A new enzyme-linked immunosorbent assay for a total anti-T lymphocyte globulin determination: Development, analytical validation, and clinical applications

Michela Montagna, Giorgio La Nasa, Maria E. Bernardo, Eugenia Piras, Maria A. Avanzini, Mario Regazzi, Franco Locatelli

Research output: Contribution to journalArticlepeer-review


Background: Anti-T lymphocyte globulin (ATLG) modulates the alloreactivity of T lymphocytes, reducing the risk of immunological posttransplant complications, in particular rejection and graft-versushost disease, after allogeneic hematopoietic stem cell transplantation (HSCT). We developed and validated a new enzyme-linked immunosorbent assay (ELISA) method to measure serum levels of total ATLG and evaluate the pharmacokinetics (PK) of the drug in children with b-Thalassemia, receiving allogeneic HSCT. Methods: Diluted serum samples were incubated with Goat-anti- Rabbit IgG antibody coated on a microtiter plate and then, with Goat-anti-Human IgG labeled with horseradish peroxidase. After incubation and washings, substrate solution was added and absorbance was read at 492 nm. ATLG concentrations in samples were determined by interpolation from a standard curve (range: 200-0.095 ng/mL), prepared by diluting a known amount of ATLG in phosphate-buffered saline (PBS). Low, medium, and high-quality control concentrations were 1.56, 6.25, and 25 ng/ mL, respectively. This method was developed and validated within the acceptance criteria in compliance with the Guidelines for a biological method validation: the sensitivity of the method was 0.095 ng/mL. We analyzed serum samples from 14 children with b-Thalassemia who received ATLG (Grafalon) at a dose of 10 mg/kg administered as intravenous (IV) infusion on days 25, 24, and 23 before HSCT (day 0). Blood sampling for PK evaluation was performed on days 25, 24, and 23 before and after drug infusion; and then from day 22 to +56. Results: The median total ATLG levels pre-IVand post-IV were 0 and 118 mcg/mL on day 25; 85.9 and 199.2 mcg/mL on day 24; 153 and 270.9 mcg/mL on day 23, respectively. The median PK values of CL was 0.0029 (range: 0.0028-0.0057) L$kg21$d21, Vd was 0.088 (range: 0.025-0.448) L/kg and t1/2 was 20.2 (range: 5.8- 50.2) days. Conclusions: These data suggest that given the marked interindividual variability of total ATLG disposition, the development of a validated ELISA method and the possibility to measure PK parameters in paediatric populations are essential steps to optimize drug therapeutic regimens.

Original languageEnglish
Pages (from-to)282-289
Number of pages8
JournalTherapeutic Drug Monitoring
Issue number3
Publication statusPublished - Jun 1 2017


  • ATG
  • Paediatric
  • Pharmacokinetics
  • Thalassemia

ASJC Scopus subject areas

  • Pharmacology
  • Pharmacology (medical)


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