A novel high-resolution melting analysis strategy for detecting cystic fibrosis-causing variants.

Cystic fibrosis (CF), an autosomal recessive disease, is caused by variants in both alleles of the CF transmembrane conductance regulator (CFTR) gene. A new assay based on allele-specific polymerase chain reaction and high-resolution melting analysis was developed for the detection of 18 CF-causing CFTR variants previously identified in Cuba and Latin America. The assay is also useful for zygosity determination of mutated alleles and includes internal controls. The reaction mixtures were normalized and evaluated using blood samples collected on filter paper. The evaluation of analytical parameters demonstrated the specificity and sensitivity of the method to detect the included CFTR variants. Internal and external validations yielded a 100% agreement between the new assay and the used reference tests. This assay can complement CF newborn screening not only in Cuba but also in Latin America.

An enzyme immunoassay to determine human chorionic gonadotropin (HCG) in serum and urine samples using an ultra-microanalytical system.

The determination of Human Chorionic Gonadotropin (HCG) in biological fluids is of great interest in the early pregnancy diagnostics, the evaluation of pregnancy disorders, as a tumor marker, as a screening procedure for anti-doping control, and many other purposes. A simple sandwich-type UltraMicro Enzyme-Linked ImmunoSorbent Assay (UMELISA) has been developed for the measurement of HCG in serum and urine samples. Strips coated with a high affinity MAb directed against HCG are used as solid phase, to ensure the specificity of the assay. The HCG assay was completed in 1.5 h, with a measuring range of 0.76-400 mIU/mL. The intra- and inter-assay coefficients of variation were lower than 10 %, depending on the HCG concentrations evaluated. Recovery percentages were 96.43-97.16 % (serum) and 98.10-99.04 % (urine). The assay detected intact HCG, nicked HCG, HCG ß, and nicked HCG ß, and did not recognize any of the interfering molecules tested. Regression analysis showed a good correlation with Elecsys in serum (n = 1459, r = 0.952, ρc = 0.948) and urine (n = 869, r = 0.988, ρc = 0.978). A good correlation was also found with 84 RIQAS samples analyzed with the kits Elecsys (r = 0.969, ρc = 0.957), Architect (r = 0.982, ρc = 0.970), Dimension (r = 0.989, ρc = 0.977), and Bioscience (r = 0.992, ρc = 0.980), all with a p < 0.01. Comparison with transvaginal ultrasonography in early pregnancy detection showed a specificity and a sensitivity of 100 % (n = 2385, κ = 1). The analytical performance characteristics of UMELISA HCG endorse its use for the quantification of HCG in serum and urine samples. This assay will make a cost-effective diagnostic kit accessible to low-income countries and is now available in the Cuban Public Health System.

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.

Generation and characterization of monoclonal antibodies against thyroid-stimulating hormone for newborn screening of congenital hypothyroidism.

Congenital hypothyroidism (CH) is one of the most frequent inherited-metabolic diseases in the world, and the main cause of treatable mental retardation in children. Because signs and symptoms of this disease are often scarce and not easily recognizable, newborns are screened for the early CH detection at birth. The Center of Immunoassay (CIE) has developed the UMELISA® TSH Neonatal and UMELISA® TSH to determine neonatal thyroid-stimulating hormone (TSH) levels in dried blood and serum samples. Both reagent kits use the same polystyrene plates coated with anti-ß-TSH monoclonal antibodies (MAbs), but one of these is commercially acquired. Obtaining appropriate anti-TSH MAbs at the CIE would guarantee economic independence and security in the production of these kits. Immunization of mice with TSH led to the generation of 7G11E3, an anti-ß-TSH IgG1-secreting hybridoma. The high affinity of 7G11E3 MAb and its characteristic epitopic recognition explain its better performance when adsorbed to UMELISA® plates for capturing low amounts of TSH in comparison with the studied MAbs. Performance of assays using polystyrene plates coated with 7G11E3 MAb was studied. Recovery percentages (100.0-106.7% for UMELISA® TSH NEONATAL and 97.3-99.0% for UMELISA® TSH) and intra (5.2-7.9% for UMELISA® TSH NEONATAL and 3.2-5.3% for UMELISA® TSH) and inter (6.6-7.7% for UMELISA® TSH NEONATAL and 5.2-8.0% for UMELISA® TSH) coefficients of variation were similar to the ones described for the commercial kits. Limits of detection and quantification were 1.0 and 3.8 mIU/L for UMELISA® TSH NEONATAL, and 0.3 and 0.6 mIU/L for UMELISA® TSH, respectively. The results also showed high overall concordance between assays (n = 2 019, ρc = 0.90 for UMELISA® TSH NEONATAL and n = 200, ρc = 0.94 for UMELISA® TSH). The 7G11E3 MAb meets the requirements for its use in the plates of UMELISA® TSH kits for CH newborn screening in Cuba. Abbreviations: CECMED, Center for the State Control of Medicaments and Medical Equipment and Devices; CH, congenital hypothyroidism; CIE, Center of Immunoassay; CLSI, Clinical and Laboratory Standards Institute; CV coefficient of variation; DBS, dried blood spots; LOB, limit of blank; LOD, limit of detection; LOQ, limit of quantitation; SD, standard deviation; Sr, repeatability standard deviation; SUMA, Ultra Micro Analytic System; UMELISA, ultramicro enzyme-linked immunosorbent assay.

Generation of monoclonal antibodies against 17α-hydroxyprogesterone for newborn screening of congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by the deficiency of one of the five enzymes involved in the biosynthesis of corticosteroids. The most common form of the disease is the lack of 21-hydroxylase which provokes an accumulation of high levels of 17α-hydroxyprogesterone (17-OHP), the main biochemical marker for illness detection. Given the significance of neonatal diagnosis for ensuring a timely treatment to patients suffering from CAH, newborn screening is worldwide performed for the determination of 17-OHP from dried blood spots on filter paper. The non-specificity of antisera employed in immunoassays and the cross-reaction with fetal adrenal hormones produce an overestimation in the 17-OHP quantification. Immunization of mice with 17-OHP-3-(O-carboxymethyl) oxime-bovine serum albumin led to the generation of 15 anti-17-OHP IgG1-and-IgG2b-secreting hybridomas. The 6E2G9 monoclonal antibody presents cross-reactivity values similar to those achieved by rabbit antibodies employed in the solid phase of UMELISA® 17-OH Progesterona Neonatal, assay for the newborn screening of CAH in Cuba. Additionally, the use of 6E2G9 in the evaluation of dried blood spots samples from newborns on filter paper showed a decrease in the mean 17-OHP levels, thus demonstrating it can replace the conventional rabbit antisera.