Newborn Screening Program for Cystic Fibrosis in Cuba: Three Years' Experience

ABSTRACT In Cuba, newborn screening (NBS) for cystic fibrosis (CF) was introduced in January 2019. The results from the first three years of the CF NBS program are presented. An IRT/IRT protocol was followed using a cut-off value of 50 ng/mL. In this period 281,717 neonates were screened, 2,197 samples had increased IRT values, and a second sample was necessary (recall rate=0.78%). In 686 (0.24%) neonates, IRT was still elevated, and they were referred for clinical evaluation. Twenty-one children were confirmed by sweat test and molecular biology. Eighteen newborns presented variant F508del. A false negative case was reported. Demographic data of 32,764 neonates were collected. The average age of sampling was six days with results available at 11 days of life, but 1.7% of the samples were collected 20 days after birth. The mean IRT value was 12.7±11.7 ng/mL (ranging 0-283 ng/mL) with a calculated 98.5 percentile value of 42.4 ng/mL. On average, the samples were processed five days after collection and two days after they were received at the laboratory. Although CF NBS program in Cuba is just beginning, it can be predicted that CF will be one of the most frequent inherited-metabolic diseases in the Cuban population.

Pilot study for cystic fibrosis neonatal screening: the Cuban experience.

Background In Cuba, no screening program for cystic fibrosis (CF) has been implemented yet. The ultramicro enzyme-linked immunosorbent assay (UMELISA)® TIR NEONATAL has been developed for the measurement of immunoreactive trypsin (IRT) in dried blood spots on filter paper. The analytical performance of the kit was evaluated in the national network of laboratories. Methods Newborn dried blood samples (DBS) were evaluated in 16 laboratories. An IRT/IRT/DNA protocol was followed using a cut-off value of 50 ng/mL. The mean, median and percentiles of the distribution were calculated and a two-sample t-test with unequal variance was used for statistical analysis. Influence of perinatal factors on IRT levels was analyzed. Results From January to June 2018, 6470 newborns were studied, obtaining a mean IRT value of 12.09 ng/mL (ranging 0-358 ng/mL) and a median of 8.99 ng/mL. Fifty-two samples (0.78%) were above the cut-off level and 16 samples (0.24%) were elevated in the re-screening process. One of them was confirmed positive by molecular biology (phe508del/c.3120 + 1G > A), constituting the first newborn screened and diagnosed early in Cuba. Second DBS samples were collected on average at 14 days and processed in the laboratory at 16 days of birth. Significant differences were observed (p < 0.05) when evaluating the influence of gender, birth weight (BW) and gestational age (GA) on the IRT values. Lower IRT concentrations were found in samples processed after 10 days of collection. Conclusions The performance of UMELISA® TIR NEONATAL in the laboratories has been satisfactory; hence CF newborn screening (NBS) was extended throughout the country from January 2019.

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

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.

An enzyme immunoassay for determining epidermal growth factor (EGF) in human serum samples using an ultramicroanalytical system.

Human epidermal growth factor is a small peptide consisting of 53 amino acid residues, which stimulates cell proliferation and is associated with several human carcinomas. A simple sandwich-type ultramicroELISA assay (UMELISA), based on the advantages of high affinity reaction between streptavidin and biotin has been developed for the measurement of EGF in human serum samples. Strips coated with a high affinity monoclonal antibody directed against EGF are used as solid phase, to ensure the specificity of the assay. The EGF assay was completed in 18 hr, with a measuring range of 39-2500 pg/mL. The intra- and inter-assay coefficients of variation were 4.4-7.3% and 0-5.1%, respectively, depending on the EGF concentrations evaluated. Percentage recovery ranged from 96-104%. Regression analysis showed a good correlation with the commercially available Human EGF Immunoassay Quantikine® ELISA kit (n = 130, r = 0.92, P < 0.01). The analytical performance characteristics of our UMELISA EGF endorse its use for the quantification of EGF in human serum samples.

Purification of human prostatic-specific antigen (hPSA) from seminal plasma by immunoaffinity chromatography using a monoclonal antibody anti total PSA.

Human prostate-specific antigen (hPSA) is found in serum and semen in a variety of forms, is form-free and complex, and has clinical importance to prostate cancer diagnosis. Here, a simple procedure is described for the efficient purification of hPSA from seminal plasma. The semen was clarified by centrifugation, and the isolation of PSA was carried out by immunoaffinity chromatography using the monoclonal antibody anti total PSA CB-PSA.4. The recuperation of PSA from seminal plasma by this procedure was 66.4%, and a purification factor of 65.8 was reached. The specificity activity obtained was 0.79 mg PSA/mg protein, and the preparation appeared homogeneous by SDS-PAGE and immunoelectrophoresis. A molecular weight of preparation was 30.46 kDa by SDS-PAGE, similar to the commercial PSA. These results indicate that immunoaffinity purification of PSA by means of this monoclonal antibody is a simple one-step procedure for the production of biologically active, highly purified human PSA.

Specific monoclonal antibody against human trypsin.

A monoclonal antibody (MAb) directed against human trypsin-1 has been produced by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. Antibodies were screened by ultramicro enzymelinked immunosorbent assay (UMELISA). MAb was purified by affinity chromatography on protein A-Sepharose, and MAb had a high affinity for trypsin-1 with the affinity constant equal 1.79 x 10(9) L/mol. Specificity was studied by UMELISA using cross-reactant proteins; MAb gave a positive reaction with native trypsinogen-1 and with the same protein after reduction. Antibody appeared to be directed against sequential epitope. One-step purification is described. The method evolved the adsorption of the enzyme onto a Sepharose-MAb(3H9) affinity column. The collected fraction was characterized and is available for immunization of BALB/c mice and for the preparation of a standard for immunoenzymatic assay.

Monoclonal antibody against human trypsin: production, characterization, and use for diagnosis.

A monoclonal antibody (MAb) directed against human immunoaffinity purified trypsinogen has been produced by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. Antibodies were screened by ultramicro-enzyme-linked immunosorbent assay (UMELISA). The MAb was purified by affinity chromatography on protein A-sepharose, and MAb had a high affinity for trypsinogen with the affinity constant equal 2.06 x 10(9) L/mol. Specificity was studied by UMELISA using cross-reactant proteins; MAb gave a positive reaction with native trypsinogen-1 but did not react with the same protein after reduction. The antibody seem to be directed against conformational epitope. The MAb obtained was characterized immunologically and used to develop UMELISA for detection Trypsin. This monoclonal assay enabled the detection of 2.8 ng/mL.

Evaluación de un péptido quimérico NS4/NS5 para la detección de anticuerpos contra VHC / Evaluation of a chimeric peptide NS4 and NS5 for HCV antibody

Para el diagnóstico del virus de la hepatitis C, se utilizan las pruebas de Elisa, debido a su sensibilidad y especificidad. Gran parte de estas pruebas se basa en el empleo de péptidos sintéticos y, en la acutalidad, de péptidos quiméricos. En este estudio se sintetizó un péptido quimérico que comprende secuencias inmunodominantes de las regiones no estructurales NS4 y NS5 del virus de la hepatitis C. El péptido representativo de las dos secuencias está separado por un brazo espaciador de dos residuos de glicina. El peptido se evaluó como antígeno en un ensayo UMELISA, utilizando muestras positivas (n=30), muestras negativas (n=40) y el panel de Boston Biomedica Inc. (n=40). Los resultados del peptido quimérico NS4/NS5 se compararon con los resultados de los péptidos individuales NS4 y NS5 y con la mezcla de estos péptidos. Los resultados de los péptidos individuales coinciden con los datos informados en la literatura. Con la mezcla de los péptidos, no aumentó la detección de muestras positivas, pero con el péptido quimérico se logró aumentar la sensibilidad

Evaluación de diferentes péptidos de la región estructural del virus de la hepatitis C / Evaluation of different peptides in hepatitis C virus structural region

Para el diagnóstico del virus de la hepatitis C, se utilizan ampliamente las pruebas de Elisa por su sensibilidad y especificidad. Gran parte de ellos se basa en el empleo de péptidos sintéticos. Una de las zonas más conservadas y de gran antigenicidad es la región estructural del virus. en este estudio, se sintetizaron siete péptidos de zonas informadas como altamente antiénicas de la región estructural del virus de la hepatitis C. Los péptidos sintetizados representan la región del núcleo del virus. Se realizó la evaluación y comparación de los resultados de los péptidos sintetizados, para lo cual se emplearon muestras positivas (n=72) y negativas (n=42). Con los péptidos más cercanos a la región amino terminal, la reactividad fue alta, pero fue disminuyendo a medida que nos acercábamos a la región carboxilo terminal. Estos resultados coinciden con lo informado por otros autores

UltramicroELISA indirecto para la detección de anticuerpos totales a Citomegalovirus en suero humano / Indirect ultramicroElisa for the detection of total antibodies against cytomegaloviruses in human blood

We have standardized an indirect ultramicro ELISA assay for detecting antibodies to human Cytomegalovirus (CMV) using human serum samples (UMELISA CMV). The optimal concentration of coating antigen (30 ug/ml), serum dilution (1:40) and anti-human conjugate working dilution (1:1500), were determined by a check board titration method. The UMELISA CMV was compared with the latex agglutination test for antibodies to CMV (Dupont de Nemours) and with an indirect immunofluorescent method. The results have showed the high coincidence, sensitivity and especificity of the proposed assay regarding the two methods compared with, and supporting its use either for a blood donors screening or in the serological diagnosis of this infection by paired serum samples.