Evaluation of Ig. G Subclass assays on the Radim Delta P. J. B. A. Johnson-Brett, H. D. 1, Carr-Smith A. R. 2 Bradwell Binding Site Ltd. , PO Box 11712, Birmingham, B 14 4 ZB, UK, 2 Division of Immunity & Infection, The Medical School, University of Birmingham, B 15 2 TT, UK(Presented at the AACC, Los Angeles, USA, 2004 (Clin Chem 2004, 50, No. 6 Supplement B-55, p. A 51) y =0. 99 x - 142. 8 r 2 = 0. 9979 y =1. 0054 x - 38. 289 r 2 = 0. 9999 Calculated results (mg/L) Ig. G 3 Method The instrument was programmed to construct a calibration curve from dilutions of a single calibration fluid. The standard curves were validated by assay of control fluids. Samples were initially measured at the standard programmed sample dilution, and if out of range, the instrument automatically re-measured the sample at appropriate alternative dilutions between neat and 1/2000, with all dilutions being made with the instrument’s onboard pipetting system. The main assay characteristics are summarised in the table below: Ig. G 2 Ig. G 1 Observed results (mg/L) Assay imprecision expressed as % coefficient of variation was from 1. 5 – 4. 9% for within-run and 4. 3 – 7. 9% for between-run studies (Table 2). The assays were found to be linear over their measuring ranges in each instance (Figure 1). Table 2: Intra- and inter-assay precision at three antigen concentrations for the TBS Ig. G subclass assays on the Radim Delta. Interference was within ± 10% when haemoglobin, bilirubin or Intralipid were added to serum samples of known Ig. G subclass concentrations (Table 3). Ig. G 4 Observed results (mg/L) Measurement of Ig. G subclasses (Ig. GSc) is useful in the diagnosis of Ig. G deficiencies where a deficiency in one Ig. GSc is masked by normal total Ig. G levels. Assays for measurement of Ig. GSc in serum have been available for many years and are routinely used in many immunology laboratories. Here we describe development of Ig. GSc assays for use on the Radim Delta, a small, low cost but fully automated bench-top nephelometer. Results Observed results (mg/L) Introduction Observed results (mg/L) 1 The 1, Showell y =1. 0123 x + 5. 0788 r 2 = 0. 9979 y =0. 9879 x - 5. 1032 r 2 = 0. 9979 0 0 Calculated results (mg/L) BN™II is a registered trademark of Dade Behring Gmb. H, Marburg, Gmb. H. 300 2000 400 500 Good agreement was observed when these assays were compared with TBS Ig. G subclass assays for the Dade-Behring BN™II (Figure 2). 2 Ig. G 1 Radim Delta (mg/L) Ig. G 2 Radim Delta (mg/L) y =0. 9605 x - 248. 4 r 2 = 0. 9981 Ig. G 1 BNII (mg/L) Ig. G 3 y =0. 9949 x - 1. 3982 r 2 = 0. 9929 0 500 1000 1500 Ig. G 3 Latex BNII (mg/L) y =0. 9578 x + 9. 8094 r 2 = 0. 9975 Ig. G 2 BNII (mg/L) 2000 Ig. G 4 Latex Radim Delta (mg/L) Intra- and inter-assay precision were assessed at three antigen levels for all four assays. The assay linearity was calculated over an antigen concentration range of 142 – 21620 mg/L for Ig. G 1, 288 – 10251 mg/L for Ig. G 2, 3 – 746 mg/L for Ig. G 3 and 3 – 474 mg/L for Ig. G 4. Serially diluted polyclonal serum samples were assayed and the results were compared with expected results. The assay was tested for possible interference from coexisting substances by adding haemoglobin (5 g/L), bilirubin (200 mg/L) and triglyceride (Intralipid 0. 3%) to serum samples and comparing to an equivalent sample blank. Comparison was made with the TBS Ig. G subclass assays for the Dade Behring BN™II by measuring samples from normal subjects and carrying out regression analysis. 200 1500 Figure 1: The assay linearity of TBS Ig. G subclass assays for use on the Radim Delta. Ig. G 3 Latex Radim Delta (mg/L) Table 1: The assay parameters of The Binding Site (TBS) Ig. G subclass assays for use on the Radim Delta. 1000 Calculated results (mg/L) Ig. G 1 Table 3: The interference test results for TBS Ig. G subclass assays for use on the Radim Delta. The percentage differences between samples diluted with saline and with the potentially interfering substances are shown. 500 Ig. G 4 y =0. 9513 x + 0. 0193 r 2 = 0. 9947 Ig. G 4 Latex BNII (mg/L) Figure 2: Comparison of results for TBS Ig. G subclass assays on the Radim Delta with existing TBS Ig. G subclass assays for use on the Dade-Behring BN™II. Conclusion The Ig. G subclass assays for the Radim Delta provide a rapid, precise method of measuring Ig. G subclasses in serum and show good agreement with existing assays. They should find good utility in medium to large laboratories where automatic sample redilution is preferable.
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