Applied Biochemistry and Biotechnology

, Volume 186, Issue 4, pp 1034–1046 | Cite as

An Enzyme Immunoassay for Determining Immunoreactive Trypsinogen (IRT) in Dried Blood Spots on Filter Paper Using an Ultra-Microanalytical System

  • Elisa M. Castells MartínezEmail author
  • Ernesto Carlos González
  • Yileidis Tejeda
  • Amarilys Frómeta
  • Odalys Martín
  • Maryeris Espinosa
  • Lesley del Río
  • Pedro L. Pérez
  • Greilys Morejón
  • Yenitse Perea
  • Antonio Melchor


Cystic fibrosis (CF) is a severe autosomal recessive disorder. It is caused by mutations in the CF transmembrane conductance regulator gene. Early diagnosis of CF can be carried out by determining high immunoreactive trypsinogen (IRT) blood values in newborns. A simple sandwich-type ultramicroELISA assay (UMELISA®) has been developed for the measurement of IRT in dried blood spots on filter paper. Strips coated with a high affinity monoclonal antibody directed against IRT are used as solid phase, to ensure the specificity of the assay. The assay is carried out within 20 h. The useful rank of the curve is 0–500 ng/mL, and the lowest detectable concentration is 4.8 ng/mL. Intra- and inter-assay coefficients of variation were lower than 10%. The recovery mean value was 100.3 ± 11.2%. Cross-reactivity with proteins structurally related to IRT (α2-macroglobulin, α1-antitrypsin, and human chymotrypsin) was lower than the detection limit of the assay. Four thousand four hundred six newborn samples from the Cuban Newborn Screening Program were analyzed, and the mean IRT concentration was 12.8 ng/mL. Higher IRT values were obtained when samples were eluted overnight. Regression analysis showed a good correlation with the commercially available AutoDELFIA® Neonatal IRT kit (n = 3948, r = 0.885, ƙ = 0.976, p < 0.01). The analytical performance characteristics of our UMELISA® TIR Neonatal suggest that it can be used for the neonatal screening of CF.


Cystic fibrosis IRT Newborn screening UMELISA® Blood spots 



cystic fibrosis


newborn screening


immunoreactive trypsinogen


enzyme-linked immunosorbent assay


Ultra Micro Analytical System


ultramicro enzyme-linked immunosorbent assay



We thank Lic. José Luis Fernández Sierra for his valuable help in the final language revision of the manuscript.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Elisa M. Castells Martínez
    • 1
    Email author
  • Ernesto Carlos González
    • 2
  • Yileidis Tejeda
    • 3
  • Amarilys Frómeta
    • 1
  • Odalys Martín
    • 1
  • Maryeris Espinosa
    • 1
  • Lesley del Río
    • 1
  • Pedro L. Pérez
    • 1
  • Greilys Morejón
    • 4
  • Yenitse Perea
    • 1
  • Antonio Melchor
    • 5
  1. 1.Department of Neonatal ScreeningCenter of ImmunoassayHavanaCuba
  2. 2.R&D LaboratoryGK Pharmaceuticals CMOGuayamaPuerto Rico
  3. 3.ETAC UniversityTlalnepantlaMexico
  4. 4.Department of Monoclonal AntibodiesCenter of ImmunoassayHavanaCuba
  5. 5.Center of ImmunoassayHavanaCuba

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