by combining them with iodinated anti-IgG* antibodies directed against the immunoglobulin ofthe species in which the antiserum to the polypeptide hormone was raised. The antihormone IgG-anti-IgG complex can then be used as a reagent in a labelled antibody (immunoradiometric) assay. In addition to being more economical in the use of antisera to polypeptide hormones the method allows a preparation of one iodinated material to be used for the establishment of a number of different assays. *
Abbreviation: IgG, immunoglobulin G.
6fl8
Materials General Carrier-free 125I at a concentration of lOOmCi/ml and specificradioactivity ofapproximately 10mCi/,ug was obtained as preparation 1MS3 from The Radiochemical Centre, Amersham, Bucks., U.K. Whatman CC41 powdered cellulose was obtained from W. and R. Balston Ltd., Maidstone, Kent, U.K.; bovine plasma albumin (fraction V) from Armour Pharmaceutical Co. Ltd., Eastbourne, Sussex, U.K., and Folin & Ciocalteu's Reagent and m-nitrobenzyloxymethylpyridinium chloride from BDH Chemicals Ltd., Poole, Dorset, U.K. Other chemicals were of analytical grade. Disposable polyethylene and polypropylene microcentrifuge tubes were obtained from Beckman-RIIC Ltd., Croydon, Surrey, U.K. Hormones
Human growth hormone was obtained as W.H.O. First I.R.P. standard from the Medical Research Council Division of Biological Standards, National Institute for Medical Research, Hampstead Laboratories, London NW3 6RB, U.K. Crystalline bovine insulin (25 i.u./mg) was obtained from Sigma (London) Chemical Co. Ltd., Kingstonupon-Thames, Surrey, U.K. Porcine glucagon and highly purified bovine parathyroid hormone were the gift of Dr. J. S. Woodhead of this department.
Antisera Antisera to human growth hormone were raised in rabbits by Dr. G. M. Addison of this department and in guinea pigs by Dr. A. R. Boyns of this department. Antisera to bovine insulin were raised in guinea pigs by the method of Robinson & Wright (1961) and pooled. Guinea-pig anti-(bovine parathyroid hormone) serum and rabbit anti-glucagon serum were kindly given by Dr. J. S. Woodhead of this department. Donkey anti-(rabbit serum) serum (lot K.661 1) and rabbit anti-(guinea-pig serum) serum (lot K.6192) were obtained from Wellcome Reagents Ltd., Wellcome Research Laboratories, Beckenham, Kent, U.K. Methods and Results Assay ofprotein concentrations The determination of hormone and antibody protein concentrations were carried out on immunoadsorbents by the method of Lowry et al. (1951), with a bovine plasma albumin standard and after removal of the insoluble matrix by centrifugation before measuring the change in absorbance.
P. BECK AND C. N. HALES
Measurement ofradioactivity The radioactivity of samples containing 125I was counted in a Wallac Decem GTL 300 automatic gamma counter (counting efficiency approx. 50%). Preparation of immunoadsorbents Immunoadsorbents were made by coupling proteins to diazonium derivatives of powdered cellulose by the method of Miles & Hales (1968b). Immunoadsorbents were prepared containing non-immune rabbit IgG, non-immune guinea-pig IgG, human growth hormone, crystalline bovine insulin, porcine glucagon and bovine parathyroid hormone. The protein/cellulose ratios obtained for each of these preparations were respectively: non-immune rabbit IgG, 270mg/g of matrix; non-immune guinea-pig IgG, 250mg/g of matrix; human growth hormone, 100mg/g of matrix; insulin, 240mg/g of matrix; glucagon, 88mg/g of matrix and bovine parathyroid hormone, 27mg/g of matrix. All the immunoadsorbents were suspended in 0.05M-veronal buffer, pH8.0, containing 5g of bovine plasma albumin/litre, 6g of NaCl/litre, 200mg of sodium azide/litre and 25mg of non-immune horse IgG/litre (veronalalbumin-azide buffer), at a concentration of 1 mg of cellulose matrix/ml of buffer.
Preparation and properties of "25I-labelled anti-IgG antibodies Anti-IgG antibodies were isolated from appropriate antisera on to IgG immunoadsorbents and were iodinated while thus bound by a modification of the chloramine-T method of Greenwood et al. (1963) as previously described (Addison etal., 1971). Rabbit or guinea-pig IgG immunoadsorbents (0.5-1 mg of cellulose base) were saturated with the corresponding anti-IgG by incubating with 0.5-1 ml of antiserum for 3-4 days at 4°C. After washing six times with 0.05M-sodium phosphate buffer, pH7.4, containing 9g of NaCl/litre, the protein uptake on to the immunoadsorbent was measured. An amount of the IgG immunoadsorbent-anti-IgG complex containing 50-2004ug of anti-IgG protein was concentrated by centrifugation and aspiration of the supernatant to a volume of 10-201l for iodination, which was carried out by using 25,ug of chloramine-T and 1-2mCi of 1251. The 1251-labelled anti-IgG-IgG immunoadsorbent complex was then washed in a filter funnel containing a double thickness of filter paper, with 200ml of 0.05 M-veronal-albumin-azide buffer, pH 8.0. Low-affinity antibodies were then eluted with 100ml of HC1, pH 3.0, and finally highaffinity antibodies were eluted with two portions of 4ml of HCl, pH2.0, directly into equal volumes of double-strength(0.1 M)veronal-albumin-azide buffer; a further 100ml of HCI, pH 2.0, was washed through 1975
609
IMMUNOASSAY WITH 125I-LABELLED ANTI-IgG ANTIBODY
the filter paper to give maximum recovery ofantibody still bound. Samples (10,ul) of these fractions were counted for radioactivity in order to calculate the approximate efficiency of the iodination. More than 80 % ofthe radioactivity was recovered in the washings with 28-35% present in the small pH 2.0 fractions and 10-15% in the large-volume pH2.0 wash. The specific radioactivities (calculated by assuming that the radioactivity in the proteins eluted with dilute HCI was bound to the antibody protein) were between 1.0 and lOmCi/mg of antibody. The iodinated antibodies in the small pH 2.0 fractions were subsequently recombined with the appropriate IgG immunoadsorbent and stored in portions at -20°C as described (Addison & Hales, 1971a,b). Up to 92% of the radioactivity in these fractions could be bound to IgG immunoadsorbent with various preparations by using anti-(rabbit IgG) and anti-(guinea-pig IgG); the amount binding to a similarly prepared bovine plasma albumin immunoadsorbent was always less than 5 %. The '25I-labelled anti-IgG antibodies were later eluted and assayed against IgG standards in an immunoradiometric assay (Miles & Hales, 1968c). A typical standard curve obtained in such an assay is shown in Fig. 1. The incubation time was 18 h at
4°C, the total radioactivity was 33 d.p.s. and the binding to immunoadsorbent in the absence of added antigen was 86 %. Preparation ofhuman growth hormone double antibody The principle of the preparation is that human growth hormone immunoadsorbent is used to isolate specific anti-(human growth hormone) antibodies from a rabbit antiserum. The 125i1 labelled anti-(rabbit IgG) is eluted from its immunoadsorbent and incubated with the rabbit anti-(human growth hormone) antibody-human growth hormone immunoadsorbent complex. A subsequent acid elution splits off both antibodies, which are then allowed to recombine, off immunoadsorbent, to give an anti-hormone IgG-anti-IgG complex which can be used as a reagent in an immunoradiometric (labelled antibody) assay (Fig. 2).
Anti-
Anti-lgG
hormone IgG Antigen (hormone) immunoadsorbent Hormone
antibody
tgG immunoadsorbent tgG
I
+> 28t > +
Cellulose- _ >X ._.
90
700 ) r
600
rI sotation of
Isolation of santi-hormome leo
anti-tgG tgG
607
Inmunoassay of Serum Polypeptide Hormones by Using '25I-Labelled Anti-(Immunoglobulin G) Antibodies By PETER BECK and C. NICHOLAS HALES Department ofMedical Biochemistry, The Welsh National School ofMedicine, Heath Park, Cardiff CF4 4XN, U.K. (Received 20 August 1974) 1. A technique for indirectly labelling antibodies to polypeptide hormones, by combining them with radioactively labelled anti-(immunoglobulin G) is described. (a) 125I-labelled anti-(rabbit immunoglobulin G) and anti-(guinea-pig immunoglobulin G) antibodies with high specific radioactivity were prepared after purification of the antibodies on immunoadsorbents containing the respective antigens. (b) Rabbit immunoglobulin G antibodies to human growth hormone, porcine glucagon and guinea-pig immunoglobulin G antibodies to bovine insulin and bovine parathyroid hormone were combined with immunoadsorbents containing the respective polypeptide hormone antigen. (c) The immunoglubulin G antibodies to the polypeptide hormones were reacted with 1251-labelled anti-(immunoglobulin G) antibodies directed against the appropriate species of immunoglobulin G, and the anti-hormone antibodies were combined with the hormone-containing immunoadsorbent. (d) 1251-labelledanti-(immunoglobulinG)antibodiesandanti-hormone antibodies were simultaneously eluted from the hormone-containing immunoadsorbent by dilute HCI, pH 2.0. After elution the anti-(immunoglobulin G) antibodies and antihormone antibodies were allowed to recombine at pH 8.0 and 4°C. 2. The resultant immunoglobulin G-anti-immunoglobulin G complex was used in immunoradiometric (labelled antibody) and two-site assays of the respective polypeptide hormone. 3. By using these immunoassays, concentrations down to 90pg of human growth hormone/ml, lOOpg of bovine insulin/ml, 80pg of bovine parathyroid hormone/ml and 15Opg of glucagon/ml were readily detected. Assays of human plasma for growth hormone and insulin by these methods showed good agreement with results obtained by using a directly '251-labelled anti-hormone antibody in an immunoradiometric assay of human growth hormone or by radioimmunoassay of human insulin. 4. The method described allows immunoradiometric or two-site assays to be performed starting with as little as 450ng of polypeptide hormone-antibody protein. An additional advantage of the method is that a single iodination of the readily available antibodies to immunoglubulin G allows the establishment of several polypeptide hormone assays. The preparation and properties of purified antibodies labelled to high specific radioactivity with radioactive iodine have been described (Miles & Hales, 1968b). The theoretical considerations leading to the use of labelled antibodies in sensitive assay systems have been discussed (Miles & Hales, 1968a; Rodbard & Weiss, 1973) and assays using such labelled antibodies have now been described for many polypeptide hormones as well as for other proteins of both low and high molecular weight (Addison et al., 1971). An important factor limiting the general application of the technique is the requirement for approximately 10g of purified antibody to achieve a satisfactory iodination. Antisera suitable for sensitive and specific immunoassays are often available in only small amounts because of Vol. 145
difficulties in raising specific high-affinity antibodies to certain polypeptides. This paper describes
a
technique for indirectly labelling antibodies to polypeptide hormones by combining them with iodinated anti-IgG* antibodies directed against the immunoglobulin ofthe species in which the antiserum to the polypeptide hormone was raised. The antihormone IgG-anti-IgG complex can then be used as a reagent in a labelled antibody (immunoradiometric) assay. In addition to being more economical in the use of antisera to polypeptide hormones the method allows a preparation of one iodinated material to be used for the establishment of a number of different assays. *
Abbreviation: IgG, immunoglobulin G.
6fl8
Materials General Carrier-free 125I at a concentration of lOOmCi/ml and specificradioactivity ofapproximately 10mCi/,ug was obtained as preparation 1MS3 from The Radiochemical Centre, Amersham, Bucks., U.K. Whatman CC41 powdered cellulose was obtained from W. and R. Balston Ltd., Maidstone, Kent, U.K.; bovine plasma albumin (fraction V) from Armour Pharmaceutical Co. Ltd., Eastbourne, Sussex, U.K., and Folin & Ciocalteu's Reagent and m-nitrobenzyloxymethylpyridinium chloride from BDH Chemicals Ltd., Poole, Dorset, U.K. Other chemicals were of analytical grade. Disposable polyethylene and polypropylene microcentrifuge tubes were obtained from Beckman-RIIC Ltd., Croydon, Surrey, U.K. Hormones
Human growth hormone was obtained as W.H.O. First I.R.P. standard from the Medical Research Council Division of Biological Standards, National Institute for Medical Research, Hampstead Laboratories, London NW3 6RB, U.K. Crystalline bovine insulin (25 i.u./mg) was obtained from Sigma (London) Chemical Co. Ltd., Kingstonupon-Thames, Surrey, U.K. Porcine glucagon and highly purified bovine parathyroid hormone were the gift of Dr. J. S. Woodhead of this department.
Antisera Antisera to human growth hormone were raised in rabbits by Dr. G. M. Addison of this department and in guinea pigs by Dr. A. R. Boyns of this department. Antisera to bovine insulin were raised in guinea pigs by the method of Robinson & Wright (1961) and pooled. Guinea-pig anti-(bovine parathyroid hormone) serum and rabbit anti-glucagon serum were kindly given by Dr. J. S. Woodhead of this department. Donkey anti-(rabbit serum) serum (lot K.661 1) and rabbit anti-(guinea-pig serum) serum (lot K.6192) were obtained from Wellcome Reagents Ltd., Wellcome Research Laboratories, Beckenham, Kent, U.K. Methods and Results Assay ofprotein concentrations The determination of hormone and antibody protein concentrations were carried out on immunoadsorbents by the method of Lowry et al. (1951), with a bovine plasma albumin standard and after removal of the insoluble matrix by centrifugation before measuring the change in absorbance.
P. BECK AND C. N. HALES
Measurement ofradioactivity The radioactivity of samples containing 125I was counted in a Wallac Decem GTL 300 automatic gamma counter (counting efficiency approx. 50%). Preparation of immunoadsorbents Immunoadsorbents were made by coupling proteins to diazonium derivatives of powdered cellulose by the method of Miles & Hales (1968b). Immunoadsorbents were prepared containing non-immune rabbit IgG, non-immune guinea-pig IgG, human growth hormone, crystalline bovine insulin, porcine glucagon and bovine parathyroid hormone. The protein/cellulose ratios obtained for each of these preparations were respectively: non-immune rabbit IgG, 270mg/g of matrix; non-immune guinea-pig IgG, 250mg/g of matrix; human growth hormone, 100mg/g of matrix; insulin, 240mg/g of matrix; glucagon, 88mg/g of matrix and bovine parathyroid hormone, 27mg/g of matrix. All the immunoadsorbents were suspended in 0.05M-veronal buffer, pH8.0, containing 5g of bovine plasma albumin/litre, 6g of NaCl/litre, 200mg of sodium azide/litre and 25mg of non-immune horse IgG/litre (veronalalbumin-azide buffer), at a concentration of 1 mg of cellulose matrix/ml of buffer.
Preparation and properties of "25I-labelled anti-IgG antibodies Anti-IgG antibodies were isolated from appropriate antisera on to IgG immunoadsorbents and were iodinated while thus bound by a modification of the chloramine-T method of Greenwood et al. (1963) as previously described (Addison etal., 1971). Rabbit or guinea-pig IgG immunoadsorbents (0.5-1 mg of cellulose base) were saturated with the corresponding anti-IgG by incubating with 0.5-1 ml of antiserum for 3-4 days at 4°C. After washing six times with 0.05M-sodium phosphate buffer, pH7.4, containing 9g of NaCl/litre, the protein uptake on to the immunoadsorbent was measured. An amount of the IgG immunoadsorbent-anti-IgG complex containing 50-2004ug of anti-IgG protein was concentrated by centrifugation and aspiration of the supernatant to a volume of 10-201l for iodination, which was carried out by using 25,ug of chloramine-T and 1-2mCi of 1251. The 1251-labelled anti-IgG-IgG immunoadsorbent complex was then washed in a filter funnel containing a double thickness of filter paper, with 200ml of 0.05 M-veronal-albumin-azide buffer, pH 8.0. Low-affinity antibodies were then eluted with 100ml of HC1, pH 3.0, and finally highaffinity antibodies were eluted with two portions of 4ml of HCl, pH2.0, directly into equal volumes of double-strength(0.1 M)veronal-albumin-azide buffer; a further 100ml of HCI, pH 2.0, was washed through 1975
609
IMMUNOASSAY WITH 125I-LABELLED ANTI-IgG ANTIBODY
the filter paper to give maximum recovery ofantibody still bound. Samples (10,ul) of these fractions were counted for radioactivity in order to calculate the approximate efficiency of the iodination. More than 80 % ofthe radioactivity was recovered in the washings with 28-35% present in the small pH 2.0 fractions and 10-15% in the large-volume pH2.0 wash. The specific radioactivities (calculated by assuming that the radioactivity in the proteins eluted with dilute HCI was bound to the antibody protein) were between 1.0 and lOmCi/mg of antibody. The iodinated antibodies in the small pH 2.0 fractions were subsequently recombined with the appropriate IgG immunoadsorbent and stored in portions at -20°C as described (Addison & Hales, 1971a,b). Up to 92% of the radioactivity in these fractions could be bound to IgG immunoadsorbent with various preparations by using anti-(rabbit IgG) and anti-(guinea-pig IgG); the amount binding to a similarly prepared bovine plasma albumin immunoadsorbent was always less than 5 %. The '25I-labelled anti-IgG antibodies were later eluted and assayed against IgG standards in an immunoradiometric assay (Miles & Hales, 1968c). A typical standard curve obtained in such an assay is shown in Fig. 1. The incubation time was 18 h at
4°C, the total radioactivity was 33 d.p.s. and the binding to immunoadsorbent in the absence of added antigen was 86 %. Preparation ofhuman growth hormone double antibody The principle of the preparation is that human growth hormone immunoadsorbent is used to isolate specific anti-(human growth hormone) antibodies from a rabbit antiserum. The 125i1 labelled anti-(rabbit IgG) is eluted from its immunoadsorbent and incubated with the rabbit anti-(human growth hormone) antibody-human growth hormone immunoadsorbent complex. A subsequent acid elution splits off both antibodies, which are then allowed to recombine, off immunoadsorbent, to give an anti-hormone IgG-anti-IgG complex which can be used as a reagent in an immunoradiometric (labelled antibody) assay (Fig. 2).
Anti-
Anti-lgG
hormone IgG Antigen (hormone) immunoadsorbent Hormone
antibody
tgG immunoadsorbent tgG
I
+> 28t > +
Cellulose- _ >X ._.
90
700 ) r
600
rI sotation of
Isolation of santi-hormome leo
anti-tgG tgG
