Immunoassay of serum polypeptide hormones by using 125I-labelled anti(-immunoglobulin G) antibodies

Abstract

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 immunoglobulin G antibodies to the polypeptide hormones were reacted with 125-I-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) 125I-labelled anti-(immunoglobulin G) antibodies and anti-hormone antibodies were simultaneously eluted from the hormone-containing immunoadsorbent by dilute HCl, pH 2.0. After elution the anti-(immunoglobulin G) antibodies and antihormone antibodies were allowed to recombine at pH 8.0 and 4 degrees 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, 100 pg of bovine insulin/ml, 80 pg of bovine parathyroid hormone/ml and 150 pg 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 125I-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 450 ng of polypeptide hormone-antibody protein. An additional advantage of the method is that a single iodination of the readily available antibodies to immunoglobulin G allows the establishemnt of several polypeptide hormone assays

Type 2 (non-insulin-dependent) diabetes mellitus: the thrifty phenotype hypothesis

“…A potential solution to the assay problem lay in the exploitation ofimmunoassay techniques involving the use of labelled antibodies termed"immunoradiometric" assays. These were developed in a variety of configurations in one of our laboratories over the years [10][11][12][13][14] leading to what was termed an "indirect two siteimmunoradiometric assay" ofhumanproinsulin [15]. It was something of a surprise to discover subsequently, with the advent of bioengineered human proinsulin [16], that this assay did not detect intact human proinsulin but rather the sum of the partially proteolysed derivates on the pathway of conversion to insulin [17].…”

Immunoaffinity purification of intact, metabolically active, cholinergic nerve terminals from mammalian brain

“…Total protein in subcellular fractions was measured by the method of Bradford (1976), and protein covalently coupled to cellulose was measured by the method of Lowry et al (1951), with sheep IgG as standard. The protein content adsorbed on the immunoadsorbent was assayed after elution with 0.5ml of lOmM-HCl in 0.25Msucrose at 4°C for 5min (Beck & Hales, 1975).…”

Sensitive and specific two-site immunoradiometric assays for human insulin, proinsulin, 65-66 split and 32-33 split proinsulins

“…At that time a major disadvantage of procedures using labelled antibodies was the need for a relatively large amount of pure antibody. In an attempt to reduce this problem, we devised the indirect two-site immunoradiometric assay (Beck & Hales, 1975) and used this approach to establish an assay for human proinsulin (Rainbow et al, 1979). However, we subsequently discovered that this assay detected partially processed proinsulins and did not recognize intact proinsulin (Gray et al, 1984).…”