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P086. Development and validation of ELISA to measure serum anti TNFa levels

L. Greathead1, P. Kelleher1, A. Steel2, 1Chelsea and Westminster Hospital, Immunology, London, United Kingdom, 2Chelsea and Westminster Hospital, Gastroenterology, London, United Kingdom

Background

Use of biologic therapies in Crohn's disease has become routine. The ability to measure biologic drug levels has led to increasing availability of commercial and in house assays. Strategies to utilise drug level monitoring are yet to be subjected to randomised clinical trials. Standardisation of assays measuring biologically active compounds is hampering progress.

Methods

An in house ELISA to measure drug levels of infliximab and adalimumab was established. Technical performance of the assay was established using

  1. inter-assay patient samples,
  2. inter assay spiked normal sera,
  3. intra-assay performance across a range of drug levels,
  4. sensitivity and specificity established with 63 disease control samples with positive autoantibodies (CCP, ANA, TTG, LKS or Rf)
  5. Recovery of drug from spiked normal sera across the range of the standard curve
  6. Doubling dilutions were used to assess linearity of the curve in patients with high levels of drug
  7. Comparison to commercially available kit

Results

Inter-assay co-efficient of variation 10% for IFX at 28 ug/ml, 20% for IFX 1.7 ug/ml, for ADA 9% at 6.6 ug/ml and 14% at 2.4 ug/ml. For spiked normal sera CV ranged 6–13% across concentrations 0.08–25 ug/ml for IFX and 6–26% for ADA.

Intra-assay CV 2% for IFX 10 ug/ml up to 17% for IFX below 1 ug/ml, for ADA figures were 4% and 14% respectively.

Specificity analysis showed mean±2SD of 0.11 ug/ml for IFX and 0.15 for ADA. Sensitivity analysis showed recovery of drug from normal sera 91–106%.

Comparison with commercial ELISA kit showed expected high levels of correlation (Pearsons r = 0.9678, p < 0.0001), however Bland–Altman analysis showed significant bias, with the commercial kit producing higher results.

Nine patients with positive IgG autoantibodies on LKS screen were analysed with in house and commercial assay. None had ever been treated with anti TNF agents. Significant positive results were reported by the commercial assay and spiked normal sera reported higher values.

Figure: Bland–Altman analysis.

Table: IgG autoantibodies and anti TNFa measurements.
SeraCommercial assayIn house assay
Control 25 ug/ml42.0425.76
Control 10 ug/ml16.348.73
Control 2 ug/ml5.052.19
Control 0.4 ug/ml2.950.42
Control 0.08 ug/ml2.790.093
LKS+1.860.042
LKS+5.160.026
LKS+4.04<0.01
LKS+3.71<0.01

Conclusion

In house development of ELISA for therapeutic drug level monitoring is possible. Standardisation of the technologies is important as wide variations exist. The presence of autoantibodies could significantly alter assay performance.