Insulin levels in samples are routinely measured using radioimmunoassays, including Scintillation Proximity Assays (SPA). Protein A beads have previously been used to capture insulin and primary antibody, but this format was subject to interference by other nonspecific antibodies in test serum samples. By using secondary antibody coated beads, such as anti-guinea pig IgG antibody beads, insulin in serum samples can be measured directly without the need for pre-treatment of the sample.
Insulin plays an important role in the regulation of metabolism; its secretion is stimulated by a high concentration of blood glucose. In addition to regulating the uptake of blood glucose into the cells, insulin also promotes the synthesis of protein, lipid and glycogen, and inhibits the degradation of these compounds(1). The lack of insulin results in diabetes mellitus.
Insulin is routinely measured by immunoassay and this method has been extended to the use of SPA technology with protein A beads. However, protein A beads are not suitable for assaying serum samples due to interference from other non-specific antibodies. This problem can be overcome through the use of secondary antibody coated SPA beads.
Antibody bound insulin reacts with either protein A or anti-guinea pig IgG antibody which is coupled to PVT SPA beads. Any [125I]insulin that is bound to the primary antibody is in close enough proximity to generate a light signal from the SPA bead. However, unbound [125I]insulin is unable to stimulate the bead, and therefore is not detected (Figure 1). As this eliminates the need for a washing step, the assay is amenable to high throughput screening and processing of multiple samples in a microplate format.
Figure 1. SPA radioimmunoassay concept.
To compare the two different bead types, protein A (Amersham Biosciences, RPNQ0019) and antiguinea pig IgG antibody (Ab) coated beads (Amersham Biosciences, RPNQ0178) were used in an SPA radioimmumoassay, with guinea pig anti-insulin antibody to generate dose-response curves and to determine the concentration of insulin in test serum samples. The standard used was porcine insulin reconstituted in 0.01N HCl to 1mg/ml. This was diluted in assay buffer (PBS containing 0.1% (w/v) BSA, 0.1% (v/v) TweenTM 20, 0.05% (w/v) sodium azide, pH 7.5) to cover the range 0.0068- 400ng/ml. The zero wells consisted of assay buffer without standard. Insulin samples from rat serum (25μl), or standard, (25μl, 0.00084-50ng/ml equivalent to 0.00017-10ng/well) was incubated with 50μl guinea pig anti-rat insulin antizerum, 50μl [125I]insulin (Amersham Biosciences, IM166, approximately 10,000 cpm) and 75μl of either protein A or anti-guinea pig IgG antibody PVT SPA beads (0.25mg).
Non-specific binding was determined in the absence of specific guinea pig antizerum. The plates were sealed and shaken at room temperature for 1 hour, before incubation at room temperature without agitation until equilibrium was reached. The amount of [125I]insulin bound to the beads was determined by counting in a Wallac 1450 MicroBeta™ liquid scintillation counter and a time course was carried out to determine when the assay reached equilibrium (Figure 2).
Figure 2. Time course obtained using the insulin SPA radioimmunoassay and protein A (•) or antiguinea pig IgG antibody (����) PVT beads. Assay conditions as described in the text. Results are means ± SEM (n=3).
With both bead types, the assay reached equilibrium after 20 hours. Dose-response curves prepared using protein A PVT beads were compared with those prepared using anti-guinea pig IgG antibody PVT beads. Typical results are shown in Figure 3.
Figure 3. Standard curves obtained using the insulin SPA radioimmunoassay and protein A (•) or antiguinea pig IgG antibody (����) PVT beads. Assay conditions as described in the text. Results are means ± SEM (n=3).
No significant differences in curve shape parameters were observed when assays were carried out with either bead type. EC50 values were determined for porcine insulin using both protein A and secondary antibody coated beads, values of 0.14ng/well and 0.15ng/well were obtained respectively.
To further compare the two bead types, the concentration of insulin in rat serum samples was determined (Tables 1 and 2).
Table 1.Determination of the insulin concentration of rat serum samples using protein A beads.
Assay conditions as described in the text. Results are means ± SD, (n=5).
Sample ng insulin/ml %CV
Table 2. Determination of the insulin concentration of rat serum samples using anti-guinea pig IgG Ab beads. Assay conditions as described in the text. Results are means ± SD, (n=5).
Different results were obtained using the two bead types so the samples were assayed using a non- SPA radioimmunoassay, the Biotrak™ rat insulin assay (Amersham Biosciences, RPA 547). This assay system involves simple magnetic separation of antibody bound tracer from tracer in the free fraction by the use of an Amerlex™-M second antibody preparation. Results were compared to those obtained using the two SPA bead types to determine which was closer to the independently derived value (Table 3).
Table 3.Determination of the insulin concentration of rat serum samples using the Biotrak rat insulin assay. Results are means ± SD, (n=3).
When neat rat serum samples were assayed, the results obtained using anti-guinea pig IgG Ab beads were closer to the values obtained using the non-SPA RIA, than results obtained using protein A beads.
This highlights the problem of using protein A SPA beads with serum samples as the assay is prone to significant interference from other antibodies. This can be avoided by purifying the serum to remove the contaminating IgG, or by diluting the serum samples. Dilution is only possible if the concentration of insulin in the diluted sample is within the range of the assay. These extra steps are not amenable to high throughput screening and can be avoided if protein A beads are replaced in the assay by a secondary antibody coated SPA bead that is compatible with the primary antibody.
The introduction of anti-guinea pig IgG antibody SPA beads allows serum samples to be assayed directly when using guinea pig anti-insulin antibodies. This eliminates the need for purification or dilution of the test serum, and there is little or no interference from antibodies within the serum.
These experiments were performed in collaboration with S. Kahl and J. Swartling at Lilly Research Laboratories, (USA) who also kindly supplied the porcine insulin.
References
- O’Riordan, J.L.H., Malan, P.G. and Gould, R.P., eds Essentials of Endocrinology Blackwell Scientific Publications, Oxford (1988).