Evaluation of IgG Subclass assays on the Radim Delta

1. Evaluation of IgG Subclass assays on the Radim Delta P.J. Showell1, B.A. Johnson-Brett, H.D. Carr-Smith1, A.R. Bradwell2 1The Binding Site Ltd., PO Box 11712, Birmingham, B14 4ZB, UK, 2Division of Immunity & Infection, The Medical School, University of Birmingham, Birmingham, B15 2TT, UK(Presented at the AACC, Los Angeles, USA, 2004 (Clin Chem 2004, 50, No.6 Supplement B-55, pA51) Introduction Measurement of IgG subclasses (IgGSc) is useful in the diagnosis of IgG deficiencies where a deficiency in one IgGSc is masked by normal total IgG levels. Assays for measurement of IgGSc in serum have been available for many years and are routinely used in many immunology laboratories. Here we describe development of IgGSc assays for use on the Radim Delta, a small, low cost but fully automated bench-top nephelometer. • Results • 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 (Figure1). IgG2 IgG1 y =0.99x - 142.8 r2 = 0.9979 y =1.0054x - 38.289 r2 = 0.9999 Observed results (mg/L) Observed results (mg/L) Calculated results (mg/L) Calculated results (mg/L) IgG3 IgG4 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 on-board pipetting system. The main assay characteristics are summarised in the table below: y =0.9879x - 5.1032 r2 = 0.9979 y =1.0123x + 5.0788 r2 = 0.9979 Observed results (mg/L) Observed results (mg/L) Observed results (mg/L) • Table 2: Intra- and inter-assay precision at three antigen concentrations for the TBS IgG subclass assays on the Radim Delta. 0 500 1000 1500 2000 0 100 200 300 400 500 Calculated results (mg/L) Calculated results (mg/L) • Figure 1: The assay linearity of TBS IgG subclass assays for use on the Radim Delta. • Interference was within ±10% when haemoglobin, bilirubin or Intralipid were added to serum samples of known IgG subclass concentrations (Table 3). • Good agreement was observed when these assays were compared with TBS IgG subclass assays for the Dade-Behring BN™II (Figure 2). 2 IgG2 IgG1 y =0.9605x - 248.4 r2 = 0.9981 y =0.9578x + 9.8094 r2 = 0.9975 IgG1 Radim Delta (mg/L) IgG2 Radim Delta (mg/L) IgG1 BNII (mg/L) IgG2 BNII (mg/L) • Table 3: The interference test results for TBS IgG subclass assays for use on the Radim Delta. The percentage differences between samples diluted with saline and with the potentially interfering substances are shown. • Table 1: The assay parameters of The Binding Site (TBS) IgG subclass assays for use on the Radim Delta. IgG3 IgG4 y =0.9949x - 1.3982 r2 = 0.9929 y =0.9513x + 0.0193 r2 = 0.9947 • 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 – 21620mg/L for IgG1, 288 – 10251mg/L for IgG2, 3 – 746mg/L for IgG3 and 3 – 474mg/L for IgG4. Serially diluted polyclonal serum samples were assayed and the results were compared with expected results. The assay was tested for possible interference from co-existing substances by adding haemoglobin (5g/L), bilirubin (200mg/L) and triglyceride (Intralipid 0.3%) to serum samples and comparing to an equivalent sample blank. Comparison was made with the TBS IgG subclass assays for the Dade Behring BN™II by measuring samples from normal subjects and carrying out regression analysis. IgG3 Latex Radim Delta (mg/L) IgG4 Latex Radim Delta (mg/L) 0 500 1000 1500 2000 IgG3 Latex BNII (mg/L) IgG4 Latex BNII (mg/L) • Figure 2: Comparison of results for TBS IgG subclass assays on the Radim Delta with existing TBS IgG subclass assays for use on the Dade-Behring BN™II. • Conclusion • The IgG subclass assays for the Radim Delta provide a rapid, precise method of measuring IgG 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. BN™II is a registered trademark of Dade Behring GmbH, Marburg, GmbH.