Generation and characterization of murine monoclonal antibodies against immunoreactive trypsinogen for newborn screening of cystic fibrosis.

Cystic fibrosis (CF) is a multisystem disorder that reduces quality of life and survival in affected individuals. In newborns, the release of pancreatic enzymes into the blood raises the levels of immunoreactive trypsinogen (IRT), the main marker for CF screening, which is detected in dried blood samples on filter paper by immunoenzymatic assays. In Cuba, CF has an estimated incidence of 1/9862 live births and should be included in the national basic newborn screening (NBS) panel given its benefits in terms of nutrition, lung function and survival. The Immunoassay Center develops and produces diagnostic kits allowing the establishment of large-scale NBS programs for inherited metabolic disorders in Cuba and other Latin American countries. IRT-specific monoclonal antibodies (MAbs) obtained at the Immunoassay Center are essential for developing an affordable immunoassay for IRT to support CF NBS in our low-income country. An immunization scheme with trypsinogen-1 originated two IgG1-producing murine hybridomas. 4C9C9 and 4C9E11 MAbs recognized different determinants on both trypsin-1 and trypsin-2 molecules. Both antibodies identified conformational epitopes on the molecule of trypsin-1 and of its zymogen. As 4C9E11 MAb cross-reacted with proteins structurally and functionally related to trypsinogen, it was used as revealing antibody in a sandwich-type UMELISA® assay for IRT determination with 4C9C9 MAb for capture. This combination, aside from detecting several commercially available trypsins, adequately quantified IRT from dried blood samples on filter paper of newborns. The evaluation of the assay's accuracy yielded percentage recoveries ranging 93.3-109.2% for commercial controls. The properties of the studied MAbs demonstrate their suitability for being used in a sandwich-type UMELISA® assay for the CF NBS in Cuba.

Performance characteristics of a fast real-time PCR assay for hepatitis B virus DNA quantification.

Detection of hepatitis B virus (HBV) DNA is particularly important for detection of early acute and of occult HBV infection. On the other hand, HBV DNA detection and quantification are essential to diagnose and treat chronic HBV infection. In this study, we evaluated the performance of the real-time PCR SUMASIGNAL VHB (un paso) (Immunoassay Center, Cuba). The clinical and analytical specificity of the assay was 100%. Intra-assay and inter-assay coefficients of variation ranged from 0.50 to 2.53% and from 1.23 to 3.03%, respectively. A strong correlation (r=0.926; P<0.001) with the COBAS® AmpliPrep/COBAS® TaqMan® HBV Test, v2.0 (Roche Molecular Systems, Inc.) was obtained. The limit of detection using the third WHO international standard for HBV DNA was 377.47 IU/mL. The test was able to detect the most prevalent HBV genotypes (A-G) equally well. In conclusion, the SUMASIGNAL VHB (un paso) is a sensitive, specific, precise and accurate assay for the quantification of serum and plasma HBV DNA. Thus, this simple and fast real-time PCR test can be used as an aid in diagnosing an HBV infection and monitoring drug efficacy.