Comparative Study Of Both Versions Of An Immunoassay ...

Adalimumab (ADA,® Humira, Abbott Laboratories, North Chicago, Illinois, USA) is a fully human monoclonal antibody that specifically binds to tumor necrosis factor α (TNΦ–α) neutralizing its biological function and modulating its response.Despite its proven efficacy widely adopted in different clinical indications, some patients do not respond or have a loss of response over time. One possible explanation is that, at steady state, serum ADA levels do not necessarily ensure that effectiveness is achieved. In some cases this has been associated with the presence of anti-ADA antibodies that form complexes with,1,2 ADA, increasing its clearance. Furthermore, quantification of therapeutic levels of ADA at the end of the dosing interval in non-responders provides valuable information in the subsequent selection of the new treatment.3 Also, the development of dose-response curves can lead to dose spacing of this drug in patients in clinical remission.4

Until now, decisions in these cases were based solely on the clinical course of the patient. However, there is consistent and gradually increasing literature showing that the drug level measurements and anti-drug antibodies are clinically relevant for the individualization of treatment.5

As of 2 years ago an enzyme immunoassay (ELISA) for the quantification of serum-free ADA and anti-ADA antibodies (Promonitor® Proteomika SL, distributed by Menarini Diagnostics SA®) is marketed in our country with precision, linearity and clinical validation criteria suitable for therapeutic drug monitoring of ADA.6,7 Recently, the manufacturer has released a new version with significant changes regarding the practicability of the analytical assay.

The objective of this paper is to describe the results of the comparative study between the 2 versions of ELISA marketed for therapeutic drug monitoring of ADA in patients with rheumatoid arthritis (RA).

We have selected 140 serum samples from patients with RA treated with ADA40mg every 14 days, with different drug concentrations and anti-drug antibodies, so that the entire analytical range of the new technique is covered (from 0.024 to 12mg/L and 3.5–2000AU/ml). For each patient a sample of 5mLserum was obtained before subcutaneous drug administration and stored frozen at -80° C until analysis in duplicate with 2 versions of ELISA, following the conditions specified by the manufacturer.

In the first version of the assay (V1), for the determination of levels of ADA, a plate was coated with TNΦ–α immobilized by a monoclonal antibody in a first incubation. And for the determination of anti-ADA antibodies, the samples were added to the wells with prior drug immobilization. After incubation with the patient sample, in both cases, the detection was carried out using a biotin-labeled monoclonal antibody and the concentration was determined by colorimetric reaction (450nm). Calibration curves were constructed with 10-fold dilutions of the standards (0.156–40ng/mL for ADA and 0.4–100AU/ml for anti-ADA antibodies), and each sample underwent 6 serial dilutions (1/10–1/10.240), in order to assure readings within the linear part of the calibration curve.

In the updated version (V2), the calibration range is larger: 1.25–60ng/mL and 3.13–200AU/mL for the quantification of ADA and anti-ADA antibodies, respectively. Dilutions per patient were reduced to 2 (1/10 and 1/200 for ADA, and undiluted and 1/10 for anti-ADA antibodies) and the labeled enzyme becomes peroxidase conjugated with streptavidin.

Interassay precision was calculated using the coefficient of variation. The Student's t test was employed for paired samples and was conducted to compare the concentrations of ADA between the 2 analyses with the same version of the test and using a Kappa statistic of agreement assessed following the categorization of results. With the correlation analysis, the relationship between the measurements with the two versions of the test was performed. The concordance correlation coefficient (CCC),8 and confidence intervals were calculated, assessing the average difference over the entire range of magnitudes measured by the Bland–Altman plot.9

The reproducibility of the new version of the assay was determined by processing 20 samples in 3 different nonconsecutive days using 2 different lots of reagent. Interassay imprecision was, on average, of 12.5%, showing an acceptable reproducibility.

Discrepancies between repetitions were evaluated by analyzing 30 samples of ADA in duplicate with each of the versions of the assay. Significant differences between the two measurements with V1 (P Copyright © 2013. Elsevier España, S.L.. All rights reserved

Subscribe to our newsletter

• Print
• Send to a friend
• Export reference
• Mendeley
• Statistics
• 

• Guide for authors
• Submit an article
• Language Editing services

• Español
• English

• Home
• All contents
  • Ahead of print
  • Latest issue
  • All issues
  • Supplements
  • Subscribe to our newsletter
• Publish your article
  • Guide for authors
  • Submit an article
  • Ethics in publishing
  • Diversity pledge
  • Open Access Option
  • Language Editing services
• About the journal
  • Aims and scope
  • Editorial Board
  • Subscribe
  • Contact
  • Advertising
• Metrics
  • Most often read
  • All metrics

• Léalo en español
• Download PDF
• Bibliography

¿Es usted profesional sanitario apto para prescribir o dispensar medicamentos?

Are you a health professional able to prescribe or dispense drugs?

Ayúdenos a conocerle mejor, indíquenos por favor su especialidad:

Help us get to know you better by telling us your specialty: